INSECTICIDAL PROTEINS AND METHODS FOR THEIR USE

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

A sequence listing having the file name “5273USPCN_sequence_listing.txt” created on Jun. 1, 2018, and having a size of 1,771 kilobytes, is filed in ASCI format as a “text” file concurrently with the specification. The sequence listing is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This disclosure relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These pesticidal proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants.

BACKGROUND OF THE INVENTION

Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards and biopesticides provide greater target specificity than is characteristic of traditional broad-spectrum chemical insecticides. In addition, biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus. Microbial insecticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bt. These genetically engineered crops are now widely used in agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods. While they have proven to be very successful commercially, these genetically engineered, insect-resistant crop plants provide resistance to only a narrow range of the economically important insect pests. In some cases, insects can develop resistance to different insecticidal compounds, which raises the need to identify alternative biological control agents for pest control.

Accordingly, there remains a need for new pesticidal proteins with different ranges of insecticidal activity against insect pests, e.g., insecticidal proteins which are active against a variety of insects in the order Lepidoptera and the order Coleoptera including but not limited to insect pests that have developed resistance to existing insecticides.

SUMMARY OF THE INVENTION

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include nucleic acid molecules encoding sequences for pesticidal and insecticidal polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also include the pesticidal polypeptide sequences and antibodies to those polypeptides. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant. Compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds.

In particular, isolated or recombinant nucleic acid molecules are provided encoding Pseudomonas Insecticidal Protein-47 (PIP-47) polypeptides including amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Additionally, amino acid sequences corresponding to the PIP-47 polypeptides are encompassed. Provided are an isolated or recombinant nucleic acid molecule capable of encoding a PIP-47 polypeptide of SEQ ID NO: 2 as well as amino acid substitutions, deletions, insertions, fragments thereof and combinations thereof. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed. Also provided are isolated or recombinant PIP-47 polypeptides of SEQ ID NO: 2 as well as amino acid substitutions, deletions, insertions, fragments thereof and combinations thereof.

Methods are provided for producing the polypeptides and for using those polypeptides for controlling or killing a Lepidopteran, Coleopteran, nematode, fungi, and/or Dipteran pests. The transgenic plants of the embodiments express one or more of the pesticidal sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional genes for insect resistance, for example, one or more additional genes for controlling Coleopteran, Lepidopteran, Hemipteran or nematode pests. It will be understood by one of skill in the art that the transgenic plant may comprise any gene imparting an agronomic trait of interest.

Methods for detecting the nucleic acids and polypeptides of the embodiments in a sample are also included. A kit for detecting the presence of a PIP-47 polypeptide or detecting the presence of a nucleotide sequence encoding a PIP-47 polypeptide in a sample is provided. The kit may be provided along with all reagents and control samples necessary for carrying out a method for detecting the intended agent, as well as instructions for use.

The compositions and methods of the embodiments are useful for the production of organisms with enhanced pest resistance or tolerance. These organisms and compositions comprising the organisms are desirable for agricultural purposes. The compositions of the embodiments are also useful for generating altered or improved proteins that have pesticidal activity or for detecting the presence of PIP-47 polypeptides or nucleic acids in products or organisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B shows the amino acid sequence alignment of PIP-47Aa (SEQ ID NO: 1), PIP-47Ba (SEQ ID NO: 4), PIP-47Bb (SEQ ID NO: 6), PIP-47Fa (SEQ ID NO: 8) and the distant homologs: Plu2048 (SEQ ID NO: 10); Plu2046 (SEQ ID NO: 12); YP003041397 (SEQ ID NO: 14); YP003041380 (SEQ ID NO: 16); ZP17107944 (SEQ ID NO: 18); and ZP05881874 (SEQ ID NO: 20). The amino acid positions relative to PIP-47Aa (SEQ ID NO: 2) subjected to saturation mutagenesis are indicated by an arrow (↓) above the position. Three motifs: motif 1—positions 77-87 relative to SEQ ID NO: 2; motif 2—positions 107-120 relative to SEQ ID NO: 2; and motif 3—positions 233-247 relative to SEQ ID NO: 2 are identified above the PIP-47Aa sequence (SEQ ID NO: 2) and the positions are underlined in the PIP-47Aa sequence (SEQ ID NO: 2).

FIG. 2 shows an alignment of the amino acid sequences of PIP-47Aa (SEQ ID NO: 2), PIP-47Ba (SEQ ID NO: 4) and PIP-47Bb (SEQ ID NO: 6). The amino acid diversity between PIP-47Ba (SEQ ID NO: 4) and PIP-47Bb (SEQ ID NO: 6), and PIP-47Aa (SEQ ID NO: 2) is indicated with shading.

FIG. 3 shows an alignment of the amino acid sequences of PIP-47Aa (SEQ ID NO: 2), PIP-47Ba (SEQ ID NO: 4), PIP-47Bb (SEQ ID NO: 6), and PIP-47Fa (SEQ ID NO: 8). The amino acid diversity is indicated with shading.

FIG. 4 shows the T0 GH efficacy results for events generated from the PHP61246 and PHP61185 constructs. Efficacy for events derived both constructs was observed relative to negative control events as measured by root protection from Western corn rootworm. Root protection was measured according to the number of nodes of roots injured (CRWNIS=corn rootworm node injury score) using the method developed by Oleson, et al. (2005) [J. Econ Entomol. 98(1):1-8]. The root injury score is measured from “0” to “3” with “0” indicating no visible root injury, “1” indicating 1 node of root damage, “2” indicating 2 nodes or root damage, and “3” indicating a maximum score of 3 nodes of root damage.

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure.

As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the protein” includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

The present disclosure is drawn to compositions and methods for controlling pests. The methods involve transforming organisms with nucleic acid sequences encoding a PIP-47 polypeptides. In particular, the nucleic acid sequences of the embodiments are useful for preparing plants and microorganisms that possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions are pesticidal nucleic acids and proteins of bacterial species. The nucleic acid sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes, and for the generation of altered PIP-47 polypeptides by methods known in the art, such as site directed mutagenesis, domain swapping or DNA shuffling. The PIP-47 polypeptides find use in controlling or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with pesticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker; and velvet bean caterpillar e.g., Anticarsia gemmatalis Hübner and Coleoptera species including but not limited to Western corn rootworm (Diabrotica virgifera)—WCRW, Southern corn rootworm (Diabrotica undecimpunctata howardi)— SCRW, and Northern corn rootworm (Diabrotica barberi)—NCRW.

By “pesticidal toxin” or “pesticidal protein” is used herein to refer to a toxin that has toxic activity against one or more pests, including, but not limited to, members of the Lepidoptera, Diptera, Hemiptera and Coleoptera orders or the Nematoda phylum or a protein that has homology to such a protein. Pesticidal proteins have been isolated from organisms including, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidal proteins include but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13); from Pseudomonas protegens strain CHA0 and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal, 3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069); U.S. Pat. No. 6,048,838, and U.S. Pat. No. 6,379,946; a PIP-1 polypeptide of U.S. Ser. No. 13/792,861; an AflP-1A and/or AflP-1B polypeptide of U.S. Ser. No. 13/800,233; a PHI-4 polypeptide of U.S. Ser. No. 13/839,702; and δ-endotoxins including, but not limited to, the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51 and Cry55 classes of δ-endotoxin genes and the B. thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins include, but are not limited to Cry1Aa1 (Accession # AAA22353); Cry1Aa2 (Accession # Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4 (Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6 (Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8 (Accession #126149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession # AAD55382); Cry1Aa11 (Accession # CAA70856); Cry1Aa12 (Accession # AAP80146); Cry1Aa13 (Accession # AAM44305); Cry1Aa14 (Accession # AAP40639); Cry1Aa15 (Accession # AAY66993); Cry1Aa16 (Accession # HQ439776); Cry1Aa17 (Accession # HQ439788); Cry1Aa18 (Accession # HQ439790); Cry1Aa19 (Accession # HQ685121); Cry1Aa20 (Accession # JF340156); Cry1Aa21 (Accession # JN651496); Cry1Aa22 (Accession # KC158223); Cry1Ab1 (Accession # AAA22330); Cry1Ab2 (Accession # AAA22613); Cry1Ab3 (Accession # AAA22561); Cry1Ab4 (Accession # BAA00071); Cry1Ab5 (Accession # CAA28405); Cry1Ab6 (Accession # AAA22420); Cry1Ab7 (Accession # CAA31620); Cry1Ab8 (Accession # AAA22551); Cry1Ab9 (Accession # CAA38701); Cry1Ab10 (Accession # A29125); Cry1Ab11 (Accession #112419); Cry1Ab12 (Accession # AAC64003); Cry1Ab13 (Accession # AAN76494); Cry1Ab14 (Accession # AAG16877); Cry1Ab15 (Accession # AAO13302); Cry1Ab16 (Accession # AAK55546); Cry1Ab17 (Accession # AAT46415); Cry1Ab18 (Accession # AAQ88259); Cry1Ab19 (Accession # AAW31761); Cry1Ab20 (Accession # ABB72460); Cry1Ab21 (Accession # ABS18384); Cry1Ab22 (Accession # ABW87320); Cry1Ab23 (Accession # HQ439777); Cry1Ab24 (Accession # HQ439778); Cry1Ab25 (Accession # HQ685122); Cry1Ab26 (Accession # HQ847729); Cry1Ab27 (Accession # JN135249); Cry1Ab28 (Accession # JN135250); Cry1Ab29 (Accession # JN135251); Cry1Ab30 (Accession # JN135252); Cry1Ab31 (Accession # JN135253); Cry1Ab32 (Accession # JN135254); Cry1Ab33 (Accession # AAS93798); Cry1Ab34 (Accession # KC156668); Cry1Ab-like (Accession # AAK14336); Cry1Ab-like (Accession # AAK14337); Cry1Ab-like (Accession # AAK14338); Cry1Ab-like (Accession # ABG88858); Cry1Ac1 (Accession # AAA22331); Cry1Ac2 (Accession # AAA22338); Cry1Ac3 (Accession # CAA38098); Cry1Ac4 (Accession # AAA73077); Cry1Ac5 (Accession # AAA22339); Cry1Ac6 (Accession # AAA86266); Cry1Ac7 (Accession # AAB46989); Cry1Ac8 (Accession # AAC44841); Cry1Ac9 (Accession # AAB49768); Cry1Ac10 (Accession # CAA05505); Cry1Ac11 (Accession # CAA10270); Cry1Ac12 (Accession #112418); Cry1Ac13 (Accession # AAD38701); Cry1Ac14 (Accession # AAQ06607); Cry1Ac15 (Accession # AAN07788); Cry1Ac16 (Accession # AAU87037); Cry1Ac17 (Accession # AAX18704); Cry1Ac18 (Accession # AAY88347); Cry1Ac19 (Accession # ABD37053); Cry1Ac20 (Accession # ABB89046); Cry1Ac21 (Accession # AAY66992); Cry1Ac22 (Accession # ABZ01836); Cry1Ac23 (Accession # CAO30431); Cry1Ac24 (Accession # ABL01535); Cry1Ac25 (Accession # FJ513324); Cry1Ac26 (Accession # FJ617446); Cry1Ac27 (Accession # FJ617447); Cry1Ac28 (Accession # ACM90319); Cry1Ac29 (Accession # DQ438941); Cry1Ac30 (Accession # GQ227507); Cry1Ac31 (Accession # GU446674); Cry1Ac32 (Accession # HM061081); Cry1Ac33 (Accession # GQ866913); Cry1Ac34 (Accession # HQ230364); Cry1Ac35 (Accession # JF340157); Cry1Ac36 (Accession # JN387137); Cry1Ac37 (Accession # JQ317685); Cry1Ad1 (Accession # AAA22340); Cry1Ad2 (Accession # CAA01880); Cry1Ae1 (Accession # AAA22410); Cry1Af1 (Accession # AAB82749); Cry1Ag1 (Accession # AAD46137); Cry1Ah1 (Accession # AAQ14326); Cry1Ah2 (Accession # ABB76664); Cry1Ah3 (Accession # HQ439779); Cry1Ai1 (Accession # AAO39719); Cry1Ai2 (Accession # HQ439780); Cry1A-like (Accession # AAK14339); Cry1Ba1 (Accession # CAA29898); Cry1Ba2 (Accession # CAA65003); Cry1Ba3 (Accession # AAK63251); Cry1Ba4 (Accession # AAK51084); Cry1Ba5 (Accession # AB020894); Cry1Ba6 (Accession # ABL60921); Cry1Ba7 (Accession # HQ439781); Cry1Bb1 (Accession # AAA22344); Cry1Bb2 (Accession # HQ439782); Cry1Bc1 (Accession # CAA86568); Cry1Bd1 (Accession # AAD10292); Cry1Bd2 (Accession # AAM93496); Cry1Be1 (Accession # AAC32850); Cry1Be2 (Accession # AAQ52387); Cry1Be3 (Accession # ACV96720); Cry1Be4 (Accession # HM070026); Cry1Bf1 (Accession # CAC50778); Cry1Bf2 (Accession # AAQ52380); Cry1Bg1 (Accession # AAO39720); Cry1Bh1 (Accession # HQ589331); Cry1Bi1 (Accession # KC156700); Cry1Ca1 (Accession # CAA30396); Cry1Ca2 (Accession # CAA31951); Cry1Ca3 (Accession # AAA22343); Cry1Ca4 (Accession # CAA01886); Cry1Ca5 (Accession # CAA65457); Cry1Ca6 [1] (Accession # AAF37224); Cry1Ca7 (Accession # AAG50438); Cry1Ca8 (Accession # AAM00264); Cry1Ca9 (Accession # AAL79362); Cry1Ca10 (Accession # AAN16462); Cry1Ca11 (Accession # AAX53094); Cry1Ca12 (Accession # HM070027); Cry1Ca13 (Accession # HQ412621); Cry1Ca14 (Accession # JN651493); Cry1Cb1 (Accession # M97880); Cry1Cb2 (Accession # AAG35409); Cry1Cb3 (Accession # ACD50894); Cry1Cb-like (Accession # AAX63901); Cry1Da1 (Accession # CAA38099); Cry1Da2 (Accession #176415); Cry1Da3 (Accession # HQ439784); Cry1Db1 (Accession # CAA80234); Cry1Db2 (Accession # AAK48937); Cry1Dc1 (Accession # ABK35074); Cry1Ea1 (Accession # CAA37933); Cry1Ea2 (Accession # CAA39609); Cry1Ea3 (Accession # AAA22345); Cry1Ea4 (Accession # AADQ4732); Cry1Ea5 (Accession # A15535); Cry1Ea6 (Accession # AAL50330); Cry1Ea7 (Accession # AAW72936); Cry1Ea8 (Accession # ABX11258); Cry1Ea9 (Accession # HQ439785); Cry1Ea10 (Accession # ADR00398); Cry1Ea11 (Accession # JQ652456); Cry1Eb1 (Accession # AAA22346); Cry1Fa1 (Accession # AAA22348); Cry1Fa2 (Accession # AAA22347); Cry1Fa3 (Accession # HM070028); Cry1Fa4 (Accession # HM439638); Cry1Fb1 (Accession # CAA80235); Cry1Fb2 (Accession # BAA25298); Cry1Fb3 (Accession # AAF21767); Cry1Fb4 (Accession # AAC10641); Cry1Fb5 (Accession # AAO13295); Cry1Fb6 (Accession # ACD50892); Cry1Fb7 (Accession # ACD50893); Cry1Ga1 (Accession # CAA80233); Cry1Ga2 (Accession # CAA70506); Cry1Gb1 (Accession # AAD10291); Cry1Gb2 (Accession # AAO13756); Cry1Gc1 (Accession # AAQ52381); Cry1Ha1 (Accession # CAA80236); Cry1Hb1 (Accession # AAA79694); Cry1Hb2 (Accession # HQ439786); Cry1H-like (Accession # AAF01213); Cry1Ia1 (Accession # CAA44633); Cry1Ia2 (Accession # AAA22354); Cry1Ia3 (Accession # AAC36999); Cry1Ia4 (Accession # AAB00958); Cry1Ia5 (Accession # CAA70124); Cry1Ia6 (Accession # AAC26910); Cry1Ia7 (Accession # AAM73516); Cry1Ia8 (Accession # AAK66742); Cry1Ia9 (Accession # AAQ08616); Cry1Ia10 (Accession # AAP86782); Cry1Ia11 (Accession # CAC85964); Cry1Ia12 (Accession # AAV53390); Cry1Ia13 (Accession # ABF83202); Cry1Ia14 (Accession # ACG63871); Cry1Ia15 (Accession # FJ617445); Cry1Ia16 (Accession # FJ617448); Cry1Ia17 (Accession # GU989199); Cry1Ia18 (Accession # ADK23801); Cry1Ia19 (Accession # HQ439787); Cry1Ia20 (Accession # JQ228426); Cry1Ia21 (Accession # JQ228424); Cry1Ia22 (Accession # JQ228427); Cry1Ia23 (Accession # JQ228428); Cry1Ia24 (Accession # JQ228429); Cry1Ia25 (Accession # JQ228430); Cry1Ia26 (Accession # JQ228431); Cry1Ia27 (Accession # JQ228432); Cry1Ia28 (Accession # JQ228433); Cry1Ia29 (Accession # JQ228434); Cry1Ia30 (Accession # JQ317686); Cry1Ia31 (Accession # JX944038); Cry1Ia32 (Accession # JX944039); Cry1Ia33 (Accession # JX944040); Cry1Ib1 (Accession # AAA82114); Cry1Ib2 (Accession # ABW88019); Cry1Ib3 (Accession # ACD75515); Cry1Ib4 (Accession # HM051227); Cry1Ib5 (Accession # HM070028); Cry1Ib6 (Accession # ADK38579); Cry1Ib7 (Accession # JN571740); Cry1Ib8 (Accession # JN675714); Cry1Ib9 (Accession # JN675715); Cry1Ib10 (Accession # JN675716); Cry1Ib11 (Accession # JQ228423); Cry1Ic1 (Accession # AAC62933); Cry1Ic2 (Accession # AAE71691); Cry1Id1 (Accession # AAD44366); Cry1Id2 (Accession # JQ228422); Cry1Ie1 (Accession # AAG43526); Cry1Ie2 (Accession # HM439636); Cry1Ie3 (Accession # KC156647); Cry1Ie4 (Accession # KC156681); Cry1If1 (Accession # AAQ52382); Cry1Ig1 (Accession # KC156701); Cry1I-like (Accession # AAC31094); Cry1I-like (Accession # ABG88859); Cry1Ja1 (Accession # AAA22341); Cry1Ja2 (Accession # HM070030); Cry1Ja3 (Accession # JQ228425); Cry1Jb1 (Accession # AAA98959); Cry1Jc1 (Accession # AAC31092); Cry1Jc2 (Accession # AAQ52372); Cry1Jd1 (Accession # CAC50779); Cry1Ka1 (Accession # AAB00376); Cry1Ka2 (Accession # HQ439783); Cry1La1 (Accession # AAS60191); Cry1La2 (Accession # HM070031); Cry1Ma1 (Accession # FJ884067); Cry1Ma2 (Accession # KC156659); Cry1Na1 (Accession # KC156648); Cry1Nb1 (Accession # KC156678); Cry1-like (Accession # AAC31091); Cry2Aa1 (Accession # AAA22335); Cry2Aa2 (Accession # AAA83516); Cry2Aa3 (Accession # D86064); Cry2Aa4 (Accession # AAC04867); Cry2Aa5 (Accession # CAA10671); Cry2Aa6 (Accession # CAA10672); Cry2Aa7 (Accession # CAA10670); Cry2Aa8 (Accession # AAO13734); Cry2Aa9 (Accession # AAO13750); Cry2Aa10 (Accession # AAQ04263); Cry2Aa11 (Accession # AAQ52384); Cry2Aa12 (Accession # AB183671); Cry2Aa13 (Accession # ABL01536); Cry2Aa14 (Accession # ACF04939); Cry2Aa15 (Accession # JN426947); Cry2Ab1 (Accession # AAA22342); Cry2Ab2 (Accession # CAA39075); Cry2Ab3 (Accession # AAG36762); Cry2Ab4 (Accession # AAO13296); Cry2Ab5 (Accession # AAQ04609); Cry2Ab6 (Accession # AAP59457); Cry2Ab7 (Accession # AAZ66347); Cry2Ab8 (Accession # ABC95996); Cry2Ab9 (Accession # ABC74968); Cry2Ab10 (Accession # EF157306); Cry2Ab11 (Accession # CAM84575); Cry2Ab12 (Accession # ABM21764); Cry2Ab13 (Accession # ACG76120); Cry2Ab14 (Accession # ACG76121); Cry2Ab15 (Accession # HM037126); Cry2Ab16 (Accession # GQ866914); Cry2Ab17 (Accession # HQ439789); Cry2Ab18 (Accession # JN135255); Cry2Ab19 (Accession # JN135256); Cry2Ab20 (Accession # JN135257); Cry2Ab21 (Accession # JN135258); Cry2Ab22 (Accession # JN135259); Cry2Ab23 (Accession # JN135260); Cry2Ab24 (Accession # JN135261); Cry2Ab25 (Accession # JN415485); Cry2Ab26 (Accession # JN426946); Cry2Ab27 (Accession # JN415764); Cry2Ab28 (Accession # JN651494); Cry2Ac1 (Accession # CAA40536); Cry2Ac2 (Accession # AAG35410); Cry2Ac3 (Accession # AAQ52385); Cry2Ac4 (Accession # ABC95997); Cry2Ac5 (Accession # ABC74969); Cry2Ac6 (Accession # ABC74793); Cry2Ac7 (Accession # CAL18690); Cry2Ac8 (Accession # CAM09325); Cry2Ac9 (Accession # CAM09326); Cry2Ac10 (Accession # ABN15104); Cry2Ac11 (Accession # CAM83895); Cry2Ac12 (Accession # CAM83896); Cry2Ad1 (Accession # AAF09583); Cry2Ad2 (Accession # ABC86927); Cry2Ad3 (Accession # CAK29504); Cry2Ad4 (Accession # CAM32331); Cry2Ad5 (Accession # CAO78739); Cry2Ae1 (Accession # AAQ52362); Cry2Af1 (Accession # AB030519); Cry2Af2 (Accession # GQ866915); Cry2Ag1 (Accession # ACH91610); Cry2Ah1 (Accession # EU939453); Cry2Ah2 (Accession # ACL80665); Cry2Ah3 (Accession # GU073380); Cry2Ah4 (Accession # KC156702); Cry2Ai1 (Accession # FJ788388); Cry2Aj (Accession #); Cry2Ak1 (Accession # KC156660); Cry2Ba1 (Accession # KC156658); Cry3Aa1 (Accession # AAA22336); Cry3Aa2 (Accession # AAA22541); Cry3Aa3 (Accession # CAA68482); Cry3Aa4 (Accession # AAA22542); Cry3Aa5 (Accession # AAA50255); Cry3Aa6 (Accession # AAC43266); Cry3Aa7 (Accession # CAB41411); Cry3Aa8 (Accession # AAS79487); Cry3Aa9 (Accession # AAW05659); Cry3Aa10 (Accession # AAU29411); Cry3Aa11 (Accession # AAW82872); Cry3Aa12 (Accession # ABY49136); Cry3Ba1 (Accession # CAA34983); Cry3Ba2 (Accession # CAA00645); Cry3Ba3 (Accession # JQ397327); Cry3Bb1 (Accession # AAA22334); Cry3Bb2 (Accession # AAA74198); Cry3Bb3 (Accession #115475); Cry3Ca1 (Accession # CAA42469); Cry4Aa1 (Accession # CAA68485); Cry4Aa2 (Accession # BAA00179); Cry4Aa3 (Accession # CAD30148); Cry4Aa4 (Accession # AFB18317); Cry4A-like (Accession # AAY96321); Cry4Ba1 (Accession # CAA30312); Cry4Ba2 (Accession # CAA30114); Cry4Ba3 (Accession # AAA22337); Cry4Ba4 (Accession # BAA00178); Cry4Ba5 (Accession # CAD30095); Cry4Ba-like (Accession # ABC47686); Cry4Ca1 (Accession # EU646202); Cry4Cb1 (Accession # FJ403208); Cry4Cb2 (Accession # FJ597622); Cry4Cc1 (Accession # FJ403207); Cry5Aa1 (Accession # AAA67694); Cry5Ab1 (Accession # AAA67693); Cry5Ac1 (Accession #134543); Cry5Ad1 (Accession # ABQ82087); Cry5Ba1 (Accession # AAA68598); Cry5Ba2 (Accession # ABW88931); Cry5Ba3 (Accession # AFJ04417); Cry5Ca1 (Accession # HM461869); Cry5Ca2 (Accession # ZP_04123426); Cry5Da1 (Accession # HM461870); Cry5Da2 (Accession # ZP_04123980); Cry5Ea1 (Accession # HM485580); Cry5Ea2 (Accession # ZP_04124038); Cry6Aa1 (Accession # AAA22357); Cry6Aa2 (Accession # AAM46849); Cry6Aa3 (Accession # ABH03377); Cry6Ba1 (Accession # AAA22358); Cry7Aa1 (Accession # AAA22351); Cry7Ab1 (Accession # AAA21120); Cry7Ab2 (Accession # AAA21121); Cry7Ab3 (Accession # ABX24522); Cry7Ab4 (Accession # EU380678); Cry7Ab5 (Accession # ABX79555); Cry7Ab6 (Accession # ACI44005); Cry7Ab7 (Accession # ADB89216); Cry7Ab8 (Accession # GU145299); Cry7Ab9 (Accession # ADD92572); Cry7Ba1 (Accession # ABB70817); Cry7Bb1 (Accession # KC156653); Cry7Ca1 (Accession # ABR67863); Cry7Cb1 (Accession # KC156698); Cry7Da1 (Accession # ACQ99547); Cry7Da2 (Accession # HM572236); Cry7Da3 (Accession # KC156679); Cry7Ea1 (Accession # HM035086); Cry7Ea2 (Accession # HM132124); Cry7Ea3 (Accession # EEM19403); Cry7Fa1 (Accession # HM035088); Cry7Fa2 (Accession # EEM19090); Cry7Fb1 (Accession # HM572235); Cry7Fb2 (Accession # KC156682); Cry7Ga1 (Accession # HM572237); Cry7Ga2 (Accession # KC156669); Cry7Gb1 (Accession # KC156650); Cry7Gc1 (Accession # KC156654); Cry7Gd1 (Accession # KC156697); Cry7Ha1 (Accession # KC156651); Cry7Ia1 (Accession # KC156665); Cry7Ja1 (Accession # KC156671); Cry7Ka1 (Accession # KC156680); Cry7Kb1 (Accession # BAM99306); Cry7La1 (Accession # BAM99307); Cry8Aa1 (Accession # AAA21117); Cry8Ab1 (Accession # EU044830); Cry8Ac1 (Accession # KC156662); Cry8Ad1 (Accession # KC156684); Cry8Ba1 (Accession # AAA21118); Cry8Bb1 (Accession # CAD57542); Cry8Bc1 (Accession # CAD57543); Cry8Ca1 (Accession # AAA21119); Cry8Ca2 (Accession # AAR98783); Cry8Ca3 (Accession # EU625349); Cry8Ca4 (Accession # ADB54826); Cry8Da1 (Accession # BAC07226); Cry8Da2 (Accession # BD133574); Cry8Da3 (Accession # BD133575); Cry8Db1 (Accession # BAF93483); Cry8Ea1 (Accession # AAQ73470); Cry8Ea2 (Accession # EU047597); Cry8Ea3 (Accession # KC855216); Cry8Fa1 (Accession # AAT48690); Cry8Fa2 (Accession # HQ174208); Cry8Fa3 (Accession # AFH78109); Cry8Ga1 (Accession # AAT46073); Cry8Ga2 (Accession # ABC42043); Cry8Ga3 (Accession # FJ198072); Cry8Ha1 (Accession # AAW81032); Cry81a1 (Accession # EU381044); Cry81a2 (Accession # GU073381); Cry81a3 (Accession # HM044664); Cry81a4 (Accession # KC156674); Cry81b1 (Accession # GU325772); Cry81b2 (Accession # KC156677); Cry8Ja1 (Accession # EU625348); Cry8Ka1 (Accession # FJ422558); Cry8Ka2 (Accession # ACN87262); Cry8Kb1 (Accession # HM123758); Cry8Kb2 (Accession # KC156675); Cry8La1 (Accession # GU325771); Cry8Ma1 (Accession # HM044665); Cry8Ma2 (Accession # EEM86551); Cry8Ma3 (Accession # HM210574); Cry8Na1 (Accession # HM640939); Cry8Pa1 (Accession # HQ388415); Cry8Qa1 (Accession # HQ441166); Cry8Qa2 (Accession # KC152468); Cry8Ra1 (Accession # AFP87548); Cry8Sa1 (Accession # JQ740599); Cry8Ta1 (Accession # KC156673); Cry8-like (Accession # FJ770571); Cry8-like (Accession # ABS53003); Cry9Aa1 (Accession # CAA41122); Cry9Aa2 (Accession # CAA41425); Cry9Aa3 (Accession # GQ249293); Cry9Aa4 (Accession # GQ249294); Cry9Aa5 (Accession # JX174110); Cry9Aa like (Accession # AAQ52376); Cry9Ba1 (Accession # CAA52927); Cry9Ba2 (Accession # GU299522); Cry9Bb1 (Accession # AAV28716); Cry9Ca1 (Accession # CAA85764); Cry9Ca2 (Accession # AAQ52375); Cry9Da1 (Accession # BAA19948); Cry9Da2 (Accession # AAB97923); Cry9Da3 (Accession # GQ249293); Cry9Da4 (Accession # GQ249297); Cry9Db1 (Accession # AAX78439); Cry9Dc1 (Accession # KC156683); Cry9Ea1 (Accession # BAA34908); Cry9Ea2 (Accession # AAO12908); Cry9Ea3 (Accession # ABM21765); Cry9Ea4 (Accession # ACE88267); Cry9Ea5 (Accession # ACF04743); Cry9Ea6 (Accession # ACG63872); Cry9Ea7 (Accession # FJ380927); Cry9Ea8 (Accession # GQ249292); Cry9Ea9 (Accession # JN651495); Cry9Eb1 (Accession # CAC50780); Cry9Eb2 (Accession # GQ249298); Cry9Eb3 (Accession # KC156646); Cry9Ec1 (Accession # AAC63366); Cry9Ed1 (Accession # AAX78440); Cry9Ee1 (Accession # GQ249296); Cry9Ee2 (Accession # KC156664); Cry9Fa1 (Accession # KC156692); Cry9Ga1 (Accession # KC156699); Cry9-like (Accession # AAC63366); Cry10Aa1 (Accession # AAA22614); Cry10Aa2 (Accession # E00614); Cry10Aa3 (Accession # CAD30098); Cry10Aa4 (Accession # AFB18318); Cry10A-like (Accession # DQ167578); Cry11Aa1 (Accession # AAA22352); Cry11Aa2 (Accession # AAA22611); Cry11Aa3 (Accession # CAD30081); Cry11Aa4 (Accession # AFB18319); Cry11Aa-like (Accession # DQ166531); Cry11Ba1 (Accession # CAA60504); Cry11Bb1 (Accession # AAC97162); Cry11Bb2 (Accession # HM068615); Cry12Aa1 (Accession # AAA22355); Cry13Aa1 (Accession # AAA22356); Cry14Aa1 (Accession # AAA21516); Cry14Ab1 (Accession # KC156652); Cry15Aa1 (Accession # AAA22333); Cry16Aa1 (Accession # CAA63860); Cry17Aa1 (Accession # CAA67841); Cry18Aa1 (Accession # CAA67506); Cry18Ba1 (Accession # AAF89667); Cry18Ca1 (Accession # AAF89668); Cry19Aa1 (Accession # CAA68875); Cry19Ba1 (Accession # BAA32397); Cry19Ca1 (Accession # AFM37572); Cry20Aa1 (Accession # AAB93476); Cry20Ba1 (Accession # ACS93601); Cry20Ba2 (Accession # KC156694); Cry20-like (Accession # GQ144333); Cry21 Aa1 (Accession #132932); Cry21 Aa2 (Accession #166477); Cry21Ba1 (Accession # BAC06484); Cry21Ca1 (Accession # JF521577); Cry21Ca2 (Accession # KC156687); Cry21 Da1 (Accession # JF521578); Cry22Aa1 (Accession #134547); Cry22Aa2 (Accession # CAD43579); Cry22Aa3 (Accession # ACD93211); Cry22Ab1 (Accession # AAK50456); Cry22Ab2 (Accession # CAD43577); Cry22Ba1 (Accession # CAD43578); Cry22Bb1 (Accession # KC156672); Cry23Aa1 (Accession # AAF76375); Cry24Aa1 (Accession # AAC61891); Cry24Ba1 (Accession # BAD32657); Cry24Ca1 (Accession # CAJ43600); Cry25Aa1 (Accession # AAC61892); Cry26Aa1 (Accession # AAD25075); Cry27Aa1 (Accession # BAA82796); Cry28Aa1 (Accession # AAD24189); Cry28Aa2 (Accession # AAG00235); Cry29Aa1 (Accession # CAC80985); Cry30Aa1 (Accession # CAC80986); Cry30Ba1 (Accession # BAD00052); Cry30Ca1 (Accession # BAD67157); Cry30Ca2 (Accession # ACU24781); Cry30Da1 (Accession # EF095955); Cry30Db1 (Accession # BAE80088); Cry30Ea1 (Accession # ACC95445); Cry30Ea2 (Accession # FJ499389); Cry30Fa1 (Accession # ACI22625); Cry30Ga1 (Accession # ACG60020); Cry30Ga2 (Accession # HQ638217); Cry31 Aa1 (Accession # BAB11757); Cry31 Aa2 (Accession # AAL87458); Cry31 Aa3 (Accession # BAE79808); Cry31 Aa4 (Accession # BAF32571); Cry31 Aa5 (Accession # BAF32572); Cry31 Aa6 (Accession # BAI44026); Cry31Ab1 (Accession # BAE79809); Cry31 Ab2 (Accession # BAF32570); Cry31 Ac1 (Accession # BAF34368); Cry31 Ac2 (Accession # AB731600); Cry31Ad1 (Accession # BAI44022); Cry32Aa1 (Accession # AAG36711); Cry32Aa2 (Accession # GU063849); Cry32Ab1 (Accession # GU063850); Cry32Ba1 (Accession # BAB78601); Cry32Ca1 (Accession # BAB78602); Cry32Cb1 (Accession # KC156708); Cry32Da1 (Accession # BAB78603); Cry32Ea1 (Accession # GU324274); Cry32Ea2 (Accession # KC156686); Cry32Eb1 (Accession # KC156663); Cry32Fa1 (Accession # KC156656); Cry32Ga1 (Accession # KC156657); Cry32Ha1 (Accession # KC156661); Cry32Hb1 (Accession # KC156666); Cry321a1 (Accession # KC156667); Cry32Ja1 (Accession # KC156685); Cry32Ka1 (Accession # KC156688); Cry32La1 (Accession # KC156689); Cry32Ma1 (Accession # KC156690); Cry32Mb1 (Accession # KC156704); Cry32Na1 (Accession # KC156691); Cry32Oa1 (Accession # KC156703); Cry32Pa1 (Accession # KC156705); Cry32Qa1 (Accession # KC156706); Cry32Ra1 (Accession # KC156707); Cry32Sa1 (Accession # KC156709); Cry32Ta1 (Accession # KC156710); Cry32Ua1 (Accession # KC156655); Cry33Aa1 (Accession # AAL26871); Cry34Aa1 (Accession # AAG50341); Cry34Aa2 (Accession # AAK64560); Cry34Aa3 (Accession # AAT29032); Cry34Aa4 (Accession # AAT29030); Cry34Ab1 (Accession # AAG41671); Cry34Ac1 (Accession # AAG50118); Cry34Ac2 (Accession # AAK64562); Cry34Ac3 (Accession # AAT29029); Cry34Ba1 (Accession # AAK64565); Cry34Ba2 (Accession # AAT29033); Cry34Ba3 (Accession # AAT29031); Cry35Aa1 (Accession # AAG50342); Cry35Aa2 (Accession # AAK64561); Cry35Aa3 (Accession # AAT29028); Cry35Aa4 (Accession # AAT29025); Cry35Ab1 (Accession # AAG41672); Cry35Ab2 (Accession # AAK64563); Cry35Ab3 (Accession # AY536891); Cry35Ac1 (Accession # AAG50117); Cry35Ba1 (Accession # AAK64566); Cry35Ba2 (Accession # AAT29027); Cry35Ba3 (Accession # AAT29026); Cry36Aa1 (Accession # AAK64558); Cry37Aa1 (Accession # AAF76376); Cry38Aa1 (Accession # AAK64559); Cry39Aa1 (Accession # BAB72016); Cry40Aa1 (Accession # BAB72018); Cry40Ba1 (Accession # BAC77648); Cry40Ca1 (Accession # EU381045); Cry40Da1 (Accession # ACF15199); Cry41Aa1 (Accession # BAD35157); Cry41Ab1 (Accession # BAD35163); Cry41Ba1 (Accession # HM461871); Cry41Ba2 (Accession # ZP_04099652); Cry42Aa1 (Accession # BAD35166); Cry43Aa1 (Accession # BAD15301); Cry43Aa2 (Accession # BAD95474); Cry43Ba1 (Accession # BAD15303); Cry43Ca1 (Accession # KC156676); Cry43Cb1 (Accession # KC156695); Cry43Cc1 (Accession # KC156696); Cry43-like (Accession # BAD15305); Cry44Aa (Accession # BAD08532); Cry45Aa (Accession # BAD22577); Cry46Aa (Accession # BAC79010); Cry46Aa2 (Accession # BAG68906); Cry46Ab (Accession # BAD35170); Cry47Aa (Accession # AAY24695); Cry48Aa (Accession # CAJ18351); Cry48Aa2 (Accession # CAJ86545); Cry48Aa3 (Accession # CAJ86546); Cry48Ab (Accession # CAJ86548); Cry48Ab2 (Accession # CAJ86549); Cry49Aa (Accession # CAH56541); Cry49Aa2 (Accession # CAJ86541); Cry49Aa3 (Accession # CAJ86543); Cry49Aa4 (Accession # CAJ86544); Cry49Ab1 (Accession # CAJ86542); Cry50Aa1 (Accession # BAE86999); Cry50Ba1 (Accession # GU446675); Cry50Ba2 (Accession # GU446676); Cry51Aa1 (Accession # AB114444); Cry51Aa2 (Accession # GU570697); Cry52Aa1 (Accession # EF613489); Cry52Ba1 (Accession # FJ361760); Cry53Aa1 (Accession # EF633476); Cry53Ab1 (Accession # FJ361759); Cry54Aa1 (Accession # ACA52194); Cry54Aa2 (Accession # GQ140349); Cry54Ba1 (Accession # GU446677); Cry55Aa1 (Accession # ABW88932); Cry54Ab1 (Accession # JQ916908); Cry55Aa2 (Accession # AAE33526); Cry56Aa1 (Accession # ACU57499); Cry56Aa2 (Accession # GQ483512); Cry56Aa3 (Accession # JX025567); Cry57Aa1 (Accession # ANC87261); Cry58Aa1 (Accession # ANC87260); Cry59Ba1 (Accession # JN790647); Cry59Aa1 (Accession # ACR43758); Cry60Aa1 (Accession # ACU24782); Cry60Aa2 (Accession # EA057254); Cry60Aa3 (Accession # EEM99278); Cry60Ba1 (Accession # GU810818); Cry60Ba2 (Accession # EA057253); Cry60Ba3 (Accession # EEM99279); Cry61Aa1 (Accession # HM035087); Cry61Aa2 (Accession # HM132125); Cry61Aa3 (Accession # EEM19308); Cry62Aa1 (Accession # HM054509); Cry63Aa1 (Accession # BAI44028); Cry64Aa1 (Accession # BAJ05397); Cry65Aa1 (Accession # HM461868); Cry65Aa2 (Accession # ZP_04123838); Cry66Aa1 (Accession # HM485581); Cry66Aa2 (Accession # ZP_04099945); Cry67Aa1 (Accession # HM485582); Cry67Aa2 (Accession # ZP_04148882); Cry68Aa1 (Accession # HQ113114); Cry69Aa1 (Accession # HQ401006); Cry69Aa2 (Accession # JQ821388); Cry69Ab1 (Accession # JN209957); Cry70Aa1 (Accession # JN646781); Cry70Ba1 (Accession # ADO51070); Cry70Bb1 (Accession # EEL67276); Cry71Aa1 (Accession # JX025568); Cry72Aa1 (Accession # JX025569); Cyt1Aa (GenBank Accession Number X03182); Cyt1Ab (GenBank Accession Number X98793); Cyt1B (GenBank Accession Number U37196); Cyt2A (GenBank Accession Number Z14147); and Cyt2B (GenBank Accession Number U52043).

Examples of δ-endotoxins also include but are not limited to Cry1A proteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of cry proteins such as Cry1A, Cry3A) of U.S. Pat. Nos. 8,304,604, 8.304,605 and 8,476,226; Cry1B of U.S. patent application Ser. No. 10/525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9F families; a Cry15 protein of Naimov, et al., (2008) Applied and Environmental Microbiology, 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 and cryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006/0191034, 2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US Patent Application Publication Number 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006/033867; AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US Patent Application Publication Number 2004/0250311; AXMI-006 of US Patent Application Publication Number 2004/0216186; AXMI-007 of US Patent Application Publication Number 2004/0210965; AXMI-009 of US Patent Application Number 2004/0210964; AXMI-014 of US Patent Application Publication Number 2004/0197917; AXMI-004 of US Patent Application Publication Number 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US Patent Application Publication Number 2011/0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of US Patent Application Publication Number 2011/0263488; AXMI-R1 and related proteins of US Patent Application Publication Number 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO 2011/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent Application Publication Number 2010/0298211; AXMI-066 and AXMI-076 of US Patent Application Publication Number 2009/0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US Patent Application Publication Number 2010/0005543, cry proteins such as Cry1A and Cry3A having modified proteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011/0064710. Other Cry proteins are well known to one skilled in the art (see, Crickmore, et al., “Bacillus thuringiensis toxin nomenclature” (2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed on the world-wide web using the “www” prefix). The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA. (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database which can be accessed on the world-wide web using the “www” prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & Cry1Fa (US2012/0317682); Cry1BE & Cry1F (US2012/0311746); Cry1CA & Cry1AB (US2012/0311745); Cry1F & CryCa (US2012/0317681); Cry1DA & Cry1BE (US2012/0331590); Cry1DA & Cry1Fa (US2012/0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1Fa & Cry2Aa and Cry1I & Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269); Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); and Cry3A and Cry1Ab or Vip3Aa (US20130116170). Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of U.S. Pat. No. 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins of U.S. Pat. Nos. 5,877,012, 6,107,279 6,137,033, 7,244,820, 7,615,686, and 8,237,020 and the like. Other VIP proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can be accessed on the world-wide web using the “www” prefix). Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein “potentiators” derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins (“Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi. Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but not limited to lycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

In some embodiments the PIP-47 polypeptides include amino acid sequences deduced from the full-length nucleic acid sequences disclosed herein and amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism the protein is expressed in or in the pest after ingestion of the protein.

Thus, provided herein are novel isolated or recombinant nucleic acid sequences that confer pesticidal activity. Also provided are the amino acid sequences of PIP-47 polypeptides. The protein resulting from translation of these PIP-47 polypeptide genes allows cells to control or kill pests that ingest it.

Bacterial Strains

One aspect pertains to bacterial strains that express a PIP-47 polypeptide. In some embodiments the bacterial strain is a Pseudomonas putida strain. In some embodiments the bacterial strain is a biologically pure culture of an Pseudomonas putida strain SS62E1, deposited on Aug. 13, 2013 under accession # NRRL B-50845 with the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, (nrrl.ncaur.usda.gov, which can be accessed on the world-wide web using the “www” prefix). The deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. § 112. Access to this deposit will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicant(s) will make available to the public, pursuant to 37 C.F.R. § 1.808, sample(s) of the deposit of with the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604. This deposit will be maintained in the NRRL depository, which is a public depository, for a period of 30 years or 5 years after the most recent request or for the enforceable life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. The deposits will irrevocably and without restriction or condition be available to the public upon issuance of a patent. Additionally, Applicant(s) have satisfied all the requirements of 37 C.F.R. §§ 1.801-1.809, including providing an indication of the viability of the sample upon deposit. Applicant(s) have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicant(s) do not waive any infringement of their rights granted under this patent. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.

Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect pertains to isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding PIP-47 polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology. As used herein, the term “nucleic acid molecule” refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in vitro. A “recombinant” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is in a recombinant bacterial or plant host cell. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecule encoding a PIP-47 polypeptide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

In some embodiments an isolated nucleic acid molecule encoding a PIP-47 polypeptide has one or more change in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments the change in the native or genomic nucleic acid sequence includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to the amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more intron; deletion of one or more upstream or downstream regulatory regions; and deletion of the 5′ and/or 3′ untranslated region associated with the genomic nucleic acid sequence. In some embodiments the nucleic acid molecule encoding a PIP-47 polypeptide is a non-genomic sequence.

A variety of polynucleotides that encode a PIP-47 polypeptides or related proteins are contemplated. Such polynucleotides are useful for production of PIP-47 polypeptides in host cells when operably linked to suitable promoter, transcription termination and/or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode PIP-47 polypeptides or related proteins.

One source of polynucleotides that encode PIP-47 polypeptides or related proteins is a Pseudomonas putida strain which contains the PIP-47Aa polynucleotide of SEQ ID NO: 1 encoding the PIP-47Aa polypeptide of SEQ ID NO: 2, a Pseudomonas putida strain which contains the PIP-47Ba polynucleotide of SEQ ID NO: 3 encoding the PIP-47Bb polypeptide of SEQ ID NO: 4, a Pseudomonas fulva strain which contains the PIP-47Bb polynucleotide of SEQ ID NO: 5 encoding the PIP-47Bb polypeptide of SEQ ID NO: 6 or a Pseudomonas chlororaphis strain which contains the PIP-47Fa polynucleotide of SEQ ID NO: 7 encoding the PIP-47Fa polypeptide of SEQ ID NO: 8. These polynucleotide sequences were isolated from a Pseudomonas host and are thus suitable for expression of the encoded PIP-47 polypeptide in other bacterial hosts. For example, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 can be used to express PIP-47 polypeptides in bacterial hosts that include but are not limited to Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas and Rhizobium bacterial host cells. The polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode PIP-47 polypeptides or related proteins. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from Pseudomonas or other related bacteria.

Polynucleotides that encode a PIP-47 polypeptide can also be synthesized de novo from a PIP-47 polypeptide sequence. The sequence of the polynucleotide gene can be deduced from a PIP-47 polypeptide sequence through use of the genetic code. Computer programs such as “BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can be used to convert a peptide sequence to the corresponding nucleotide sequence encoding the peptide. Examples of PIP-47 polypeptide sequences that can be used to obtain corresponding nucleotide encoding sequences include, but are not limited to, the PIP-47 polypeptide of sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8. Furthermore, synthetic PIP-47 polynucleotide sequences of the disclosure can be designed so that they will be expressed in plants. U.S. Pat. No. 5,500,365 describes a method for synthesizing plant genes to improve the expression level of the protein encoded by the synthesized gene. This method relates to the modification of the structural gene sequences of the exogenous transgene, to cause them to be more efficiently transcribed, processed, translated and expressed by the plant. Features of genes that are expressed well in plants include elimination of sequences that can cause undesired intron splicing or polyadenylation in the coding region of a gene transcript while retaining substantially the amino acid sequence of the toxic portion of the insecticidal protein. A similar method for obtaining enhanced expression of transgenes in monocotyledonous plants is disclosed in U.S. Pat. No. 5,689,052.

In some embodiments the nucleic acid molecule encoding a PIP-47 polypeptide is a polynucleotide having the sequence set forth in SEQ ID NO: 1; SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and variants, fragments and complements thereof. “Complement” is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. “Polynucleotide sequence variants” is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.

In some embodiments a nucleic acid molecule encoding the PIP-47 polypeptide is a non-genomic nucleic acid sequence. As used herein a “non-genomic nucleic acid sequence” or “non-genomic nucleic acid molecule” refers to a nucleic acid molecule that has one or more change in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments the change to a native or genomic nucleic acid molecule includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; codon optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more intron associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5′ and/or 3′ untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5′ and/or 3′ untranslated region; and modification of a polyadenylation site. In some embodiments the non-genomic nucleic acid molecule is a cDNA. In some embodiments the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

In some embodiments the non-genomic nucleic acid molecule encoding a PIP-47 polypeptide is not the polynucleotide having the sequence set forth in SEQ ID NO: 1; SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

In some embodiments the non-genomic nucleic acid molecule encoding a PIP-47 polypeptide does not encode the polypeptide of SEQ ID NO: 2; SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.

In some embodiments the non-genomic nucleic acid molecule encoding a PIP-47 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, wherein the polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encoding a PIP-47 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NO: 4, wherein the polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encoding a PIP-47 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NO: 6, wherein the polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encoding a PIP-47 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, to the amino acid sequence of SEQ ID NO: 8, wherein the polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence having at least 80% identity, to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 wherein the PIP-47 polypeptide has at least one amino acid change compared to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and the PIP-47 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence having at least 80% identity, to the amino acid sequence of SEQ ID NO: 2, wherein the PIP-47 polypeptide has at least one amino acid change compared to SEQ ID NO: 2 and the PIP-47 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence having at least 80% identity, to the amino acid sequence of SEQ ID NO: 4, wherein the PIP-47 polypeptide has at least one amino acid change compared to SEQ ID NO: 4 and the PIP-47 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence having at least 80% identity, to the amino acid sequence of SEQ ID NO: 6, wherein the PIP-47 polypeptide has at least one amino acid change compared to SEQ ID NO: 6 and the PIP-47 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence having at least 80% identity, to the amino acid sequence of SEQ ID NO: 8, wherein the PIP-47 polypeptide has at least one amino acid change compared to SEQ ID NO: 8 and the PIP-47 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions compared to the native amino acid at the corresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 4 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions compared to the native amino acid at the corresponding position of SEQ ID NO: 4.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 6 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions compared to the native amino acid at the corresponding position of SEQ ID NO: 6.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 8 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions compared to the native amino acid at the corresponding position of SEQ ID NO: 8.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 481 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, at positions designated by Xaa, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 482 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions, at positions designated by Xaa, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 483 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, at positions designated by Xaa, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 484 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, at positions designated by Xaa, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 485 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, at positions designated by Xaa, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 486 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, at positions designated by Xaa, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes a PIP-47 polypeptide comprising an amino acid sequence of SEQ ID NO: 487 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 amino acid substitutions, at positions designated by Xaa, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the nucleic acid molecule encoding a PIP-47 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 481, wherein Xaa at position 47 is Thr, Gly, Ala, Leu, Met, Pro, Ser, Cys, Asn, Asp, Glu, Lys, Arg, Tyr, Ile or Trp; Xaa at position 73 is Tyr, Gly, Cys or Glu; Xaa at position 77 is Ala, Ser, Cys, Gln, Pro or Asn; Xaa at position 78 is Asp, Gly, Ala, Val, Leu, Trp, Ser, Cys, Gln, Glu, Arg, Phe, Ile, Tyr, Met, Asn, His, Thr or Lys; Xaa at position 79 is Ala, Gly, Tyr, Val, Leu, Trp, Gln, Glu, Lys, Arg, Asn, Asp, Met, Phe, Ile, Cys, Ser or Thr; Xaa at position 80 is Asn, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Tyr, Phe, Asp or Met; Xaa at position 81 is Val, Gly, Ala, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Ile, Asp, Pro or Met; Xaa at position 82 is Ser, Gly, Ala, Val, Trp, Thr, Cys, Gln, Glu, Lys, Asn, His, Phe, Ile, Asp or Met; Xaa at position 83 is Pro, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Phe, Ile or Asp; Xaa at position 84 is Gly, Tyr, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Arg, Asn, Phe, Ile, Asp, Pro or Met; Xaa at position 85 is Ser, Gly, Ala, Val, Leu, Trp, Tyr, Thr, Cys, Gln, Glu, Arg, Phe, Asp, Pro or Met; Xaa at position 86 is Ala, Gly, Tyr, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, His, Phe, Ile, Asp, Pro or Met; Xaa at position 95 is Gln, Ala, Val, Ser, Thr, Asn or His; Xaa at position 107 is Tyr, Cys, Phe, Gly, His or Leu; Xaa at position 108 is Asn, Ala, Cys, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr or Trp; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Ala, Glu, Phe, Met, Asn, Pro, Ser, Thr, Val or Trp; Xaa at position 111 is Asp, Ala, Glu, Phe, Gly, His, Asn, Gln, Ser, Val or Tyr; Xaa at position 112 is Gln, Cys, Pro or Val; Xaa at position 113 is Pro, Cys or Gly; Xaa at position 114 is Thr, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Val, Trp or Tyr; Xaa at position 115 is Asp, Ala, Glu, Phe, Gly, His, Ile, Leu, Pro, Gln, Arg, Ser, Thr or Tyr; Xaa at position 116 is Gln, Ala, Cys, Phe, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val or Trp; Xaa at position 117 is Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 118 is Pro, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 119 is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 120 is Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position 142 is Leu, Gly, Val, Met, Ser, Thr, Cys, Asn, Gln, Asp, Glu or His; Xaa at position 170 is Gly, Ala, Val, Ile, Leu, Ser, Lys or Arg; Xaa at position 205 is Gln, Ala, Val, Pro, Ser, Thr, Cys, Tyr, Asn, Asp or Arg; Xaa at position 221 is Tyr, Ala, Val, Leu, Ser, Gln, Met, Ile or His; Xaa at position 233 is Lys, Glu or Arg; Xaa at position 234 is Gly, Ala, Cys, Phe, His, Lys, Leu, Met, Pro, Arg, Ser, Val or Tyr; Xaa at position 235 is Thr, Ala, Cys, Glu, Phe, Gly, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Trp; Xaa at position 236 is Ile, Ala, Cys, Phe, Leu, Pro, Thr or Val; Xaa at position 237 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Val or Trp; Xaa at position 238 is Phe, Ala, Asp, Leu, Pro, Thr, Val, Trp or Tyr; Xaa at position 239 is Gln, Cys, Gly, Arg, Tyr, Ser, Pro, Trp, Glu, Thr, Met, Ala, Lys, His, Ile, Val, Leu or Asp; Xaa at position 240 is Ser, Ala, Glu, Gly, His, Leu, Met, Asn, Pro, Gln, Thr or Val; Xaa at position 241 is Ser, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 242 is Tyr, Cys, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 243 is Thr, Asp, Glu, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val or Tyr; Xaa at position 244 is Leu, Ala, Cys, Phe, Gly, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 245 is Ser, Ala, Cys, Asp, Gly, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 246 is Gln, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro or Arg; Xaa at position 247 is Ala, Asp, Phe, Lys, Met, Ser or Val; and Xaa at position 279 is Thr, Ser, Cys, Asn or Glu; and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide.

In some embodiments the nucleic acid molecule encoding a PIP-47 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 485, Xaa at position 2 is His or Ser; Xaa at position 3 is Ala or Thr; Xaa at position 4 is Pro or Gln; Xaa at position 5 is Gly, Asn or deleted; Xaa at position 6 is Ala, His or deleted; Xaa at position 7 is Ile, Ala, Lys or deleted; Xaa at position 8 is Pro, His or deleted; Xaa at position 9 is Ser or Thr; Xaa at position 10 is Glu, Val or Thr; Xaa at position 11 is Lys or Glu; Xaa at position 12 is Glu or Lys; Xaa at position 13 is Ser, Gly, Val or Leu; Xaa at position 14 is Ala or Lys; Xaa at position 15 is His or Ala; Xaa at position 16 is Ala or Trp; Xaa at position 17 is Trp or Leu; Xaa at position 18 is Leu or Asn; Xaa at position 19 is Thr, Glu or deleted; Xaa at position 20 is Glu, Thr, Gly, Ser or deleted; Xaa at position 21 is Thr, Ala, Glu, Lys, His or deleted; Xaa at position 22 is Lys, Glu, Thr or Ala; Xaa at position 23 is Ala, Ser or Arg; Xaa at position 24 is Asn, Arg, Lys or Val; Xaa at position 25 is Ala, Val, Lys or Glu; Xaa at position 26 is Lys, Ser, Leu or Asn; Xaa at position 27 is Ser or Phe; Xaa at position 28 is Thr or Ala; Xaa at position 29 is Ala, Thr, Gln or Ile; Xaa at position 30 is Leu, Thr, Val or Ala; Xaa at position 31 is Ala, Arg, Asn or His; Xaa at position 32 is Gly or Pro; Xaa at position 34 is Ile or Phe; Xaa at position 35 is Phe or Tyr; Xaa at position 36 is Ala, Val or Ser; Xaa at position 37 is Gln, Ala, Arg, Leu or His; Xaa at position 38 is Asp or Glu; Xaa at position 39 is Tyr, Ala, Arg; Xaa at position 40 is Asn, Ser or His; Xaa at position 41 is Ala, Arg or His; Xaa at position 42 is Gln, Ser or His; Xaa at position 43 is Leu, Gln or Ser; Xaa at position 44 is Leu, Phe or Val; Xaa at position 45 is Thr or Ser; Xaa at position 46 is Ala, Glu, Pro or Ser; Xaa at position 47 is Thr, Gly, Ala, Leu, Met, Pro, Ser, Cys, Asn, Asp, Glu, Lys, Arg, Tyr, Ile or Trp; Xaa at position 48 is Gly or Leu; Xaa at position 49 is Gln, Ala, Arg or Leu; Xaa at position 50 is Ser, Thr or Ala; Xaa at position 51 is Met or Pro; Xaa at position 52 is Ala, Arg or Gly; Xaa at position 53 is Ser or Thr; Xaa at position 57 is Ala or Ile; Xaa at position 58 is Ile, Asp or Ala; Xaa at position 62 is Phe or Tyr; Xaa at position 63 is Ser, Gly or Ala; Xaa at position 65 is Ala or Thr; Xaa at position 66 is Lys, Gly, Ala or Ser; Xaa at position 67 is Gly or Ser; Xaa at position 68 is Thr, Gly, Ala or Leu; Xaa at position 71 is Gly, Asp, Ala or Asn; Xaa at position 72 is Ser, Gly, Ala, Arg or Asn; Xaa at position 73 is Tyr, Gly, Cys or Glu; Xaa at position 74 is Ala, Val or Leu; Xaa at position 75 is Lys, Gln or Asn; Xaa at position 76 is Asp or Ala; Xaa at position 77 is Ala, Ser, Cys, Gln, Pro or Asn; Xaa at position 78 is Asp, Gly, Ala, Val, Leu, Trp, Ser, Cys, Gln, Glu, Arg, Phe, Ile, Tyr, Met, Asn, His, Thr or Lys; Xaa at position 79 is Ala, Gly, Tyr, Val, Leu, Trp, Gln, Glu, Lys, Arg, Asn, Asp, Met, Phe, Ile, Cys, Ser or Thr; Xaa at position 80 is Asn, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Tyr, Phe, Asp or Met; Xaa at position 81 is Val, Gly, Ala, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Ile, Asp, Pro or Met; Xaa at position 82 is Ser, Gly, Ala, Val, Trp, Thr, Cys, Gln, Glu, Lys, Asn, His, Phe, Ile, Asp or Met; Xaa at position 83 is Pro, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Phe, Ile or Asp; Xaa at position 84 is Gly, Tyr, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Arg, Asn, Phe, Ile, Asp, Pro or Met; Xaa at position 85 is Ser, Gly, Ala, Val, Leu, Trp, Tyr, Thr, Cys, Gln, Glu, Arg, Phe, Asp, Pro, Lys or Met; Xaa at position 86 is Ala, Gly, Tyr, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, His, Phe, Ile, Asp, Pro or Met; Xaa at position 88 is Val, Glu or Thr; Xaa at position 90 is Ile, Phe or Leu; Xaa at position 91 is Tyr, Gln, Cys or His; Xaa at position 92 is Glu, Asp or Lys; Xaa at position 95 is Gln, Ala, Val, Ser, Thr, Asn or His; Xaa at position 96 is Thr or Ser; Xaa at position 97 is Ala, Ser or Cys; Xaa at position 98 is Ile or Val; Xaa at position 99 is Asp or Asn; Xaa at position 100 is Ile, Thr or Met; Xaa at position 103 is Arg or Thr; Xaa at position 106 is Gly or Glu; Xaa at position 107 is Tyr, Cys, Phe, Gly, His, Trp or Leu; Xaa at position 108 is Asn, Ala, Cys, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr or Trp; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Ala, Glu, Phe, Met, Asn, Pro, Ser, Thr, Val or Trp; Xaa at position 111 is Asp, Ala, Glu, Phe, Gly, His, Asn, Gln, Ser, Val or Tyr; Xaa at position 112 is Gln, Cys, Pro or Val; Xaa at position 113 is Pro, Cys or Gly; Xaa at position 114 is Thr, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Val, Trp or Tyr; Xaa at position 115 is Asp, Ala, Glu, Phe, Gly, His, Ile, Leu, Pro, Gln, Arg, Ser, Thr or Tyr; Xaa at position 116 is Gln, Ala, Cys, Phe, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val or Trp; Xaa at position 117 is Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 118 is Pro, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 119 is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 120 is Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position 121 is Ala, Thr or Lys; Xaa at position 124 is Arg or Tyr; Xaa at position 130 is Phe or Leu; Xaa at position 132 is Gln or Lys; Xaa at position 133 is Gln or Glu; Xaa at position 135 is Ala or Ser; Xaa at position 136 is Glu, Ser, Ala, Arg or Asn; Xaa at position 137 is Ile or Asn; Xaa at position 141 is Ser or Thr; Xaa at position 142 is Leu, Gly, Val, Met, Ser, Thr, Cys, Asn, Gln, Asp, Glu, His or Pro; Xaa at position 143 is Leu or Glu; Xaa at position 144 is Ala or Ser; Xaa at position 146 is Gln or Ser; Xaa at position 147 is Val or Ser; Xaa at position 148 is Thr or Ser; Xaa at position 149 is Gln or Leu; Xaa at position 150 is Ile or Val; Xaa at position 151 is Gln, Ala, Arg or Met; Xaa at position 153 is Lys or Gln; Xaa at position 156 is Asp or Gly; Xaa at position 158 is Asn or Asp; Xaa at position 161 is Val or Ile; Xaa at position 164 is Phe or Cys; Xaa at position 165 is Val or Ala; Xaa at position 168 is Phe or Ser; Xaa at position 169 is Ile or His; Xaa at position 170 is Gly, Ala, Val, Ile, Leu, Ser, Lys, Arg or Asp; Xaa at position 172 is Lys, Glu or Ala; Xaa at position 173 is Asn, Thr or Ala; Xaa at position 174 is Gln, Ala or Lys; Xaa at position 176 is Val or Ile; Xaa at position 178 is Gln or Glu; Xaa at position 179 is Ile or Thr; Xaa at position 181 is Gln, Ala, Arg or Lys; Xaa at position 182 is Ser, Phe or Gly; Xaa at position 184 is Ser or Lys; Xaa at position 185 is Ser, Glu or Asn; Xaa at position 188 is Asn, Ser or Lys; Xaa at position 189 is Ala or Val; Xaa at position 193 is Tyr, Glu or Cys; Xaa at position 194 is Ser, Phe or Cys; Xaa at position 196 is Gln or Lys; Xaa at position 198 is Glu or Asn; Xaa at position 200 is Gln or Glu; Xaa at position 202 is Asn or Phe; Xaa at position 203 is Phe or Leu; Xaa at position 205 is Gln, Ala, Val, Pro, Ser, Thr, Cys, Tyr, Asn, Asp or Arg; Xaa at position 206 is Asn or His; Xaa at position 207 is Ile or Thr; Xaa at position 208 is Leu or Met; Xaa at position 212 is Asp, Asn or Ile; Xaa at position 213 is Ser, Gly or Ala; Xaa at position 221 is Tyr, Ala, Val, Leu, Ser, Gln, Met, Ile or His; Xaa at position 222 is Ala or Pro; Xaa at position 226 is Thr or Ser; Xaa at position 228 is Lys or Val; Xaa at position 230 is Ser, Thr or Ala; Xaa at position 231 is Ser, Asp or Asn; Xaa at position 232 is His, Gln or Lys; Xaa at position 233 is Lys, Glu or Arg; Xaa at position 234 is Gly, Ala, Cys, Phe, His, Lys, Leu, Met, Pro, Arg, Ser, Val or Tyr; Xaa at position 235 is Thr, Ala, Cys, Glu, Phe, Gly, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Trp; Xaa at position 236 is Ile, Ala, Cys, Phe, Leu, Pro, Thr or Val; Xaa at position 237 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Val, Trp or Asn; Xaa at position 238 is Phe, Ala, Asp, Leu, Pro, Thr, Val, Trp or Tyr; Xaa at position 239 is Gln, Cys, Gly, Arg, Tyr, Ser, Pro, Trp, Glu, Thr, Met, Ala, Lys, His, Ile, Val, Leu or Asp; Xaa at position 240 is Ser, Ala, Glu, Gly, His, Leu, Met, Asn, Pro, Gln, Thr or Val; Xaa at position 241 is Ser, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 242 is Tyr, Cys, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 243 is Thr, Asp, Glu, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val or Tyr; Xaa at position 244 is Leu, Ala, Cys, Phe, Gly, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 245 is Ser, Ala, Cys, Asp, Gly, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 246 is Gln, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro or Arg; Xaa at position 247 is Ala, Asp, Phe, Lys, Met, Ser or Val; Xaa at position 248 is Ile, Thr, Val or Lys; Xaa at position 250 is Gln or Thr; Xaa at position 253 is Val or Gly; Xaa at position 254 is Glu or Gly; Xaa at position 255 is Ser or Gly; Xaa at position 257 is Asn or Glu; Xaa at position 260 is Lys, Ala, Arg; Xaa at position 262 is Val, Thr or Ala; Xaa at position 264 is Ser, Pro, Ala or Tyr; Xaa at position 265 is Lys, Asp, Glu, Ala, Arg or Asn; Xaa at position 267 is Gln or Leu; Xaa at position 270 is Asp or Lys; Xaa at position 272 is Gln, Asp, Glu or Asn; Xaa at position 273 is Gln, Thr or Lys; Xaa at position 274 is Trp or Thr; Xaa at position 275 is Leu, Pro or His; Xaa at position 276 is Gly or Asp; Xaa at position 277 is Asp, Ser or Asn; Xaa at position 279 is Thr, Ser, Cys, Asn, Glu or Ile; Xaa at position 280 is Thr or Pro; Xaa at position 281 is Gln or Pro; Xaa at position 282 is Val, Gly, Ala or Arg; Xaa at position 283 is Ala, Arg, Gln or Lys; Xaa at position 284 is Glu, Ala or Pro; Xaa at position 287 is Lys or Ser; and Xaa at position 295 is Ser or Gly; and wherein an amino acid is optionally inserted between position 41 and 42 relative to SEQ ID NO: 485; and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide relative to SEQ ID NO: 485.

In some embodiments the nucleic acid molecule encoding a PIP-47 polypeptide is a polynucleotide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 487, Xaa at position 2 is His, Ser or Thr; Xaa at position 3 is Ala, Thr or Ser; Xaa at position 4 is Pro, Gln or Asn; Xaa at position 5 is Gly, Asn, Gln or deleted; Xaa at position 6 is Ala, His, Ser or deleted; Xaa at position 7 is Ile, Ala, Lys, Leu, Val, Arg or deleted; Xaa at position 8 is Pro, His or deleted; Xaa at position 9 is Ser or Thr; Xaa at position 10 is Glu, Val, Thr, Asp, Ile, Leu or Ser; Xaa at position 11 is Lys, Glu, Arg or Asp; Xaa at position 12 is Glu, Lys, Asp or Arg; Xaa at position 13 is Ser, Gly, Val, Leu, Thr or Val; Xaa at position 14 is Ala, Lys or Arg; Xaa at position 15 is His or Ala; Xaa at position 16 is Ala, Trp, Phe or Tyr; Xaa at position 17 is Trp, Leu, Tyr, Phe, Ile or Val; Xaa at position 18 is Leu, Asn, Ile, Val or Gln; Xaa at position 19 is Thr, Glu, Ser, Asp or deleted; Xaa at position 20 is Glu, Thr, Gly, Ser, Asp or deleted; Xaa at position 21 is Thr, Ala, Glu, Lys, His, Ser, Asp, Arg or deleted; Xaa at position 22 is Lys, Glu, Thr, Ala, Arg, Asp or Ser; Xaa at position 23 is Ala, Ser, Arg, Thr or Lys; Xaa at position 24 is Asn, Arg, Lys, Val, Gln, Ile or Leu; Xaa at position 25 is Ala, Val, Lys, Glu, Ile, Leu, Arg or Asn; Xaa at position 26 is Lys, Ser, Leu, Asn, Arg, Thr, Ile, Val or Gln; Xaa at position 27 is Ser, Phe, Thr, Tyr or Trp; Xaa at position 28 is Thr, Ala or Ser; Xaa at position 29 is Ala, Thr, Gln, Ile, Ser, Asn, Leu or Val; Xaa at position 30 is Leu, Thr, Val, Ala, Ile or Ser; Xaa at position 31 is Ala, Arg, Asn, His, Lys or Gln; Xaa at position 32 is Gly or Pro; Xaa at position 34 is Ile, Phe, Leu or Val; Xaa at position 35 is Phe, Tyr or Trp; Xaa at position 36 is Ala, Val, Ser, Leu, Ile or Thr; Xaa at position 37 is Gln, Ala, Arg, Leu, His, Asn, Lys, Ile or Val; Xaa at position 38 is Asp or Glu; Xaa at position 39 is Tyr, Ala, Arg, Phe, Trp, His or Lys; Xaa at position 40 is Asn, Ser, His, Gln or Thr; Xaa at position 41 is Ala, Arg, His or Lys; Xaa at position 42 is Gln, Ser, His, Asn or Thr; Xaa at position 43 is Leu, Gln, Ser, Asn or Thr; Xaa at position 44 is Leu, Phe, Val, Ile, Trp or Tyr; Xaa at position 45 is Thr or Ser; Xaa at position 46 is Ala, Glu, Pro, Ser, Asp or Thr; Xaa at position 47 is Thr, Gly, Ala, Leu, Met, Pro, Ser, Cys, Asn, Asp, Glu, Lys, Arg, Tyr, Ile or Trp; Xaa at position 48 is Gly, Leu, Ile or Val; Xaa at position 49 is Gln, Ala, Arg, Leu, Asn, Lys, Ile or Val; Xaa at position 50 is Ser, Thr or Ala; Xaa at position 51 is Met or Pro; Xaa at position 52 is Ala, Arg, Gly or Lys; Xaa at position 53 is Ser or Thr; Xaa at position 57 is Ala, Ile, Leu or Val; Xaa at position 58 is Ile, Asp, Ala, Leu, Val or Glu; Xaa at position 62 is Phe, Tyr or Trp; Xaa at position 63 is Ser, Gly, Ala or Thr; Xaa at position 65 is Ala, Thr or Ser; Xaa at position 66 is Lys, Gly, Ala, Ser, Arg or Thr; Xaa at position 67 is Gly, Ser or Thr; Xaa at position 68 is Thr, Gly, Ala, Leu, Ile, Val or Ser; Xaa at position 71 is Gly, Asp, Ala, Asn, Glu or Gln; Xaa at position 72 is Ser, Gly, Ala, Arg, Asn, Thr, Lys or Gln; Xaa at position 73 is Tyr, Gly, Cys or Glu; Xaa at position 74 is Ala, Val, Leu or Ile; Xaa at position 75 is Lys, Gln, Asn or Arg; Xaa at position 76 is Asp, Ala or Glu; Xaa at position 77 is Ala, Ser, Cys, Gln, Pro or Asn; Xaa at position 78 is Asp, Gly, Ala, Val, Leu, Trp, Ser, Cys, Gln, Glu, Arg, Phe, Ile, Tyr, Met, Asn, His, Thr or Lys; Xaa at position 79 is Ala, Gly, Tyr, Val, Leu, Trp, Gln, Glu, Lys, Arg, Asn, Asp, Met, Phe, Ile, Cys, Ser or Thr; Xaa at position 80 is Asn, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Tyr, Phe, Asp or Met; Xaa at position 81 is Val, Gly, Ala, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Ile, Asp, Pro or Met; Xaa at position 82 is Ser, Gly, Ala, Val, Trp, Thr, Cys, Gln, Glu, Lys, Asn, His, Phe, Ile, Asp or Met; Xaa at position 83 is Pro, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Phe, Ile or Asp; Xaa at position 84 is Gly, Tyr, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Arg, Asn, Phe, Ile, Asp, Pro or Met; Xaa at position 85 is Ser, Gly, Ala, Val, Leu, Trp, Tyr, Thr, Cys, Gln, Glu, Arg, Phe, Asp, Pro, Lys or Met; Xaa at position 86 is Ala, Gly, Tyr, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, His, Phe, Ile, Asp, Pro or Met; Xaa at position 78 is Asp, Asn, Glu or Gln; Xaa at position 79 is Ala, Asn, Lys, Gln or Arg; Xaa at position 80 is Asn or Asp; Xaa at position 81 is Val, Ala, Ile or Val; Xaa at position 84 is Gly, Ser or The; Xaa at position 85 is Ser, Thr, Lys or Arg; Xaa at position 86 is Ala, Val, Ile or Leu; Xaa at position 88 is Val, Glu, Thr, Ile, Leu, Asp or Ser; Xaa at position 90 is Ile, Phe, Leu or Val; Xaa at position 91 is Tyr, Gln, Cys, His, Trp, Phe or Asn; Xaa at position 92 is Glu, Asp, Lys or Arg; Xaa at position 95 is Gln, Ala, Val, Ser, Thr, Asn or His; Xaa at position 96 is Thr or Ser; Xaa at position 97 is Ala, Ser, Cys or Thr; Xaa at position 98 is Ile, Val or Leu; Xaa at position 99 is Asp, Asn, Glu or Gln; Xaa at position 100 is Ile, Thr, Met, Leu, Val or Ser; Xaa at position 103 is Arg, Thr, Lys or Ser; Xaa at position 106 is Gly, Glu or Asp; Xaa at position 107 is Tyr, Cys, Phe, Gly, His, Trp or Leu; Xaa at position 108 is Asn, Ala, Cys, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr or Trp; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Ala, Glu, Phe, Met, Asn, Pro, Ser, Thr, Val or Trp; Xaa at position 111 is Asp, Ala, Glu, Phe, Gly, His, Asn, Gln, Ser, Val or Tyr; Xaa at position 112 is Gln, Cys, Pro or Val; Xaa at position 113 is Pro, Cys or Gly; Xaa at position 114 is Thr, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Val, Trp or Tyr; Xaa at position 115 is Asp, Ala, Glu, Phe, Gly, His, Ile, Leu, Pro, Gln, Arg, Ser, Thr or Tyr; Xaa at position 116 is Gln, Ala, Cys, Phe, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val or Trp; Xaa at position 117 is Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 118 is Pro, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 119 is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 120 is Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position 107 is Tyr, Trp or Phe; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Asn, Ile, Val or Gln; Xaa at position 112 is Gln, Pro or Asn; Xaa at position 115 is Asp, Gly, Ala or Glu; Xaa at position 116 is Gln, Ala, Arg, Asn or Lys; Xaa at position 117 is Lys, Ala, Arg, Asn or Gln; Xaa at position 119 is Lys, His or Arg; Xaa at position 120 is Ser, Thr or Ala; Xaa at position 121 is Ala, Thr, Lys, Ser or Arg; Xaa at position 124 is Arg, Tyr, Lys, His, Trp or Phe; Xaa at position 130 is Phe, Leu, Ile or Val; Xaa at position 132 is Gln, Lys, Asn or Arg; Xaa at position 133 is Gln, Glu, Asn or Asp; Xaa at position 135 is Ala, Ser or Thr; Xaa at position 136 is Glu, Ser, Ala, Arg, Asn, Asp, Thr, Lys or Gln; Xaa at position 137 is Ile, Asn, Leu, Val or Gln; Xaa at position 141 is Ser or Thr; Xaa at position 142 is Leu, Gly, Val, Met, Ser, Thr, Cys, Asn, Gln, Asp, Glu, His or Pro; Xaa at position 143 is Leu, Glu, Ile, Val or Asp; Xaa at position 144 is Ala, Ser or Thr; Xaa at position 146 is Gln, Ser, Asn or Thr; Xaa at position 147 is Val, Ser, Ile, Leu or Thr; Xaa at position 148 is Thr or Ser; Xaa at position 149 is Gln, Leu, Asn, Ile or Val; Xaa at position 150 is Ile, Val or Leu; Xaa at position 151 is Gln, Ala, Arg, Met, Asn or Lys; Xaa at position 153 is Lys, Gln, Arg or Asn; Xaa at position 156 is Asp, Gly or Glu; Xaa at position 158 is Asn, Asp, Gln or Glu; Xaa at position 161 is Val, Ile or Leu; Xaa at position 164 is Phe or Cys; Xaa at position 165 is Val, Ala, Ile or Leu; Xaa at position 168 is Phe, Ser or Thr; Xaa at position 169 is Ile, His, Leu or Val; Xaa at position 170 is Gly, Ala, Val, Ile, Leu, Ser, Lys, Arg or Asp; Xaa at position 172 is Lys, Glu, Ala, Arg or Asp; Xaa at position 173 is Asn, Thr, Ala, Gln or Ser; Xaa at position 174 is Gln, Ala, Lys, Asn or Arg; Xaa at position 176 is Val, Ile or Leu; Xaa at position 178 is Gln, Glu, Asn or Asp; Xaa at position 179 is Ile, Thr, Leu, Val or Ser; Xaa at position 181 is Gln, Ala, Arg, Lys or Asn; Xaa at position 182 is Ser, Phe or Gly; Xaa at position 184 is Ser, Lys, Thr or Arg; Xaa at position 185 is Ser, Glu, Asn, Thr, Gln or Asp; Xaa at position 188 is Asn, Ser, Lys, Gln, Thr or Arg; Xaa at position 189 is Ala, Val, Ile or Leu; Xaa at position 193 is Tyr, Glu, Cys, Trp, Phe or Asp; Xaa at position 194 is Ser, Phe, Cys or Thr; Xaa at position 196 is Gln, Lys, Asn or Arg; Xaa at position 198 is Glu, Asn, Asp or Gln; Xaa at position 200 is Gln or Glu; Xaa at position 202 is Asn, Phe or Gln; Xaa at position 203 is Phe, Leu, Ile or Val; Xaa at position 205 is Gln, Ala, Val, Pro, Ser, Thr, Cys, Tyr, Asn, Asp or Arg; Xaa at position 206 is Asn, His or Gln; Xaa at position 207 is Ile, Thr, Leu, Val or Ser; Xaa at position 208 is Leu, Met, Ile or Val; Xaa at position 212 is Asp, Asn, Ile, Glu, Gln, Leu or Val; Xaa at position 213 is Ser, Gly, Ala or Thr; Xaa at position 221 is Tyr, Ala, Val, Leu, Ser, Gln, Met, Ile or His; Xaa at position 222 is Ala or Pro; Xaa at position 226 is Thr or Ser; Xaa at position 228 is Lys, Val, Arg, Ile or Leu; Xaa at position 230 is Ser, Thr or Ala; Xaa at position 231 is Ser, Asp, Asn, Thr, Glu or Gln; Xaa at position 232 is His, Gln, Lys, Asn or Arg; Xaa at position 233 is Lys, Glu or Arg; Xaa at position 234 is Gly, Ala, Cys, Phe, His, Lys, Leu, Met, Pro, Arg, Ser, Val or Tyr; Xaa at position 235 is Thr, Ala, Cys, Glu, Phe, Gly, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Trp; Xaa at position 236 is Ile, Ala, Cys, Phe, Leu, Pro, Thr or Val; Xaa at position 237 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Val, Trp or Asn; Xaa at position 238 is Phe, Ala, Asp, Leu, Pro, Thr, Val, Trp or Tyr; Xaa at position 239 is Gln, Cys, Gly, Arg, Tyr, Ser, Pro, Trp, Glu, Thr, Met, Ala, Lys, His, Ile, Val, Leu or Asp; Xaa at position 240 is Ser, Ala, Glu, Gly, His, Leu, Met, Asn, Pro, Gln, Thr or Val; Xaa at position 241 is Ser, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 242 is Tyr, Cys, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 243 is Thr, Asp, Glu, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val or Tyr; Xaa at position 244 is Leu, Ala, Cys, Phe, Gly, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 245 is Ser, Ala, Cys, Asp, Gly, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 246 is Gln, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro or Arg; Xaa at position 247 is Ala, Asp, Phe, Lys, Met, Ser or Val; Xaa at position 248 is Ile, Thr, Val, Lys, Leu, Val, Ser or Arg; Xaa at position 250 is Gln, Thr, Asn or Ser; Xaa at position 253 is Val, Gly, Ile or Leu; Xaa at position 254 is Glu, Gly or Asp; Xaa at position 255 is Ser, Gly or Thr; Xaa at position 257 is Asn, Glu, Asp or Gln; Xaa at position 260 is Lys, Ala or Arg; Xaa at position 262 is Val, Thr, Ala, Ile, Leu or Ser; Xaa at position 264 is Ser, Pro, Ala, Tyr, Trp or Phe; Xaa at position 265 is Lys, Asp, Glu, Ala, Arg, Asn or Gln; Xaa at position 267 is Gln, Leu, Asn, Ile or Val; Xaa at position 270 is Asp, Lys, Glu or Arg; Xaa at position 272 is Gln, Asp, Glu or Asn; Xaa at position 273 is Gln, Thr, Lys, Asn, Ser or Arg; Xaa at position 274 is Trp, Thr, Tyr, Phe or Ser; Xaa at position 275 is Leu, Pro, His, Ile or Val; Xaa at position 276 is Gly, Asp or Glu; Xaa at position 277 is Asp, Ser, Asn or Thr; Xaa at position 279 is Thr, Ser, Cys, Asn, Glu or Ile; Xaa at position 280 is Thr, Pro or Ser; Xaa at position 281 is Gln, Pro or Asn; Xaa at position 282 is Val, Gly, Ala, Arg, Ile, Leu or Lys; Xaa at position 283 is Ala, Arg, Gln, Lys or Asn; Xaa at position 284 is Glu, Ala, Pro or Asp; Xaa at position 287 is Lys, Ser, Arg or Thr; and Xaa at position 295 is Ser, Gly or Thr; and wherein an amino acid is optionally inserted between position 41 and 42 relative to SEQ ID NO: 487; and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide relative to SEQ ID NO: 487.

In some embodiments the nucleic acid molecules encode a PIP-47 polypeptide having a nucleotide sequence encoding a PIP-47 polypeptide comprises an amino acid motif as represented by positions 234-247 of SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486 or SEQ ID NO: 487.

In some embodiments the nucleic acid molecules encode a PIP-47 polypeptide having a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 2

In some embodiments exemplary nucleic acid molecules encode a PIP-47 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, SEQ ID NO: 916-SEQ ID NO: 921, and active fragments thereof, as well as amino acid substitutions deletions, insertions and fragments thereof and combinations thereof.

In some embodiments the nucleic acid molecules encode a PIP-47 polypeptide of Table 8, Table 10, Table 11, Table 12, Table 13, Table 14, Table 15, Table 16, Table 17, Table 18, and/or Table 19 combinations of the amino acid substitutions thereof, and deletions and/or insertions thereof.

Also provided are nucleic acid molecules that encode transcription and/or translation products that are subsequently spliced to ultimately produce functional PIP-47 polypeptides. Splicing can be accomplished in vitro or in vivo, and can involve cis- or trans-splicing. The substrate for splicing can be polynucleotides (e.g., RNA transcripts) or polypeptides. An example of cis-splicing of a polynucleotide is where an intron inserted into a coding sequence is removed and the two flanking exon regions are spliced to generate a PIP-47 polypeptide encoding sequence. An example of trans splicing would be where a polynucleotide is encrypted by separating the coding sequence into two or more fragments that can be separately transcribed and then spliced to form the full-length pesticidal encoding sequence. The use of a splicing enhancer sequence, which can be introduced into a construct, can facilitate splicing either in cis or trans-splicing of polypeptides (U.S. Pat. Nos. 6,365,377 and 6,531,316). Thus, in some embodiments the polynucleotides do not directly encode a full-length PIP-47 polypeptide, but rather encode a fragment or fragments of a PIP-47 polypeptide. These polynucleotides can be used to express a functional PIP-47 polypeptide through a mechanism involving splicing, where splicing can occur at the level of polynucleotide (e.g., intron/exon) and/or polypeptide (e.g., intein/extein). This can be useful, for example, in controlling expression of pesticidal activity, since a functional pesticidal polypeptide will only be expressed if all required fragments are expressed in an environment that permits splicing processes to generate functional product. In another example, introduction of one or more insertion sequences into a polynucleotide can facilitate recombination with a low homology polynucleotide; use of an intron or intein for the insertion sequence facilitates the removal of the intervening sequence, thereby restoring function of the encoded variant.

Nucleic acid molecules that are fragments of these nucleic acid sequences encoding PIP-47 polypeptides are also encompassed by the embodiments. “Fragment” as used herein refers to a portion of the nucleic acid sequence encoding a PIP-47 polypeptide. A fragment of a nucleic acid sequence may encode a biologically active portion of a PIP-47 polypeptide or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of a nucleic acid sequence encoding a PIP-47 polypeptide comprise at least about 150, 180, 210, 240, 270, 300, 330 or 360, contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence encoding a PIP-47 polypeptide disclosed herein, depending upon the intended use. “Contiguous nucleotides” is used herein to refer to nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the PIP-47 polypeptide and, hence, retain insecticidal activity. “Retains PIP-47 activity” is used herein to refer to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the insecticidal activity of the full-length PIP-47Aa polypeptide of SEQ ID NO: 2. In one embodiment, the insecticidal activity is Lepidoptera activity. In one embodiment, the insecticidal activity is against a Coleopteran species. In one embodiment, the insecticidal activity is against one or more insect pests of the corn rootworm complex: western corn rootworm, Diabrotica virgifera; northern corn rootworm, D. barberi: Southern corn rootworm or spotted cucumber beetle; Diabrotica undecimpunctata howardi, and the Mexican corn rootworm, D. virgifera zeae. In one embodiment, the insecticidal activity is against a Diabrotica species.

In some embodiments a fragment of a nucleic acid sequence encoding a PIP-47 polypeptide encoding a biologically active portion of a protein will encode at least about 15, 20, 30, 50, 75, 100, 125, contiguous amino acids or up to the total number of amino acids present in a full-length PIP-47 polypeptide of the embodiments. In some embodiments, the fragment is an N-terminal and/or a C-terminal truncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N-terminus and/or C-terminus relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 or variants thereof, e.g., by proteolysis, insertion of a start codon, deletion of the codons encoding the deleted amino acids with the concomitant insertion of a stop codon or by insertion of a stop codon in the coding sequence. In some embodiments, the fragments encompassed herein result from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids from the N-terminus relative to SEQ ID NO: 2, 28, 32 or 36 or variants thereof, e.g., by proteolysis or by insertion of a start codon in the coding sequence. In some embodiments, the fragments encompassed herein result from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 amino acids relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 or variants thereof, e.g., by proteolysis or by insertion of a start codon in the coding sequence.

In some embodiments a PIP-47 polypeptide is encoded by a nucleic acid sequence sufficiently homologous to the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. “Sufficiently homologous” is used herein to refer to an amino acid or nucleic acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding homology of proteins encoded by two nucleic acid sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. In some embodiments the sequence homology is against the full length sequence of the polynucleotide encoding a PIP-47 polypeptide or against the full length sequence of a PIP-47 polypeptide. In some embodiments the PIP-47 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence identity compared to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. In some embodiments the sequence identity is against the full length sequence of the polynucleotide encoding a PIP-47 polypeptide or against the full length sequence of a PIP-47 polypeptide. In some embodiments the sequence identity is calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.) with all default parameters. In some embodiments the sequence identity is across the entire length of polypeptide calculated using ClustalW algorithm in the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.) with all default parameters.

To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are the same length. In another embodiment, the comparison is across the entirety of the reference sequence (e.g., across the entirety of one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul, et al., (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleic acid sequences homologous to pesticidal nucleic acid molecules of the embodiments. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to pesticidal protein molecules of the embodiments. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, et al., (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See, Altschul, et al., (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection.

Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins, et al., (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting example of a software program useful for analysis of ClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CAB/OS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software to determine sequence identity or similarity using the following default parameters: % identity and % similarity for a nucleic acid sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmpii scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used. “Equivalent program” is used herein to refer to any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

The embodiments also encompass nucleic acid molecules encoding PIP-47 polypeptide variants. “Variants” of the PIP-47 polypeptide encoding nucleic acid sequences include those sequences that encode the PIP-47 polypeptides disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the PIP-47 polypeptides disclosed as discussed below.

The present disclosure provides isolated or recombinant polynucleotides that encode any of the PIP-47 polypeptides disclosed herein. Those having ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding PIP-47 polypeptides of the present disclosure exist. Table 1 is a codon table that provides the synonymous codons for each amino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine. Thus, at every position in the nucleic acids of the disclosure where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.

TABLE 1

Alanine
Ala
A
GCA GCC GCG GCU

Cysteine
Cys
C
UGC UGU

Aspartic acid
Asp
D
GAC GAU

Glutamic acid
Glu
E
GAA GAG

Phenylalanine
Phe
F
UUC UUU

Glycine
Gly
G
GGA GGC GGG GGU

Histidine
His

CAC CAU

Isoleucine
Ile
I
AUA AUC AUU

Lysine
Lys
K
AAA AAG

Leucine
Leu
L
UUA UUG CUA CUC CUG CUU

Methionine
Met
M
AUG

Asparagine
Asn
N
AAC AAU

Proline
Pro
P
CCA CCC CCG CCU

Glutamine
Gln
Q
CAA CAG

Arginine
Arg
R
AGA AGG CGA CGC CGG CGU

Serine
Ser
S
AGC AGU UCA UCC UCG UCU

Threonine
Thr
T
ACA ACC ACG ACU

Valine
Val
V
GUA GUC GUG GUU

Tryptophan
Trp
W
UGG

Tyrosine
Tyr
Y
UAC UAU

The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleic acid sequences thereby leading to changes in the amino acid sequence of the encoded PIP-47 polypeptides, without altering the biological activity of the proteins. Thus, variant nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions and/or deletions into the corresponding nucleic acid sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ability to confer pesticidal activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.

The polynucleotides of the disclosure and fragments thereof are optionally used as substrates for a variety of recombination and recursive recombination reactions, in addition to standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., to produce additional pesticidal polypeptide homologues and fragments thereof with desired properties. A variety of such reactions are known, including those developed by the inventors and their co-workers. Methods for producing a variant of any nucleic acid listed herein comprising recursively recombining such polynucleotide with a second (or more) polynucleotide, thus forming a library of variant polynucleotides are also embodiments of the disclosure, as are the libraries produced, the cells comprising the libraries and any recombinant polynucleotide produces by such methods. Additionally, such methods optionally comprise selecting a variant polynucleotide from such libraries based on pesticidal activity, as is wherein such recursive recombination is done in vitro or in vivo.

A variety of diversity generating protocols, including nucleic acid recursive recombination protocols are available and fully described in the art. The procedures can be used separately, and/or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well as variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e.g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and/or organisms with new and/or improved characteristics.

While distinctions and classifications are made in the course of the ensuing discussion for clarity, it will be appreciated that the techniques are often not mutually exclusive. Indeed, the various methods can be used singly or in combination, in parallel or in series, to access diverse sequence variants.

The result of any of the diversity generating procedures described herein can be the generation of one or more nucleic acids, which can be selected or screened for nucleic acids with or which confer desirable properties or that encode proteins with or which confer desirable properties. Following diversification by one or more of the methods herein or otherwise available to one of skill, any nucleic acids that are produced can be selected for a desired activity or property, e.g. pesticidal activity or, such activity at a desired pH, etc. This can include identifying any activity that can be detected, for example, in an automated or automatable format, by any of the assays in the art, see, e.g., discussion of screening of insecticidal activity, infra. A variety of related (or even unrelated) properties can be evaluated, in serial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures for generating modified nucleic acid sequences, e.g., those coding for polypeptides having pesticidal activity or fragments thereof, are found in the following publications and the references cited therein: Soong, et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) Tumor Targeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang, et al., (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999) Curr Opin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol 17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al., (1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA 94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733; Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) Nat Biotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373-386; Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene, 164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995) Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 and Stemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith, (1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science 229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) “The efficiency of oligonucleotide directed mutagenesis” in Nucleic Acids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA 82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass, et al., (1988) Science 242:240-245); oligonucleotide-directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500; Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller and Smith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modified DNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764; Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988) Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, et al., (1988) Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, et al., (1984) Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter, (1987) Methods in Enzymol 154:382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115), restriction-selection and restriction-purification (Wells, et al., (1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total gene synthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar and Khorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315-323 and Grundstrom, et al., (1985) Nucl Acids Res 13:3305-3316), double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech 4:450-455). Additional details on many of the above methods can be found in Methods Enzymol Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.

Additional details regarding various diversity generating methods can be found in the following US patents, PCT Publications and applications and EPO publications: U.S. Pat. No. 5,723,323, U.S. Pat. No. 5,763,192, U.S. Pat. No. 5,814,476, U.S. Pat. No. 5,817,483, U.S. Pat. No. 5,824,514, U.S. Pat. No. 5,976,862, U.S. Pat. No. 5,605,793, U.S. Pat. No. 5,811,238, U.S. Pat. No. 5,830,721, U.S. Pat. No. 5,834,252, U.S. Pat. No. 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO 1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368, EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO 1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832, WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO 2000/18906, WO 2000/04190, WO 2000/42561, WO 2000/42559, WO 2000/42560, WO 2001/23401 and PCT/US01/06775.

The nucleotide sequences of the embodiments can also be used to isolate corresponding sequences from other organisms, particularly other bacteria, particularly a Pseudomonas species and more particularly a Pseudomonas putida, a Pseudomonas fulva or a Pseudomonas chlororaphis strain. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments. Such sequences include sequences that are orthologs of the disclosed sequences. The term “orthologs” refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

To identify potential PIP-47 polypeptides from bacterial collections, the bacterial cell lysates can be screened with antibodies generated against a PIP-47 polypeptide using Western blotting and/or ELISA methods. This type of assays can be performed in a high throughput fashion. Positive samples can be further analyzed by various techniques such as antibody based protein purification and identification. Methods of generating antibodies are well known in the art as discussed infra.

Alternatively, mass spectrometry based protein identification method can be used to identify homologs of PIP-47 polypeptides using protocols in the literatures (Scott Patterson, (1998), 10.22, 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc). Specifically, LC-MS/MS based protein identification method is used to associate the MS data of given cell lysate or desired molecular weight enriched samples (excised from SDS-PAGE gel of relevant molecular weight bands to PIP-47) with sequence information of PIP-47 (e.g., SEQ ID NO: 2)) and its homologs. Any match in peptide sequences indicates the potential of having the homologous proteins in the samples. Additional techniques (protein purification and molecular biology) can be used to isolate the protein and identify the sequences of the homologs.

In hybridization methods, all or part of the pesticidal nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides and may be labeled with a detectable group such as 32P or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known PIP-47 polypeptide-encoding nucleic acid sequence disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleic acid sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of nucleic acid sequence encoding a PIP-47 polypeptide of the disclosure or a fragment or variant thereof. Methods for the preparation of probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra, herein incorporated by reference.

For example, an entire nucleic acid sequence, encoding a PIP-47 polypeptide, disclosed herein or one or more portions thereof may be used as a probe capable of specifically hybridizing to corresponding nucleic acid sequences encoding PIP-47 polypeptide-like sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique and are preferably at least about 10 nucleotides in length or at least about 20 nucleotides in length. Such probes may be used to amplify corresponding pesticidal sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringent conditions. “Stringent conditions” or “stringent hybridization conditions” is used herein to refer to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≥90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Proteins and Variants and Fragments Thereof

PIP-47 polypeptides are also encompassed by the disclosure. “Pseudomonas Insecticidal Protein-47”, “PIP-47 polypeptide” or “PIP-47 protein” as used herein interchangeably refers to a polypeptide having pesticidal activity including but not limited to fungicidal activity, and insecticidal activity against one or more insect pests of the Lepidoptera and/or Coleoptera orders, and is sufficiently homologous to the protein of SEQ ID NO: 2. A variety of PIP-47 polypeptides are contemplated. One source of PIP-47 polypeptides or related proteins is a Pseudomonas putida strain which contains the PIP-47Aa polynucleotide of SEQ ID NO: 1 encoding the PIP-47Aa polypeptide of SEQ ID NO: 2, a Pseudomonas putida strain which contains the PIP-47Ba polynucleotide of SEQ ID NO: 3 encoding the PIP-47Bb polypeptide of SEQ ID NO: 4, a Pseudomonas fulva strain which contains the PIP-47Bb polynucleotide of SEQ ID NO: 5 encoding the PIP-47Bb polypeptide of SEQ ID NO: 6 or a Pseudomonas chlororaphis strain which contains the PIP-47Fa polynucleotide of SEQ ID NO: 7 encoding the PIP-47Fa polypeptide of SEQ ID NO: 8.

In some embodiments a PIP-47 polypeptide is sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. “Sufficiently homologous” is used herein to refer to an amino acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence homology compared to a reference sequence using one of the alignment programs described herein using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding homology of proteins taking into account amino acid similarity and the like. In some embodiments the sequence homology is against the full length sequence a PIP-47 polypeptide. In some embodiments the PIP-47 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. In some embodiments the sequence identity is against the full length sequence of a PIP-47 polypeptide. In some embodiments the sequence identity is calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.) with all default parameters. In some embodiments the sequence identity is across the entire length of polypeptide calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.) with all default parameters.

As used herein, the terms “protein,” “peptide molecule,” or “polypeptide” includes any molecule that comprises five or more amino acids. It is well known in the art that protein, peptide or polypeptide molecules may undergo modification, including post-translational modifications, such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation or oligomerization. Thus, as used herein, the terms “protein,” “peptide molecule” or “polypeptide” includes any protein that is modified by any biological or non-biological process. The terms “amino acid” and “amino acids” refer to all naturally occurring L-amino acids.

A “recombinant protein” is used herein to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell. A PIP-47 polypeptide that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10% or 5% (by dry weight) of non-pesticidal protein (also referred to herein as a “contaminating protein”).

“Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to a PIP-47 polypeptide and that exhibit insecticidal activity. “Fragments” or “biologically active portions” of PIP-47 polypeptides includes fragments comprising amino acid sequences sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, SEQ ID NO: 916-SEQ ID NO: 921, respectively. A biologically active portion of a PIP-47 polypeptide can be a polypeptide that is, for example, 10, 25, 50, 75, 100, 125, 132 or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for insecticidal activity. As used here, a fragment comprises at least 8 contiguous amino acids of a PIP-47 polypeptide. In some embodiments a PIP-47 polypeptide fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, SEQ ID NO: 916-SEQ ID NO: 921. In some embodiments a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 2. In some embodiments a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 18. In some embodiments, the PIP-47 polypeptide fragment is an N-terminal and/or a C-terminal truncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N-terminus and/or C-terminus relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or variants thereof including but not limited to: SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQ ID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO: 416, SEQ ID NO: 417, SEQ ID NO: 418, SEQ ID NO: 419, SEQ ID NO: 420, SEQ ID NO: 629, SEQ ID NO: 630, SEQ ID NO: 631, SEQ ID NO: 632, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635, SEQ ID NO: 636, SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO: 641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 644, SEQ ID NO: 645, SEQ ID NO: 646, SEQ ID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, SEQ ID NO: 651, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, SEQ ID NO: 699, SEQ ID NO: 700, SEQ ID NO: 701, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO: 719, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 797, SEQ ID NO: 798, SEQ ID NO: 799, SEQ ID NO: 800, SEQ ID NO: 801, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 805, SEQ ID NO: 806, SEQ ID NO: 807, SEQ ID NO: 808, SEQ ID NO: 809, SEQ ID NO: 810, SEQ ID NO: 811, SEQ ID NO: 812, SEQ ID NO: 813, SEQ ID NO: 814, SEQ ID NO: 814, SEQ ID NO: 815, SEQ ID NO: 816, SEQ ID NO: 817, SEQ ID NO: 818, SEQ ID NO: 819, SEQ ID NO: 820, SEQ ID NO: 821, SEQ ID NO: 822, SEQ ID NO: 823, SEQ ID NO: 824, SEQ ID NO: 825, SEQ ID NO: 826, SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ ID NO: 830, SEQ ID NO: 831, SEQ ID NO: 832, SEQ ID NO: 833, SEQ ID NO: 834, SEQ ID NO: 835, SEQ ID NO: 836, SEQ ID NO: 837, SEQ ID NO: 838, SEQ ID NO: 839, SEQ ID NO: 840, SEQ ID NO: 841, SEQ ID NO: 842, SEQ ID NO: 843, SEQ ID NO: 844, SEQ ID NO: 845, SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, SEQ ID NO: 850, SEQ ID NO: 851, SEQ ID NO: 852, SEQ ID NO: 853, SEQ ID NO: 854, SEQ ID NO: 855, SEQ ID NO: 856, SEQ ID NO: 857, SEQ ID NO: 858, SEQ ID NO: 859, SEQ ID NO: 860, SEQ ID NO: 861, SEQ ID NO: 862, SEQ ID NO: 863, SEQ ID NO: 864, SEQ ID NO: 865, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 890, SEQ ID NO: 891, SEQ ID NO: 892, SEQ ID NO: 893, SEQ ID NO: 894, SEQ ID NO: 895, SEQ ID NO: 896, SEQ ID NO: 897, SEQ ID NO: 898, SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, SEQ ID NO: 903, SEQ ID NO: 904, SEQ ID NO: 905, SEQ ID NO: 906, SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO: 916, SEQ ID NO: 917, SEQ ID NO: 918, SEQ ID NO: 919, SEQ ID NO: 920 and SEQ ID NO: 921, e.g., by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon, and/or insertion of a stop codon.

In some embodiments, the PIP-47 polypeptide fragments encompassed herein result from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or variants thereof including, but not limited to any one of SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, SEQ ID NO: 916-SEQ ID NO: 921, e.g., by proteolysis or by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon.

In some embodiments, the PIP-47 polypeptide fragments encompassed herein result from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or variants thereof including, but not limited to any one of SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, SEQ ID NO: 916-SEQ ID NO: 921. In some embodiments the truncation is of the first 4 amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 or variants thereof including, but not limited to any one of SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, SEQ ID NO: 916-SEQ ID NO: 921.

“Variants” as used herein refers to proteins or polypeptides having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the parental amino acid sequence. In some embodiments a PIP-47 polypeptide variant does not have the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8.

In some embodiments a PIP-47 polypeptide has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity across the entire length of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. In some embodiments a PIP-47 polypeptide has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity across the entire length of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. In some embodiments a PIP-47 polypeptide has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity across the entire length of the amino acid sequence of SEQ ID NO: 2. In some embodiments a PIP-47 polypeptide has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity across the entire length of the amino acid sequence of SEQ ID NO: 4. In some embodiments a PIP-47 polypeptide has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity across the entire length of the amino acid sequence of SEQ ID NO: 6. In some embodiments a PIP-47 polypeptide has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity across the entire length of the amino acid sequence of SEQ ID NO: 8. In some embodiments a PIP-47 polypeptide comprises an amino acid sequence having at least 50% identity, to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, wherein the polypeptide has insecticidal activity. In some embodiments a PIP-47 polypeptide comprises an amino acid sequence having at least 80% identity, to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, wherein the polypeptide has insecticidal activity. In some embodiments a PIP-47 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide has insecticidal activity. In some embodiments a PIP-47 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 4, wherein the polypeptide has insecticidal activity. In some embodiments a PIP-47 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 6, wherein the polypeptide has insecticidal activity. In some embodiments a PIP-47 polypeptide comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 8, wherein the polypeptide has insecticidal activity.

In some embodiments the sequence identity is across the entire length of polypeptide calculated using ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.) with all default parameters.

In some embodiments the PIP-47 polypeptide comprises an amino acid Motif 1 as represented by positions 77-87 of SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486 or SEQ ID NO: 487.

In some embodiments the PIP-47 polypeptide comprises an amino acid Motif 2 as represented by positions 107-120 of SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486 or SEQ ID NO: 487.

In some embodiments the PIP-47 polypeptide comprises an amino acid Motif 3 as represented by positions 234-247 of SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486 or SEQ ID NO: 487.

In some embodiments the PIP-47 polypeptide comprises an amino acid Motif 1 as represented by positions 77-87; an amino acid Motif 2 as represented by positions 107-120; and an amino acid Motif 3 as represented by positions 234-247 of SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486 or SEQ ID NO: 487.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 481, wherein Xaa at position 47 is Thr, Gly, Ala, Leu, Met, Pro, Ser, Cys, Asn, Asp, Glu, Lys, Arg, Tyr, Ile or Trp; Xaa at position 73 is Tyr, Gly, Cys or Glu; Xaa at position 77 is Ala, Ser, Cys, Gln, Pro or Asn; Xaa at position 78 is Asp, Gly, Ala, Val, Leu, Trp, Ser, Cys, Gln, Glu, Arg, Phe, Ile, Tyr, Met, Asn, His, Thr or Lys; Xaa at position 79 is Ala, Gly, Tyr, Val, Leu, Trp, Gln, Glu, Lys, Arg, Asn, Asp, Met, Phe, Ile, Cys, Ser or Thr; Xaa at position 80 is Asn, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Tyr, Phe, Asp or Met; Xaa at position 81 is Val, Gly, Ala, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Ile, Asp, Pro or Met; Xaa at position 82 is Ser, Gly, Ala, Val, Trp, Thr, Cys, Gln, Glu, Lys, Asn, His, Phe, Ile, Asp or Met; Xaa at position 83 is Pro, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Phe, Ile or Asp; Xaa at position 84 is Gly, Tyr, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Arg, Asn, Phe, Ile, Asp, Pro or Met; Xaa at position 85 is Ser, Gly, Ala, Val, Leu, Trp, Tyr, Thr, Cys, Gln, Glu, Arg, Phe, Asp, Pro or Met; Xaa at position 86 is Ala, Gly, Tyr, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, His, Phe, Ile, Asp, Pro or Met; Xaa at position 95 is Gln, Ala, Val, Ser, Thr, Asn or His; Xaa at position 107 is Tyr, Cys, Phe, Gly, His or Leu; Xaa at position 108 is Asn, Ala, Cys, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr or Trp; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Ala, Glu, Phe, Met, Asn, Pro, Ser, Thr, Val or Trp; Xaa at position 111 is Asp, Ala, Glu, Phe, Gly, His, Asn, Gln, Ser, Val or Tyr; Xaa at position 112 is Gln, Cys, Pro or Val; Xaa at position 113 is Pro, Cys or Gly; Xaa at position 114 is Thr, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Val, Trp or Tyr; Xaa at position 115 is Asp, Ala, Glu, Phe, Gly, His, Ile, Leu, Pro, Gln, Arg, Ser, Thr or Tyr; Xaa at position 116 is Gln, Ala, Cys, Phe, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val or Trp; Xaa at position 117 is Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 118 is Pro, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 119 is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 120 is Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position 142 is Leu, Gly, Val, Met, Ser, Thr, Cys, Asn, Gln, Asp, Glu or His; Xaa at position 170 is Gly, Ala, Val, Ile, Leu, Ser, Lys or Arg; Xaa at position 205 is Gln, Ala, Val, Pro, Ser, Thr, Cys, Tyr, Asn, Asp or Arg; Xaa at position 221 is Tyr, Ala, Val, Leu, Ser, Gln, Met, Ile or His; Xaa at position 233 is Lys, Glu or Arg; Xaa at position 234 is Gly, Ala, Cys, Phe, His, Lys, Leu, Met, Pro, Arg, Ser, Val or Tyr; Xaa at position 235 is Thr, Ala, Cys, Glu, Phe, Gly, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Trp; Xaa at position 236 is Ile, Ala, Cys, Phe, Leu, Pro, Thr or Val; Xaa at position 237 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Val or Trp; Xaa at position 238 is Phe, Ala, Asp, Leu, Pro, Thr, Val, Trp or Tyr; Xaa at position 239 is Gln, Cys, Gly, Arg, Tyr, Ser, Pro, Trp, Glu, Thr, Met, Ala, Lys, His, Ile, Val, Leu or Asp; Xaa at position 240 is Ser, Ala, Glu, Gly, His, Leu, Met, Asn, Pro, Gln, Thr or Val; Xaa at position 241 is Ser, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 242 is Tyr, Cys, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 243 is Thr, Asp, Glu, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val or Tyr; Xaa at position 244 is Leu, Ala, Cys, Phe, Gly, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 245 is Ser, Ala, Cys, Asp, Gly, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 246 is Gln, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro or Arg; Xaa at position 247 is Ala, Asp, Phe, Lys, Met, Ser or Val; and Xaa at position 279 is Thr, Ser, Cys, Asn or Glu; and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 481 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 481, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 481 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 481, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 482, wherein Xaa at position 2 is His or Asn; Xaa at position 4 is Pro or deleted; Xaa at position 5 is Gly or deleted; Xaa at position 6 is Ala or deleted; Xaa at position 7 is Ile, Ala or deleted; Xaa at position 9 is Ser or Thr; Xaa at position 10 is Glu or Val; Xaa at position 13 is Ser or Val; Xaa at position 19 is Thr, Glu or deleted; Xaa at position 20 is Glu, Gly or deleted; Xaa at position 21 is Thr, Gly or deleted; Xaa at position 22 is Lys or Thr; Xaa at position 24 is Asn, Lys or Arg; Xaa at position 25 is Ala or Val; Xaa at position 26 is Lys or Asn; Xaa at position 29 is Ala, Thr or Ile; Xaa at position 30 is Leu, Ala or Val; Xaa at position 31 is Arg or His; Xaa at position 37 is Gln or His; Xaa at position 38 is Asp or Glu; Xaa at position 41 is Arg or His; Xaa at position 42 is Gln or His; Xaa at position 43 is Leu or Gln; Xaa at position 46 is Ala, Ser or Pro; Xaa at position 47 is Thr, Gly, Ala, Leu, Met, Pro, Ser, Cys, Asn, Asp, Glu, Lys, Arg, Tyr, Ile or Trp; Xaa at position 49 is Gln, Arg or Leu; Xaa at position 50 is Ser or Ala; Xaa at position 53 is Ser or Thr; Xaa at position 62 is Phe or Tyr; Xaa at position 66 is Lys, Ala or Ser; Xaa at position 68 is Thr or Ala; Xaa at position 71 is Gly or Asp; Xaa at position 73 is Tyr, Gly, Cys or Glu; Xaa at position 75 is Lys or Asn; Xaa at position 77 is Ala, Ser, Cys, Gln, Pro or Asn; Xaa at position 78 is Asp, Gly, Ala, Val, Leu, Trp, Ser, Cys, Gln, Glu, Arg, Phe, Ile, Tyr, Met, Asn, His, Thr or Lys; Xaa at position 79 is Ala, Gly, Tyr, Val, Leu, Trp, Gln, Glu, Lys, Arg, Asn, Asp, Met, Phe, Ile, Cys, Ser or Thr; Xaa at position 80 is Asn, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Tyr, Phe, Asp or Met; Xaa at position 81 is Val, Gly, Ala, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Ile, Asp, Pro or Met; Xaa at position 82 is Ser, Gly, Ala, Val, Trp, Thr, Cys, Gln, Glu, Lys, Asn, His, Phe, Ile, Asp or Met; Xaa at position 83 is Pro, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Phe, Ile or Asp; Xaa at position 84 is Gly, Lys, Tyr, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Arg, Asn, Phe, Ile, Asp, Pro or Met; Xaa at position 85 is Ser, Gly, Ala, Val, Leu, Trp, Tyr, Thr, Cys, Gln, Glu, Arg, Phe, Asp, Pro or Met; Xaa at position 86 is Ala, Gly, Tyr, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, His, Phe, Ile, Asp, Pro or Met; Xaa at position 95 is Gln, Ala, Val, Ser, Thr, Asn or His; Xaa at position 97 is Ala or Ser; Xaa at position 106 is Gly or Glu; Xaa at position 107 is Tyr, Cys, Phe, Gly, His or Leu; Xaa at position 108 is Asn, Ala, Cys, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr or Trp; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Ala, Glu, Phe, Met, Asn, Pro, Ser, Thr, Val or Trp; Xaa at position 111 is Asp, Ala, Glu, Phe, Gly, His, Asn, Gln, Ser, Val or Tyr; Xaa at position 112 is Gln, Cys, Pro or Val; Xaa at position 113 is Pro, Cys or Gly; Xaa at position 114 is Thr, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Val, Trp or Tyr; Xaa at position 115 is Asp, Ala, Glu, Phe, Gly, His, Ile, Leu, Pro, Gln, Arg, Ser, Thr or Tyr; Xaa at position 116 is Gln, Ala, Cys, Phe, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val or Trp; Xaa at position 117 is Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 118 is Pro, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 119 is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 120 is Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position 121 is Ala or Lys; Xaa at position 136 is Glu or Asn; Xaa at position 142 is Leu, Gly, Val, Met, Ser, Thr, Cys, Asn, Gln, Asp, Glu or His; Xaa at position 161 is Val or Ile; Xaa at position 170 is Gly, Ala, Val, Ile, Leu, Ser, Lys or Arg; Xaa at position 172 is Lys or Ala; Xaa at position 173 is Asn or Ala; Xaa at position 174 is Gln or Lys; Xaa at position 181 is Gln or Lys; Xaa at position 205 is Gln, Ala, Val, Pro, Ser, Thr, Cys, Tyr, Asn, Asp or Arg; Xaa at position 212 is Asp, Ile or Asn; Xaa at position 213 is Ser, Ala or Gly; Xaa at position 221 is Tyr, Ala, Val, Leu, Ser, Gln, Met, Ile or His; Xaa at position 230 is Ser or Thr; Xaa at position 232 is His or Lys; Xaa at position 233 is Lys, Glu or Arg; Xaa at position 234 is Gly, Ala, Cys, Phe, His, Lys, Leu, Met, Pro, Arg, Ser, Val or Tyr; Xaa at position 235 is Thr, Ala, Cys, Glu, Phe, Gly, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Trp; Xaa at position 236 is Ile, Ala, Cys, Phe, Leu, Pro, Thr or Val; Xaa at position 237 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Val or Trp; Xaa at position 238 is Phe, Ala, Asp, Leu, Pro, Thr, Val, Trp or Tyr; Xaa at position 239 is Gln, Cys, Gly, Arg, Tyr, Ser, Pro, Trp, Glu, Thr, Met, Ala, Lys, His, Ile, Val, Leu or Asp; Xaa at position 240 is Ser, Ala, Glu, Gly, His, Leu, Met, Asn, Pro, Gln, Thr or Val; Xaa at position 241 is Ser, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 242 is Tyr, Cys, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 243 is Thr, Asp, Glu, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val or Tyr; Xaa at position 244 is Leu, Ala, Cys, Phe, Gly, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 245 is Ser, Ala, Cys, Asp, Gly, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 246 is Gln, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro or Arg; Xaa at position 247 is Ala, Asp, Phe, Lys, Met, Ser or Val; Xaa at position 248 is Ile or Val; Xaa at position 257 is Asn or Glu; Xaa at position 260 is Lys or Arg; Xaa at position 262 is Val or Thr; Xaa at position 264 is Ser or Ala; Xaa at position 272 is Gln or Glu; Xaa at position 276 is Gly or Asp; Xaa at position 279 is Thr, Ser, Cys, Asn or Glu; Xaa at position 281 is Gln or Pro; Xaa at position 283 is Arg or Lys; Xaa at position 284 is Glu or Pro; Xaa at position 287 is Lys or Ser; and Xaa at position 295 is Ser or Gly; and wherein an amino acid is optionally inserted between position 41 and 42 relative to SEQ ID NO: 482, and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide relative to SEQ ID NO: 482.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 482 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 482, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 482 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 482, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 483, wherein Xaa at position 2 is His, Asn, Arg, Lys or Gln; Xaa at position 4 is Pro or deleted; Xaa at position 5 is Gly or deleted; Xaa at position 6 is Ala or deleted; Xaa at position 7 is Ile, Ala, Leu, Val or deleted; Xaa at position 9 is Ser or Thr; Xaa at position 10 is Glu, Val, Asp, Ala, Ile or Leu; Xaa at position 13 is Ser, Val, Thr, Ile, Leu or Ala; Xaa at position 19 is Thr, Glu, Ser, Asp or deleted; Xaa at position 20 is Glu, Gly, Asp or deleted; Xaa at position 21 is Thr, Gly, Ser or deleted; Xaa at position 22 is Lys, Thr, Arg or Ser; Xaa at position 24 is Asn, Lys, Arg or Gln; Xaa at position 25 is Ala, Val, Ile or Leu; Xaa at position 26 is Lys, Asn, Arg or Gln; Xaa at position 29 is Ala, Thr, Ile, Ser, Leu, Val or Met; Xaa at position 30 is Leu, Ala, Val, Ile; Xaa at position 31 is Arg, His or Lys; Xaa at position 37 is Gln, His, Asn, Arg or Lys; Xaa at position 38 is Asp or Glu; Xaa at position 41 is Arg, His or Lys; Xaa at position 42 is Gln, His, Asn; Xaa at position 43 is Leu, Gln, Ile, Val or Asn; Xaa at position 46 is Ala, Ser, Pro or Thr; Xaa at position 47 is Thr, Gly, Ala, Leu, Met, Pro, Ser, Cys, Asn, Asp, Glu, Lys, Arg, Tyr, Ile or Trp; Xaa at position 49 is Gln, Arg, Leu, Asn, Lys, Ile or Val; Xaa at position 50 is Ser, Ala, Thr; Xaa at position 53 is Ser or Thr; Xaa at position 62 is Phe or Tyr; Xaa at position 66 is Lys, Ala, Ser, Arg or Thr; Xaa at position 68 is Thr, Ala, Ser; Xaa at position 71 is Gly, Asp or Glu; Xaa at position 73 is Tyr, Gly, Cys or Glu; Xaa at position 75 is Lys, Asn, Arg or Gln; Xaa at position 78 is Asp, Asn, Glu or Gln; Xaa at position 79 is Ala, Lys, Asn, Arg or Gln; Xaa at position 85 is Ser, Lys, Thr or Arg; Xaa at position 95 is Gln, Ala, Val, Ser, Thr, Asn or His; Xaa at position 97 is Ala, Ser or Thr; Xaa at position 106 is Gly, Glu or Asp; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 117 is Lys or Arg; Xaa at position 120 is Ser, Ala or Thr; Xaa at position 121 is Ala, Lys or Arg; Xaa at position 136 is Glu, Asn, Gln or Asp; Xaa at position 142 is Leu, Gly, Val, Met, Ser, Thr, Cys, Asn, Gln, Asp, Glu or His; Xaa at position 161 is Val, Ile or Met; Xaa at position 170 is Gly, Ala, Val, Ile, Leu, Ser, Lys or Arg; Xaa at position 172 is Lys, Ala or Arg; Xaa at position 173 is Asn, Ala or Gln; Xaa at position 174 is Gln, Lys, Asn or Arg; Xaa at position 181 is Gln, Lys, Asn or Arg; Xaa at position 205 is Gln, Ala, Val, Pro, Ser, Thr, Cys, Tyr, Asn, Asp or Arg; Xaa at position 212 is Asp, Ile, Asn, Glu, Gln, Leu or Val; Xaa at position 213 is Ser, Ala, Gly or Thr; Xaa at position 221 is Tyr, Ala, Val, Leu, Ser, Gln, Met, Ile or His; Xaa at position 230 is Ser or Thr; Xaa at position 232 is His, Lys or Arg; Xaa at position 233 is Lys, Glu or Arg; Xaa at position 234 is Gly, Ala, Cys, Phe, His, Lys, Leu, Met, Pro, Arg, Ser, Val or Tyr; Xaa at position 235 is Thr, Ala, Cys, Glu, Phe, Gly, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Trp; Xaa at position 236 is Ile, Ala, Cys, Phe, Leu, Pro, Thr or Val; Xaa at position 237 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Val or Trp; Xaa at position 238 is Phe, Ala, Asp, Leu, Pro, Thr, Val, Trp or Tyr; Xaa at position 239 is Gln, Cys, Gly, Arg, Tyr, Ser, Pro, Trp, Glu, Thr, Met, Ala, Lys, His, Ile, Val, Leu or Asp; Xaa at position 240 is Ser, Ala, Glu, Gly, His, Leu, Met, Asn, Pro, Gln, Thr or Val; Xaa at position 241 is Ser, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 242 is Tyr, Cys, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 243 is Thr, Asp, Glu, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val or Tyr; Xaa at position 244 is Leu, Ala, Cys, Phe, Gly, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 245 is Ser, Ala, Cys, Asp, Gly, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 246 is Gln, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro or Arg; Xaa at position 247 is Ala, Asp, Phe, Lys, Met, Ser or Val; Xaa at position 248 is Ile, Val or Leu; Xaa at position 257 is Asn, Glu, Gln or Asp; Xaa at position 260 is Lys or Arg; Xaa at position 262 is Val, Thr, Ile, Leu or Ser; Xaa at position 264 is Ser, Ala or Thr; Xaa at position 272 is Gln, Glu, Asn or Asp; Xaa at position 276 is Gly, Asp or Glu; Xaa at position 279 is Thr, Ser, Cys, Asn or Glu; Xaa at position 281 is Gln, Pro or Asn; Xaa at position 283 is Arg or Lys; Xaa at position 284 is Glu, Pro or Asp; Xaa at position 287 is Lys, Ser, Arg or Thr; and Xaa at position 295 is Ser, Gly or Thr; and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide and/or an amino acid is inserted between position 41 and 42 relative to SEQ ID NO: 483.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 483 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 483, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 483 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 483, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 484 wherein Xaa at position 2 is His or Ser; Xaa at position 3 is Ala or Thr; Xaa at position 4 is Pro or Gln; Xaa at position 5 is Gly, Asn or deleted; Xaa at position 6 is Ala, His or deleted; Xaa at position 7 is Ile, Ala, Lys or deleted; Xaa at position 8 is Pro, His or deleted; Xaa at position 9 is Ser or Thr; Xaa at position 10 is Glu, Val or Thr; Xaa at position 11 is Lys or Glu; Xaa at position 12 is Glu or Lys; Xaa at position 13 is Ser, Gly, Val or Leu; Xaa at position 14 is Ala or Lys; Xaa at position 15 is His or Ala; Xaa at position 16 is Ala or Trp; Xaa at position 17 is Trp or Leu; Xaa at position 18 is Leu or Asn; Xaa at position 19 is Thr, Glu or deleted; Xaa at position 20 is Glu, Thr, Gly, Ser or deleted; Xaa at position 21 is Thr, Ala, Glu, Lys, His or deleted; Xaa at position 22 is Lys, Glu, Thr or Ala; Xaa at position 23 is Ala, Ser or Arg; Xaa at position 24 is Asn, Arg, Lys or Val; Xaa at position 25 is Ala, Val, Lys or Glu; Xaa at position 26 is Lys, Ser, Leu or Asn; Xaa at position 27 is Ser or Phe; Xaa at position 28 is Thr or Ala; Xaa at position 29 is Ala, Thr, Gln or Ile; Xaa at position 30 is Leu, Thr, Val or Ala; Xaa at position 31 is Ala, Arg, Asn or His; Xaa at position 32 is Gly or Pro; Xaa at position 34 is Ile or Phe; Xaa at position 35 is Phe or Tyr; Xaa at position 36 is Ala, Val or Ser; Xaa at position 37 is Gln, Ala, Arg, Leu or His; Xaa at position 38 is Asp or Glu; Xaa at position 39 is Tyr, Ala, Arg; Xaa at position 40 is Asn, Ser or His; Xaa at position 41 is Ala, Arg or His; Xaa at position 42 is Gln, Ser or His; Xaa at position 43 is Leu, Gln or Ser; Xaa at position 44 is Leu, Phe or Val; Xaa at position 45 is Thr or Ser; Xaa at position 46 is Ala, Glu, Pro or Ser; Xaa at position 47 is Thr or Ser; Xaa at position 48 is Gly or Leu; Xaa at position 49 is Gln, Ala, Arg or Leu; Xaa at position 50 is Ser, Thr or Ala; Xaa at position 51 is Met or Pro; Xaa at position 52 is Ala, Arg or Gly; Xaa at position 53 is Ser or Thr; Xaa at position 57 is Ala or Ile; Xaa at position 58 is Ile, Asp or Ala; Xaa at position 62 is Phe or Tyr; Xaa at position 63 is Ser, Gly or Ala; Xaa at position 65 is Ala or Thr; Xaa at position 66 is Lys, Gly, Ala or Ser; Xaa at position 67 is Gly or Ser; Xaa at position 68 is Thr, Gly, Ala or Leu; Xaa at position 71 is Gly, Asp, Ala or Asn; Xaa at position 72 is Ser, Gly, Ala, Arg or Asn; Xaa at position 74 is Ala, Val or Leu; Xaa at position 75 is Lys, Gln or Asn; Xaa at position 76 is Asp or Ala; Xaa at position 78 is Asp or Asn; Xaa at position 79 is Ala, Asn or Lys; Xaa at position 80 is Asn or Asp; Xaa at position 81 is Val or Ala; Xaa at position 84 is Gly or Ser; Xaa at position 85 is Ser, Thr or Lys; Xaa at position 86 is Ala or Val; Xaa at position 88 is Val, Glu or Thr; Xaa at position 90 is Ile, Phe or Leu; Xaa at position 91 is Tyr, Gln, Cys or His; Xaa at position 92 is Glu, Asp or Lys; Xaa at position 96 is Thr or Ser; Xaa at position 97 is Ala, Ser or Cys; Xaa at position 98 is Ile or Val; Xaa at position 99 is Asp or Asn; Xaa at position 100 is Ile, Thr or Met; Xaa at position 103 is Arg or Thr; Xaa at position 106 is Gly or Glu; Xaa at position 107 is Tyr or Trp; Xaa at position 110 is Leu or Asn; Xaa at position 112 is Gln or Pro; Xaa at position 115 is Asp, Gly or Ala; Xaa at position 116 is Gln, Ala or Arg; Xaa at position 117 is Lys, Ala, Arg or Asn; Xaa at position 119 is Lys or His; Xaa at position 120 is Ser, Thr or Ala; Xaa at position 121 is Ala, Thr or Lys; Xaa at position 124 is Arg or Tyr; Xaa at position 130 is Phe or Leu; Xaa at position 132 is Gln or Lys; Xaa at position 133 is Gln or Glu; Xaa at position 135 is Ala or Ser; Xaa at position 136 is Glu, Ser, Ala, Arg or Asn; Xaa at position 137 is Ile or Asn; Xaa at position 141 is Ser or Thr; Xaa at position 142 is Leu or Pro; Xaa at position 143 is Leu or Glu; Xaa at position 144 is Ala or Ser; Xaa at position 146 is Gln or Ser; Xaa at position 147 is Val or Ser; Xaa at position 148 is Thr or Ser; Xaa at position 149 is Gln or Leu; Xaa at position 150 is Ile or Val; Xaa at position 151 is Gln, Ala, Arg or Met; Xaa at position 153 is Lys or Gln; Xaa at position 156 is Asp or Gly; Xaa at position 158 is Asn or Asp; Xaa at position 161 is Val or Ile; Xaa at position 164 is Phe or Cys; Xaa at position 165 is Val or Ala; Xaa at position 168 is Phe or Ser; Xaa at position 169 is Ile or His; Xaa at position 170 is Gly or Asp; Xaa at position 172 is Lys, Glu or Ala; Xaa at position 173 is Asn, Thr or Ala; Xaa at position 174 is Gln, Ala or Lys; Xaa at position 176 is Val or Ile; Xaa at position 178 is Gln or Glu; Xaa at position 179 is Ile or Thr; Xaa at position 181 is Gln, Ala, Arg or Lys; Xaa at position 182 is Ser, Phe or Gly; Xaa at position 184 is Ser or Lys; Xaa at position 185 is Ser, Glu or Asn; Xaa at position 188 is Asn, Ser or Lys; Xaa at position 189 is Ala or Val; Xaa at position 193 is Tyr, Glu or Cys; Xaa at position 194 is Ser, Phe or Cys; Xaa at position 196 is Gln or Lys; Xaa at position 198 is Glu or Asn; Xaa at position 200 is Gln or Glu; Xaa at position 202 is Asn or Phe; Xaa at position 203 is Phe or Leu; Xaa at position 206 is Asn or His; Xaa at position 207 is Ile or Thr; Xaa at position 208 is Leu or Met; Xaa at position 212 is Asp, Asn or Ile; Xaa at position 213 is Ser, Gly or Ala; Xaa at position 222 is Ala or Pro; Xaa at position 226 is Thr or Ser; Xaa at position 228 is Lys or Val; Xaa at position 230 is Ser, Thr or Ala; Xaa at position 231 is Ser, Asp or Asn; Xaa at position 232 is His, Gln or Lys; Xaa at position 235 is Thr or Ala; Xaa at position 236 is Ile or Leu; Xaa at position 237 is Thr or Asn; Xaa at position 243 is Thr, Ser or Leu; Xaa at position 245 is Ser or Thr; Xaa at position 246 is Gln or Pro; Xaa at position 247 is Ala or Ser; Xaa at position 248 is Ile, Thr, Val or Lys; Xaa at position 250 is Gln or Thr; Xaa at position 253 is Val or Gly; Xaa at position 254 is Glu or Gly; Xaa at position 255 is Ser or Gly; Xaa at position 257 is Asn or Glu; Xaa at position 260 is Lys, Ala, Arg; Xaa at position 262 is Val, Thr or Ala; Xaa at position 264 is Ser, Pro, Ala or Tyr; Xaa at position 265 is Lys, Asp, Glu, Ala, Arg or Asn; Xaa at position 267 is Gln or Leu; Xaa at position 270 is Asp or Lys; Xaa at position 272 is Gln, Asp, Glu or Asn; Xaa at position 273 is Gln, Thr or Lys; Xaa at position 274 is Trp or Thr; Xaa at position 275 is Leu, Pro or His; Xaa at position 276 is Gly or Asp; Xaa at position 277 is Asp, Ser or Asn; Xaa at position 279 is Thr or Ile; Xaa at position 280 is Thr or Pro; Xaa at position 281 is Gln or Pro; Xaa at position 282 is Val, Gly, Ala or Arg; Xaa at position 283 is Ala, Arg, Gln or Lys; Xaa at position 284 is Glu, Ala or Pro; Xaa at position 287 is Lys or Ser; and Xaa at position 295 is Ser or Gly, and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide and/or an amino acid is inserted between position 41 and 42 relative to SEQ ID NO: 484.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 484 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 484, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 484 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 484, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 485, Xaa at position 2 is His or Ser; Xaa at position 3 is Ala or Thr; Xaa at position 4 is Pro or Gln; Xaa at position 5 is Gly, Asn or deleted; Xaa at position 6 is Ala, His or deleted; Xaa at position 7 is Ile, Ala, Lys or deleted; Xaa at position 8 is Pro, His or deleted; Xaa at position 9 is Ser or Thr; Xaa at position 10 is Glu, Val or Thr; Xaa at position 11 is Lys or Glu; Xaa at position 12 is Glu or Lys; Xaa at position 13 is Ser, Gly, Val or Leu; Xaa at position 14 is Ala or Lys; Xaa at position 15 is His or Ala; Xaa at position 16 is Ala or Trp; Xaa at position 17 is Trp or Leu; Xaa at position 18 is Leu or Asn; Xaa at position 19 is Thr, Glu or deleted; Xaa at position 20 is Glu, Thr, Gly, Ser or deleted; Xaa at position 21 is Thr, Ala, Glu, Lys, His or deleted; Xaa at position 22 is Lys, Glu, Thr or Ala; Xaa at position 23 is Ala, Ser or Arg; Xaa at position 24 is Asn, Arg, Lys or Val; Xaa at position 25 is Ala, Val, Lys or Glu; Xaa at position 26 is Lys, Ser, Leu or Asn; Xaa at position 27 is Ser or Phe; Xaa at position 28 is Thr or Ala; Xaa at position 29 is Ala, Thr, Gln or Ile; Xaa at position 30 is Leu, Thr, Val or Ala; Xaa at position 31 is Ala, Arg, Asn or His; Xaa at position 32 is Gly or Pro; Xaa at position 34 is Ile or Phe; Xaa at position 35 is Phe or Tyr; Xaa at position 36 is Ala, Val or Ser; Xaa at position 37 is Gln, Ala, Arg, Leu or His; Xaa at position 38 is Asp or Glu; Xaa at position 39 is Tyr, Ala, Arg; Xaa at position 40 is Asn, Ser or His; Xaa at position 41 is Ala, Arg or His; Xaa at position 42 is Gln, Ser or His; Xaa at position 43 is Leu, Gln or Ser; Xaa at position 44 is Leu, Phe or Val; Xaa at position 45 is Thr or Ser; Xaa at position 46 is Ala, Glu, Pro or Ser; Xaa at position 47 is Thr, Gly, Ala, Leu, Met, Pro, Ser, Cys, Asn, Asp, Glu, Lys, Arg, Tyr, Ile or Trp; Xaa at position 48 is Gly or Leu; Xaa at position 49 is Gln, Ala, Arg or Leu; Xaa at position 50 is Ser, Thr or Ala; Xaa at position 51 is Met or Pro; Xaa at position 52 is Ala, Arg or Gly; Xaa at position 53 is Ser or Thr; Xaa at position 57 is Ala or Ile; Xaa at position 58 is Ile, Asp or Ala; Xaa at position 62 is Phe or Tyr; Xaa at position 63 is Ser, Gly or Ala; Xaa at position 65 is Ala or Thr; Xaa at position 66 is Lys, Gly, Ala or Ser; Xaa at position 67 is Gly or Ser; Xaa at position 68 is Thr, Gly, Ala or Leu; Xaa at position 71 is Gly, Asp, Ala or Asn; Xaa at position 72 is Ser, Gly, Ala, Arg or Asn; Xaa at position 73 is Tyr, Gly, Cys or Glu; Xaa at position 74 is Ala, Val or Leu; Xaa at position 75 is Lys, Gln or Asn; Xaa at position 76 is Asp or Ala; Xaa at position 77 is Ala, Ser, Cys, Gln, Pro or Asn; Xaa at position 78 is Asp, Gly, Ala, Val, Leu, Trp, Ser, Cys, Gln, Glu, Arg, Phe, Ile, Tyr, Met, Asn, His, Thr or Lys; Xaa at position 79 is Ala, Gly, Tyr, Val, Leu, Trp, Gln, Glu, Lys, Arg, Asn, Asp, Met, Phe, Ile, Cys, Ser or Thr; Xaa at position 80 is Asn, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Tyr, Phe, Asp or Met; Xaa at position 81 is Val, Gly, Ala, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Ile, Asp, Pro or Met; Xaa at position 82 is Ser, Gly, Ala, Val, Trp, Thr, Cys, Gln, Glu, Lys, Asn, His, Phe, Ile, Asp or Met; Xaa at position 83 is Pro, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Phe, Ile or Asp; Xaa at position 84 is Gly, Tyr, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Arg, Asn, Phe, Ile, Asp, Pro or Met; Xaa at position 85 is Ser, Gly, Ala, Val, Leu, Trp, Tyr, Thr, Cys, Gln, Glu, Arg, Phe, Asp, Pro, Lys or Met; Xaa at position 86 is Ala, Gly, Tyr, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, His, Phe, Ile, Asp, Pro or Met; Xaa at position 88 is Val, Glu or Thr; Xaa at position 90 is Ile, Phe or Leu; Xaa at position 91 is Tyr, Gln, Cys or His; Xaa at position 92 is Glu, Asp or Lys; Xaa at position 95 is Gln, Ala, Val, Ser, Thr, Asn or His; Xaa at position 96 is Thr or Ser; Xaa at position 97 is Ala, Ser or Cys; Xaa at position 98 is Ile or Val; Xaa at position 99 is Asp or Asn; Xaa at position 100 is Ile, Thr or Met; Xaa at position 103 is Arg or Thr; Xaa at position 106 is Gly or Glu; Xaa at position 107 is Tyr, Cys, Phe, Gly, His, Trp or Leu; Xaa at position 108 is Asn, Ala, Cys, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr or Trp; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Ala, Glu, Phe, Met, Asn, Pro, Ser, Thr, Val or Trp; Xaa at position 111 is Asp, Ala, Glu, Phe, Gly, His, Asn, Gln, Ser, Val or Tyr; Xaa at position 112 is Gln, Cys, Pro or Val; Xaa at position 113 is Pro, Cys or Gly; Xaa at position 114 is Thr, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Val, Trp or Tyr; Xaa at position 115 is Asp, Ala, Glu, Phe, Gly, His, Ile, Leu, Pro, Gln, Arg, Ser, Thr or Tyr; Xaa at position 116 is Gln, Ala, Cys, Phe, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val or Trp; Xaa at position 117 is Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 118 is Pro, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 119 is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 120 is Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position 121 is Ala, Thr or Lys; Xaa at position 124 is Arg or Tyr; Xaa at position 130 is Phe or Leu; Xaa at position 132 is Gln or Lys; Xaa at position 133 is Gln or Glu; Xaa at position 135 is Ala or Ser; Xaa at position 136 is Glu, Ser, Ala, Arg or Asn; Xaa at position 137 is Ile or Asn; Xaa at position 141 is Ser or Thr; Xaa at position 142 is Leu, Gly, Val, Met, Ser, Thr, Cys, Asn, Gln, Asp, Glu, His or Pro; Xaa at position 143 is Leu or Glu; Xaa at position 144 is Ala or Ser; Xaa at position 146 is Gln or Ser; Xaa at position 147 is Val or Ser; Xaa at position 148 is Thr or Ser; Xaa at position 149 is Gln or Leu; Xaa at position 150 is Ile or Val; Xaa at position 151 is Gln, Ala, Arg or Met; Xaa at position 153 is Lys or Gln; Xaa at position 156 is Asp or Gly; Xaa at position 158 is Asn or Asp; Xaa at position 161 is Val or Ile; Xaa at position 164 is Phe or Cys; Xaa at position 165 is Val or Ala; Xaa at position 168 is Phe or Ser; Xaa at position 169 is Ile or His; Xaa at position 170 is Gly, Ala, Val, Ile, Leu, Ser, Lys, Arg or Asp; Xaa at position 172 is Lys, Glu or Ala; Xaa at position 173 is Asn, Thr or Ala; Xaa at position 174 is Gln, Ala or Lys; Xaa at position 176 is Val or Ile; Xaa at position 178 is Gln or Glu; Xaa at position 179 is Ile or Thr; Xaa at position 181 is Gln, Ala, Arg or Lys; Xaa at position 182 is Ser, Phe or Gly; Xaa at position 184 is Ser or Lys; Xaa at position 185 is Ser, Glu or Asn; Xaa at position 188 is Asn, Ser or Lys; Xaa at position 189 is Ala or Val; Xaa at position 193 is Tyr, Glu or Cys; Xaa at position 194 is Ser, Phe or Cys; Xaa at position 196 is Gln or Lys; Xaa at position 198 is Glu or Asn; Xaa at position 200 is Gln or Glu; Xaa at position 202 is Asn or Phe; Xaa at position 203 is Phe or Leu; Xaa at position 205 is Gln, Ala, Val, Pro, Ser, Thr, Cys, Tyr, Asn, Asp or Arg; Xaa at position 206 is Asn or His; Xaa at position 207 is Ile or Thr; Xaa at position 208 is Leu or Met; Xaa at position 212 is Asp, Asn or Ile; Xaa at position 213 is Ser, Gly or Ala; Xaa at position 221 is Tyr, Ala, Val, Leu, Ser, Gln, Met, Ile or His; Xaa at position 222 is Ala or Pro; Xaa at position 226 is Thr or Ser; Xaa at position 228 is Lys or Val; Xaa at position 230 is Ser, Thr or Ala; Xaa at position 231 is Ser, Asp or Asn; Xaa at position 232 is His, Gln or Lys; Xaa at position 233 is Lys, Glu or Arg; Xaa at position 234 is Gly, Ala, Cys, Phe, His, Lys, Leu, Met, Pro, Arg, Ser, Val or Tyr; Xaa at position 235 is Thr, Ala, Cys, Glu, Phe, Gly, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Trp; Xaa at position 236 is Ile, Ala, Cys, Phe, Leu, Pro, Thr or Val; Xaa at position 237 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Val, Trp or Asn; Xaa at position 238 is Phe, Ala, Asp, Leu, Pro, Thr, Val, Trp or Tyr; Xaa at position 239 is Gln, Cys, Gly, Arg, Tyr, Ser, Pro, Trp, Glu, Thr, Met, Ala, Lys, His, Ile, Val, Leu or Asp; Xaa at position 240 is Ser, Ala, Glu, Gly, His, Leu, Met, Asn, Pro, Gln, Thr or Val; Xaa at position 241 is Ser, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 242 is Tyr, Cys, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 243 is Thr, Asp, Glu, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val or Tyr; Xaa at position 244 is Leu, Ala, Cys, Phe, Gly, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 245 is Ser, Ala, Cys, Asp, Gly, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 246 is Gln, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro or Arg; Xaa at position 247 is Ala, Asp, Phe, Lys, Met, Ser or Val; Xaa at position 248 is Ile, Thr, Val or Lys; Xaa at position 250 is Gln or Thr; Xaa at position 253 is Val or Gly; Xaa at position 254 is Glu or Gly; Xaa at position 255 is Ser or Gly; Xaa at position 257 is Asn or Glu; Xaa at position 260 is Lys, Ala, Arg; Xaa at position 262 is Val, Thr or Ala; Xaa at position 264 is Ser, Pro, Ala or Tyr; Xaa at position 265 is Lys, Asp, Glu, Ala, Arg or Asn; Xaa at position 267 is Gln or Leu; Xaa at position 270 is Asp or Lys; Xaa at position 272 is Gln, Asp, Glu or Asn; Xaa at position 273 is Gln, Thr or Lys; Xaa at position 274 is Trp or Thr; Xaa at position 275 is Leu, Pro or His; Xaa at position 276 is Gly or Asp; Xaa at position 277 is Asp, Ser or Asn; Xaa at position 279 is Thr, Ser, Cys, Asn, Glu or Ile; Xaa at position 280 is Thr or Pro; Xaa at position 281 is Gln or Pro; Xaa at position 282 is Val, Gly, Ala or Arg; Xaa at position 283 is Ala, Arg, Gln or Lys; Xaa at position 284 is Glu, Ala or Pro; Xaa at position 287 is Lys or Ser; and Xaa at position 295 is Ser or Gly; and wherein an amino acid is optionally inserted between position 41 and 42 relative to SEQ ID NO: 485; and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 relative to SEQ ID NO: 485.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 485 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 485, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 485 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 485, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 486, wherein Xaa at position 2 is His, Ser or Thr; Xaa at position 3 is Ala, Thr or Ser; Xaa at position 4 is Pro, Gln or Asn; Xaa at position 5 is Gly, Asn, Gln or deleted; Xaa at position 6 is Ala, His, Ser or deleted; Xaa at position 7 is Ile, Ala, Lys, Leu, Val, Arg or deleted; Xaa at position 8 is Pro, His or deleted; Xaa at position 9 is Ser or Thr; Xaa at position 10 is Glu, Val, Thr, Asp, Ile, Leu or Ser; Xaa at position 11 is Lys, Glu, Arg or Asp; Xaa at position 12 is Glu, Lys, Asp or Arg; Xaa at position 13 is Ser, Gly, Val, Leu, Thr or Val; Xaa at position 14 is Ala, Lys or Arg; Xaa at position 15 is His or Ala; Xaa at position 16 is Ala, Trp, Phe or Tyr; Xaa at position 17 is Trp, Leu, Tyr, Phe, Ile or Val; Xaa at position 18 is Leu, Asn, Ile, Val or Gln; Xaa at position 19 is Thr, Glu, Ser, Asp or deleted; Xaa at position 20 is Glu, Thr, Gly, Ser, Asp or deleted; Xaa at position 21 is Thr, Ala, Glu, Lys, His, Ser, Asp, Arg or deleted; Xaa at position 22 is Lys, Glu, Thr, Ala, Arg, Asp or Ser; Xaa at position 23 is Ala, Ser, Arg, Thr or Lys; Xaa at position 24 is Asn, Arg, Lys, Val, Gln, Ile or Leu; Xaa at position 25 is Ala, Val, Lys, Glu, Ile, Leu, Arg or Asn; Xaa at position 26 is Lys, Ser, Leu, Asn, Arg, Thr, Ile, Val or Gln; Xaa at position 27 is Ser, Phe, Thr, Tyr or Trp; Xaa at position 28 is Thr, Ala or Ser; Xaa at position 29 is Ala, Thr, Gln, Ile, Ser, Asn, Leu or Val; Xaa at position 30 is Leu, Thr, Val, Ala, Ile or Ser; Xaa at position 31 is Ala, Arg, Asn, His, Lys or Gln; Xaa at position 32 is Gly or Pro; Xaa at position 34 is Ile, Phe, Leu or Val; Xaa at position 35 is Phe, Tyr or Trp; Xaa at position 36 is Ala, Val, Ser, Leu, Ile or Thr; Xaa at position 37 is Gln, Ala, Arg, Leu, His, Asn, Lys, Ile or Val; Xaa at position 38 is Asp or Glu; Xaa at position 39 is Tyr, Ala, Arg, Phe, Trp, His or Lys; Xaa at position 40 is Asn, Ser, His, Gln or Thr; Xaa at position 41 is Ala, Arg, His or Lys; Xaa at position 42 is Gln, Ser, His, Asn or Thr; Xaa at position 43 is Leu, Gln, Ser, Asn or Thr; Xaa at position 44 is Leu, Phe, Val, Ile, Trp or Tyr; Xaa at position 45 is Thr or Ser; Xaa at position 46 is Ala, Glu, Pro, Ser, Asp or Thr; Xaa at position 47 is Thr or Ser; Xaa at position 48 is Gly, Leu, Ile or Val; Xaa at position 49 is Gln, Ala, Arg, Leu, Asn, Lys, Ile or Val; Xaa at position 50 is Ser, Thr or Ala; Xaa at position 51 is Met or Pro; Xaa at position 52 is Ala, Arg, Gly or Lys; Xaa at position 53 is Ser or Thr; Xaa at position 57 is Ala, Ile, Leu or Val; Xaa at position 58 is Ile, Asp, Ala, Leu, Val or Glu; Xaa at position 62 is Phe, Tyr or Trp; Xaa at position 63 is Ser, Gly, Ala or Thr; Xaa at position 65 is Ala, Thr or Ser; Xaa at position 66 is Lys, Gly, Ala, Ser, Arg or Thr; Xaa at position 67 is Gly, Ser or Thr; Xaa at position 68 is Thr, Gly, Ala, Leu, Ile, Val or Ser; Xaa at position 71 is Gly, Asp, Ala, Asn, Glu or Gln; Xaa at position 72 is Ser, Gly, Ala, Arg, Asn, Thr, Lys or Gln; Xaa at position 74 is Ala, Val, Leu or Ile; Xaa at position 75 is Lys, Gln, Asn or Arg; Xaa at position 76 is Asp, Ala or Glu; Xaa at position 78 is Asp, Asn, Glu or Gln; Xaa at position 79 is Ala, Asn, Lys, Gln or Arg; Xaa at position 80 is Asn or Asp; Xaa at position 81 is Val, Ala, Ile or Val; Xaa at position 84 is Gly, Ser or The; Xaa at position 85 is Ser, Thr, Lys or Arg; Xaa at position 86 is Ala, Val, Ile or Leu; Xaa at position 88 is Val, Glu, Thr, Ile, Leu, Asp or Ser; Xaa at position 90 is Ile, Phe, Leu or Val; Xaa at position 91 is Tyr, Gln, Cys, His, Trp, Phe or Asn; Xaa at position 92 is Glu, Asp, Lys or Arg; Xaa at position 96 is Thr or Ser; Xaa at position 97 is Ala, Ser, Cys or Thr; Xaa at position 98 is Ile, Val or Leu; Xaa at position 99 is Asp, Asn, Glu or Gln; Xaa at position 100 is Ile, Thr, Met, Leu, Val or Ser; Xaa at position 103 is Arg, Thr, Lys or Ser; Xaa at position 106 is Gly, Glu or Asp; Xaa at position 107 is Tyr, Trp or Phe; Xaa at position 110 is Leu, Asn, Ile, Val or Gln; Xaa at position 112 is Gln, Pro or Asn; Xaa at position 115 is Asp, Gly, Ala or Glu; Xaa at position 116 is Gln, Ala, Arg, Asn or Lys; Xaa at position 117 is Lys, Ala, Arg, Asn or Gln; Xaa at position 119 is Lys, His or Arg; Xaa at position 120 is Ser, Thr or Ala; Xaa at position 121 is Ala, Thr, Lys, Ser or Arg; Xaa at position 124 is Arg, Tyr, Lys, His, Trp or Phe; Xaa at position 130 is Phe, Leu, Ile or Val; Xaa at position 132 is Gln, Lys, Asn or Arg; Xaa at position 133 is Gln, Glu, Asn or Asp; Xaa at position 135 is Ala, Ser or Thr; Xaa at position 136 is Glu, Ser, Ala, Arg, Asn, Asp, Thr, Lys or Gln; Xaa at position 137 is Ile, Asn, Leu, Val or Gln; Xaa at position 141 is Ser or Thr; Xaa at position 142 is Leu, Pro, Ile or Val; Xaa at position 143 is Leu, Glu, Ile, Val or Asp; Xaa at position 144 is Ala, Ser or Thr; Xaa at position 146 is Gln, Ser, Asn or Thr; Xaa at position 147 is Val, Ser, Ile, Leu or Thr; Xaa at position 148 is Thr or Ser; Xaa at position 149 is Gln, Leu, Asn, Ile or Val; Xaa at position 150 is Ile, Val or Leu; Xaa at position 151 is Gln, Ala, Arg, Met, Asn or Lys; Xaa at position 153 is Lys, Gln, Arg or Asn; Xaa at position 156 is Asp, Gly or Glu; Xaa at position 158 is Asn, Asp, Gln or Glu; Xaa at position 161 is Val, Ile or Leu; Xaa at position 164 is Phe or Cys; Xaa at position 165 is Val, Ala, Ile or Leu; Xaa at position 168 is Phe, Ser or Thr; Xaa at position 169 is Ile, His, Leu or Val; Xaa at position 170 is Gly, Asp or Glu; Xaa at position 172 is Lys, Glu, Ala, Arg or Asp; Xaa at position 173 is Asn, Thr, Ala, Gln or Ser; Xaa at position 174 is Gln, Ala, Lys, Asn or Arg; Xaa at position 176 is Val, Ile or Leu; Xaa at position 178 is Gln, Glu, Asn or Asp; Xaa at position 179 is Ile, Thr, Leu, Val or Ser; Xaa at position 181 is Gln, Ala, Arg, Lys or Asn; Xaa at position 182 is Ser, Phe or Gly; Xaa at position 184 is Ser, Lys, Thr or Arg; Xaa at position 185 is Ser, Glu, Asn, Thr, Gln or Asp; Xaa at position 188 is Asn, Ser, Lys, Gln, Thr or Arg; Xaa at position 189 is Ala, Val, Ile or Leu; Xaa at position 193 is Tyr, Glu, Cys, Trp, Phe or Asp; Xaa at position 194 is Ser, Phe, Cys or Thr; Xaa at position 196 is Gln, Lys, Asn or Arg; Xaa at position 198 is Glu, Asn, Asp or Gln; Xaa at position 200 is Gln or Glu; Xaa at position 202 is Asn, Phe or Gln; Xaa at position 203 is Phe, Leu, Ile or Val; Xaa at position 206 is Asn, His or Gln; Xaa at position 207 is Ile, Thr, Leu, Val or Ser; Xaa at position 208 is Leu, Met, Ile or Val; Xaa at position 212 is Asp, Asn, Ile, Glu, Gln, Leu or Val; Xaa at position 213 is Ser, Gly, Ala or Thr; Xaa at position 222 is Ala or Pro; Xaa at position 226 is Thr or Ser; Xaa at position 228 is Lys, Val, Arg, Ile or Leu; Xaa at position 230 is Ser, Thr or Ala; Xaa at position 231 is Ser, Asp, Asn, Thr, Glu or Gln; Xaa at position 232 is His, Gln, Lys, Asn or Arg; Xaa at position 235 is Thr, Ala or Ser; Xaa at position 236 is Ile, Leu or Val; Xaa at position 237 is Thr, Asn, Ser or Gln; Xaa at position 243 is Thr, Ser, Leu, Ile or Val; Xaa at position 245 is Ser or Thr; Xaa at position 246 is Gln, Pro or Asn; Xaa at position 247 is Ala, Ser or Thr; Xaa at position 248 is Ile, Thr, Val, Lys, Leu, Val, Ser or Arg; Xaa at position 250 is Gln, Thr, Asn or Ser; Xaa at position 253 is Val, Gly, Ile or Leu; Xaa at position 254 is Glu, Gly or Asp; Xaa at position 255 is Ser, Gly or Thr; Xaa at position 257 is Asn, Glu, Asp or Gln; Xaa at position 260 is Lys, Ala or Arg; Xaa at position 262 is Val, Thr, Ala, Ile, Leu or Ser; Xaa at position 264 is Ser, Pro, Ala, Tyr, Trp or Phe; Xaa at position 265 is Lys, Asp, Glu, Ala, Arg, Asn or Gln; Xaa at position 267 is Gln, Leu, Asn, Ile or Val; Xaa at position 270 is Asp, Lys, Glu or Arg; Xaa at position 272 is Gln, Asp, Glu or Asn; Xaa at position 273 is Gln, Thr, Lys, Asn, Ser or Arg; Xaa at position 274 is Trp, Thr, Tyr, Phe or Ser; Xaa at position 275 is Leu, Pro, His, Ile or Val; Xaa at position 276 is Gly, Asp or Glu; Xaa at position 277 is Asp, Ser, Asn or Thr; Xaa at position 279 is Thr, Ile, Ser, Leu or Val; Xaa at position 280 is Thr, Pro or Ser; Xaa at position 281 is Gln, Pro or Asn; Xaa at position 282 is Val, Gly, Ala, Arg, Ile, Leu or Lys; Xaa at position 283 is Ala, Arg, Gln, Lys or Asn; Xaa at position 284 is Glu, Ala, Pro or Asp; Xaa at position 287 is Lys, Ser, Arg or Thr; and Xaa at position 295 is Ser, Gly or Thr; and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide and/or an amino acid is inserted between position 41 and 42 relative to SEQ ID NO: 486.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 486 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 486, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 486 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 486, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 487, Xaa at position 2 is His, Ser or Thr; Xaa at position 3 is Ala, Thr or Ser; Xaa at position 4 is Pro, Gln or Asn; Xaa at position 5 is Gly, Asn, Gln or deleted; Xaa at position 6 is Ala, His, Ser or deleted; Xaa at position 7 is Ile, Ala, Lys, Leu, Val, Arg or deleted; Xaa at position 8 is Pro, His or deleted; Xaa at position 9 is Ser or Thr; Xaa at position 10 is Glu, Val, Thr, Asp, Ile, Leu or Ser; Xaa at position 11 is Lys, Glu, Arg or Asp; Xaa at position 12 is Glu, Lys, Asp or Arg; Xaa at position 13 is Ser, Gly, Val, Leu, Thr or Val; Xaa at position 14 is Ala, Lys or Arg; Xaa at position 15 is His or Ala; Xaa at position 16 is Ala, Trp, Phe or Tyr; Xaa at position 17 is Trp, Leu, Tyr, Phe, Ile or Val; Xaa at position 18 is Leu, Asn, Ile, Val or Gln; Xaa at position 19 is Thr, Glu, Ser, Asp or deleted; Xaa at position 20 is Glu, Thr, Gly, Ser, Asp or deleted; Xaa at position 21 is Thr, Ala, Glu, Lys, His, Ser, Asp, Arg or deleted; Xaa at position 22 is Lys, Glu, Thr, Ala, Arg, Asp or Ser; Xaa at position 23 is Ala, Ser, Arg, Thr or Lys; Xaa at position 24 is Asn, Arg, Lys, Val, Gln, Ile or Leu; Xaa at position 25 is Ala, Val, Lys, Glu, Ile, Leu, Arg or Asn; Xaa at position 26 is Lys, Ser, Leu, Asn, Arg, Thr, Ile, Val or Gln; Xaa at position 27 is Ser, Phe, Thr, Tyr or Trp; Xaa at position 28 is Thr, Ala or Ser; Xaa at position 29 is Ala, Thr, Gln, Ile, Ser, Asn, Leu or Val; Xaa at position 30 is Leu, Thr, Val, Ala, Ile or Ser; Xaa at position 31 is Ala, Arg, Asn, His, Lys or Gln; Xaa at position 32 is Gly or Pro; Xaa at position 34 is Ile, Phe, Leu or Val; Xaa at position 35 is Phe, Tyr or Trp; Xaa at position 36 is Ala, Val, Ser, Leu, Ile or Thr; Xaa at position 37 is Gln, Ala, Arg, Leu, His, Asn, Lys, Ile or Val; Xaa at position 38 is Asp or Glu; Xaa at position 39 is Tyr, Ala, Arg, Phe, Trp, His or Lys; Xaa at position 40 is Asn, Ser, His, Gln or Thr; Xaa at position 41 is Ala, Arg, His or Lys; Xaa at position 42 is Gln, Ser, His, Asn or Thr; Xaa at position 43 is Leu, Gln, Ser, Asn or Thr; Xaa at position 44 is Leu, Phe, Val, Ile, Trp or Tyr; Xaa at position 45 is Thr or Ser; Xaa at position 46 is Ala, Glu, Pro, Ser, Asp or Thr; Xaa at position 47 is Thr, Gly, Ala, Leu, Met, Pro, Ser, Cys, Asn, Asp, Glu, Lys, Arg, Tyr, Ile or Trp; Xaa at position 48 is Gly, Leu, Ile or Val; Xaa at position 49 is Gln, Ala, Arg, Leu, Asn, Lys, Ile or Val; Xaa at position 50 is Ser, Thr or Ala; Xaa at position 51 is Met or Pro; Xaa at position 52 is Ala, Arg, Gly or Lys; Xaa at position 53 is Ser or Thr; Xaa at position 57 is Ala, Ile, Leu or Val; Xaa at position 58 is Ile, Asp, Ala, Leu, Val or Glu; Xaa at position 62 is Phe, Tyr or Trp; Xaa at position 63 is Ser, Gly, Ala or Thr; Xaa at position 65 is Ala, Thr or Ser; Xaa at position 66 is Lys, Gly, Ala, Ser, Arg or Thr; Xaa at position 67 is Gly, Ser or Thr; Xaa at position 68 is Thr, Gly, Ala, Leu, Ile, Val or Ser; Xaa at position 71 is Gly, Asp, Ala, Asn, Glu or Gln; Xaa at position 72 is Ser, Gly, Ala, Arg, Asn, Thr, Lys or Gln; Xaa at position 73 is Tyr, Gly, Cys or Glu; Xaa at position 74 is Ala, Val, Leu or Ile; Xaa at position 75 is Lys, Gln, Asn or Arg; Xaa at position 76 is Asp, Ala or Glu; Xaa at position 77 is Ala, Ser, Cys, Gln, Pro or Asn; Xaa at position 78 is Asp, Gly, Ala, Val, Leu, Trp, Ser, Cys, Gln, Glu, Arg, Phe, Ile, Tyr, Met, Asn, His, Thr or Lys; Xaa at position 79 is Ala, Gly, Tyr, Val, Leu, Trp, Gln, Glu, Lys, Arg, Asn, Asp, Met, Phe, Ile, Cys, Ser or Thr; Xaa at position 80 is Asn, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Tyr, Phe, Asp or Met; Xaa at position 81 is Val, Gly, Ala, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Ile, Asp, Pro or Met; Xaa at position 82 is Ser, Gly, Ala, Val, Trp, Thr, Cys, Gln, Glu, Lys, Asn, His, Phe, Ile, Asp or Met; Xaa at position 83 is Pro, Gly, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, Phe, Ile or Asp; Xaa at position 84 is Gly, Tyr, Ala, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Arg, Asn, Phe, Ile, Asp, Pro or Met; Xaa at position 85 is Ser, Gly, Ala, Val, Leu, Trp, Tyr, Thr, Cys, Gln, Glu, Arg, Phe, Asp, Pro, Lys or Met; Xaa at position 86 is Ala, Gly, Tyr, Val, Leu, Trp, Ser, Thr, Cys, Gln, Glu, Lys, Arg, Asn, His, Phe, Ile, Asp, Pro or Met; Xaa at position 78 is Asp, Asn, Glu or Gln; Xaa at position 79 is Ala, Asn, Lys, Gln or Arg; Xaa at position 80 is Asn or Asp; Xaa at position 81 is Val, Ala, Ile or Val; Xaa at position 84 is Gly, Ser or The; Xaa at position 85 is Ser, Thr, Lys or Arg; Xaa at position 86 is Ala, Val, Ile or Leu; Xaa at position 88 is Val, Glu, Thr, Ile, Leu, Asp or Ser; Xaa at position 90 is Ile, Phe, Leu or Val; Xaa at position 91 is Tyr, Gln, Cys, His, Trp, Phe or Asn; Xaa at position 92 is Glu, Asp, Lys or Arg; Xaa at position 95 is Gln, Ala, Val, Ser, Thr, Asn or His; Xaa at position 96 is Thr or Ser; Xaa at position 97 is Ala, Ser, Cys or Thr; Xaa at position 98 is Ile, Val or Leu; Xaa at position 99 is Asp, Asn, Glu or Gln; Xaa at position 100 is Ile, Thr, Met, Leu, Val or Ser; Xaa at position 103 is Arg, Thr, Lys or Ser; Xaa at position 106 is Gly, Glu or Asp; Xaa at position 107 is Tyr, Cys, Phe, Gly, His, Trp or Leu; Xaa at position 108 is Asn, Ala, Cys, Glu, Phe, Gly, Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr or Trp; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Ala, Glu, Phe, Met, Asn, Pro, Ser, Thr, Val or Trp; Xaa at position 111 is Asp, Ala, Glu, Phe, Gly, His, Asn, Gln, Ser, Val or Tyr; Xaa at position 112 is Gln, Cys, Pro or Val; Xaa at position 113 is Pro, Cys or Gly; Xaa at position 114 is Thr, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Gln, Arg, Ser, Val, Trp or Tyr; Xaa at position 115 is Asp, Ala, Glu, Phe, Gly, His, Ile, Leu, Pro, Gln, Arg, Ser, Thr or Tyr; Xaa at position 116 is Gln, Ala, Cys, Phe, His, Ile, Leu, Asn, Pro, Arg, Ser, Thr, Val or Trp; Xaa at position 117 is Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 118 is Pro, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 119 is Lys, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 120 is Ser, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position 107 is Tyr, Trp or Phe; Xaa at position 109 is Pro, Ala, Leu, Met, Cys, Tyr, Glu or Lys; Xaa at position 110 is Leu, Asn, Ile, Val or Gln; Xaa at position 112 is Gln, Pro or Asn; Xaa at position 115 is Asp, Gly, Ala or Glu; Xaa at position 116 is Gln, Ala, Arg, Asn or Lys; Xaa at position 117 is Lys, Ala, Arg, Asn or Gln; Xaa at position 119 is Lys, His or Arg; Xaa at position 120 is Ser, Thr or Ala; Xaa at position 121 is Ala, Thr, Lys, Ser or Arg; Xaa at position 124 is Arg, Tyr, Lys, His, Trp or Phe; Xaa at position 130 is Phe, Leu, Ile or Val; Xaa at position 132 is Gln, Lys, Asn or Arg; Xaa at position 133 is Gln, Glu, Asn or Asp; Xaa at position 135 is Ala, Ser or Thr; Xaa at position 136 is Glu, Ser, Ala, Arg, Asn, Asp, Thr, Lys or Gln; Xaa at position 137 is Ile, Asn, Leu, Val or Gln; Xaa at position 141 is Ser or Thr; Xaa at position 142 is Leu, Gly, Val, Met, Ser, Thr, Cys, Asn, Gln, Asp, Glu, His or Pro; Xaa at position 143 is Leu, Glu, Ile, Val or Asp; Xaa at position 144 is Ala, Ser or Thr; Xaa at position 146 is Gln, Ser, Asn or Thr; Xaa at position 147 is Val, Ser, Ile, Leu or Thr; Xaa at position 148 is Thr or Ser; Xaa at position 149 is Gln, Leu, Asn, Ile or Val; Xaa at position 150 is Ile, Val or Leu; Xaa at position 151 is Gln, Ala, Arg, Met, Asn or Lys; Xaa at position 153 is Lys, Gln, Arg or Asn; Xaa at position 156 is Asp, Gly or Glu; Xaa at position 158 is Asn, Asp, Gln or Glu; Xaa at position 161 is Val, Ile or Leu; Xaa at position 164 is Phe or Cys; Xaa at position 165 is Val, Ala, Ile or Leu; Xaa at position 168 is Phe, Ser or Thr; Xaa at position 169 is Ile, His, Leu or Val; Xaa at position 170 is Gly, Ala, Val, Ile, Leu, Ser, Lys, Arg or Asp; Xaa at position 172 is Lys, Glu, Ala, Arg or Asp; Xaa at position 173 is Asn, Thr, Ala, Gln or Ser; Xaa at position 174 is Gln, Ala, Lys, Asn or Arg; Xaa at position 176 is Val, Ile or Leu; Xaa at position 178 is Gln, Glu, Asn or Asp; Xaa at position 179 is Ile, Thr, Leu, Val or Ser; Xaa at position 181 is Gln, Ala, Arg, Lys or Asn; Xaa at position 182 is Ser, Phe or Gly; Xaa at position 184 is Ser, Lys, Thr or Arg; Xaa at position 185 is Ser, Glu, Asn, Thr, Gln or Asp; Xaa at position 188 is Asn, Ser, Lys, Gln, Thr or Arg; Xaa at position 189 is Ala, Val, Ile or Leu; Xaa at position 193 is Tyr, Glu, Cys, Trp, Phe or Asp; Xaa at position 194 is Ser, Phe, Cys or Thr; Xaa at position 196 is Gln, Lys, Asn or Arg; Xaa at position 198 is Glu, Asn, Asp or Gln; Xaa at position 200 is Gln or Glu; Xaa at position 202 is Asn, Phe or Gln; Xaa at position 203 is Phe, Leu, Ile or Val; Xaa at position 205 is Gln, Ala, Val, Pro, Ser, Thr, Cys, Tyr, Asn, Asp or Arg; Xaa at position 206 is Asn, His or Gln; Xaa at position 207 is Ile, Thr, Leu, Val or Ser; Xaa at position 208 is Leu, Met, Ile or Val; Xaa at position 212 is Asp, Asn, Ile, Glu, Gln, Leu or Val; Xaa at position 213 is Ser, Gly, Ala or Thr; Xaa at position 221 is Tyr, Ala, Val, Leu, Ser, Gln, Met, Ile or His; Xaa at position 222 is Ala or Pro; Xaa at position 226 is Thr or Ser; Xaa at position 228 is Lys, Val, Arg, Ile or Leu; Xaa at position 230 is Ser, Thr or Ala; Xaa at position 231 is Ser, Asp, Asn, Thr, Glu or Gln; Xaa at position 232 is His, Gln, Lys, Asn or Arg; Xaa at position 233 is Lys, Glu or Arg; Xaa at position 234 is Gly, Ala, Cys, Phe, His, Lys, Leu, Met, Pro, Arg, Ser, Val or Tyr; Xaa at position 235 is Thr, Ala, Cys, Glu, Phe, Gly, Lys, Leu, Met, Asn, Gln, Arg, Ser, Val or Trp; Xaa at position 236 is Ile, Ala, Cys, Phe, Leu, Pro, Thr or Val; Xaa at position 237 is Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Gln, Arg, Ser, Val, Trp or Asn; Xaa at position 238 is Phe, Ala, Asp, Leu, Pro, Thr, Val, Trp or Tyr; Xaa at position 239 is Gln, Cys, Gly, Arg, Tyr, Ser, Pro, Trp, Glu, Thr, Met, Ala, Lys, His, Ile, Val, Leu or Asp; Xaa at position 240 is Ser, Ala, Glu, Gly, His, Leu, Met, Asn, Pro, Gln, Thr or Val; Xaa at position 241 is Ser, Ala, Cys, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 242 is Tyr, Cys, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 243 is Thr, Asp, Glu, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val or Tyr; Xaa at position 244 is Leu, Ala, Cys, Phe, Gly, Lys, Pro, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 245 is Ser, Ala, Cys, Asp, Gly, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position 246 is Gln, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro or Arg; Xaa at position 247 is Ala, Asp, Phe, Lys, Met, Ser or Val; Xaa at position 248 is Ile, Thr, Val, Lys, Leu, Val, Ser or Arg; Xaa at position 250 is Gln, Thr, Asn or Ser; Xaa at position 253 is Val, Gly, Ile or Leu; Xaa at position 254 is Glu, Gly or Asp; Xaa at position 255 is Ser, Gly or Thr; Xaa at position 257 is Asn, Glu, Asp or Gln; Xaa at position 260 is Lys, Ala or Arg; Xaa at position 262 is Val, Thr, Ala, Ile, Leu or Ser; Xaa at position 264 is Ser, Pro, Ala, Tyr, Trp or Phe; Xaa at position 265 is Lys, Asp, Glu, Ala, Arg, Asn or Gln; Xaa at position 267 is Gln, Leu, Asn, Ile or Val; Xaa at position 270 is Asp, Lys, Glu or Arg; Xaa at position 272 is Gln, Asp, Glu or Asn; Xaa at position 273 is Gln, Thr, Lys, Asn, Ser or Arg; Xaa at position 274 is Trp, Thr, Tyr, Phe or Ser; Xaa at position 275 is Leu, Pro, His, Ile or Val; Xaa at position 276 is Gly, Asp or Glu; Xaa at position 277 is Asp, Ser, Asn or Thr; Xaa at position 279 is Thr, Ser, Cys, Asn, Glu or Ile; Xaa at position 280 is Thr, Pro or Ser; Xaa at position 281 is Gln, Pro or Asn; Xaa at position 282 is Val, Gly, Ala, Arg, Ile, Leu or Lys; Xaa at position 283 is Ala, Arg, Gln, Lys or Asn; Xaa at position 284 is Glu, Ala, Pro or Asp; Xaa at position 287 is Lys, Ser, Arg or Thr; and Xaa at position 295 is Ser, Gly or Thr; and wherein an amino acid is optionally inserted between position 41 and 42 relative to SEQ ID NO: 487; and wherein, 1 to 14 amino acids are optionally deleted from the N-terminus of the PIP-47 polypeptide relative to SEQ ID NO: 487.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 487 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 487, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments a PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 487 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acid substitutions at residues designated by Xaa in SEQ ID NO: 287, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments exemplary PIP-47 polypeptides are encoded by the polynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, any one of SEQ ID NO: 21-SEQ ID NO: 220, SEQ ID NO: 530-SEQ ID NO:628, SEQ ID NO: 866-SEQ ID NO:877 or SEQ ID NO: 910-SEQ ID NO: 915. In some embodiments the PIP-47 polypeptide is encoded by the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

In some embodiments exemplary PIP-47 polypeptides are set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 345, SEQ ID NO: 346, SEQ ID NO: 347, SEQ ID NO: 348, SEQ ID NO: 349, SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 357, SEQ ID NO: 358, SEQ ID NO: 359, SEQ ID NO: 360, SEQ ID NO: 361, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 372, SEQ ID NO: 373, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, SEQ ID NO: 406, SEQ ID NO: 407, SEQ ID NO: 408, SEQ ID NO: 409, SEQ ID NO: 410, SEQ ID NO: 411, SEQ ID NO: 412, SEQ ID NO: 413, SEQ ID NO: 414, SEQ ID NO: 415, SEQ ID NO: 416, SEQ ID NO: 417, SEQ ID NO: 418, SEQ ID NO: 419, SEQ ID NO: 420, SEQ ID NO: 629, SEQ ID NO: 630, SEQ ID NO: 631, SEQ ID NO: 632, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635, SEQ ID NO: 636, SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO: 641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 644, SEQ ID NO: 645, SEQ ID NO: 646, SEQ ID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, SEQ ID NO: 651, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, SEQ ID NO: 699, SEQ ID NO: 700, SEQ ID NO: 701, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO: 719, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 797, SEQ ID NO: 798, SEQ ID NO: 799, SEQ ID NO: 800, SEQ ID NO: 801, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 805, SEQ ID NO: 806, SEQ ID NO: 807, SEQ ID NO: 808, SEQ ID NO: 809, SEQ ID NO: 810, SEQ ID NO: 811, SEQ ID NO: 812, SEQ ID NO: 813, SEQ ID NO: 814, SEQ ID NO: 814, SEQ ID NO: 815, SEQ ID NO: 816, SEQ ID NO: 817, SEQ ID NO: 818, SEQ ID NO: 819, SEQ ID NO: 820, SEQ ID NO: 821, SEQ ID NO: 822, SEQ ID NO: 823, SEQ ID NO: 824, SEQ ID NO: 825, SEQ ID NO: 826, SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ ID NO: 830, SEQ ID NO: 831, SEQ ID NO: 832, SEQ ID NO: 833, SEQ ID NO: 834, SEQ ID NO: 835, SEQ ID NO: 836, SEQ ID NO: 837, SEQ ID NO: 838, SEQ ID NO: 839, SEQ ID NO: 840, SEQ ID NO: 841, SEQ ID NO: 842, SEQ ID NO: 843, SEQ ID NO: 844, SEQ ID NO: 845, SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, SEQ ID NO: 850, SEQ ID NO: 851, SEQ ID NO: 852, SEQ ID NO: 853, SEQ ID NO: 854, SEQ ID NO: 855, SEQ ID NO: 856, SEQ ID NO: 857, SEQ ID NO: 858, SEQ ID NO: 859, SEQ ID NO: 860, SEQ ID NO: 861, SEQ ID NO: 862, SEQ ID NO: 863, SEQ ID NO: 864, SEQ ID NO: 865, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 890, SEQ ID NO: 891, SEQ ID NO: 892, SEQ ID NO: 893, SEQ ID NO: 894, SEQ ID NO: 895, SEQ ID NO: 896, SEQ ID NO: 897, SEQ ID NO: 898, SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, SEQ ID NO: 903, SEQ ID NO: 904, SEQ ID NO: 905, SEQ ID NO: 906, SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO: 916, SEQ ID NO: 917, SEQ ID NO: 918, SEQ ID NO: 919, SEQ ID NO: 920 and SEQ ID NO: 921.

In some embodiments the PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO; 2, SEQ ID NO; 4, SEQ ID NO; 6 or SEQ ID NO: 8.

In some embodiments exemplary PIP-47 polypeptides are the polypeptides shown in Table 8, Table 10, Table 12, Table 13, Table 14, Table 15, Table 16, Table 17, Table 18 and/or Table 19 and any combinations of the amino acid substitutions thereof as well as deletions and or insertions and fragments thereof.

In some embodiments a PIP-47 polypeptide has a calculated molecular weight of between about 26 kD and about 40 kD, between about 28 kD and about 40 kD, between about 30 kD and about 37 kD, between about 32 kD and about 37 kD, between about 34 kD and about 36 kD, and about 35 kD.

In some embodiments the PIP-47 polypeptide has a modified physical property. As used herein, the term “physical property” refers to any parameter suitable for describing the physical-chemical characteristics of a protein. As used herein, “physical property of interest” and “property of interest” are used interchangeably to refer to physical properties of proteins that are being investigated and/or modified. Examples of physical properties include, but are not limited to net surface charge and charge distribution on the protein surface, net hydrophobicity and hydrophobic residue distribution on the protein surface, surface charge density, surface hydrophobicity density, total count of surface ionizable groups, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second virial coefficient. Examples of physical properties also include, but are not limited to solubility, folding, stability, and digestibility. In some embodiments the PIP-47 polypeptide has increased digestibility of proteolytic fragments in an insect gut. Models for digestion by simulated simulated gastric fluids are known to one skilled in the art (Fuchs, R. L. and J. D. Astwood. Food Technology 50: 83-88, 1996; Astwood, J. D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu T J et al J. Agric Food Chem. 50: 7154-7160, 2002).

In some embodiments variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the disclosure are biologically active, that is they continue to possess the desired biological activity (i.e. pesticidal activity) of the native protein. In some embodiment the variant will have at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 80% or more of the insecticidal activity of the native protein. In some embodiments, the variants may have improved activity over the native protein.

Bacterial genes quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. On rare occasions, translation in bacterial systems can initiate at a TTG codon, though in this event the TTG encodes a methionine. Furthermore, it is not often determined a priori which of these codons are used naturally in the bacterium. Thus, it is understood that use of one of the alternate methionine codons may also lead to generation of pesticidal proteins. These pesticidal proteins are encompassed in the present disclosure and may be used in the methods of the present disclosure. It will be understood that, when expressed in plants, it will be necessary to alter the alternate start codon to ATG for proper translation.

In another aspect the PIP-47 polypeptide may be expressed as a precursor protein with an intervening sequence that catalyzes multi-step, post translational protein splicing. Protein splicing involves the excision of an intervening sequence from a polypeptide with the concomitant joining of the flanking sequences to yield a new polypeptide (Chong, et al., (1996) J. Biol. Chem., 271:22159-22168). This intervening sequence or protein splicing element, referred to as inteins, which catalyze their own excision through three coordinated reactions at the N-terminal and C-terminal splice junctions: an acyl rearrangement of the N-terminal cysteine or serine; a transesterfication reaction between the two termini to form a branched ester or thioester intermediate and peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein (Evans, et al., (2000) J. Biol. Chem., 275:9091-9094. The elucidation of the mechanism of protein splicing has led to a number of intein-based applications (Comb, et al., U.S. Pat. No. 5,496,714; Comb, et al., U.S. Pat. No. 5,834,247; Camarero and Muir, (1999) J. Amer. Chem. Soc. 121:5597-5598; Chong, et al., (1997) Gene 192:271-281, Chong, et al., (1998) Nucleic Acids Res. 26:5109-5115; Chong, et al., (1998) J. Biol. Chem. 273:10567-10577; Cotton, et al., (1999) J. Am. Chem. Soc. 121:1100-1101; Evans, et al., (1999) J. Biol. Chem. 274:18359-18363; Evans, et al., (1999) J. Biol. Chem. 274:3923-3926; Evans, et al., (1998) Protein Sci. 7:2256-2264; Evans, et al., (2000) J. Biol. Chem. 275:9091-9094; Iwai and Pluckthun, (1999) FEBS Lett. 459:166-172; Mathys, et al., (1999) Gene 231:1-13; Mills, et al., (1998) Proc. Natl. Acad. Sci. USA 95:3543-3548; Muir, et al., (1998) Proc. Natl. Acad. Sci. USA 95:6705-6710; Otomo, et al., (1999) Biochemistry 38:16040-16044; Otomo, et al., (1999) J. BiolmoL NMR 14:105-114; Scott, et al., (1999) Proc. Natl. Acad. Sci. USA 96:13638-13643; Severinov and Muir, (1998) J. Biol. Chem. 273:16205-16209; Shingledecker, et al., (1998) Gene 207:187-195; Southworth, et al., (1998) EMBO J. 17:918-926; Southworth, et al., (1999) Biotechniques 27:110-120; Wood, et al., (1999) Nat. Biotechnol. 17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA 95:9226-9231; Wu, et al., (1998b) Biochim Biophys Acta 1387:422-432; Xu, et al., (1999) Proc. Natl. Acad. Sci. USA 96:388-393; Yamazaki, et al., (1998) J. Am. Chem. Soc., 120:5591-5592). For the application of inteins in plant transgenes, see, Yang, et al., (Transgene Res 15:583-593 (2006)) and Evans, et al., (Annu. Rev. Plant Biol. 56:375-392 (2005)).

In another aspect the PIP-47 polypeptide may be encoded by two separate genes where the intein of the precursor protein comes from the two genes, referred to as a split-intein, and the two portions of the precursor are joined by a peptide bond formation. This peptide bond formation is accomplished by intein-mediated trans-splicing. For this purpose, a first and a second expression cassette comprising the two separate genes further code for inteins capable of mediating protein trans-splicing. By trans-splicing, the proteins and polypeptides encoded by the first and second fragments may be linked by peptide bond formation. Trans-splicing inteins may be selected from the nucleolar and organellar genomes of different organisms including eukaryotes, archaebacteria and eubacteria. Inteins that may be used for are listed at neb.com/neb/inteins.html, which can be accessed on the world-wide web using the “www” prefix). The nucleotide sequence coding for an intein may be split into a 5′ and a 3′ part that code for the 5′ and the 3′ part of the intein, respectively. Sequence portions not necessary for intein splicing (e.g. homing endonuclease domain) may be deleted. The intein coding sequence is split such that the 5′ and the 3′ parts are capable of trans-splicing. For selecting a suitable splitting site of the intein coding sequence, the considerations published by Southworth, et al., (1998) EMBO J. 17:918-926 may be followed. In constructing the first and the second expression cassette, the 5′ intein coding sequence is linked to the 3′ end of the first fragment coding for the N-terminal part of the PIP-47 polypeptide and the 3′ intein coding sequence is linked to the 5′ end of the second fragment coding for the C-terminal part of the PIP-47 polypeptide.

In general, the trans-splicing partners can be designed using any split intein, including any naturally-occurring or artificially-split split intein. Several naturally-occurring split inteins are known, for example: the split intein of the DnaE gene of Synechocystis sp. PCC6803 (see, Wu, et al., (1998) Proc Natl Acad Sci USA. 95(16):9226-31 and Evans, et al., (2000) J Biol Chem. 275(13):9091-4 and of the DnaE gene from Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett. 580(7):1853-8). Non-split inteins have been artificially split in the laboratory to create new split inteins, for example: the artificially split Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta. 1387:422-32) and split Sce VMA intein (see, Brenzel, et al., (2006) Biochemistry. 45(6):1571-8) and an artificially split fungal mini-intein (see, Elleuche, et al., (2007) Biochem Biophys Res Commun. 355(3):830-4). There are also intein databases available that catalogue known inteins (see for example the online-database available at: bioinformatics.weizmann.ac.il/{tilde over ( )} pietro/inteins/Inteinstable.html, which can be accessed on the world-wide web using the “www” prefix).

Naturally-occurring non-split inteins may have endonuclease or other enzymatic activities that can typically be removed when designing an artificially-split split intein. Such mini-inteins or minimized split inteins are well known in the art and are typically less than 200 amino acid residues long (see, Wu, et al., (1998) Biochim Biophys Acta. 1387:422-32). Suitable split inteins may have other purification enabling polypeptide elements added to their structure, provided that such elements do not inhibit the splicing of the split intein or are added in a manner that allows them to be removed prior to splicing. Protein splicing has been reported using proteins that comprise bacterial intein-like (BIL) domains (see, Amitai, et al., (2003) Mol Microbiol. 47:61-73) and hedgehog (Hog) auto-processing domains (the latter is combined with inteins when referred to as the Hog/intein superfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem. 279:32001-7) and domains such as these may also be used to prepare artificially-split inteins. In particular, non-splicing members of such families may be modified by molecular biology methodologies to introduce or restore splicing activity in such related species. Recent studies demonstrate that splicing can be observed when a N-terminal split intein component is allowed to react with a C-terminal split intein component not found in nature to be its “partner”; for example, splicing has been observed utilizing partners that have as little as 30 to 50% homology with the “natural” splicing partner (see, Dassa, et al., (2007) Biochemistry. 46(1):322-30). Other such mixtures of disparate split intein partners have been shown to be unreactive one with another (see, Brenzel, et al., (2006) Biochemistry. 45(6):1571-8). However, it is within the ability of a person skilled in the relevant art to determine whether a particular pair of polypeptides is able to associate with each other to provide a functional intein, using routine methods and without the exercise of inventive skill.

In another aspect the PIP-47 polypeptide is a circular permuted variant. In certain embodiments the PIP-47 polypeptide is a circular permuted variant of the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or any one of SEQ ID NO: 629, SEQ ID NO: 630, SEQ ID NO: 631, SEQ ID NO: 632, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635, SEQ ID NO: 636, SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO: 641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 644, SEQ ID NO: 645, SEQ ID NO: 646, SEQ ID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, SEQ ID NO: 651, SEQ ID NO: 652, SEQ ID NO: 653, SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689, SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, SEQ ID NO: 699, SEQ ID NO: 700, SEQ ID NO: 701, SEQ ID NO: 702, SEQ ID NO: 703, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 706, SEQ ID NO: 707, SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710, SEQ ID NO: 711, SEQ ID NO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716, SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO: 719, SEQ ID NO: 720, SEQ ID NO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 797, SEQ ID NO: 798, SEQ ID NO: 799, SEQ ID NO: 800, SEQ ID NO: 801, SEQ ID NO: 802, SEQ ID NO: 803, SEQ ID NO: 804, SEQ ID NO: 805, SEQ ID NO: 806, SEQ ID NO: 807, SEQ ID NO: 808, SEQ ID NO: 809, SEQ ID NO: 810, SEQ ID NO: 811, SEQ ID NO: 812, SEQ ID NO: 813, SEQ ID NO: 814, SEQ ID NO: 814, SEQ ID NO: 815, SEQ ID NO: 816, SEQ ID NO: 817, SEQ ID NO: 818, SEQ ID NO: 819, SEQ ID NO: 820, SEQ ID NO: 821, SEQ ID NO: 822, SEQ ID NO: 823, SEQ ID NO: 824, SEQ ID NO: 825, SEQ ID NO: 826, SEQ ID NO: 827, SEQ ID NO: 828, SEQ ID NO: 829, SEQ ID NO: 830, SEQ ID NO: 831, SEQ ID NO: 832, SEQ ID NO: 833, SEQ ID NO: 834, SEQ ID NO: 835, SEQ ID NO: 836, SEQ ID NO: 837, SEQ ID NO: 838, SEQ ID NO: 839, SEQ ID NO: 840, SEQ ID NO: 841, SEQ ID NO: 842, SEQ ID NO: 843, SEQ ID NO: 844, SEQ ID NO: 845, SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, SEQ ID NO: 850, SEQ ID NO: 851, SEQ ID NO: 852, SEQ ID NO: 853, SEQ ID NO: 854, SEQ ID NO: 855, SEQ ID NO: 856, SEQ ID NO: 857, SEQ ID NO: 858, SEQ ID NO: 859, SEQ ID NO: 860, SEQ ID NO: 861, SEQ ID NO: 862, SEQ ID NO: 863, SEQ ID NO: 864, SEQ ID NO: 865, SEQ ID NO: 888, SEQ ID NO: 889, SEQ ID NO: 890, SEQ ID NO: 891, SEQ ID NO: 892, SEQ ID NO: 893, SEQ ID NO: 894, SEQ ID NO: 895, SEQ ID NO: 896, SEQ ID NO: 897, SEQ ID NO: 898, SEQ ID NO: 899, SEQ ID NO: 900, SEQ ID NO: 901, SEQ ID NO: 902, SEQ ID NO: 903, SEQ ID NO: 904, SEQ ID NO: 905, SEQ ID NO: 906, SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ ID NO: 916, SEQ ID NO: 917, SEQ ID NO: 918, SEQ ID NO: 919, SEQ ID NO: 920 and SEQ ID NO: 921.

The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and Erfle, (1990) J. Bacteriol. 172:3837-3841; Schimming, et al., (1992) Eur. J. Biochem. 204:13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett. 260:127-130; MacGregor, et al., (1996) FEBS Lett. 378:263-266). The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). In creating a circular permuted variant a new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain. The length of the amino acid sequence of the linker can be selected empirically or with guidance from structural information or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be prepared for testing using a design whose length is varied in order to span a range from 0 to 50 Å and whose sequence is chosen in order to be consistent with surface exposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol. 20:483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157:105-132; solvent exposed surface area, Lee and Richards, (1971) J. Mol. Biol. 55:379-400) and the ability to adopt the necessary conformation without deranging the configuration of the pesticidal polypeptide (conformationally flexible; Karplus and Schulz, (1985) Naturwissenschaften 72:212-213). Assuming an average of translation of 2.0 to 3.8 Å per residue, this would mean the length to test would be between 0 to 30 residues, with 0 to 15 residues being the preferred range. Exemplary of such an empirical series would be to construct linkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated n times, where n is 1, 2, 3 or 4. Those skilled in the art will recognize that there are many such sequences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor short (cf., Sandhu, (1992) Critical Rev. Biotech. 12:437-462); if they are too long, entropy effects will likely destabilize the three-dimensional fold, and may also make folding kinetically impractical, and if they are too short, they will likely destabilize the molecule because of torsional or steric strain. Those skilled in the analysis of protein structural information will recognize that using the distance between the chain ends, defined as the distance between the c-alpha carbons, can be used to define the length of the sequence to be used or at least to limit the number of possibilities that must be tested in an empirical selection of linkers. They will also recognize that it is sometimes the case that the positions of the ends of the polypeptide chain are ill-defined in structural models derived from x-ray diffraction or nuclear magnetic resonance spectroscopy data, and that when true, this situation will therefore need to be taken into account in order to properly estimate the length of the linker required. From those residues whose positions are well defined are selected two residues that are close in sequence to the chain ends, and the distance between their c-alpha carbons is used to calculate an approximate length for a linker between them. Using the calculated length as a guide, linkers with a range of number of residues (calculated using 2 to 3.8 Å per residue) are then selected. These linkers may be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues may be chosen to be flexible and hydrophilic as described above; or optionally the original sequence may be substituted for using a series of linkers, one example being the Gly-Gly-Gly-Ser cassette approach mentioned above; or optionally a combination of the original sequence and new sequence having the appropriate total length may be used. Sequences of pesticidal polypeptides capable of folding to biologically active states can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while using the linker sequence as described above. Amino and carboxyl termini are selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel amino acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus. However, those skilled in the art will recognize that selections of termini anywhere within the region may function, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence. It is a central tenet of molecular biology that the primary amino acid sequence of a protein dictates folding to the three-dimensional structure necessary for expression of its biological function. Methods are known to those skilled in the art to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein Crystals or nuclear magnetic resonance spectroscopy of protein solutions. Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops; Kabsch and Sander, (1983) Biopolymers 22:2577-2637; the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, (1984) Ann. Rev. Biochem. 53:537-572) and the static and dynamic distribution of conformations along the polypeptide chain (Alber and Mathews, (1987) Methods Enzymol. 154:511-533). In some cases additional information is known about solvent exposure of residues; one example is a site of post-translational attachment of carbohydrate which is necessarily on the surface of the protein. When experimental structural information is not available or is not feasible to obtain, methods are also available to analyze the primary amino acid sequence in order to make predictions of protein tertiary and secondary structure, solvent accessibility and the occurrence of turns and loops. Biochemical methods are also sometimes applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile and Salvatore, (1993) Eur. J. Biochem. 218:603-621). Thus using either the experimentally derived structural information or predictive methods (e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics 22:81-99) the parental amino acid sequence is inspected to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The occurrence of sequences within regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so-called hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini. In contrast, those regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity, are the preferred sites for location of the extremes of the polypeptide chain. Continuous stretches of amino acid sequence that are preferred based on the above criteria are referred to as a breakpoint region. Polynucleotides encoding circular permuted PIP-47 polypeptides with new N-terminus/C-terminus which contain a linker region separating the original C-terminus and N-terminus can be made essentially following the method described in Mullins, et al., (1994) J. Am. Chem. Soc. 116:5529-5533. Multiple steps of polymerase chain reaction (PCR) amplifications are used to rearrange the DNA sequence encoding the primary amino acid sequence of the protein. Polynucleotides encoding circular permuted PIP-47 polypeptides with new N-terminus/C-terminus which contain a linker region separating the original C-terminus and N-terminus can be made based on the tandem-duplication method described in Horlick, et al., (1992) Protein Eng. 5:427-431. Polymerase chain reaction (PCR) amplification of the new N-terminus/C-terminus genes is performed using a tandemly duplicated template DNA.

In another aspect fusion proteins are provided that include within its amino acid sequence an amino acid sequence comprising a PIP-47 polypeptide including but not limited to the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or any one of SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, SEQ ID NO: 916-SEQ ID NO: 921, and active fragments thereof.

Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. Polynucleotides encoding a PIP-47 polypeptide may be fused to signal sequences which will direct the localization of the PIP-47 polypeptide to particular compartments of a prokaryotic or eukaryotic cell and/or direct the secretion of the PIP-47 polypeptide of the embodiments from a prokaryotic or eukaryotic cell. For example, in E. coli, one may wish to direct the expression of the protein to the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) to which the PIP-47 polypeptide may be fused in order to direct the expression of the polypeptide to the periplasmic space of bacteria include, but are not limited to, the pelB signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit and the signal sequence of alkaline phosphatase. Several vectors are commercially available for the construction of fusion proteins which will direct the localization of a protein, such as the pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs. In a specific embodiment, the PIP-47 polypeptide may be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria (see, U.S. Pat. Nos. 5,576,195 and 5,846,818). Plant plastid transit peptide/polypeptide fusions are well known in the art (see, U.S. Pat. No. 7,193,133). Apoplast transit peptides such as rice or barley alpha-amylase secretion signal are also well known in the art. The plastid transit peptide is generally fused N-terminal to the polypeptide to be targeted (e.g., the fusion partner). In one embodiment, the fusion protein consists essentially of the plastid transit peptide and the PIP-47 polypeptide to be targeted. In another embodiment, the fusion protein comprises the plastid transit peptide and the polypeptide to be targeted. In such embodiments, the plastid transit peptide is preferably at the N-terminus of the fusion protein. However, additional amino acid residues may be N-terminal to the plastid transit peptide providing that the fusion protein is at least partially targeted to a plastid. In a specific embodiment, the plastid transit peptide is in the N-terminal half, N-terminal third or N-terminal quarter of the fusion protein. Most or all of the plastid transit peptide is generally cleaved from the fusion protein upon insertion into the plastid. The position of cleavage may vary slightly between plant species, at different plant developmental stages, as a result of specific intercellular conditions or the particular combination of transit peptide/fusion partner used. In one embodiment, the plastid transit peptide cleavage is homogenous such that the cleavage site is identical in a population of fusion proteins. In another embodiment, the plastid transit peptide is not homogenous, such that the cleavage site varies by 1-10 amino acids in a population of fusion proteins. The plastid transit peptide can be recombinantly fused to a second protein in one of several ways. For example, a restriction endonuclease recognition site can be introduced into the nucleotide sequence of the transit peptide at a position corresponding to its C-terminal end and the same or a compatible site can be engineered into the nucleotide sequence of the protein to be targeted at its N-terminal end. Care must be taken in designing these sites to ensure that the coding sequences of the transit peptide and the second protein are kept “in frame” to allow the synthesis of the desired fusion protein. In some cases, it may be preferable to remove the initiator methionine codon of the second protein when the new restriction site is introduced. The introduction of restriction endonuclease recognition sites on both parent molecules and their subsequent joining through recombinant DNA techniques may result in the addition of one or more extra amino acids between the transit peptide and the second protein. This generally does not affect targeting activity as long as the transit peptide cleavage site remains accessible and the function of the second protein is not altered by the addition of these extra amino acids at its N-terminus. Alternatively, one skilled in the art can create a precise cleavage site between the transit peptide and the second protein (with or without its initiator methionine) using gene synthesis (Stemmer, et al., (1995) Gene 164:49-53) or similar methods. In addition, the transit peptide fusion can intentionally include amino acids downstream of the cleavage site. The amino acids at the N-terminus of the mature protein can affect the ability of the transit peptide to target proteins to plastids and/or the efficiency of cleavage following protein import. This may be dependent on the protein to be targeted. See, e.g., Comai, et al., (1988) J. Biol. Chem. 263(29):15104-9.

In some embodiments fusion proteins are provide comprising a PIP-47 polypeptide, and an insecticidal polypeptide joined by an amino acid linker.

In some embodiments fusion proteins are provided represented by a formula selected from the group consisting of:

R¹-L-R², R²-L-R¹, R¹-R²or R²-R¹

wherein R¹is a PIP-47 polypeptide or the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or any one of SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, and SEQ ID NO: 916-SEQ ID NO: 921, R²is an insecticidal polypeptide. The R¹polypeptide is fused either directly or through a linker (L) segment to the R²polypeptide. The term “directly” defines fusions in which the polypeptides are joined without a peptide linker. Thus “L” represents a chemical bound or polypeptide segment to which both R¹and R²are fused in frame, most commonly L is a linear peptide to which R¹and R²are bound by amide bonds linking the carboxy terminus of R¹to the amino terminus of L and carboxy terminus of L to the amino terminus of R². By “fused in frame” is meant that there is no translation termination or disruption between the reading frames of R¹and R². The linking group (L) is generally a polypeptide of between 1 and 500 amino acids in length. The linkers joining the two molecules are preferably designed to (1) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two proteins, (3) have minimal hydrophobic or charged characteristic which could interact with the functional protein domains and (4) provide steric separation of R¹and R²such that R¹and R²could interact simultaneously with their corresponding receptors on a single cell. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the fusions.

In some embodiments the linkers comprise sequences selected from the group of formulas: (Gly₃Ser)_n, (Gly₄Ser)_n, (Gly₅Ser)_n, (Gly_nSer)_nor (AlaGlySer)_nwhere n is an integer. One example of a highly-flexible linker is the (GlySer)-rich spacer region present within the pill protein of the filamentous bacteriophages, e.g. bacteriophages M13 or fd (Schaller, et al., 1975). This region provides a long, flexible spacer region between two domains of the pill surface protein. Also included are linkers in which an endopeptidase recognition sequence is included. Such a cleavage site may be valuable to separate the individual components of the fusion to determine if they are properly folded and active in vitro. Examples of various endopeptidases include, but are not limited to, Plasmin, Enterokinase, Kallikerin, Urokinase, Tissue Plasminogen activator, clostripain, Chymosin, Collagenase, Russell's Viper Venom Protease, Postproline cleavage enzyme, V8 protease, Thrombin and factor Xa. In some embodiments the linker comprises the amino acids EEKKN (SEQ ID NO: 488) from the multi-gene expression vehicle (MGEV), which is cleaved by vacuolar proteases as disclosed in US Patent Application Publication Number US 2007/0277263. In other embodiments, peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides. Especially useful are those hinge regions where the cysteines are replaced with serines. Linkers of the present disclosure include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines. The fusion proteins are not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere adversely with the folding and function of the individual molecules of the fusion.

In another aspect chimeric PIP-47 polypeptides are provided that are created through joining two or more portions of PIP-47 genes, which originally encoded separate PIP-47 proteins to create a chimeric gene. The translation of the chimeric gene results in a single chimeric PIP-47 polypeptide with regions, motifs or domains derived from each of the original polypeptides. In certain embodiments the chimeric protein comprises portions, motifs or domains of PIP-47Aa (SEQ ID NO: 2), PIP-47Ba (SEQ ID NO: 4), PIP-47Bb (SEQ ID NO: 6) and PIP-47Fa (SEQ ID NO: 8) in any combination.

It is recognized that DNA sequences may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by the wild-type (or native) pesticidal protein. In some embodiments a PIP-47 polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations and insertions of one or more amino acids, including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or more amino acid substitutions, deletions and/or insertions or combinations thereof compared to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486 or SEQ ID NO: 487. In some embodiments a PIP-47 polypeptide comprises the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids from the N-terminus of the PIP-47 polypeptide relative to the amino acid position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or any one of SEQ ID NO: 221-SEQ ID NO: 420, SEQ ID NO: 629-SEQ ID NO: 727, SEQ ID NO: 797-SEQ ID NO: 865, SEQ ID NO: 888-SEQ ID NO: 909, and SEQ ID NO: 916-SEQ ID NO: 921.

Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a PIP-47 polypeptide can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis and/or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess the desired pesticidal activity. However, it is understood that the ability of a PIP-47 polypeptide to confer pesticidal activity may be improved by the use of such techniques upon the compositions of this disclosure.

For example, conservative amino acid substitutions may be made at one or more, predicted, nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a PIP-47 polypeptide without altering the biological activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic, acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); small aliphatic, nonpolar or slightly polar residues (e.g., Alanine, serine, threonine, proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cystine); beta-branched side chains (e.g., threonine, valine, isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

Amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins). However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(1):105-32). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, ibid). These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9) and arginine (−4.5). In making such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions or alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions. Such fusion proteins are often used to (1) increase expression of a protein of interest (2) introduce a binding domain, enzymatic activity or epitope to facilitate either protein purification, protein detection or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, mitochondria or chloroplasts of plants or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.

In some embodiments, the PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 2 having amino acid substitutions compared to the native amino acid of SEQ ID NO: 2 at one or more residues selected from positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 57, 58, 62, 63, 65, 66, 67, 68, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 88, 90, 91, 92, 95, 96, 97, 98, 99, 100, 103, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 124, 130, 132, 133, 135, 136, 137, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151, 153, 156, 158, 161, 164, 165, 168, 169, 170, 172, 173, 174, 176, 178, 179, 181, 182, 184, 185, 188, 189, 193, 194, 196, 198, 200, 202, 203, 205, 206, 207, 208, 212, 213, 221, 222, 226, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 253, 254, 255, 257, 260, 262, 264, 265, 267, 270, 272, 273, 274, 275, 276, 277, 279, 280, 281, 282, 283, 284, 287 or 295 of SEQ ID NO: 2, in any combination, and optionally the PIP-47 polypeptide further comprises a deletion of 1 to 5 amino acids, an insertion of 1 to 5 amino acids, addition of one or more amino acids at the N-terminus or addition of one or more amino acids at the C-terminus relative to SEQ ID NO: 2, in any combination.

In some embodiments, the PIP-47 polypeptide comprises an amino acid sequence of SEQ ID NO: 2 having amino acid substitutions compared to the native amino acid of SEQ ID NO: 2 at 1 to 60 residues selected from positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 57, 58, 62, 63, 65, 66, 67, 68, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 88, 90, 91, 92, 95, 96, 97, 98, 99, 100, 103, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 124, 130, 132, 133, 135, 136, 137, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151, 153, 156, 158, 161, 164, 165, 168, 169, 170, 172, 173, 174, 176, 178, 179, 181, 182, 184, 185, 188, 189, 193, 194, 196, 198, 200, 202, 203, 205, 206, 207, 208, 212, 213, 221, 222, 226, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 250, 253, 254, 255, 257, 260, 262, 264, 265, 267, 270, 272, 273, 274, 275, 276, 277, 279, 280, 281, 282, 283, 284, 287 or 295 of SEQ ID NO: 2, in any combination, and optionally the PIP-47 polypeptide further comprises a deletion of 1 to 5 amino acids, an insertion of 1 to 5 amino acids, addition of one or more amino acids at the N-terminus or addition of one or more amino acids at the C-terminus relative to SEQ ID NO: 2, in any combination.

In specific embodiments, the substitution is an alanine for the native amino acid at the recited position(s). Also encompassed are the nucleic acid sequence(s) encoding the variant protein or polypeptide.

Variant nucleotide and amino acid sequences of the disclosure also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different PIP-47 polypeptide coding regions can be used to create a new PIP-47 polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating altered PIP-47 polypeptides. Domains may be swapped between PIP-47 polypeptides, resulting in hybrid or chimeric toxins with improved insecticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov, et al., (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, et al., (1991) J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol. Chem. 265:20923-20930; Rang, et al., 91999) Appl. Environ. Microbiol. 65:2918-2925).

Both DNA shuffling and site directed mutagenesis were used to define polypeptide sequences that possess pesticidal activity. In Example 8 DNA shuffling was used to generate a library of active variants by recombination of the diversity present in PIP-47Aa (SEQ ID NO: 2). The person skilled in the art will be able to use comparisons to other proteins or functional assays to further define motifs. High throughput screening can be used to test variations of those motifs to determine the role of specific residues. Given that knowledge for several motifs, one can then define the requirements for a functional protein. Knowledge of the motifs allows the skilled artisan to design sequence variations that would not impact function.

Alignment of homologues of PIP-47 homologs (FIGS. 1, 2 & 3) allowed identification of residues that are highly conserved among natural homologues in this family (FIG. 1). In Example 9 and 10, saturation mutagenesis was used to make and test substitutions at selected amino acid positions in the motifs at conserved residues. These mutants were tested for activity and a number of active substitutions not present among the homologues were identified providing an understanding of the functional constraints at these residues.

Compositions

Compositions comprising a PIP-47 polypeptide are also embraced. In some embodiments the composition comprises a PIP-47 polypeptide. In some embodiments the composition comprises a PIP-47 fusion protein.

Antibodies

Antibodies to a PIP-47 polypeptide of the embodiments or to variants or fragments thereof are also encompassed. The antibodies of the disclosure include polyclonal and monoclonal antibodies as well as fragments thereof which retain their ability to bind to PIP-47 proteins found in the insect gut. An antibody, monoclonal antibody or fragment thereof is said to be capable of binding a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody, monoclonal antibody or fragment thereof. The term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as fragments or binding regions or domains thereof (such as, for example, Fab and F(ab).sub.2 fragments) which are capable of binding hapten. Such fragments are typically produced by proteolytic cleavage, such as papain or pepsin. Alternatively, hapten-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry. Methods for the preparation of the antibodies of the present disclosure are generally known in the art. For example, see, Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory, N.Y. (1988), as well as the references cited therein. Standard reference works setting forth the general principles of immunology include: Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, N.Y. (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, N.Y. (1980) and Campbell, “Monoclonal Antibody Technology,” In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon, et al., (eds.), Elsevier, Amsterdam (1984). See also, U.S. Pat. Nos. 4,196,265; 4,609,893; 4,713,325; 4,714,681; 4,716,111; 4,716,117 and 4,720,459. PIP-47 polypeptide polypeptide antibodies or antigen-binding portions thereof can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256:495. Other techniques for producing monoclonal antibody can also be employed such as viral or oncogenic transformation of B lymphocytes. An animal system for preparing hybridomas is a murine system. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. The antibody and monoclonal antibodies of the disclosure can be prepared by utilizing a PIP-47 polypeptide polypeptide as antigens.

A kit for detecting the presence of a PIP-47 polypeptide polypeptide or detecting the presence of a nucleotide sequence encoding a PIP-47 polypeptide polypeptide, in a sample is provided. In one embodiment, the kit provides antibody-based reagents for detecting the presence of a PIP-47 polypeptide polypeptide in a tissue sample. In another embodiment, the kit provides labeled nucleic acid probes useful for detecting the presence of one or more polynucleotides encoding PIP-47 polypeptide(s). The kit is provided along with appropriate reagents and controls for carrying out a detection method, as well as instructions for use of the kit.

Receptor Identification and Isolation

Receptors to the PIP-47 polypeptide of the embodiments or to variants or fragments thereof, are also encompassed. Methods for identifying receptors are well known in the art (see, Hofmann, et. al., (1988) Eur. J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282) can be employed to identify and isolate the receptor that recognizes the PIP-47 polypeptides using the brush-border membrane vesicles from susceptible insects. In addition to the radioactive labeling method listed in the cited literatures, PIP-47 polypeptide can be labeled with fluorescent dye and other common labels such as streptavidin. Brush-border membrane vesicles (BBMV) of susceptible insects such as soybean looper and stink bugs can be prepared according to the protocols listed in the references and separated on SDS-PAGE gel and blotted on suitable membrane. Labeled PIP-47 polypeptides can be incubated with blotted membrane of BBMV and labeled the PIP-47 polypeptides can be identified with the labeled reporters. Identification of protein band(s) that interact with the PIP-47 polypeptides can be detected by N-terminal amino acid gas phase sequencing or mass spectrometry based protein identification method (Patterson, (1998) 10.22, 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc). Once the protein is identified, the corresponding gene can be cloned from genomic DNA or cDNA library of the susceptible insects and binding affinity can be measured directly with the PIP-47 polypeptides. Receptor function for insecticidal activity by the PIP-47 polypeptides can be verified by accomplished by RNAi type of gene knock out method (Rajagopal, et al., (2002) J. Biol. Chem. 277:46849-46851).

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as an organism selected from plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a DNA molecule of the embodiments, an expression cassette comprising the DNA molecule or a vector comprising the expression cassette, which may be stably incorporated into the genome of the transformed organism.

The sequences of the embodiments are provided in DNA constructs for expression in the organism of interest. The construct will include 5′ and 3′ regulatory sequences operably linked to a sequence of the embodiments. The term “operably linked” as used herein refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and where necessary to join two protein coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sites for insertion of the PIP-47 polypeptide gene sequence to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host. The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign or heterologous to the host organism and/or to the sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term “foreign” as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter is “foreign” or “heterologous” to the sequence of the embodiments, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked sequence of the embodiments. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.

In some embodiments the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term an “enhancer” refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants (US Patent Application Publication Number 2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96) and the enhancers of U.S. Pat. No. 7,803,992 may also be used, each of which is incorporated by reference. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.

The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize-preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391 and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep 37:677-684, 2010, herein incorporated by reference. A Zea maize codon usage table can be also found at kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4577, which can be accessed using the www prefix. Table 2 shows a maize optimal codon analysis (adapted from Liu H et al. Mol Bio Rep 37:677-684, 2010).

TABLE 2

Amino

High

Low

Amino

High

Low

Acid
Codon
Count
RSCU
Count
RSCU
Acid
Codon
Count
RSCU
Count
RSCU

Phe
UUU
115
0.04
2,301
1.22
Ala
GCU
629
0.17
3,063
1.59

UUC*
5,269
1.96
1,485
0.78

GCC*
8,057
2.16
1,136
0.59

Ser
UCU
176
0.13
2,498
1.48

GCA
369
0.1
2,872
1.49

UCC*
3,489
2.48
1,074
0.63

GCG*
5,835
1.57
630
0.33

UCA
104
0.07
2,610
1.54
Tyr
UAU
71
0.04
1,632
1.22

UCG*
1,975
1.4
670
0.4

UAC*
3,841
1.96
1,041
0.78

AGU
77
0.05
1,788
1.06
His
CAU
131
0.09
1,902
1.36

AGC*
2,617
1.86
1,514
0.89

CAC*
2,800
1.91
897
0.64

Leu
UUA
10
0.01
1,326
0.79
Cys
UGU
52
0.04
1,233
1.12

UUG
174
0.09
2,306
1.37

UGC*
2,291
1.96
963
0.88

CUU
223
0.11
2,396
1.43
Gln
CAA
99
0.05
2,312
1.04

CUC*
5,979
3.08
1,109
0.66

CAG*
3,557
1.95
2,130
0.96

CUA
106
0.05
1,280
0.76
Arg
CGU
153
0.12
751
0.74

CUG*
5,161
2.66
1,646
0.98

CGC*
4,278
3.25
466
0.46

Pro
CCU
427
0.22
1,900
1.47

CGA
92
0.07
659
0.65

CCC*
3,035
1.59
601
0.47

CGG*
1,793
1.36
631
0.62

CCA
311
0.16
2,140
1.66

AGA
83
0.06
1,948
1.91

CCG*
3,846
2.02
513
0.4

AGG*
1,493
1.14
1,652
1.62

Ile
AUU
138
0.09
2,388
1.3
Asn
AAU
131
0.07
3,074
1.26

AUC*
4,380
2.85
1,353
0.74

AAC*
3,814
1.93
1,807
0.74

AUA
88
0.06
1,756
0.96
Lys
AAA
130
0.05
3,215
0.98

Thr
ACU
136
0.09
1,990
1.43

AAG*
5,047
1.95
3,340
1.02

ACC*
3,398
2.25
991
0.71
Asp
GAU
312
0.09
4,217
1.38

ACA
133
0.09
2,075
1.5

GAC*
6,729
1.91
1,891
0.62

ACG*
2,378
1.57
495
0.36
Gly
GGU
363
0.13
2,301
1.35

Val
GUU
182
0.07
2,595
1.51

GGC*
7,842
2.91
1,282
0.75

GUC*
4,584
1.82
1,096
0.64

GGA
397
0.15
2,044
1.19

GUA
74
0.03
1,325
0.77

GGG*
2,186
0.81
1,215
0.71

GUG*
5,257
2.08
1,842
1.07
Glu
GAA
193
0.06
4,080
1.1

GAG*
6,010
1.94
3,307
0.9

Codon usage was compared using Chi squared contingency test to identify optimal codons. Codons that occur significantly more often (P\0.01) are indicated with an asterisk.

A Glycine max codon usage table is shown in Table 3 and can also be found at kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3847&aa=1&style=N, which can be accessed using the www prefix.

TABLE 3

TTT
F
21.2
(10493)
TCT
S
18.4
(9107)

TTC
F
21.2
(10487)
TCC
S
12.9
(6409)

TTA
L
9.2
(4545)
TCA
S
15.6
(7712)

TTG
L
22.9
(11340)
TCG
S
4.8
(2397)

CTT
L
23.9
(11829)
CCT
P
18.9
(9358)

CTC
L
17.1
(8479)
CCC
P
10.1
(5010)

CTA
L
8.5
(4216)
CCA
P
19.1
(9461)

CTG
L
12.7
(6304)
CCG
P
4.7
(2312)

ATT
I
25.1
(12411)
ACT
T
17.1
(8490)

ATC
I
16.3
(8071)
ACC
T
14.3
(7100)

ATA
I
12.9
(6386)
ACA
T
14.9
(7391)

ATG
M
22.7
(11218)
ACG
T
4.3
(2147)

GTT
V
26.1
(12911)
GCT
A
26.7
(13201)

GTC
V
11.9
(5894)
GCC
A
16.2
(8026)

GTA
V
7.7
(3803)
GCA
A
21.4
(10577)

GTG
V
21.4
(10610)
GCG
A
6.3
(3123)

TAT
Y
15.7
(7779)
TGT
C
8.1
(3995)

TAC
Y
14.9
(7367)
TGC
C
8.0
(3980)

TAA
*
0.9
(463)
TGA
*
1.0
(480)

TAG
*
0.5
(263)
TGG
W
13.0
(6412)

CAT
H
14.0
(6930)
CGT
R
6.6
(3291)

CAC
H
11.6
(5759)
CGC
R
6.2
(3093)

CAA
Q
20.5
(10162)
CGA
R
4.1
(2018)

CAG
Q
16.2
(8038)
CGG
R
3.1
(1510)

AAT
N
22.4
(11088)
AGT
S
12.6
(6237)

AAC
N
22.8
(11284)
AGC
S
11.3
(5594)

AAA
K
26.9
(13334)
AGA
R
14.8
(7337)

AAG
K
35.9
(17797)
AGG
R
13.3
(6574)

GAT
D
32.4
(16040)
GGT
G
20.9
(10353)

GAC
D
20.4
(10097)
GGC
G
13.4
(6650)

GAA
E
33.2
(16438)
GGA
G
22.3
(11022)

GAG
E
33.2
(16426)
GGG
G
13.0
(6431)

In some embodiments the recombinant nucleic acid molecule encoding a PIP-47 polypeptide has maize optimized codons.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term “host cell” as used herein refers to a cell which contains a vector and supports the replication and/or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a maize host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Such constructs may also contain a “signal sequence” or “leader sequence” to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum or Golgi apparatus.

“Signal sequence” as used herein refers to a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Insecticidal toxins of bacteria are often synthesized as protoxins, which are protolytically activated in the gut of the target pest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments, the signal sequence is located in the native sequence or may be derived from a sequence of the embodiments. “Leader sequence” as used herein refers to any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Nuclear-encoded proteins targeted to the chloroplast thylakoid lumen compartment have a characteristic bipartite transit peptide, composed of a stromal targeting signal peptide and a lumen targeting signal peptide. The stromal targeting information is in the amino-proximal portion of the transit peptide. The lumen targeting signal peptide is in the carboxyl-proximal portion of the transit peptide, and contains all the information for targeting to the lumen. Recent research in proteomics of the higher plant chloroplast has achieved in the identification of numerous nuclear-encoded lumen proteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al. Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta 1503:350-356, 2001), the lumen targeting signal peptide of which can potentially be used in accordance with the present disclosure. About 80 proteins from Arabidopsis, as well as homologous proteins from spinach and garden pea, are reported by Kieselbach et al., Photosynthesis Research, 78:249-264, 2003. In particular, Table 2 of this publication, which is incorporated into the description herewith by reference, discloses 85 proteins from the chloroplast lumen, identified by their accession number (see also US Patent Application Publication 2009/09044298). In addition, the recently published draft version of the rice genome (Goff et al, Science 296:92-100, 2002) is a suitable source for lumen targeting signal peptide which may be used in accordance with the present disclosure.

Suitable chloroplast transit peptides (CTP) are well known to one skilled in the art also include chimeric CTPs comprising but not limited to, an N-terminal domain, a central domain or a C-terminal domain from a CTP from Oryza sativa 1-deoxy-D xyulose-5-Phosphate Synthase oryza sativa-Superoxide dismutase oryza sativa-soluble starch synthase oryza sativa-NADP-dependent Malic acid enzyme oryza sativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 oryza sativa-L-Ascorbate peroxidase 5 oryza sativa-Phosphoglucan water dikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malate dehydrogenase, Zea Mays Thioredoxin M-type US Patent Application Publication 2012/0304336).

The PIP-47 polypeptide gene to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.

In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant MoL Biol. 12:619-632 and Christensen, et al., (1992) Plant MoL Biol. 18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Depending on the desired outcome, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath. 28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al., (1989) MoL Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992) Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol. 22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene (Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, herein incorporated by reference.

Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. Plant Pathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and Van Loon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999/43819, herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen, et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201; Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible) and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced PIP-47 polypeptide expression within a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994) Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire, et al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi, (1991) describe their analysis of the promoters of the highly expressed roIC and rolD root-inducing genes of Agrobacterium rhizogenes (see, Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2′ gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1′ gene fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772) and roIB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Arabidopsis thaliana root-preferred regulatory sequences are disclosed in US20130117883.

“Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). See, Thompson, et al., (1989) BioEssays 10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529, herein incorporated by reference). Gamma-zein and Glb-1 are endosperm-specific promoters. For dicots, seed-specific promoters include, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku and Goldberg, (1989) Plant Cell 1:1079-1093), bean β-phaseolin, napin, β-conglycinin, glycinin 1, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also, WO 2000/12733, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference. In dicots, seed specific promoters include but are not limited to seed coat promoter from Arabidopsis, pBAN; and the early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S. Pat. Nos. 7,294,760 and 7,847,153). A promoter that has “preferred” expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue-preferred promoters show expression almost exclusively in the particular tissue.

Where low level expression is desired, weak promoters will be used. Generally, the term “weak promoter” as used herein refers to a promoter that drives expression of a coding sequence at a low level. By low level expression at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts is intended. Alternatively, it is recognized that the term “weak promoters” also encompasses promoters that drive expression in only a few cells and not in others to give a total low level of expression. Where a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoter of the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, those disclosed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611, herein incorporated by reference.

The above list of promoters is not meant to be limiting. Any appropriate promoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820); streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol. 7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol. 15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423); glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patent application Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin (DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc. NatL Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) MoL Microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc. NatL Acad. Sci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. NatL Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) MoL Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. NatL Acad. Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill, et al., (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the embodiments.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide or polynucleotide into a plant. “Introducing” is as used herein means presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the embodiments do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

“Stable transformation” is as used herein means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant. “Plant” as used herein refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells and pollen).

Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al., (1988) Biotechnology 6:923-926) and Lecl transformation (WO 00/28058). For potato transformation see, Tu, et al., (1998) Plant Molecular Biology 37:829-838 and Chong, et al., (2000) Transgenic Research 9:71-78. Additional transformation procedures can be found in Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al, (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al, (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

In specific embodiments, the sequences of the embodiments can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the PIP-47 polypeptide or variants and fragments thereof directly into the plant or the introduction of the PIP-47 polypeptide transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the PIP-47 polypeptide polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).

Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO 1999/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide of the embodiments can be contained in transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.

Plant transformation vectors may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as “binary vectors”. Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux, (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with appropriate selection, one identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.

Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g., Hiei, et al., (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park, (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformed material contains many cells; both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants.

The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the embodiments may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired PIP-47 polypeptide. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a PIP-47 polypeptide of the embodiments, may have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences that encode for them are encompassed by the embodiments. Methods for providing plants with nucleotide constructs and producing the encoded proteins in the plants, which involve viral DNA or RNA molecules are known in the art. See, for example, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367 and 5,316,931; herein incorporated by reference.

Methods for transformation of chloroplasts are known in the art. See, for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride, et al., (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The embodiments further relate to plant-propagating material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers that may be employed in practicing the embodiments include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). Plants of the embodiments include crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewing's fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithh); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum); carpet grass (Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalurn (Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.

Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, (2001) supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or “blot” is then probed with, for example, radiolabeled 32P target DNA fragment to confirm the integration of introduced gene into the plant genome according to standard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, (2001) supra). Expression of RNA encoded by the pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a pesticidal gene, by methods known in the art (Sambrook and Russell, (2001) supra).

Western blot, biochemical assays and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the PIP-47 polypeptide.

Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more insecticidal polynucleotides disclosed herein with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences. Transgenic plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene and co-transformation of genes into a single plant cell. As used herein, the term “stacked” includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid). In one non-limiting example, “stacked traits” comprise a molecular stack where the sequences are physically adjacent to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. Co-transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO 1999/25853, all of which are herein incorporated by reference.

In some embodiments the polynucleotides encoding the PIP-47 polypeptides disclosed herein, alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like). Thus, the polynucleotide embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.

Transgenes useful for stacking include but are not limited to:

1. Transgenes that Confer Resistance to Insects or Disease and that Encode:

(A) Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant variety can be transformed with cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example, Jones, et al., (1994) Science 266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae), McDowell and Woffenden, (2003) Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) Transgenic Res. 11(6):567-82. A plant resistant to a disease is one that is more resistant to a pathogen as compared to the wild type plant.

(B) Genes encoding a Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser, et al., (1986) Gene 48:109, who disclose the cloning and nucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC® Accession Numbers 40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillus thuringiensis transgenes being genetically engineered are given in the following patents and patent applications and hereby are incorporated by reference for this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594, 6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826, 7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556, 7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862, 7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846, 7,858,849 and WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581 and WO 1997/40162.

Genes encoding pesticidal proteins may also be stacked including but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonas protegens strain CHA0 and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386: GenBank Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et al., (2010) J. Agric. Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxinology Journal 3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069), U.S. Pat. No. 6,048,838, and U.S. Pat. No. 6,379,946; a PIP-1 polypeptide of U.S. Ser. No. 13/792,861; an AflP-1A and/or AflP-1B polypeptide of U.S. Ser. No. 13/800,233; a PHI-4 polypeptide of U.S. Ser. No. 13/839,702; and δ-endotoxins including, but not limited to, the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51 and Cry55 classes of δ-endotoxin genes and the B. thuringiensis cytolytic Cyt1 and Cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins include, but are not limited to Cry1Aa1 (Accession # AAA22353); Cry1Aa2 (Accession # Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4 (Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6 (Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8 (Accession #126149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession # AAD55382); Cry1Aa11 (Accession # CAA70856); Cry1Aa12 (Accession # AAP80146); Cry1Aa13 (Accession # AAM44305); Cry1Aa14 (Accession # AAP40639); Cry1Aa15 (Accession # AAY66993); Cry1Aa16 (Accession # HQ439776); Cry1Aa17 (Accession # HQ439788); Cry1Aa18 (Accession # HQ439790); Cry1Aa19 (Accession # HQ685121); Cry1Aa20 (Accession # JF340156); Cry1Aa21 (Accession # JN651496); Cry1Aa22 (Accession # KC158223); Cry1Ab1 (Accession # AAA22330); Cry1Ab2 (Accession # AAA22613); Cry1Ab3 (Accession # AAA22561); Cry1Ab4 (Accession # BAA00071); Cry1Ab5 (Accession # CAA28405); Cry1Ab6 (Accession # AAA22420); Cry1Ab7 (Accession # CAA31620); Cry1Ab8 (Accession # AAA22551); Cry1Ab9 (Accession # CAA38701); Cry1Ab10 (Accession # A29125); Cry1Ab11 (Accession #112419); Cry1Ab12 (Accession # AAC64003); Cry1Ab13 (Accession # AAN76494); Cry1Ab14 (Accession # AAG16877); Cry1Ab15 (Accession # AAO13302); Cry1Ab16 (Accession # AAK55546); Cry1Ab17 (Accession # AAT46415); Cry1Ab18 (Accession # AAQ88259); Cry1Ab19 (Accession # AAW31761); Cry1Ab20 (Accession # ABB72460); Cry1Ab21 (Accession # ABS18384); Cry1Ab22 (Accession # ABW87320); Cry1Ab23 (Accession # HQ439777); Cry1Ab24 (Accession # HQ439778); Cry1Ab25 (Accession # HQ685122); Cry1Ab26 (Accession # HQ847729); Cry1Ab27 (Accession # JN135249); Cry1Ab28 (Accession # JN135250); Cry1Ab29 (Accession # JN135251); Cry1Ab30 (Accession # JN135252); Cry1Ab31 (Accession # JN135253); Cry1Ab32 (Accession # JN135254); Cry1Ab33 (Accession # AAS93798); Cry1Ab34 (Accession # KC156668); Cry1Ab-like (Accession # AAK14336); Cry1Ab-like (Accession # AAK14337); Cry1Ab-like (Accession # AAK14338); Cry1Ab-like (Accession # ABG88858); Cry1Ac1 (Accession # AAA22331); Cry1Ac2 (Accession # AAA22338); Cry1Ac3 (Accession # CAA38098); Cry1Ac4 (Accession # AAA73077); Cry1Ac5 (Accession # AAA22339); Cry1Ac6 (Accession # AAA86266); Cry1Ac7 (Accession # AAB46989); Cry1Ac8 (Accession # AAC44841); Cry1Ac9 (Accession # AAB49768); Cry1Ac10 (Accession # CAA05505); Cry1Ac11 (Accession # CAA10270); Cry1Ac12 (Accession #112418); Cry1Ac13 (Accession # AAD38701); Cry1Ac14 (Accession # AAQ06607); Cry1Ac15 (Accession # AAN07788); Cry1Ac16 (Accession # AAU87037); Cry1Ac17 (Accession # AAX18704); Cry1Ac18 (Accession # AAY88347); Cry1Ac19 (Accession # ABD37053); Cry1Ac20 (Accession # ABB89046); Cry1Ac21 (Accession # AAY66992); Cry1Ac22 (Accession # ABZ01836); Cry1Ac23 (Accession # CAO30431); Cry1Ac24 (Accession # ABL01535); Cry1Ac25 (Accession # FJ513324); Cry1Ac26 (Accession # FJ617446); Cry1Ac27 (Accession # FJ617447); Cry1Ac28 (Accession # ACM90319); Cry1Ac29 (Accession # DQ438941); Cry1Ac30 (Accession # GQ227507); Cry1Ac31 (Accession # GU446674); Cry1Ac32 (Accession # HM061081); Cry1Ac33 (Accession # GQ866913); Cry1Ac34 (Accession # HQ230364); Cry1Ac35 (Accession # JF340157); Cry1Ac36 (Accession # JN387137); Cry1Ac37 (Accession # JQ317685); Cry1Ad1 (Accession # AAA22340); Cry1Ad2 (Accession # CAA01880); Cry1Ae1 (Accession # AAA22410); Cry1Af1 (Accession # AAB82749); Cry1Ag1 (Accession # AAD46137); Cry1Ah1 (Accession # AAQ14326); Cry1Ah2 (Accession # ABB76664); Cry1Ah3 (Accession # HQ439779); Cry1Ai1 (Accession # AAO39719); Cry1Ai2 (Accession # HQ439780); Cry1A-like (Accession # AAK14339); Cry1Ba1 (Accession # CAA29898); Cry1Ba2 (Accession # CAA65003); Cry1Ba3 (Accession # AAK63251); Cry1Ba4 (Accession # AAK51084); Cry1Ba5 (Accession # AB020894); Cry1Ba6 (Accession # ABL60921); Cry1Ba1 (Accession # HQ439781); Cry1Bb1 (Accession # AAA22344); Cry1Bb2 (Accession # HQ439782); Cry1Bc1 (Accession # CAA86568); Cry1Bd1 (Accession # AAD10292); Cry1Bd2 (Accession # AAM93496); Cry1Be1 (Accession # AAC32850); Cry1Be2 (Accession # AAQ52387); Cry1Be3 (Accession # ACV96720); Cry1Be4 (Accession # HM070026); Cry1Bf1 (Accession # CAC50778); Cry1Bf2 (Accession # AAQ52380); Cry1Bg1 (Accession # AAO39720); Cry1Bh1 (Accession # HQ589331); Cry1Bi1 (Accession # KC156700); Cry1Ca1 (Accession # CAA30396); Cry1Ca2 (Accession # CAA31951); Cry1Ca3 (Accession # AAA22343); Cry1Ca4 (Accession # CAA01886); Cry1Ca5 (Accession # CAA65457); Cry1Ca6 [1] (Accession # AAF37224); Cry1Ca1 (Accession # AAG50438); Cry1Ca8 (Accession # AAM00264); Cry1Ca9 (Accession # AAL79362); Cry1Ca10 (Accession # AAN16462); Cry1Ca11 (Accession # AAX53094); Cry1Ca12 (Accession # HM070027); Cry1Ca13 (Accession # HQ412621); Cry1Ca14 (Accession # JN651493); Cry1Cb1 (Accession # M97880); Cry1Cb2 (Accession # AAG35409); Cry1Cb3 (Accession # ACD50894); Cry1Cb-like (Accession # AAX63901); Cry1Da1 (Accession # CAA38099); Cry1Da2 (Accession #176415); Cry1Da3 (Accession # HQ439784); Cry1Db1 (Accession # CAA80234); Cry1Db2 (Accession # AAK48937); Cry1Dc1 (Accession # ABK35074); Cry1Ea1 (Accession # CAA37933); Cry1Ea2 (Accession # CAA39609); Cry1Ea3 (Accession # AAA22345); Cry1Ea4 (Accession # AADQ4732); Cry1Ea5 (Accession # A15535); Cry1Ea6 (Accession # AAL50330); Cry1Ea7 (Accession # AAW72936); Cry1Ea8 (Accession # ABX11258); Cry1Ea9 (Accession # HQ439785); Cry1Ea10 (Accession # ADR00398); Cry1Ea11 (Accession # JQ652456); Cry1Eb1 (Accession # AAA22346); Cry1Fa1 (Accession # AAA22348); Cry1Fa2 (Accession # AAA22347); Cry1Fa3 (Accession # HM070028); Cry1Fa4 (Accession # HM439638); Cry1Fb1 (Accession # CAA80235); Cry1Fb2 (Accession # BAA25298); Cry1Fb3 (Accession # AAF21767); Cry1Fb4 (Accession # AAC10641); Cry1Fb5 (Accession # AAO13295); Cry1Fb6 (Accession # ACD50892); Cry1Fb7 (Accession # ACD50893); Cry1Ga1 (Accession # CAA80233); Cry1Ga2 (Accession # CAA70506); Cry1Gb1 (Accession # AAD10291); Cry1Gb2 (Accession # AAO13756); Cry1Gc1 (Accession # AAQ52381); Cry1Ha1 (Accession # CAA80236); Cry1Hb1 (Accession # AAA79694); Cry1Hb2 (Accession # HQ439786); Cry1H-like (Accession # AAF01213); Cry1Ia1 (Accession # CAA44633); Cry1Ia2 (Accession # AAA22354); Cry1Ia3 (Accession # AAC36999); Cry1Ia4 (Accession # AAB00958); Cry1Ia5 (Accession # CAA70124); Cry1Ia6 (Accession # AAC26910); Cry1Ia7 (Accession # AAM73516); Cry1Ia8 (Accession # AAK66742); Cry1Ia9 (Accession # AAQ08616); Cry1Ia10 (Accession # AAP86782); Cry1Ia11 (Accession # CAC85964); Cry1Ia12 (Accession # AAV53390); Cry1Ia13 (Accession # ABF83202); Cry1Ia14 (Accession # ACG63871); Cry1Ia15 (Accession # FJ617445); Cry1Ia16 (Accession # FJ617448); Cry1Ia17 (Accession # GU989199); Cry1Ia18 (Accession # ADK23801); Cry1Ia19 (Accession # HQ439787); Cry1Ia20 (Accession # JQ228426); Cry1Ia21 (Accession # JQ228424); Cry1Ia22 (Accession # JQ228427); Cry1Ia23 (Accession # JQ228428); Cry1Ia24 (Accession # JQ228429); Cry1Ia25 (Accession # JQ228430); Cry1Ia26 (Accession # JQ228431); Cry1Ia27 (Accession # JQ228432); Cry1Ia28 (Accession # JQ228433); Cry1Ia29 (Accession # JQ228434); Cry1Ia30 (Accession # JQ317686); Cry1Ia31 (Accession # JX944038); Cry1Ia32 (Accession # JX944039); Cry1Ia33 (Accession # JX944040); Cry1Ib1 (Accession # AAA82114); Cry1Ib2 (Accession # ABW88019); Cry1Ib3 (Accession # ACD75515); Cry1Ib4 (Accession # HM051227); Cry1Ib5 (Accession # HM070028); Cry1Ib6 (Accession # ADK38579); Cry1Ib7 (Accession # JN571740); Cry1Ib8 (Accession # JN675714); Cry1Ib9 (Accession # JN675715); Cry1Ib10 (Accession # JN675716); Cry1Ib11 (Accession # JQ228423); Cry1Ic1 (Accession # AAC62933); Cry1Ic2 (Accession # AAE71691); Cry1Id1 (Accession # AAD44366); Cry1Id2 (Accession # JQ228422); Cry1Ie1 (Accession # AAG43526); Cry1Ie2 (Accession # HM439636); Cry1Ie3 (Accession # KC156647); Cry1Ie4 (Accession # KC156681); Cry1If1 (Accession # AAQ52382); Cry1Ig1 (Accession # KC156701); Cry1I-like (Accession # AAC31094); Cry1I-like (Accession # ABG88859); Cry1Ja1 (Accession # AAA22341); Cry1Ja2 (Accession # HM070030); Cry1Ja3 (Accession # JQ228425); Cry1Jb1 (Accession # AAA98959); Cry1Jc1 (Accession # AAC31092); Cry1Jc2 (Accession # AAQ52372); Cry1Jd1 (Accession # CAC50779); Cry1Ka1 (Accession # AAB00376); Cry1Ka2 (Accession # HQ439783); Cry1La1 (Accession # AAS60191); Cry1La2 (Accession # HM070031); Cry1Ma1 (Accession # FJ884067); Cry1Ma2 (Accession # KC156659); Cry1Na1 (Accession # KC156648); Cry1Nb1 (Accession # KC156678); Cry1-like (Accession # AAC31091); Cry2Aa1 (Accession # AAA22335); Cry2Aa2 (Accession # AAA83516); Cry2Aa3 (Accession # D86064); Cry2Aa4 (Accession # AAC04867); Cry2Aa5 (Accession # CAA10671); Cry2Aa6 (Accession # CAA10672); Cry2Aa7 (Accession # CAA10670); Cry2Aa8 (Accession # AAO13734); Cry2Aa9 (Accession # AAO13750); Cry2Aa10 (Accession # AAQ04263); Cry2Aa11 (Accession # AAQ52384); Cry2Aa12 (Accession # AB183671); Cry2Aa13 (Accession # ABL01536); Cry2Aa14 (Accession # ACF04939); Cry2Aa15 (Accession # JN426947); Cry2Ab1 (Accession # AAA22342); Cry2Ab2 (Accession # CAA39075); Cry2Ab3 (Accession # AAG36762); Cry2Ab4 (Accession # AAO13296); Cry2Ab5 (Accession # AAQ04609); Cry2Ab6 (Accession # AAP59457); Cry2Ab7 (Accession # AAZ66347); Cry2Ab8 (Accession # ABC95996); Cry2Ab9 (Accession # ABC74968); Cry2Ab10 (Accession # EF157306); Cry2Ab11 (Accession # CAM84575); Cry2Ab12 (Accession # ABM21764); Cry2Ab13 (Accession # ACG76120); Cry2Ab14 (Accession # ACG76121); Cry2Ab15 (Accession # HM037126); Cry2Ab16 (Accession # GQ866914); Cry2Ab17 (Accession # HQ439789); Cry2Ab18 (Accession # JN135255); Cry2Ab19 (Accession # JN135256); Cry2Ab20 (Accession #JN135257); Cry2Ab21 (Accession #JN135258); Cry2Ab22 (Accession # JN135259); Cry2Ab23 (Accession # JN135260); Cry2Ab24 (Accession # JN135261); Cry2Ab25 (Accession #JN415485); Cry2Ab26 (Accession # JN426946); Cry2Ab27 (Accession # JN415764); Cry2Ab28 (Accession # JN651494); Cry2Ac1 (Accession # CAA40536); Cry2Ac2 (Accession # AAG35410); Cry2Ac3 (Accession # AAQ52385); Cry2Ac4 (Accession # ABC95997); Cry2Ac5 (Accession # ABC74969); Cry2Ac6 (Accession # ABC74793); Cry2Ac7 (Accession # CAL18690); Cry2Ac8 (Accession # CAM09325); Cry2Ac9 (Accession # CAM09326); Cry2Ac10 (Accession # ABN15104); Cry2Ac11 (Accession # CAM83895); Cry2Ac12 (Accession # CAM83896); Cry2Ad1 (Accession # AAF09583); Cry2Ad2 (Accession # ABC86927); Cry2Ad3 (Accession # CAK29504); Cry2Ad4 (Accession # CAM32331); Cry2Ad5 (Accession # CAO78739); Cry2Ae1 (Accession # AAQ52362); Cry2Af1 (Accession # AB030519); Cry2Af2 (Accession # GQ866915); Cry2Ag1 (Accession # ACH91610); Cry2Ah1 (Accession # EU939453); Cry2Ah2 (Accession # ACL80665); Cry2Ah3 (Accession # GU073380); Cry2Ah4 (Accession # KC156702); Cry2Ai1 (Accession # FJ788388); Cry2Aj (Accession #); Cry2Ak1 (Accession # KC156660); Cry2Ba1 (Accession # KC156658); Cry3Aa1 (Accession # AAA22336); Cry3Aa2 (Accession # AAA22541); Cry3Aa3 (Accession # CAA68482); Cry3Aa4 (Accession # AAA22542); Cry3Aa5 (Accession # AAA50255); Cry3Aa6 (Accession # AAC43266); Cry3Aa7 (Accession # CAB41411); Cry3Aa8 (Accession # AAS79487); Cry3Aa9 (Accession # AAW05659); Cry3Aa10 (Accession # AAU29411); Cry3Aa11 (Accession # AAW82872); Cry3Aa12 (Accession # ABY49136); Cry3Ba1 (Accession # CAA34983); Cry3Ba2 (Accession # CAA00645); Cry3Ba3 (Accession # JQ397327); Cry3Bb1 (Accession # AAA22334); Cry3Bb2 (Accession # AAA74198); Cry3Bb3 (Accession #115475); Cry3Ca1 (Accession # CAA42469); Cry4Aa1 (Accession # CAA68485); Cry4Aa2 (Accession # BAA00179); Cry4Aa3 (Accession # CAD30148); Cry4Aa4 (Accession # AFB18317); Cry4A-like (Accession # AAY96321); Cry4Ba1 (Accession # CAA30312); Cry4Ba2 (Accession # CAA30114); Cry4Ba3 (Accession # AAA22337); Cry4Ba4 (Accession # BAA00178); Cry4Ba5 (Accession # CAD30095); Cry4Ba-like (Accession # ABC47686); Cry4Ca1 (Accession # EU646202); Cry4Cb1 (Accession # FJ403208); Cry4Cb2 (Accession # FJ597622); Cry4Cc1 (Accession # FJ403207); Cry5Aa1 (Accession # AAA67694); Cry5Ab1 (Accession # AAA67693); Cry5Ac1 (Accession #134543); Cry5Ad1 (Accession # ABQ82087); Cry5Ba1 (Accession # AAA68598); Cry5Ba2 (Accession # ABW88931); Cry5Ba3 (Accession # AFJ04417); Cry5Ca1 (Accession # HM461869); Cry5Ca2 (Accession # ZP_04123426); Cry5Da1 (Accession # HM461870); Cry5Da2 (Accession # ZP_04123980); Cry5Ea1 (Accession # HM485580); Cry5Ea2 (Accession # ZP_04124038); Cry6Aa1 (Accession # AAA22357); Cry6Aa2 (Accession # AAM46849); Cry6Aa3 (Accession # ABH03377); Cry6Ba1 (Accession # AAA22358); Cry7Aa1 (Accession # AAA22351); Cry7Ab1 (Accession # AAA21120); Cry7Ab2 (Accession # AAA21121); Cry7Ab3 (Accession # ABX24522); Cry7Ab4 (Accession # EU380678); Cry7Ab5 (Accession # ABX79555); Cry7Ab6 (Accession # ACI44005); Cry7Ab7 (Accession # ADB89216); Cry7Ab8 (Accession # GU145299); Cry7Ab9 (Accession # ADD92572); Cry7Ba1 (Accession # ABB70817); Cry7Bb1 (Accession # KC156653); Cry7Ca1 (Accession # ABR67863); Cry7Cb1 (Accession # KC156698); Cry7Da1 (Accession # ACQ99547); Cry7Da2 (Accession # HM572236); Cry7Da3 (Accession # KC156679); Cry7Ea1 (Accession # HM035086); Cry7Ea2 (Accession # HM132124); Cry7Ea3 (Accession # EEM19403); Cry7Fa1 (Accession # HM035088); Cry7Fa2 (Accession # EEM19090); Cry7Fb1 (Accession # HM572235); Cry7Fb2 (Accession # KC156682); Cry7Ga1 (Accession # HM572237); Cry7Ga2 (Accession # KC156669); Cry7Gb1 (Accession # KC156650); Cry7Gc1 (Accession # KC156654); Cry7Gd1 (Accession # KC156697); Cry7Ha1 (Accession # KC156651); Cry7Ia1 (Accession # KC156665); Cry7Ja1 (Accession # KC156671); Cry7Ka1 (Accession # KC156680); Cry7Kb1 (Accession # BAM99306); Cry7La1 (Accession # BAM99307); Cry8Aa1 (Accession # AAA21117); Cry8Ab1 (Accession # EU044830); Cry8Ac1 (Accession # KC156662); Cry8Ad1 (Accession # KC156684); Cry8Ba1 (Accession # AAA21118); Cry8Bb1 (Accession # CAD57542); Cry8Bc1 (Accession # CAD57543); Cry8Ca1 (Accession # AAA21119); Cry8Ca2 (Accession # AAR98783); Cry8Ca3 (Accession # EU625349); Cry8Ca4 (Accession # ADB54826); Cry8Da1 (Accession # BAC07226); Cry8Da2 (Accession # BD133574); Cry8Da3 (Accession # BD133575); Cry8Db1 (Accession # BAF93483); Cry8Ea1 (Accession # AAQ73470); Cry8Ea2 (Accession # EU047597); Cry8Ea3 (Accession # KC855216); Cry8Fa1 (Accession # AAT48690); Cry8Fa2 (Accession # HQ174208); Cry8Fa3 (Accession # AFH78109); Cry8Ga1 (Accession # AAT46073); Cry8Ga2 (Accession # ABC42043); Cry8Ga3 (Accession # FJ198072); Cry8Ha1 (Accession # AAW81032); Cry81a1 (Accession # EU381044); Cry81a2 (Accession # GU073381); Cry81a3 (Accession # HM044664); Cry81a4 (Accession # KC156674); Cry81b1 (Accession # GU325772); Cry81b2 (Accession # KC156677); Cry8Ja1 (Accession # EU625348); Cry8Ka1 (Accession # FJ422558); Cry8Ka2 (Accession # ACN87262); Cry8Kb1 (Accession # HM123758); Cry8Kb2 (Accession # KC156675); Cry8La1 (Accession # GU325771); Cry8Ma1 (Accession # HM044665); Cry8Ma2 (Accession # EEM86551); Cry8Ma3 (Accession # HM210574); Cry8Na1 (Accession # HM640939); Cry8Pa1 (Accession # HQ388415); Cry8Qa1 (Accession # HQ441166); Cry8Qa2 (Accession # KC152468); Cry8Ra1 (Accession # AFP87548); Cry8Sa1 (Accession # JQ740599); Cry8Ta1 (Accession # KC156673); Cry8-like (Accession # FJ770571); Cry8-like (Accession # ABS53003); Cry9Aa1 (Accession # CAA41122); Cry9Aa2 (Accession # CAA41425); Cry9Aa3 (Accession # GQ249293); Cry9Aa4 (Accession # GQ249294); Cry9Aa5 (Accession # JX174110); Cry9Aa like (Accession # AAQ52376); Cry9Ba1 (Accession # CAA52927); Cry9Ba2 (Accession # GU299522); Cry9Bb1 (Accession # AAV28716); Cry9Ca1 (Accession # CAA85764); Cry9Ca2 (Accession # AAQ52375); Cry9Da1 (Accession # BAA19948); Cry9Da2 (Accession # AAB97923); Cry9Da3 (Accession # GQ249293); Cry9Da4 (Accession # GQ249297); Cry9Db1 (Accession # AAX78439); Cry9Dc1 (Accession # KC156683); Cry9Ea1 (Accession # BAA34908); Cry9Ea2 (Accession # AAO12908); Cry9Ea3 (Accession # ABM21765); Cry9Ea4 (Accession # ACE88267); Cry9Ea5 (Accession # ACF04743); Cry9Ea6 (Accession # ACG63872); Cry9Ea7 (Accession # FJ380927); Cry9Ea8 (Accession # GQ249292); Cry9Ea9 (Accession # JN651495); Cry9Eb1 (Accession # CAC50780); Cry9Eb2 (Accession # GQ249298); Cry9Eb3 (Accession # KC156646); Cry9Ec1 (Accession # AAC63366); Cry9Ed1 (Accession # AAX78440); Cry9Ee1 (Accession # GQ249296); Cry9Ee2 (Accession # KC156664); Cry9Fa1 (Accession # KC156692); Cry9Ga1 (Accession # KC156699); Cry9-like (Accession # AAC63366); Cry10Aa1 (Accession # AAA22614); Cry10Aa2 (Accession # E00614); Cry10Aa3 (Accession # CAD30098); Cry10Aa4 (Accession # AFB18318); Cry10A-like (Accession # DQ167578); Cry11Aa1 (Accession # AAA22352); Cry11Aa2 (Accession # AAA22611); Cry11 Aa3 (Accession # CAD30081); Cry11 Aa4 (Accession # AFB18319); Cry11 Aa-like (Accession # DQ166531); Cry11Ba1 (Accession # CAA60504); Cry11Bb1 (Accession # AAC97162); Cry11 Bb2 (Accession # HM068615); Cry12Aa1 (Accession # AAA22355); Cry13Aa1 (Accession # AAA22356); Cry14Aa1 (Accession # AAA21516); Cry14Ab1 (Accession # KC156652); Cry15Aa1 (Accession # AAA22333); Cry16Aa1 (Accession # CAA63860); Cry17Aa1 (Accession # CAA67841); Cry18Aa1 (Accession # CAA67506); Cry18Ba1 (Accession # AAF89667); Cry18Ca1 (Accession # AAF89668); Cry19Aa1 (Accession # CAA68875); Cry19Ba1 (Accession # BAA32397); Cry19Ca1 (Accession # AFM37572); Cry20Aa1 (Accession # AAB93476); Cry20Ba1 (Accession # ACS93601); Cry20Ba2 (Accession # KC156694); Cry20-like (Accession # GQ144333); Cry21 Aa1 (Accession #132932); Cry21 Aa2 (Accession #166477); Cry21Ba1 (Accession # BAC06484); Cry21Ca1 (Accession # JF521577); Cry21Ca2 (Accession # KC156687); Cry21 Da1 (Accession # JF521578); Cry22Aa1 (Accession #134547); Cry22Aa2 (Accession # CAD43579); Cry22Aa3 (Accession # ACD93211); Cry22Ab1 (Accession # AAK50456); Cry22Ab2 (Accession # CAD43577); Cry22Ba1 (Accession # CAD43578); Cry22Bb1 (Accession # KC156672); Cry23Aa1 (Accession # AAF76375); Cry24Aa1 (Accession # AAC61891); Cry24Ba1 (Accession # BAD32657); Cry24Ca1 (Accession # CAJ43600); Cry25Aa1 (Accession # AAC61892); Cry26Aa1 (Accession # AAD25075); Cry27Aa1 (Accession # BAA82796); Cry28Aa1 (Accession # AAD24189); Cry28Aa2 (Accession # AAG00235); Cry29Aa1 (Accession # CAC80985); Cry30Aa1 (Accession # CAC80986); Cry30Ba1 (Accession # BAD00052); Cry30Ca1 (Accession # BAD67157); Cry30Ca2 (Accession # ACU24781); Cry30Da1 (Accession # EF095955); Cry30Db1 (Accession # BAE80088); Cry30Ea1 (Accession # ACC95445); Cry30Ea2 (Accession # FJ499389); Cry30Fa1 (Accession # ACI22625); Cry30Ga1 (Accession # ACG60020); Cry30Ga2 (Accession # HQ638217); Cry31Aa1 (Accession # BAB11757); Cry31Aa2 (Accession # AAL87458); Cry31Aa3 (Accession # BAE79808); Cry31Aa4 (Accession # BAF32571); Cry31Aa5 (Accession # BAF32572); Cry31Aa6 (Accession # BAI44026); Cry31Ab1 (Accession # BAE79809); Cry31Ab2 (Accession # BAF32570); Cry31Ac1 (Accession # BAF34368); Cry31Ac2 (Accession # AB731600); Cry31Ad1 (Accession # BAI44022); Cry32Aa1 (Accession # AAG36711); Cry32Aa2 (Accession # GU063849); Cry32Ab1 (Accession # GU063850); Cry32Ba1 (Accession # BAB78601); Cry32Ca1 (Accession # BAB78602); Cry32Cb1 (Accession # KC156708); Cry32Da1 (Accession # BAB78603); Cry32Ea1 (Accession # GU324274); Cry32Ea2 (Accession # KC156686); Cry32Eb1 (Accession # KC156663); Cry32Fa1 (Accession # KC156656); Cry32Ga1 (Accession # KC156657); Cry32Ha1 (Accession # KC156661); Cry32Hb1 (Accession # KC156666); Cry32Ia1 (Accession # KC156667); Cry32Ja1 (Accession # KC156685); Cry32Ka1 (Accession # KC156688); Cry32La1 (Accession # KC156689); Cry32Ma1 (Accession # KC156690); Cry32Mb1 (Accession # KC156704); Cry32Na1 (Accession # KC156691); Cry32Oa1 (Accession # KC156703); Cry32Pa1 (Accession # KC156705); Cry32Qa1 (Accession # KC156706); Cry32Ra1 (Accession # KC156707); Cry32Sa1 (Accession # KC156709); Cry32Ta1 (Accession # KC156710); Cry32Ua1 (Accession # KC156655); Cry33Aa1 (Accession # AAL26871); Cry34Aa1 (Accession # AAG50341); Cry34Aa2 (Accession # AAK64560); Cry34Aa3 (Accession # AAT29032); Cry34Aa4 (Accession # AAT29030); Cry34Ab1 (Accession # AAG41671); Cry34Ac1 (Accession # AAG50118); Cry34Ac2 (Accession # AAK64562); Cry34Ac3 (Accession # AAT29029); Cry34Ba1 (Accession # AAK64565); Cry34Ba2 (Accession # AAT29033); Cry34Ba3 (Accession # AAT29031); Cry35Aa1 (Accession # AAG50342); Cry35Aa2 (Accession # AAK64561); Cry35Aa3 (Accession # AAT29028); Cry35Aa4 (Accession # AAT29025); Cry35Ab1 (Accession # AAG41672); Cry35Ab2 (Accession # AAK64563); Cry35Ab3 (Accession # AY536891); Cry35Ac1 (Accession # AAG50117); Cry35Ba1 (Accession # AAK64566); Cry35Ba2 (Accession # AAT29027); Cry35Ba3 (Accession # AAT29026); Cry36Aa1 (Accession # AAK64558); Cry37Aa1 (Accession # AAF76376); Cry38Aa1 (Accession # AAK64559); Cry39Aa1 (Accession # BAB72016); Cry40Aa1 (Accession # BAB72018); Cry40Ba1 (Accession # BAC77648); Cry40Ca1 (Accession # EU381045); Cry40Da1 (Accession # ACF15199); Cry41Aa1 (Accession # BAD35157); Cry41Ab1 (Accession # BAD35163); Cry41Ba1 (Accession # HM461871); Cry41Ba2 (Accession # ZP_04099652); Cry42Aa1 (Accession # BAD35166); Cry43Aa1 (Accession # BAD15301); Cry43Aa2 (Accession # BAD95474); Cry43Ba1 (Accession # BAD15303); Cry43Ca1 (Accession # KC156676); Cry43Cb1 (Accession # KC156695); Cry43Cc1 (Accession # KC156696); Cry43-like (Accession # BAD15305); Cry44Aa (Accession # BAD08532); Cry45Aa (Accession # BAD22577); Cry46Aa (Accession # BAC79010); Cry46Aa2 (Accession # BAG68906); Cry46Ab (Accession # BAD35170); Cry47Aa (Accession # AAY24695); Cry48Aa (Accession # CAJ18351); Cry48Aa2 (Accession # CAJ86545); Cry48Aa3 (Accession # CAJ86546); Cry48Ab (Accession # CAJ86548); Cry48Ab2 (Accession # CAJ86549); Cry49Aa (Accession # CAH56541); Cry49Aa2 (Accession # CAJ86541); Cry49Aa3 (Accession # CAJ86543); Cry49Aa4 (Accession # CAJ86544); Cry49Ab1 (Accession # CAJ86542); Cry50Aa1 (Accession # BAE86999); Cry50Ba1 (Accession # GU446675); Cry50Ba2 (Accession # GU446676); Cry51Aa1 (Accession # AB114444); Cry51Aa2 (Accession # GU570697); Cry52Aa1 (Accession # EF613489); Cry52Ba1 (Accession # FJ361760); Cry53Aa1 (Accession # EF633476); Cry53Ab1 (Accession # FJ361759); Cry54Aa1 (Accession # ACA52194); Cry54Aa2 (Accession # GQ140349); Cry54Ba1 (Accession # GU446677); Cry55Aa1 (Accession # ABW88932); Cry54Ab1 (Accession # JQ916908); Cry55Aa2 (Accession # AAE33526); Cry56Aa1 (Accession # ACU57499); Cry56Aa2 (Accession # GQ483512); Cry56Aa3 (Accession # JX025567); Cry57Aa1 (Accession # ANC87261); Cry58Aa1 (Accession # ANC87260); Cry59Ba1 (Accession # JN790647); Cry59Aa1 (Accession # ACR43758); Cry60Aa1 (Accession # ACU24782); Cry60Aa2 (Accession # EA057254); Cry60Aa3 (Accession # EEM99278); Cry60Ba1 (Accession # GU810818); Cry60Ba2 (Accession # EA057253); Cry60Ba3 (Accession # EEM99279); Cry61Aa1 (Accession # HM035087); Cry61Aa2 (Accession # HM132125); Cry61Aa3 (Accession # EEM19308); Cry62Aa1 (Accession # HM054509); Cry63Aa1 (Accession # BAI44028); Cry64Aa1 (Accession # BAJ05397); Cry65Aa1 (Accession # HM461868); Cry65Aa2 (Accession # ZP_04123838); Cry66Aa1 (Accession # HM485581); Cry66Aa2 (Accession # ZP_04099945); Cry67Aa1 (Accession # HM485582); Cry67Aa2 (Accession # ZP_04148882); Cry68Aa1 (Accession # HQ113114); Cry69Aa1 (Accession # HQ401006); Cry69Aa2 (Accession # JQ821388); Cry69Ab1 (Accession # JN209957); Cry70Aa1 (Accession # JN646781); Cry70Ba1 (Accession # ADQ51070); Cry70Bb1 (Accession # EEL67276); Cry71Aa1 (Accession # JX025568); Cry72Aa1 (Accession # JX025569).

Examples of δ-endotoxins also include but are not limited to Cry1A proteins of U.S. Pat. Nos. 5,880,275 and 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of α-helix 1 and/or α-helix 2 variants of Cry proteins such as Cry1A) of U.S. Pat. Nos. 8,304,604 and 8.304,605, Cry1B of U.S. patent application Ser. No. 10/525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188,960, 6,218,188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Ab protein of U.S. Pat. No. 7,064,249); a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008) Applied and Environmental Microbiology 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 and CryET34 protein of U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006/0191034, 2012/0278954, and PCT Publication Number WO 2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006/033867; AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018, AXMI-020, and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032; AXMI-003 of WO 2005/021585; AXMI-008 of US 2004/0250311; AXMI-006 of US 2004/0216186; AXMI-007 of US 2004/0210965; AXMI-009 of US 2004/0210964; AXMI-014 of US 2004/0197917; AXMI-004 of US 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO 2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US20110023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063, and AXMI-064 of US 2011/0263488; AXMI-R1 and related proteins of US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230, and AXMI231 of WO11/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of US 2010/0298211; AXMI-066 and AXMI-076 of US20090144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US 2010/0005543; and Cry proteins such as Cry1A and Cry3A having modified proteolytic sites of U.S. Pat. No. 8,319,019; and a Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011/0064710. Other Cry proteins are well known to one skilled in the art (see, Crickmore, et al., “Bacillus thuringiensis toxin nomenclature” (2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed on the world-wide web using the “www” prefix). The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C. at cera-gmc.org/index.php?action=gm_crop_database which can be accessed on the world-wide web using the “www” prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & Cry1Fa (US2012/0317682), Cry1BE & Cry1F (US2012/0311746), Cry1CA & Cry1AB (US2012/0311745), Cry1F & CryCa (US2012/0317681), Cry1DA & Cry1BE (US2012/0331590), Cry1DA & Cry1Fa (US2012/0331589), Cry1AB & Cry1BE (US2012/0324606), and Cry1Fa & Cry2Aa, Cry1I or Cry1E (US2012/0324605). Pesticidal proteins also include insecticidal lipases including lipid acyl hydrolases of U.S. Pat. No. 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins of U.S. Pat. Nos. 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686, and 8,237,020, and the like. Other VIP proteins are well known to one skilled in the art (see, Iifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can be accessed on the world-wide web using the “www” prefix). Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S. Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein “potentiators” derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins (“Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi. Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but are not limited to lycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

(C) A polynucleotide encoding an insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon or an antagonist or agonist thereof. See, for example, the disclosure by Hammock, et al., (1990) Nature 344:458, of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) A polynucleotide encoding an insect-specific peptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of, Regan, (1994) J. Biol. Chem. 269:9 (expression cloning yields DNA coding for insect diuretic hormone receptor); Pratt, et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin is identified in Diploptera puntata); Chattopadhyay, et al., (2004) Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J Nat Prod 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon 40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 and Vasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S. Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encoding insect-specific toxins.

(E) A polynucleotide encoding an enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.

(F) A polynucleotide encoding an enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See, PCT Application WO 1993/02197 in the name of Scott, et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC® under Accession Numbers 39637 and 67152. See also, Kramer, et al., (1993) Insect Biochem. Molec. Biol. 23:691, who teach the nucleotide sequence of a cDNA encoding tobacco hookworm chitinase and Kawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and U.S. Pat. Nos. 6,563,020; 7,145,060 and 7,087,810.

(G) A polynucleotide encoding a molecule that stimulates signal transduction. For example, see the disclosure by Botella, et al., (1994) Plant Molec. Biol. 24:757, of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol. 104:1467, who provide the nucleotide sequence of a maize calmodulin cDNA clone.

(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCT Application WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure of peptide derivatives of Tachyplesin which inhibit fungal plant pathogens) and PCT Application WO 1995/18855 and U.S. Pat. No. 5,607,914 (teaches synthetic antimicrobial peptides that confer disease resistance).

(I) A polynucleotide encoding a membrane permease, a channel former or a channel blocker. For example, see the disclosure by Jaynes, et al., (1993) Plant Sci. 89:43, of heterologous expression of a cecropin-beta lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A gene encoding a viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See, Beachy, et al., (1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

(K) A gene encoding an insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).

(L) A gene encoding a virus-specific antibody. See, for example, Tavladoraki, et al., (1993) Nature 366:469, who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.

(M) A polynucleotide encoding a developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992) Bio/Technology 10:1436. The cloning and characterization of a gene which encodes a bean endopolygalacturonase-inhibiting protein is described by Toubart, et al., (1992) Plant J. 2:367.

(N) A polynucleotide encoding a developmental-arrestive protein produced in nature by a plant. For example, Logemann, et al., (1992) Bio/Technology 10:305, have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.

- (O) Genes involved in the Systemic Acquired Resistance (SAR) Response and/or the pathogenesis related genes. Briggs, (1995) Current Biology 5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64 and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol. 101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell, et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S. patent application Ser. Nos. 09/950,933; 11/619,645; 11/657,710; 11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. LysM Receptor-like kinases for the perception of chitin fragments as a first step in plant defense response against fungal pathogens (US 2012/0110696).

(Q) Detoxification genes, such as for fumonisin, beauvericin, moniliformin and zearalenone and their structurally related derivatives. For example, see, U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255; 5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) A polynucleotide encoding a Cystatin and cysteine proteinase inhibitors. See, U.S. Pat. No. 7,205,453.

(S) Defensin genes. See, WO 2003/000863 and U.S. Pat. Nos. 6,911,577; 6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See, e.g., PCT Application WO 1996/30517; PCT Application WO 1993/19181, WO 2003/033651 and Urwin, et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin Plant Bio. 2(4):327-31; U.S. Pat. Nos. 6,284,948 and 7,301,069 and miR164 genes (WO 2012/058266).

(U) Genes that confer resistance to Phytophthora Root Rot, such as the Rps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes. See, for example, Shoemaker, et al., Phytophthora Root Rot Resistance Gene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif. (1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described in U.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

(W) Genes that confer resistance to Colletotrichum, such as described in US Patent Application Publication US 2009/0035765 and incorporated by reference for this purpose. This includes the Rcg locus that may be utilized as a single locus conversion.

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) A polynucleotide encoding resistance to a herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J. 7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449, respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824; U.S. patent application Ser. No. 11/683,737 and International Publication WO 1996/33270.

(B) A polynucleotide encoding a protein for resistance to Glyphosate (resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS which can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes. See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 5,094,945, 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and 5,491,288 and International Publications EP 1173580; WO 2001/66704; EP 1173581 and EP 1173582, which are incorporated herein by reference for this purpose. Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido-reductase enzyme as described more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose. In addition glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyltransferase. See, for example, U.S. Pat. Nos. 7,462,481; 7,405,074 and US Patent Application Publication Number US 2008/0234130. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC® Accession Number 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. EP Application Number 0 333 033 to Kumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0 242 246 and 0 242 236 to Leemans, et al.; De Greef, et al., (1989) Bio/Technology 7:61, describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 B1 and 5,879,903, which are incorporated herein by reference for this purpose. Exemplary genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop, are the Acc1-S1, Acc1-52 and Acc1-S3 genes described by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

(C) A polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell 3:169, describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC® Accession Numbers 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes, et al., (1992) Biochem. J. 285:173.

(D) A polynucleotide encoding a protein for resistance to Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants (see, e.g., Hattori, et al., (1995) Mol Gen Genet. 246:419). Other genes that confer resistance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant Physiol 106:17), genes for glutathione reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genes for various phosphotransferases (Datta, et al., (1992) Plant Mol Biol 20:619).

(E) A polynucleotide encoding resistance to a herbicide targeting Protoporphyrinogen oxidase (protox) which is necessary for the production of chlorophyll. The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373 and International Publication WO 2001/12825.

(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodes the aryloxyalkanoate dioxygenase (AAD-1) protein. The trait confers tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly referred to as “fop” herbicides such as quizalofop) herbicides. The aad-1 gene, itself, for herbicide tolerance in plants was first disclosed in WO 2005/107437 (see also, US 2009/0093366). The aad-12 gene, derived from Delftia acidovorans, which encodes the aryloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to 2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides by deactivating several herbicides with an aryloxyalkanoate moiety, including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxy auxins (e.g., fluroxypyr, triclopyr).

(G) A polynucleotide encoding a herbicide resistant dicamba monooxygenase disclosed in US Patent Application Publication 2003/0135879 for imparting dicamba tolerance;

(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance;

(I) A polynucleotide molecule encoding phytoene (crtl) described in Misawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994) Plant J. 6:481-489 for norflurazon tolerance.

3. Transgenes that Confer or Contribute to an Altered Grain Characteristic

Such as:

(A) Altered fatty acids, for example, by

(1) Down-regulation of stearoyl-ACP to increase stearic acid content of the plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).

(2) Elevating oleic acid via FAD-2 gene modification and/or decreasing linolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 1993/11245).

(3) Altering conjugated linolenic or linoleic acid content, such as in WO 2001/12800.

(4) Altering LEC1, AGP, Dek1, Superal1, mi1 ps, various Ipa genes such as Ipa1, Ipa3, hpt or hggt. For example, see, WO 2002/42424, WO 1998/22604, WO 2003/011015, WO 2002/057439, WO 2003/011015, U.S. Pat. Nos. 6,423,886, 6,197,561, 6,825,397 and US Patent Application Publication Numbers US 2003/0079247, US 2003/0204870 and Rivera-Madrid, et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.

(5) Genes encoding delta-8 desaturase for making long-chain polyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and 8,338,152), delta-9 desaturase for lowering saturated fats (U.S. Pat. No. 8,063,269), Primula Δ6-desaturase for improving omega-3 fatty acid profiles.

(6) Isolated nucleic acids and proteins associated with lipid and sugar metabolism regulation, in particular, lipid metabolism protein (LMP) used in methods of producing transgenic plants and modulating levels of seed storage compounds including lipids, fatty acids, starches or seed storage proteins and use in methods of modulating the seed size, seed number, seed weights, root length and leaf size of plants (EP 2404499).

(7) Altering expression of a High-Level Expression of Sugar-Inducible 2 (HSI2) protein in the plant to increase or decrease expression of HSI2 in the plant. Increasing expression of HSI2 increases oil content while decreasing expression of HSI2 decreases abscisic acid sensitivity and/or increases drought resistance (US Patent Application Publication Number 2012/0066794).

(8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oil content in plant seed, particularly to increase the levels of omega-3 fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (US Patent Application Publication Number 2011/0191904).

(9) Nucleic acid molecules encoding wrinkled1-like polypeptides for modulating sugar metabolism (U.S. Pat. No. 8,217,223).

(B) Altered phosphorus content, for example, by the

(1) Introduction of a phytase-encoding gene would enhance breakdown of phytate, adding more free phosphate to the transformed plant. For example, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for a disclosure of the nucleotide sequence of an Aspergillus niger phytase gene.

(2) Modulating a gene that reduces phytate content. In maize, this, for example, could be accomplished, by cloning and then re-introducing DNA associated with one or more of the alleles, such as the LPA alleles, identified in maize mutants characterized by low levels of phytic acid, such as in WO 2005/113778 and/or by altering inositol kinase activity as in WO 2002/059324, US Patent Application Publication Number 2003/0009011, WO 2003/027243, US Patent Application Publication Number 2003/0079247, WO 1999/05298, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,291,224, U.S. Pat. No. 6,391,348, WO 2002/059324, US Patent Application Publication Number 2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.

(C) Altered carbohydrates affected, for example, by altering a gene for an enzyme that affects the branching pattern of starch or, a gene altering thioredoxin such as NTR and/or TRX (see, U.S. Pat. No. 6,531,648. which is incorporated by reference for this purpose) and/or a gamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, U.S. Pat. No. 6,858,778 and US Patent Application Publication Number 2005/0160488, US Patent Application Publication Number 2005/0204418, which are incorporated by reference for this purpose). See, Shiroza, et al., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcus mutant fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen. Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrase gene), Pen, et al., (1992) Bio/Technology 10:292 (production of transgenic plants that express Bacillus licheniformis alpha-amylase), Elliot, et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequences of tomato invertase genes), Søgaard, et al., (1993) J. Biol. Chem. 268:22480 (site-directed mutagenesis of barley alpha-amylase gene) and Fisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starch branching enzyme II), WO 1999/10498 (improved digestibility and/or starch extraction through modification of UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method of producing high oil seed by modification of starch levels (AGP)). The fatty acid modification genes mentioned herein may also be used to affect starch content and/or composition through the interrelationship of the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration of tocopherol or tocotrienols. For example, see, U.S. Pat. No. 6,787,683, US Patent Application Publication Number 2004/0034886 and WO 2000/68393 involving the manipulation of antioxidant levels and WO 2003/082899 through alteration of a homogentisate geranyl geranyl transferase (hggt).

(E) Altered essential seed amino acids. For example, see, U.S. Pat. No. 6,127,600 (method of increasing accumulation of essential amino acids in seeds), U.S. Pat. No. 6,080,913 (binary methods of increasing accumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389 (high lysine), WO 1999/40209 (alteration of amino acid compositions in seeds), WO 1999/29882 (methods for altering amino acid content of proteins), U.S. Pat. No. 5,850,016 (alteration of amino acid compositions in seeds), WO 1998/20133 (proteins with enhanced levels of essential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S. Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plant amino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increased lysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophan synthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolic enzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414 (increased methionine), WO 1998/56935 (plant amino acid biosynthetic enzymes), WO 1998/45458 (engineered seed protein having higher percentage of essential amino acids), WO 1998/42831 (increased lysine), U.S. Pat. No. 5,633,436 (increasing sulfur amino acid content), U.S. Pat. No. 5,559,223 (synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants), WO 1996/01905 (increased threonine), WO 1995/15392 (increased lysine), US Patent Application Publication Number 2003/0163838, US Patent Application Publication Number 2003/0150014, US Patent Application Publication Number 2004/0068767, U.S. Pat. No. 6,803,498, WO 2001/79516.

4. Genes that Control Male-Sterility:

There are several methods of conferring genetic male sterility available, such as multiple mutant genes at separate locations within the genome that confer male sterility, as disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocations as described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. In addition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068, describe a system of nuclear male sterility which includes: identifying a gene which is critical to male fertility; silencing this native gene which is critical to male fertility; removing the native promoter from the essential male fertility gene and replacing it with an inducible promoter; inserting this genetically engineered gene back into the plant; and thus creating a plant that is male sterile because the inducible promoter is not “on” resulting in the male fertility gene not being transcribed. Fertility is restored by inducing or turning “on”, the promoter, which in turn allows the gene that confers male fertility to be transcribed.

(A) Introduction of a deacetylase gene under the control of a tapetum-specific promoter and with the application of the chemical N-Ac-PPT (WO 2001/29237).

(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO 1992/13957).

For additional examples of nuclear male and female sterility systems and genes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369; 5,824,524; 5,850,014 and 6,265,640, all of which are hereby incorporated by reference.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in the FLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system. For example, see, Lyznik, et al., (2003) Plant Cell Rep 21:925-932 and WO 1999/25821, which are hereby incorporated by reference. Other systems that may be used include the Gin recombinase of phage Mu (Maeser, et al., (1991) Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto, et al., 1983) and the R/RS system of the pSRi plasmid (Araki, et al., 1992).

6. Genes that Affect Abiotic Stress Resistance

Including but not limited to flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance and salt resistance or tolerance and increased yield under stress.

(A) For example, see: WO 2000/73475 where water use efficiency is altered through alteration of malate; U.S. Pat. Nos. 5,892,009, 5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446, 6,706,866, 6,717,034, 6,801,104, WO 2000/060089, WO 2001/026459, WO 2001/035725, WO 2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO 2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO 2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO 2004/031349, WO 2004/076638, WO 199809521.

(B) WO 199938977 describing genes, including CBF genes and transcription factors effective in mitigating the negative effects of freezing, high salinity and drought on plants, as well as conferring other positive effects on plant phenotype.

(C) US Patent Application Publication Number 2004/0148654 and WO 2001/36596 where abscisic acid is altered in plants resulting in improved plant phenotype such as increased yield and/or increased tolerance to abiotic stress.

(D) WO 2000/006341, WO 2004/090143, U.S. Pat. Nos. 7,531,723 and 6,992,237 where cytokinin expression is modified resulting in plants with increased stress tolerance, such as drought tolerance, and/or increased yield. Also see, WO 2002/02776, WO 2003/052063, JP 2002/281975, U.S. Pat. No. 6,084,153, WO 2001/64898, U.S. Pat. No. 6,177,275 and U.S. Pat. No. 6,107,547 (enhancement of nitrogen utilization and altered nitrogen responsiveness).

(E) For ethylene alteration, see, US Patent Application Publication Number 2004/0128719, US Patent Application Publication Number 2003/0166197 and WO 2000/32761.

(F) For plant transcription factors or transcriptional regulators of abiotic stress, see, e.g., US Patent Application Publication Number 2004/0098764 or US Patent Application Publication Number 2004/0078852.

(G) Genes that increase expression of vacuolar pyrophosphatase such as AVP1 (U.S. Pat. No. 8,058,515) for increased yield; nucleic acid encoding a HSFA4 or a HSFA5 (Heat Shock Factor of the class A4 or A5) polypeptides, an oligopeptide transporter protein (OPT4-like) polypeptide; a plastochron2-like (PLA2-like) polypeptide or a Wuschel related homeobox 1-like (WOX1-like) polypeptide (U. Patent Application Publication Number US 2011/0283420).

(H) Down regulation of polynucleotides encoding poly (ADP-ribose) polymerase (PARP) proteins to modulate programmed cell death (U.S. Pat. No. 8,058,510) for increased vigor.

(I) Polynucleotide encoding DTP21 polypeptides for conferring drought resistance (US Patent Application Publication Number US 2011/0277181).

(J) Nucleotide sequences encoding ACC Synthase 3 (ACS3) proteins for modulating development, modulating response to stress, and modulating stress tolerance (US Patent Application Publication Number US 2010/0287669).

(K) Polynucleotides that encode proteins that confer a drought tolerance phenotype (DTP) for conferring drought resistance (WO 2012/058528).

(L) Tocopherol cyclase (TC) genes for conferring drought and salt tolerance (US Patent Application Publication Number 2012/0272352).

(M) CAAX amino terminal family proteins for stress tolerance (U.S. Pat. No. 8,338,661).

(N) Mutations in the SAL1 encoding gene have increased stress tolerance, including increased drought resistant (US Patent Application Publication Number 2010/0257633).

(O) Expression of a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide increasing yield-related traits (US Patent Application Publication Number 2011/0061133).

(P) Modulating expression in a plant of a nucleic acid encoding a Class III Trehalose Phosphate Phosphatase (TPP) polypeptide for enhancing yield-related traits in plants, particularly increasing seed yield (US Patent Application Publication Number 2010/0024067).

Other genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, plant growth and/or plant structure, can be introduced or introgressed into plants, see e.g., WO 1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No. 6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO 1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064 (GI), WO 2000/46358 (FR1), WO 1997/29123, U.S. Pat. No. 6,794,560, U.S. Pat. No. 6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO 2004/031349 (transcription factors).

7. Genes that Confer Increased Yield

(A) A transgenic crop plant transformed by a 1-AminoCyclopropane-1-Carboxylate Deaminase-like Polypeptide (ACCDP) coding nucleic acid, wherein expression of the nucleic acid sequence in the crop plant results in the plant's increased root growth, and/or increased yield, and/or increased tolerance to environmental stress as compared to a wild type variety of the plant (U.S. Pat. No. 8,097,769).

(B) Over-expression of maize zinc finger protein gene (Zm-ZFP1) using a seed preferred promoter has been shown to enhance plant growth, increase kernel number and total kernel weight per plant (US Patent Application Publication Number 2012/0079623).

(C) Constitutive over-expression of maize lateral organ boundaries (LOB) domain protein (Zm-LOBDP1) has been shown to increase kernel number and total kernel weight per plant (US Patent Application Publication Number 2012/0079622).

(D) Enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a VIM1 (Variant in Methylation 1)-like polypeptide or a VTC2-like (GDP-L-galactose phosphorylase) polypeptide or a DUF1685 polypeptide or an ARF6-like (Auxin Responsive Factor) polypeptide (WO 2012/038893).

(E) Modulating expression in a plant of a nucleic acid encoding a Ste20-like polypeptide or a homologue thereof gives plants having increased yield relative to control plants (EP 2431472).

(F) Genes encoding nucleoside diphosphatase kinase (NDK) polypeptides and homologs thereof for modifying the plant's root architecture (US Patent Application Publication Number 2009/0064373).

8. Genes that Confer Plant Digestibility.

(A) Altering the level of xylan present in the cell wall of a plant by modulating expression of xylan synthase (U.S. Pat. No. 8,173,866).

In some embodiment the stacked trait may be a trait or event that has received regulatory approval including but not limited to the events in Table 4A-4F.

TABLE 4A

Triticum aestivum Wheat

Event
Company
Description

AP205CL
BASF Inc.
Selection for a mutagenized version of the enzyme

acetohydroxyacid synthase (AHAS), also known

as acetolactate synthase (ALS) or acetolactate

pyruvate-lyase.

AP602CL
BASF Inc.
Selection for a mutagenized version of the enzyme

acetohydroxyacid synthase (AHAS), also known

as acetolactate synthase (ALS) or acetolactate

pyruvate-lyase.

BW255-2, BW238-3
BASF Inc.
Selection for a mutagenized version of the enzyme

acetohydroxyacid synthase (AHAS), also known

as acetolactate synthase (ALS) or acetolactate

pyruvate-lyase.

BW7
BASF Inc.
Tolerance to imidazolinone herbicides induced by

chemical mutagenesis of the acetohydroxyacid

synthase (AHAS) gene using sodium azide.

MON71800
Monsanto Company
Glyphosate tolerant wheat variety produced by

inserting a modified 5-enolpyruvylshikimate-3-

phosphate synthase (EPSPS) encoding gene from

the soil bacterium Agrobacterium tumefaciens,

strain CP4.

SWP965001
Cyanamid Crop
Selection for a mutagenized version of the enzyme

Protection
acetohydroxyacid synthase (AHAS), also known

as acetolactate synthase (ALS) or acetolactate

pyruvate-lyase.

Teal 11A
BASF Inc.
Selection for a mutagenized version of the enzyme

acetohydroxyacid synthase (AHAS), also known

as acetolactate synthase (ALS) or acetolactate

pyruvate-lyase.

TABLE 4B

Helianthus annuus Sunflower

Event
Company
Description

X81359
BASF Inc.
Tolerance to imidazolinone herbicides by

selection of a naturally occurring mutant.

TABLE 4C

Glycine max L. Soybean

Event
Company
Description

A2704-12, A2704-21,
Bayer CropScience
Glufosinate ammonium herbicide tolerant soybean

A5547-35
(Aventis CropScience
produced by inserting a modified phosphinothricin

(AgrEvo))
acetyltransferase (PAT) encoding gene from the

soil bacterium Streptomyces viridochromogenes.

A5547-127
Bayer CropScience
Glufosinate ammonium herbicide tolerant soybean

(Aventis CropScience
produced by inserting a modified phosphinothricin

(AgrEvo))
acetyltransferase (PAT) encoding gene from the

soil bacterium Streptomyces viridochromogenes.

BPS-CV127-9
BASF Inc.
The introduced csr1-2 gene from Arabidopsis

thaliana encodes an acetohydroxyacid synthase

protein that confers tolerance to imidazolinone

herbicides due to a point mutation that results in a

single amino acid substitution in which the serine

residue at position 653 is replaced by asparagine

(S653N).

DP-305423
Pioneer Hi-Bred
High oleic acid soybean produced by inserting

International Inc.
additional copies of a portion of the omega-6

desaturase encoding gene, gm-fad2-1 resulting in

silencing of the endogenous omega-6 desaturase

gene (FAD2-1).

DP356043
Pioneer Hi-Bred
Soybean event with two herbicide tolerance

International Inc.
genes: glyphosate N-acetlytransferase, which

detoxifies glyphosate, and a modified acetolactate

synthase (ALS) gene which is tolerant to ALS-

inhibiting herbicides.

G94-1, G94-19, G168
DuPont Canada
High oleic acid soybean produced by inserting a

Agricultural Products
second copy of the fatty acid desaturase

(GmFad2-1) encoding gene from soybean, which

resulted in “silencing” of the endogenous host

gene.

GTS 40-3-2
Monsanto Company
Glyphosate tolerant soybean variety produced by

inserting a modified 5-enolpyruvylshikimate-3-

phosphate synthase (EPSPS) encoding gene from

the soil bacterium Agrobacterium tumefaciens.

GU262
Bayer CropScience
Glufosinate ammonium herbicide tolerant soybean

(Aventis
produced by inserting a modified phosphinothricin

CropScience(AgrEvo))
acetyltransferase (PAT) encoding gene from the

soil bacterium Streptomyces viridochromogenes.

MON87701
Monsanto Company
Resistance to Lepidopteran pests of soybean

including velvetbean caterpillar (Anticarsia

gemmatalis) and soybean looper (Pseudoplusia

includens).

MON87701 ×
Monsanto Company
Glyphosate herbicide tolerance through

MON89788

expression of the EPSPS encoding gene from A. tumefaciens

strain CP4, and resistance to

Lepidopteran pests of soybean including

velvetbean caterpillar (Anticarsia gemmatalis) and

soybean looper (Pseudoplusia includens) via

expression of the Cry1Ac encoding gene from B. thuringiensis.

MON89788
Monsanto Company
Glyphosate-tolerant soybean produced by

inserting a modified 5-enolpyruvylshikimate-3-

phosphate synthase (EPSPS) encoding aroA

(epsps) gene from Agrobacterium tumefaciens

CP4.

OT96-15
Agriculture & Agri-Food
Low linolenic acid soybean produced through

Canada
traditional cross-breeding to incorporate the novel

trait from a naturally occurring fan1 gene mutant

that was selected for low linolenic acid.

W62, W98
Bayer CropScience
Glufosinate ammonium herbicide tolerant soybean

(Aventis
produced by inserting a modified phosphinothricin

CropScience(AgrEvo))
acetyltransferase (PAT) encoding gene from the

soil bacterium Streptomyces hygroscopicus.

TABLE 4D

Medicago sativa Alfalfa

Event
Company
Description

J101, J163
Monsanto
Glyphosate herbicide tolerant alfalfa (lucerne)

Company
produced by inserting a gene encoding the

and Forage
enzyme 5-enolypyruvylshikimate-3-phosphate

Genetics
synthase (EPSPS) from the CP4 strain of

International

Agrobacterium tumefaciens.

TABLE 4E

Oryza sativa Rice

Event
Company
Description

CL121, CL141, CFX51
BASF Inc.
Tolerance to the imidazolinone herbicide,

imazethapyr, induced by chemical mutagenesis of

the acetolactate synthase (ALS) enzyme using

ethyl methanesulfonate (EMS).

IMINTA-1, IMINTA-4
BASF Inc.
Tolerance to imidazolinone herbicides induced by

chemical mutagenesis of the acetolactate

synthase (ALS) enzyme using sodium azide.

LLRICE06, LLRICE62
Aventis CropScience
Glufosinate ammonium herbicide tolerant rice

produced by inserting a modified phosphinothricin

acetyltransferase (PAT) encoding gene from the

soil bacterium Streptomyces hygroscopicus).

LLRICE601
Bayer CropScience
Glufosinate ammonium herbicide tolerant rice

(Aventis
produced by inserting a modified phosphinothricin

CropScience(AgrEvo))
acetyltransferase (PAT) encoding gene from the

soil bacterium Streptomyces hygroscopicus).

PWC16
BASF Inc.
Tolerance to the imidazolinone herbicide,

imazethapyr, induced by chemical mutagenesis of

the acetolactate synthase (ALS) enzyme using

ethyl methanesulfonate (EMS).

TABLE 4F

Zea mays L. Maize

Event
Company
Description

176
Syngenta Seeds, Inc.
Insect-resistant maize produced by inserting the

Cry1Ab gene from Bacillus thuringiensis subsp.

kurstaki. The genetic modification affords

resistance to attack by the European corn borer

(ECB).

3751IR
Pioneer Hi-Bred
Selection of somaclonal variants by culture of

International Inc.
embryos on imidazolinone containing media.

676, 678, 680
Pioneer Hi-Bred
Male-sterile and glufosinate ammonium herbicide

International Inc.
tolerant maize produced by inserting genes

encoding DNA adenine methylase and

phosphinothricin acetyltransferase (PAT) from

Escherichia coli and Streptomyces

viridochromogenes, respectively.

B16 (DLL25)
Dekalb Genetics
Glufosinate ammonium herbicide tolerant maize

Corporation
produced by inserting the gene encoding

phosphinothricin acetyltransferase (PAT) from

Streptomyces hygroscopicus.

BT11 (X4334CBR,
Syngenta Seeds, Inc.
Insect-resistant and herbicide tolerant maize

X4734CBR)

produced by inserting the Cry1Ab gene from

Bacillus thuringiensis subsp. kurstaki, and the

phosphinothricin N-acetyltransferase (PAT)

encoding gene from S. viridochromogenes.

BT11 × GA21
Syngenta Seeds, Inc.
Stacked insect resistant and herbicide tolerant

maize produced by conventional cross breeding of

parental lines BT11 (OECD unique identifier: SYN-

BTO11-1) and GA21 (OECD unique identifier:

MON-OOO21-9).

BT11 × MIR162 ×
Syngenta Seeds, Inc.
Resistance to Coleopteran pests, particularly corn

MIR604 × GA21

rootworm pests (Diabrotica spp.) and several

Lepidopteran pests of corn, including European

corn borer (ECB, Ostrinia nubilalis), corn earworm

(CEW, Helicoverpa zea), fall army worm (FAW,

Spodoptera frugiperda), and black cutworm (BCW,

Agrotis ipsilon); tolerance to glyphosate and

glufosinate-ammonium containing herbicides.

BT11 × MIR162
Syngenta Seeds, Inc.
Stacked insect resistant and herbicide tolerant

maize produced by conventional cross breeding of

parental lines BT11 (OECD unique identifier: SYN-

BTO11-1) and MIR162 (OECD unique identifier:

SYN-IR162-4). Resistance to the European Corn

Borer and tolerance to the herbicide glufosinate

ammonium (Liberty) is derived from BT11, which

contains the Cry1Ab gene from Bacillus

thuringiensis subsp. kurstaki, and the

phosphinothricin N-acetyltransferase (PAT)

encoding gene from S. viridochromogenes.

Resistance to other Lepidopteran pests, including

H. zea, S. frugiperda, A. ipsilon, and S. albicosta,

is derived from MIR162, which contains the vip3Aa

gene from Bacillus thuringiensis strain AB88.

BT11 × MIR162 ×
Syngenta Seeds, Inc.

Bacillus thuringiensis Cry1Ab delta-endotoxin

MIR604

protein and the genetic material necessary for its

production (via elements of vector pZO1502) in

Event Bt11 corn (OECD Unique Identifier: SYN-

BTO11-1) × Bacillus thuringiensis Vip3Aa20

insecticidal protein and the genetic material

necessary for its production (via elements of

vector pNOV1300) in Event MIR162 maize (OECD

Unique Identifier: SYN-IR162-4) × modified Cry3A

protein and the genetic material necessary for its

production (via elements of vector pZM26) in

Event MIR604 corn (OECD Unique Identifier:

SYN-IR6O4-5).

CBH-351
Aventis CropScience
Insect-resistant and glufosinate ammonium

herbicide tolerant maize developed by inserting

genes encoding Cry9C protein from Bacillus

thuringiensis subsp tolworthi and phosphinothricin

acetyltransferase (PAT) from Streptomyces

hygroscopicus.

DAS-06275-8
DOW AgroSciences LLC
Lepidopteran insect resistant and glufosinate

ammonium herbicide-tolerant maize variety

produced by inserting the Cry1F gene from

Bacillus thuringiensis var aizawai and the

phosphinothricin acetyltransferase (PAT) from

Streptomyces hygroscopicus.

BT11 × MIR604
Syngenta Seeds, Inc.
Stacked insect resistant and herbicide tolerant

maize produced by conventional cross breeding of

parental lines BT11 (OECD unique identifier: SYN-

BTO11-1) and MIR604 (OECD unique identifier:

SYN-IR6O5-5). Resistance to the European Corn

Borer and tolerance to the herbicide glufosinate

ammonium (Liberty) is derived from BT11, which

contains the Cry1Ab gene from Bacillus

thuringiensis subsp. kurstaki, and the

phosphinothricin N-acetyltransferase (PAT)

encoding gene from S. viridochromogenes. Corn

rootworm-resistance is derived from MIR604

which contains the mCry3A gene from Bacillus

thuringiensis.

BT11 × MIR604 × GA21
Syngenta Seeds, Inc.
Stacked insect resistant and herbicide tolerant

maize produced by conventional cross breeding of

parental lines BT11 (OECD unique identifier: SYN-

BTO11-1), MIR604 (OECD unique identifier: SYN-

IR6O5-5) and GA21 (OECD unique identifier:

MON-OOO21-9). Resistance to the European

Corn Borer and tolerance to the herbicide

glufosinate ammonium (Liberty) is derived from

BT11, which contains the Cry1Ab gene from

Bacillus thuringiensis subsp. kurstaki, and the

phosphinothricin N-acetyltransferase (PAT)

encoding gene from S. viridochromogenes. Corn

rootworm-resistance is derived from MIR604

which contains the mCry3A gene from Bacillus

thuringiensis. Tolerance to glyphosate herbicide is

derived from GA21 which contains a a modified

EPSPS gene from maize.

DAS-59122-7
DOW AgroSciences LLC
Corn rootworm-resistant maize produced by

and Pioneer Hi-Bred
inserting the Cry34Ab1 and Cry35Ab1 genes from

International Inc.

Bacillus thuringiensis strain PS149B1. The PAT

encoding gene from Streptomyces

viridochromogenes was introduced as a selectable

marker.

DAS-59122-7 × TC1507 ×
DOW AgroSciences LLC
Stacked insect resistant and herbicide tolerant

NK603
and Pioneer Hi-Bred
maize produced by conventional cross breeding of

International Inc.
parental lines DAS-59122-7 (OECD unique

identifier: DAS-59122-7) and TC1507 (OECD

unique identifier: DAS-O15O7-1) with NK603

(OECD unique identifier: MON-OO6O3-6). Corn

rootworm-resistance is derived from DAS-59122-7

which contains the Cry34Ab1 and Cry35Ab1

genes from Bacillus thuringiensis strain PS149B1.

Lepidopteran resistance and tolerance to

glufosinate ammonium herbicide is derived from

TC1507. Tolerance to glyphosate herbicide is

derived from NK603.

DBT418
Dekalb Genetics
Insect-resistant and glufosinate ammonium

Corporation
herbicide tolerant maize developed by inserting

genes encoding Cry1AC protein from Bacillus

thuringiensis subsp kurstaki and phosphinothricin

acetyltransferase (PAT) from Streptomyces

hygroscopicus

MIR604 × GA21
Syngenta Seeds, Inc.
Stacked insect resistant and herbicide tolerant

maize produced by conventional cross breeding of

parental lines MIR604 (OECD unique identifier:

SYN-IR6O5-5) and GA21 (OECD unique identifier:

MON-OOO21-9). Corn rootworm-resistance is

derived from MIR604 which contains the mCry3A

gene from Bacillus thuringiensis. Tolerance to

glyphosate herbicide is derived from GA21.

MON80100
Monsanto Company
Insect-resistant maize produced by inserting the

Cry1Ab gene from Bacillus thuringiensis subsp.

kurstaki. The genetic modification affords

resistance to attack by the European corn borer

(ECB).

MON802
Monsanto Company
Insect-resistant and glyphosate herbicide tolerant

maize produced by inserting the genes encoding

the Cry1Ab protein from Bacillus thuringiensis and

the 5-enolpyruvylshikimate-3-phosphate synthase

(EPSPS) from A. tumefaciens strain CP4.

MON809
Pioneer Hi-Bred
Resistance to European corn borer (Ostrinia

International Inc.

nubilalis) by introduction of a synthetic Cry1Ab

gene. Glyphosate resistance via introduction of the

bacterial version of a plant enzyme, 5-enolpyruvyl

shikimate-3-phosphate synthase (EPSPS).

MON810
Monsanto Company
Insect-resistant maize produced by inserting a

truncated form of the Cry1Ab gene from Bacillus

thuringiensis subsp. kurstaki HD-1. The genetic

modification affords resistance to attack by the

European corn borer (ECB).

MON810 × LY038
Monsanto Company
Stacked insect resistant and enhanced lysine

content maize derived from conventional cross-

breeding of the parental lines MON810 (OECD

identifier: MON-OO81O-6) and LY038 (OECD

identifier: REN-OOO38-3).

MON810 × MON88017
Monsanto Company
Stacked insect resistant and glyphosate tolerant

maize derived from conventional cross-breeding of

the parental lines MON810 (OECD identifier:

MON-OO81O-6) and MON88017 (OECD

identifier: MON-88O17-3). European corn borer

(ECB) resistance is derived from a truncated form

of the Cry1Ab gene from Bacillus thuringiensis

subsp. kurstaki HD-1 present in MON810. Corn

rootworm resistance is derived from the Cry3Bb1

gene from Bacillus thuringiensis subspecies

kumamotoensis strain EG4691 present in

MON88017. Glyphosate tolerance is derived from

a 5-enolpyruvylshikimate-3-phosphate synthase

(EPSPS) encoding gene from Agrobacterium

tumefaciens strain CP4 present in MON88017.

MON832
Monsanto Company
Introduction, by particle bombardment, of

glyphosate oxidase (GOX) and a modified 5-

enolpyruvyl shikimate-3-phosphate synthase

(EPSPS), an enzyme involved in the shikimate

biochemical pathway for the production of the

aromatic amino acids.

MON863
Monsanto Company
Corn rootworm resistant maize produced by

inserting the Cry3Bb1 gene from Bacillus

thuringiensis subsp. kumamotoensis.

MON863 × MON810
Monsanto Company
Stacked insect resistant corn hybrid derived from

conventional cross-breeding of the parental lines

MON863 (OECD identifier: MON-OO863-5) and

MON810 (OECD identifier: MON-OO81O-6)

MON863 × MON810 ×
Monsanto Company
Stacked insect resistant and herbicide tolerant

NK603

corn hybrid derived from conventional cross-

breeding of the stacked hybrid MON-OO863-5 ×

MON-OO81O-6 and NK603 (OECD

identifier: MON-OO6O3-6).

MON863 × NK603
Monsanto Company
Stacked insect resistant and herbicide tolerant

corn hybrid derived from conventional cross-

breeding of the parental lines MON863 (OECD

identifier: MON-OO863-5) and NK603 (OECD

identifier: MON-OO603-6).

MON87460
Monsanto Company
MON 87460 was developed to provide reduced

yield loss under water-limited conditions compared

to conventional maize. Efficacy in MON 87460 is

derived by expression of the inserted Bacillus

subtilis cold shock protein B (CspB).

MON88017
Monsanto Company
Corn rootworm-resistant maize produced by

inserting the Cry3Bb1 gene from Bacillus

thuringiensis subspecies kumamotoensis strain

EG4691. Glyphosate tolerance derived by

inserting a 5-enolpyruvylshikimate-3-phosphate

synthase (EPSPS) encoding gene from

Agrobacterium tumefaciens strain CP4.

MON89034
Monsanto Company
Maize event expressing two different insecticidal

proteins from Bacillus thuringiensis providing

resistance to number of Lepidopteran pests.

MON89034 ×
Monsanto Company
Stacked insect resistant and glyphosate tolerant

MON88017

maize derived from conventional cross-breeding of

the parental lines MON89034 (OECD identifier:

MON-89O34-3) and MON88017 (OECD

identifier: MON-88O17-3). Resistance to

Lepidopteran insects is derived from two Cry

genes present in MON89043. Corn rootworm

resistance is derived from a single Cry genes and

glyphosate tolerance is derived from the 5-

enolpyruvylshikimate-3-phosphate synthase

(EPSPS) encoding gene from Agrobacterium

tumefaciens present in MON88017.

MON89034 × NK603
Monsanto Company
Stacked insect resistant and herbicide tolerant

maize produced by conventional cross breeding of

parental lines MON89034 (OECD identifier: MON-

89O34-3) with NK603 (OECD unique identifier:

MON-OO6O3-6). Resistance to Lepidopteran

insects is derived from two Cry genes present in

MON89043. Tolerance to glyphosate herbicide is

derived from NK603.

NK603 ×
Monsanto Company
Stacked insect resistant and herbicide tolerant corn

MON810

hybrid derived from conventional cross-breeding of the

parental lines NK603 (OECD identifier: MON-OO6O3-6)

and MON810 (OECD identifier: MON-OO81O-6).

MON89034 ×
Monsanto Company and
Stacked insect resistant and herbicide tolerant maize

TC1507 ×
Mycogen Seeds c/o Dow
produced by conventional cross breeding of parental

MON88017 ×
AgroSciences LLC
lines: MON89034, TC1507, MON88017, and DAS-

DAS-59122-7

59122. Resistance to the above-ground and below-

ground insect pests and tolerance to glyphosate and

glufosinate-ammonium containing herbicides.

MS3
Bayer CropScience
Male sterility caused by expression of the barnase

(Aventis
ribonuclease gene from Bacillus amyloliquefaciens; PPT

CropScience(AgrEvo))
resistance was via PPT-acetyltransferase (PAT).

MS6
Bayer CropScience
Male sterility caused by expression of the barnase

(Aventis
ribonuclease gene from Bacillus amyloliquefaciens; PPT

CropScience(AgrEvo))
resistance was via PPT-acetyltransferase (PAT).

NK603
Monsanto Company
Introduction, by particle bombardment, of a modified 5-

enolpyruvyl shikimate-3-phosphate synthase (EPSPS),

an enzyme involved in the shikimate biochemical

pathway for the production of the aromatic amino acids.

NK603 × T25
Monsanto Company
Stacked glufosinate ammonium and glyphosate

herbicide tolerant maize hybrid derived from

conventional cross-breeding of the parental lines NK603

(OECD identifier: MON-OO6O3-6) and T25 (OECD

identifier: ACS-ZM003-2).

T25 × MON810
Bayer CropScience
Stacked insect resistant and herbicide tolerant corn

(Aventis
hybrid derived from conventional cross-breeding of the

CropScience(AgrEvo))
parental lines T25 (OECD identifier: ACS-ZMOO3-2) and

MON810 (OECD identifier: MON-OO81O-6).

TC1507
Mycogen (c/o Dow
Insect-resistant and glufosinate ammonium herbicide

AgroSciences); Pioneer
tolerant maize produced by inserting the Cry1F gene

(c/o DuPont)
from Bacillus thuringiensis var. aizawai and the

phosphinothricin N-acetyltransferase encoding gene

from Streptomyces viridochromogenes.

TC1507 ×
DOW AgroSciences LLC
Stacked insect resistant and herbicide tolerant corn

NK603

hybrid derived from conventional cross-breeding of the

parental lines 1507 (OECD identifier: DAS-O15O7-1)

and NK603 (OECD identifier: MON-OO6O3-6).

TC1507 × DAS-
DOW AgroSciences LLC
Stacked insect resistant and herbicide tolerant maize

59122-7
and Pioneer Hi-Bred
produced by conventional cross breeding of parental

International Inc.
lines TC1507 (OECD unique identifier: DAS-O15O7-1)

with DAS-59122-7 (OECD unique identifier: DAS-59122-

7). Resistance to Lepidopteran insects is derived from

TC1507 due the presence of the Cry1F gene from

Bacillus thuringiensis var. aizawai. Corn rootworm-

resistance is derived from DAS-59122-7 which contains

the Cry34Ab1 and Cry35Ab1 genes from Bacillus

thuringiensis strain PS149B1. Tolerance to glufosinate

ammonium herbicide is derived from TC1507 from the

phosphinothricin N-acetyltransferase encoding gene

from Streptomyces viridochromogenes.

Other events with regulatory approval are well known to one skilled in the art and can be found at the Center for Environmental Risk Assessment (cera-gmc.org/?action=gm_crop_database, which can be accessed using the www prefix) and at the International Service for the Acquisition of Agri-Biotech Applications (isaaa.org/gmapprovaldatabase/default.asp, which can be accessed using the www prefix).

Gene Silencing

In some embodiments the stacked trait may be in the form of silencing of one or more polynucleotides of interest resulting in suppression of one or more target pest polypeptides. In some embodiments the silencing is achieved through the use of a suppression DNA construct.

In some embodiments one or more polynucleotide encoding the polypeptides of the PIP-47 polypeptides or fragments or variants thereof may be stacked with one or more polynucleotides encoding one or more polypeptides having insecticidal activity or agronomic traits as set forth supra and optionally may further include one or more polynucleotides providing for gene silencing of one or more target polynucleotides as discussed infra.

“Suppression DNA construct” is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in “silencing” of a target gene in the plant. The target gene may be endogenous or transgenic to the plant. “Silencing,” as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality. The term “suppression” includes lower, reduce, decline, decrease, inhibit, eliminate and prevent. “Silencing” or “gene silencing” does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending upon the approach to be utilized, the region may be 100% identical or less than 100% identical (e.g., at least 50% or any integer between 51% and 100% identical) to all or part of the sense strand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.

“Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcripts capable of suppressing the expression of the target protein. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see, Vaucheret, et al., (1998) Plant J. 16:651-659 and Gura, (2000) Nature 404:804-808).

Another variation describes the use of plant viral sequences to direct the suppression of proximal mRNA encoding sequences (PCT Publication WO 1998/36083).

Recent work has described the use of “hairpin” structures that incorporate all or part, of an mRNA encoding sequence in a complementary orientation that results in a potential “stem-loop” structure for the expressed RNA (PCT Publication WO 1999/53050). In this case the stem is formed by polynucleotides corresponding to the gene of interest inserted in either sense or anti-sense orientation with respect to the promoter and the loop is formed by some polynucleotides of the gene of interest, which do not have a complement in the construct. This increases the frequency of cosuppression or silencing in the recovered transgenic plants. For review of hairpin suppression, see, Wesley, et al., (2003) Methods in Molecular Biology, Plant Functional Genomics: Methods and Protocols 236:273-286.

A construct where the stem is formed by at least 30 nucleotides from a gene to be suppressed and the loop is formed by a random nucleotide sequence has also effectively been used for suppression (PCT Publication WO 1999/61632).

The use of poly-T and poly-A sequences to generate the stem in the stem-loop structure has also been described (PCT Publication WO 2002/00894).

Yet another variation includes using synthetic repeats to promote formation of a stem in the stem-loop structure. Transgenic organisms prepared with such recombinant DNA fragments have been shown to have reduced levels of the protein encoded by the nucleotide fragment forming the loop as described in PCT Publication WO 2002/00904.

RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing (PTGS) or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire, et al., (1999) Trends Genet. 15:358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein, et al., (2001) Nature 409:363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir, et al., (2001) Genes Dev. 15:188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner, et al., (2001) Science 293:834). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementarity to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir, et al., (2001) Genes Dev. 15:188). In addition, RNA interference can also involve small RNA (e.g., miRNA) mediated gene silencing, presumably through cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see, e.g., Allshire, (2002) Science 297:1818-1819; Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, (2002) Science 297:2215-2218 and Hall, et al., (2002) Science 297:2232-2237). As such, miRNA molecules of the disclosure can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional or post-transcriptional level.

Methods and compositions are further provided which allow for an increase in RNAi produced from the silencing element. In such embodiments, the methods and compositions employ a first polynucleotide comprising a silencing element for a target pest sequence operably linked to a promoter active in the plant cell; and, a second polynucleotide comprising a suppressor enhancer element comprising the target pest sequence or an active variant or fragment thereof operably linked to a promoter active in the plant cell. The combined expression of the silencing element with suppressor enhancer element leads to an increased amplification of the inhibitory RNA produced from the silencing element over that achievable with only the expression of the silencing element alone. In addition to the increased amplification of the specific RNAi species itself, the methods and compositions further allow for the production of a diverse population of RNAi species that can enhance the effectiveness of disrupting target gene expression. As such, when the suppressor enhancer element is expressed in a plant cell in combination with the silencing element, the methods and composition can allow for the systemic production of RNAi throughout the plant; the production of greater amounts of RNAi than would be observed with just the silencing element construct alone; and, the improved loading of RNAi into the phloem of the plant, thus providing better control of phloem feeding insects by an RNAi approach. Thus, the various methods and compositions provide improved methods for the delivery of inhibitory RNA to the target organism. See, for example, US Patent Application Publication 2009/0188008.

As used herein, a “suppressor enhancer element” comprises a polynucleotide comprising the target sequence to be suppressed or an active fragment or variant thereof. It is recognize that the suppressor enhancer element need not be identical to the target sequence, but rather, the suppressor enhancer element can comprise a variant of the target sequence, so long as the suppressor enhancer element has sufficient sequence identity to the target sequence to allow for an increased level of the RNAi produced by the silencing element over that achievable with only the expression of the silencing element. Similarly, the suppressor enhancer element can comprise a fragment of the target sequence, wherein the fragment is of sufficient length to allow for an increased level of the RNAi produced by the silencing element over that achievable with only the expression of the silencing element.

It is recognized that multiple suppressor enhancer elements from the same target sequence or from different target sequences or from different regions of the same target sequence can be employed. For example, the suppressor enhancer elements employed can comprise fragments of the target sequence derived from different region of the target sequence (i.e., from the 3′UTR, coding sequence, intron, and/or 5′UTR). Further, the suppressor enhancer element can be contained in an expression cassette, as described elsewhere herein, and in specific embodiments, the suppressor enhancer element is on the same or on a different DNA vector or construct as the silencing element. The suppressor enhancer element can be operably linked to a promoter as disclosed herein. It is recognized that the suppressor enhancer element can be expressed constitutively or alternatively, it may be produced in a stage-specific manner employing the various inducible or tissue-preferred or developmentally regulated promoters that are discussed elsewhere herein.

In specific embodiments, employing both a silencing element and the suppressor enhancer element the systemic production of RNAi occurs throughout the entire plant. In further embodiments, the plant or plant parts of the disclosure have an improved loading of RNAi into the phloem of the plant than would be observed with the expression of the silencing element construct alone and, thus provide better control of phloem feeding insects by an RNAi approach. In specific embodiments, the plants, plant parts and plant cells of the disclosure can further be characterized as allowing for the production of a diversity of RNAi species that can enhance the effectiveness of disrupting target gene expression.

In specific embodiments, the combined expression of the silencing element and the suppressor enhancer element increases the concentration of the inhibitory RNA in the plant cell, plant, plant part, plant tissue or phloem over the level that is achieved when the silencing element is expressed alone.

As used herein, an “increased level of inhibitory RNA” comprises any statistically significant increase in the level of RNAi produced in a plant having the combined expression when compared to an appropriate control plant. For example, an increase in the level of RNAi in the plant, plant part or the plant cell can comprise at least about a 1%, about a 1%-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a 30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%, about a 80%-90%, about a 90%-100% or greater increase in the level of RNAi in the plant, plant part, plant cell or phloem when compared to an appropriate control. In other embodiments, the increase in the level of RNAi in the plant, plant part, plant cell or phloem can comprise at least about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold, about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40 fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70 fold, about 70 fold-80 fold, about a 80 fold-90 fold, about a 90 fold-100 fold or greater increase in the level of RNAi in the plant, plant part, plant cell or phloem when compared to an appropriate control. Examples of combined expression of the silencing element with suppressor enhancer element for the control of Stinkbugs and Lygus can be found in US Patent Application Publication 2011/0301223 and US Patent Application Publication 2009/0192117.

Some embodiments relate to down-regulation of expression of target genes in insect pest species by interfering ribonucleic acid (RNA) molecules. PCT Publication WO 2007/074405 describes methods of inhibiting expression of target genes in invertebrate pests including Colorado potato beetle. PCT Publication WO 2005/110068 describes methods of inhibiting expression of target genes in invertebrate pests including in particular Western corn rootworm as a means to control insect infestation. Furthermore, PCT Publication WO 2009/091864 describes compositions and methods for the suppression of target genes from insect pest species including pests from the Lygus genus. Nucleic acid molecules including RNAi for targeting the vacuolar ATPase H subunit, useful for controlling a coleopteran pest population and infestation as described in US Patent Application Publication 2012/0198586. PCT Publication WO 2012/055982 describes ribonucleic acid (RNA or double stranded RNA) that inhibits or down regulates the expression of a target gene that encodes: an insect ribosomal protein such as the ribosomal protein L19, the ribosomal protein L40 or the ribosomal protein S27A; an insect proteasome subunit such as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2 protein; an insect β-coatomer of the COPI vesicle, the γ-coatomer of the COPI vesicle, the β′-coatomer protein or the ζ-coatomer of the COPI vesicle; an insect Tetraspanine 2 A protein which is a putative transmembrane domain protein; an insect protein belonging to the actin family such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23 protein which is a GTPase activator involved in intracellular protein transport; an insect crinkled protein which is an unconventional myosin which is involved in motor activity; an insect crooked neck protein which is involved in the regulation of nuclear alternative mRNA splicing; an insect vacuolar H+-ATPase G-subunit protein and an insect Tbp-1 such as Tat-binding protein. US Patent Application Publications 2012/029750, US 20120297501, and 2012/0322660 describe interfering ribonucleic acids (RNA or double stranded RNA) that functions upon uptake by an insect pest species to down-regulate expression of a target gene in said insect pest, wherein the RNA comprises at least one silencing element wherein the silencing element is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the target gene. US Patent Application Publication 2012/0164205 describe potential targets for interfering double stranded ribonucleic acids for inhibiting invertebrate pests including: a Chd3 Homologous Sequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPase Homologous Sequence, a EF1α Homologous Sequence, a 26S Proteosome Subunit p28 Homologous Sequence, a Juvenile Hormone Epoxide Hydrolase Homologous Sequence, a Swelling Dependent Chloride Channel Protein Homologous Sequence, a Glucose-6-Phosphate 1-Dehydrogenase Protein Homologous Sequence, an Act42A Protein Homologous Sequence, a ADP-Ribosylation Factor 1 Homologous Sequence, a Transcription Factor IIB Protein Homologous Sequence, a Chitinase Homologous Sequences, a Ubiquitin Conjugating Enzyme Homologous Sequence, a Glyceraldehyde-3-Phosphate Dehydrogenase Homologous Sequence, an Ubiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog, and an Alpha Tubuliln Homologous Sequence.

Use in Pesticidal Control

General methods for employing strains comprising a nucleic acid sequence of the embodiments or a variant thereof, in pesticide control or in engineering other organisms as pesticidal agents are known in the art. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

Microorganism hosts that are known to occupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest may be selected. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the PIP-47 polypeptide, and desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas chlororaphis, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinelandii and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms. Host organisms of particular interest include yeast, such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae), Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp. (such as P. aeruginosa, P. fluorescens, P. chlororaphis), Erwinia spp., and Flavobacterium spp., and other such organisms, including Agrobacterium tumefaciens, E. coli, Bacillus subtilis, Bacillus cereus and the like.

Genes encoding the PIP-47 polypeptides of the embodiments can be introduced into microorganisms that multiply on plants (epiphytes) to deliver PIP-47 polypeptides to potential target pests. Epiphytes, for example, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a Bacillus cereus strain that colonizes roots can be isolated from roots of a plant (see, for example, Handelsman et al. (1991) Appl. Environ. Microbiol. 56:713-718). Genes encoding the PIP-47 polypeptides of the embodiments can be introduced into a root-colonizing Bacillus cereus by standard methods known in the art.

Genes encoding PIP-47 polypeptides can be introduced, for example, into the root-colonizing Bacillus by means of electro transformation. Specifically, genes encoding the PIP-47 polypeptides can be cloned into a shuttle vector, for example, pHT3101 (Lerecius, et al., (1989) FEMS Microbiol. Letts. 60:211-218. The shuttle vector pHT3101 containing the coding sequence for the particular PIP-47 polypeptide gene can, for example, be transformed into the root-colonizing Bacillus by means of electroporation (Lerecius, et al., (1989) FEMS Microbiol. Letts. 60:211-218).

Expression systems can be designed so that PIP-47 polypeptides are secreted outside the cytoplasm of gram-negative bacteria, such as E. coli, for example. Advantages of having PIP-47 polypeptides secreted are: (1) avoidance of potential cytotoxic effects of the PIP-47 polypeptide expressed; and (2) improvement in the efficiency of purification of the PIP-47 polypeptide, including, but not limited to, increased efficiency in the recovery and purification of the protein per volume cell broth and decreased time and/or costs of recovery and purification per unit protein.

PIP-47 polypeptides can be made to be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino-terminal end of the PIP-47 polypeptide. Signal peptides recognized by E. coli can be found in proteins already known to be secreted in E. coli, for example the OmpA protein (Ghrayeb, et al., (1984) EMBO J, 3:2437-2442). OmpA is a major protein of the E. coli outer membrane, and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified before processing as may be the case for other signal peptides, for example lipoprotein signal peptide (Duffaud, et al., (1987) Meth. Enzymol. 153:492).

PIP-47 polypeptides of the embodiments can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner that Bt strains have been used as insecticidal sprays. In the case of a PIP-47 polypeptide(s) that is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and/or deletions prevent secretion of the PIP-47 polypeptide(s) into the growth medium during the fermentation process. The PIP-47 polypeptides are retained within the cell, and the cells are then processed to yield the encapsulated PIP-47 polypeptides. Any suitable microorganism can be used for this purpose. Pseudomonas has been used to express Bt toxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide (Gaertner, et al., (1993), in: Advanced Engineered Pesticides, ed. Kim).

Alternatively, the PIP-47 polypeptides are produced by introducing a heterologous gene into a cellular host. Expression of the heterologous gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated PIP-47 polypeptides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein.

Pesticidal Compositions

In some embodiments the active ingredients can be applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be fertilizers, weed killers, Cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and/or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise the formulations may be prepared into edible “baits” or fashioned into pest “traps” to permit feeding or ingestion by a target pest of the pesticidal formulation.

Methods of applying an active ingredient or an agrochemical composition that contains at least one of the PIP-47 polypeptides produced by the bacterial strains include leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenation, extraction, filtration, centrifugation, sedimentation or concentration of a culture of cells comprising the polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present in a concentration of from about 1% to about 99% by weight.

Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteran pests may be killed or reduced in numbers in a given area by the methods of the disclosure or may be prophylactically applied to an environmental area to prevent infestation by a susceptible pest. Preferably the pest ingests or is contacted with, a pesticidally-effective amount of the polypeptide. “Pesticidally-effective amount” as used herein refers to an amount of the pesticide that is able to bring about death to at least one pest or to noticeably reduce pest growth, feeding or normal physiological development. This amount will vary depending on such factors as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop or agricultural site to be treated, the environmental conditions and the method, rate, concentration, stability, and quantity of application of the pesticidally-effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of pest infestation.

The pesticide compositions described may be made by formulating either the bacterial cell, Crystal and/or spore suspension or isolated protein component with the desired agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material or a suspension in oil (vegetable or mineral) or water or oil/water emulsions or as a wettable powder or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term “agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in U.S. Pat. No. 6,468,523, herein incorporated by reference. The plants can also be treated with one or more chemical compositions, including one or more herbicide, insecticides or fungicides. Exemplary chemical compositions include: Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran, FluaCrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox, Methomyl, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on; Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs, Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam, Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam, Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid, Oxpoconazole fumarate, Cyazof amid, Fenamidone, Zoxamide, Picoxystrobin, Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon, Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron, Triasulfuron, Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen, Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron Methyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides: Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos, Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-) Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon, Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl, Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos, Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole, Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-Amethyl](2,2-difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides: Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor, Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole, Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides: Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran, β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

In some embodiments the herbicide is Atrazine, Bromacil, Diuron, Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron, Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron, Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin, Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

In some embodiments the insecticide is Esfenvalerate, Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinations thereof.

Pesticidal and Insecticidal Activity

“Pest” includes but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds are equally effective against all pests. Compounds of the embodiments display activity against insect pests, which may include economically important agronomic, forest, greenhouse, nursery ornamentals, food and fiber, public and animal health, domestic and commercial structure, household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers and heliothines in the family Noctuidae Spodoptera frugiperda JE Smith (fall armyworm); S. exigua H{umlaut over (b)}ner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia ni Hübner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Hübner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leaf roller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); and leaf rollers, budworms, seed worms and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermüller (European grape vine moth); Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (vine moth); Bonagota salubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneura spp.

Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Collas eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée (omnivorous looper); Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermüller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenée; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae and Curculionidae (including, but not limited to: Anthonomus grandis Boheman (boll weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctata Fabricius (clover leaf weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug)); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles and leafminers in the family Chrysomelidae (including, but not limited to: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabrotica virgifera virgifera LeConte (western corn rootworm); D. barberi Smith and Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn flea beetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle); Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius (grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle); Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from the family Coccinellidae (including, but not limited to: Epilachna varivestis Mulsant (Mexican bean beetle)); chafers and other beetles from the family Scarabaeidae (including, but not limited to: Popillia japonica Newman (Japanese beetle); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer); Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer (carrot beetle)); carpet beetles from the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp.; Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae and beetles from the family Tenebrionidae.

Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicornis Loew (corn blotch leafminer); midges (including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Géhin (wheat midge); Neolasioptera murtfeldtiana Felt, (sunflower seed midge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fruit flies); maggots (including, but not limited to: Delia platura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly) and other Delia spp., Meromyza americana Fitch (wheat stem maggot); Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F. femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus (stable flies)); face flies, horn flies, blow flies, Chrysomya spp.; Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; bot flies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer flies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; biting midges, sand flies, sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the orders Hemiptera and Homoptera such as, but not limited to, adelgids from the family Adelgidae, plant bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppers from the families Cixiidae, Flatidae, Fulgoroidea, lssidae and Delphacidae, treehoppers from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, scales from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from the family Tingidae, stink bugs from the family Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugs from the family Cercopidae squash bugs from the family Coreidae and red bugs and cotton stainers from the family Pyrrhocoridae.

Agronomically important members from the order Homoptera further include, but are not limited to: Acyrthisiphon pisum Harris (pea aphid); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantaginea Paaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly apple aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopterus pruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphum euphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Therioaphis maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris (potato leafhopper); Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestes quadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stål (rice leafhopper); Nilaparvata lugens Stål (brown planthopper); Peregrinus maidis Ashmead (corn planthopper); Sogatella furcifera Horvath (white-backed planthopper); Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee (white apple leafhopper); Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasi Maskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citri Risso (citrus mealybug); Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ash mead (persimmon psylla).

Agronomically important species of interest from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Anasa tristis De Geer (squash bug); Blissus leucopterus leucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Herrich-Schaffer (cotton stainer); Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say (leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Lygocoris pabulinus Linnaeus (common green capsid); Nezara viridula Linnaeus (southern green stink bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper).

Furthermore, embodiments may be effective against Hemiptera such, Calocoris norvegicus Gmelin (strawberry bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onion plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius (four-lined plant bug); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp. and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (wheat curl mite); Petrobia latens Müller (brown wheat mite); spider mites and red mites in the family Tetranychidae, Panonychus ulmi Koch (European red mite); Tetranychus urticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Eriophyidae and other foliar feeding mites and mites important in human and animal health, i.e., dust mites in the family Epidermoptidae, follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deer tick); I. holocyclus Neumann (Australian paralysis tick); Dermacentor variabilis Say (American dog tick); Amblyomma americanum Linnaeus (lone star tick) and scab and itch mites in the families Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepisma saccharina Linnaeus (silverfish); Thermobia domestica Packard (firebrat).

Additional arthropod pests covered include: spiders in the order Araneae such as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) and the Latrodectus mactans Fabricius (black widow spider) and centipedes in the order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (house centipede).

Insect pest of interest include the superfamily of stink bugs and other related insects including but not limited to species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guildini, Euschistus servus, Acrosternum hilare, Euschistus heros, Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae (Megacopta cribraria—Bean plataspid) and the family Cydnidae (Scaptocoris castanea—Root stink bug) and Lepidoptera species including but not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker and velvet bean caterpillar e.g., Anticarsia gemmatalis Hübner.

Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang, (1990) J. Econ. Entomol. 83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone, et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No. 5,743,477, all of which are herein incorporated by reference in their entirety. Generally, the protein is mixed and used in feeding assays. See, for example Marrone, et al., (1985) J. of Economic Entomology 78:290-293. Such assays can include contacting plants with one or more pests and determining the plant's ability to survive and/or cause the death of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst and lesion nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode) and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Seed Treatment

To protect and to enhance yield production and trait technologies, seed treatment options can provide additional crop plan flexibility and cost effective control against insects, weeds and diseases. Seed material can be treated, typically surface treated, with a composition comprising combinations of chemical or biological herbicides, herbicide safeners, insecticides, fungicides, germination inhibitors and enhancers, nutrients, plant growth regulators and activators, bactericides, nematocides, avicides and/or molluscicides. These compounds are typically formulated together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. The coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Examples of the various types of compounds that may be used as seed treatments are provided in The Pesticide Manual: A World Compendium, C. D. S. Tomlin Ed., Published by the British Crop Production Council, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are not limited to, one or more of abscisic acid, acibenzolar-S-methyl, avermectin, amitrol, azaconazole, azospirillum, azadirachtin, azoxystrobin, Bacillus spp. (including one or more of cereus, firmus, megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species), bradyrhizobium spp. (including one or more of betae, canariense, elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/or yuanmingense), captan, carboxin, chitosan, clothianidin, copper, cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil, fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein, imazalil, imidacloprid, ipconazole, isoflavenoids, lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam, metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium, penthiopyrad, permethrine, picoxystrobin, prothioconazole, pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram, tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin, triticonazole and/or zinc. PCNB seed coat refers to EPA Registration Number 00293500419, containing quintozen and terrazole. TCMTB refers to 2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be tested to determine which seed treatment options and application rates may complement such varieties and transgenic traits in order to enhance yield. For example, a variety with good yield potential but head smut susceptibility may benefit from the use of a seed treatment that provides protection against head smut, a variety with good yield potential but cyst nematode susceptibility may benefit from the use of a seed treatment that provides protection against cyst nematode, and so on. Likewise, a variety encompassing a transgenic trait conferring insect resistance may benefit from the second mode of action conferred by the seed treatment, a variety encompassing a transgenic trait conferring herbicide resistance may benefit from a seed treatment with a safener that enhances the plants resistance to that herbicide, etc. Further, the good root establishment and early emergence that results from the proper use of a seed treatment may result in more efficient nitrogen use, a better ability to withstand drought and an overall increase in yield potential of a variety or varieties containing a certain trait when combined with a seed treatment.

Methods for Killing an Insect Pest and Controlling an Insect Population

In some embodiments methods are provided for killing an insect pest, comprising contacting the insect pest, either simultaneously or sequentially, with an insecticidally-effective amount of a recombinant PIP-47 polypeptide. In some embodiments methods are provided for killing an insect pest, comprising contacting the insect pest with an insecticidally-effective amount of a recombinant pesticidal protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or a variant thereof.

In some embodiments methods are provided for controlling an insect pest population, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of a recombinant PIP-47 polypeptide. In some embodiments methods are provided for controlling an insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of a recombinant pesticidal protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or a variant thereof. As used herein, “controlling a pest population” or “controls a pest” refers to any effect on a pest that results in limiting the damage that the pest causes. Controlling a pest includes, but is not limited to, killing the pest, inhibiting development of the pest, altering fertility or growth of the pest in such a manner that the pest provides less damage to the plant, decreasing the number of offspring produced, producing less fit pests, producing pests more susceptible to predator attack or deterring the pests from eating the plant.

In some embodiments methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population, either simultaneously or sequentially, with an insecticidally-effective amount of a recombinant PIP-47 polypeptide. In some embodiments methods are provided for controlling an insect pest population resistant to a pesticidal protein, comprising contacting the insect pest population with an insecticidally-effective amount of a recombinant pesticidal protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or a variant thereof.

In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof a recombinant polynucleotide encoding a PIP-47 polypeptide. In some embodiments methods are provided for protecting a plant from an insect pest, comprising expressing in the plant or cell thereof a recombinant polynucleotide encoding pesticidal protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or variants thereof.

Insect Resistance Management (IRM) Strategies

Expression of B. thuringiensis δ-endotoxins in transgenic corn plants has proven to be an effective means of controlling agriculturally important insect pests (Perlak, et al., 1990; 1993). However, insects have evolved that are resistant to B. thuringiensis δ-endotoxins expressed in transgenic plants. Such resistance, should it become widespread, would clearly limit the commercial value of germplasm containing genes encoding such B. thuringiensis δ-endotoxins.

One way to increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests is to use provide non-transgenic (i.e., non-insecticidal protein) refuges (a section of non-insecticidal crops/corn) for use with transgenic crops producing a single insecticidal protein active against target pests. The United States Environmental Protection Agency (epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which can be accessed using the www prefix) publishes the requirements for use with transgenic crops producing a single Bt protein active against target pests. In addition, the National Corn Growers Association, on their website: (ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can be accessed using the www prefix) also provides similar guidance regarding refuge requirements. Due to losses to insects within the refuge area, larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenic insecticides against target pests and contemporaneously reducing the development of insecticide-resistant pests would be to have a repository of insecticidal genes that are effective against groups of insect pests and which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic to the same insect species, each insecticide being expressed at efficacious levels would be another way to achieve control of the development of resistance. This is based on the principle that evolution of resistance against two separate modes of action is far more unlikely than only one. Roush, for example, outlines two-toxin strategies, also called “pyramiding” or “stacking,” for management of insecticidal transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353:1777-1786). Stacking or pyramiding of two different proteins each effective against the target pests and with little or no cross-resistance can allow for use of a smaller refuge. The US Environmental Protection Agency requires significantly less (generally 5%) structured refuge of non-Bt corn be planted than for single trait products (generally 20%). There are various ways of providing the IRM effects of a refuge, including various geometric planting patterns in the fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the PIP-47 polypeptides of the disclosure are useful as an insect resistance management strategy in combination (i.e., pyramided) with other pesticidal proteins include but are not limited to Bt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, and the like.

Provided are methods of controlling Lepidoptera and/or Coleoptera insect infestation(s) in a transgenic plant that promote insect resistance management, comprising expressing in the plant at least two different insecticidal proteins having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/or Coleoptera insect infestation in a transgenic plant and promoting insect resistance management the at least one of the insecticidal proteins comprise a PIP-47 polypeptide insecticidal to insects in the order Lepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/or Coleoptera insect infestation in a transgenic plant and promoting insect resistance management the at least one of the insecticidal proteins comprises a protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or variants thereof, insecticidal to insects in the order Lepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/or Coleoptera insect infestation in a transgenic plant and promoting insect resistance management comprise expressing in the transgenic plant a PIP-47 polypeptide and a Cry protein insecticidal to insects in the order Lepidoptera and/or Coleoptera having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/or Coleoptera insect infestation in a transgenic plant and promoting insect resistance management comprise in the transgenic plant a protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or variants thereof and a Cry protein insecticidal to insects in the order Lepidoptera and/or Coleoptera having different modes of action.

Also provided are methods of reducing likelihood of emergence of Lepidoptera and/or Coleoptera insect resistance to transgenic plants expressing in the plants insecticidal proteins to control the insect species, comprising expression of a PIP-47 polypeptide insecticidal to the insect species in combination with a second insecticidal protein to the insect species having different modes of action.

Also provided are methods of reducing likelihood of emergence of Lepidoptera and/or Coleoptera insect resistance to transgenic plants expressing in the plants insecticidal proteins to control the insect species, comprising expression of a protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or variants thereof, insecticidal to the insect species in combination with a second insecticidal protein to the insect species having different modes of action.

Also provided are means for effective Lepidoptera and/or Coleoptera insect resistance management of transgenic plants, comprising co-expressing at high levels in the plants two or more insecticidal proteins toxic to Lepidoptera and/or Coleoptera insects but each exhibiting a different mode of effectuating its killing activity, wherein the two or more insecticidal proteins comprise a PIP-47 polypeptide and a Cry protein. Also provided are means for effective Lepidoptera and/or Coleoptera insect resistance management of transgenic plants, comprising co-expressing at high levels in the plants two or more insecticidal proteins toxic to Lepidoptera and/or Coleoptera insects but each exhibiting a different mode of effectuating its killing activity, wherein the two or more insecticidal proteins comprise a protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or variants thereof and a Cry protein.

In addition, methods are provided for obtaining regulatory approval for planting or commercialization of plants expressing proteins insecticidal to insects in the order Lepidoptera and/or Coleoptera, comprising the step of referring to, submitting or relying on insect assay binding data showing that the PIP-47 polypeptide does not compete with binding sites for Cry proteins in such insects. In addition, methods are provided for obtaining regulatory approval for planting or commercialization of plants expressing proteins insecticidal to insects in the order Lepidoptera and/or Coleoptera, comprising the step of referring to, submitting or relying on insect assay binding data showing that the protein of SEQ ID NO: 18 and/or protein of SEQ ID NO: 20 or variant thereof does not compete with binding sites for Cry proteins in such insects.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods comprise providing a plant or plant cell expressing a polynucleotide encoding the pesticidal polypeptide sequence disclosed herein and growing the plant or a seed thereof in a field infested with a pest against which the polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity against a Lepidopteran, Coleopteran, Dipteran, Hemipteran or nematode pest, and the field is infested with a Lepidopteran, Hemipteran, Coleopteran, Dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/or quantity of biomass produced by the plant. “Biomass” as used herein refers to any measured plant product. An increase in biomass production is any improvement in the yield of the measured plant product. Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase in yield compared to a plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improved pest resistance of a plant expressing a PIP-47 polypeptide disclosed herein. Expression of the PIP-47 polypeptide results in a reduced ability of a pest to infest or feed on the plant, thus improving plant yield.

Methods of Processing

Further provided are methods of processing a plant, plant part or seed to obtain a food or feed product from a plant, plant part or seed comprising a PIP-47 polypeptide. The plants, plant parts or seeds provided herein, can be processed to yield oil, protein products and/or by-products that are derivatives obtained by processing that have commercial value. Non-limiting examples include transgenic seeds comprising a nucleic acid molecule encoding a PIP-47 polypeptide which can be processed to yield soy oil, soy products and/or soy by-products.

“Processing” refers to any physical and chemical methods used to obtain any soy product and includes, but is not limited to, heat conditioning, flaking and grinding, extrusion, solvent extraction or aqueous soaking and extraction of whole or partial seeds.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTALS
Example 1—Identification of an Insecticidal Protein Active Against Western Corn Root Worm (WCRW) from Strain SS62E1

The WCRW (Diabrotica virgifera) insecticidal protein PIP-47Aa (SEQ ID NO: 1) was identified by protein purification, liquid chromatography mass spectrometry (LC-MS/MS) and PCR cloning from Pseudomonas putida strain SS62E1 as follows:

Pseudomonas Strain SS62E1 was grown in tryptic soy broth for 2 days at 30° C. and 250 rpm. Cells were harvested by centrifugation and cell pellets were washed once with phosphate buffered saline (PBS) before storage at −70° C. For protein extraction, cells were thawed and re-suspended in 50 mM sodium acetate buffer, pH 5 (buffer A) containing protease inhibitor cocktail V from Calbiochem®. A crude cleared lysate was obtained by passing the cells through a homogenizer at 30,000 psi, followed by centrifugation at 13,800 x g for 20 min.

WCRW bioassays were conducted using 10 microliter cell lysate samples mixed with molten low-melt WCRW diet (Southland Products Inc., Lake Village, Ark.) in a 96 well format. Diabrotica virgifera virgifera neonates were placed into each well of a 96 well plate. The assay was run for 4 days at 25° C. and then was scored for insect mortality and stunting of insect growth. The scores were noted as dead, severely stunted (little or no growth but alive), stunted (growth to second instar but not equivalent to controls) or no activity.

Genomic DNA from strain SS62E1 was extracted with a Sigma Bacterial Genomic DNA Extraction Kit (Cat # NA2110-KT, Sigma-Aldrich, PO Box 14508, St. Louis, Mo. 63178) according to the manufactures' instructions. The DNA concentration was determined using a NanoDrop™ Spectrophotometer (Thermo Scientific, 3411 Silverside Road, Bancroft Building, Suite 100, Wilmington, Del. 19810) and the genomic DNA was diluted to 40 ng/ul with sterile water. A 25 ul PCR reaction was set up by combining 80 ng genomic DNA, 2 ul (5 uM) 16S ribosomal DNA primers TACCTTGTTACGACTT (SEQ ID NO: 421) and AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 422), 1 ul 10 mM dNTP, 1× Phusion HF buffer, and 1 unit of Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Cat #M0530L, 240 County Road, Ipswich, Mass. 01938-2723). The PCR reaction was run in MJ Research PTC-200 Thermo Cycler (Bio-Rad Laboratories, Inc., 1000 Alfred Nobel Drive, Hercules, Calif., 94547, USA) with the following program: 96° C. 1 min; 30 cycles of 96° C. 15 seconds, 52° C. 2 minutes and 72° C. 2 minutes; 72° C. 10 minutes; and hold on 4° C. The PCR products were purified with QiaQuick® DNA purification Kit (Cat #28104, QIAGEN Inc., 27220 Turnberry Lane, Valencia, Calif. 91355). The purified PCR sample was DNA sequenced and the resulting 16S ribosomal DNA sequence was BLAST searched against the NCBI database which indicated that SS62E1 is a Pseudomonas Putida strain. The Pseudomonas Putida strain SS62E1 was deposited on Aug. 13, 2013 under accession # NRRL B-50845 with the Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, (nrrl.ncaur.usda.gov, which can be accessed on the world-wide web using the “www” prefix).

Isolated strain SS62E1 genomic DNA was also prepared according to a library construction protocol developed by Illumina™ and sequenced using the Illumina™ Genome Analyzer IIx (Cat# SY-301-1301, Illumina Inc., 9885 Towne Center Drive, San Diego, Calif. 92121). The nucleic acid contig sequences were assembled and open reading frames were generated.

For purification of the insecticidal protein, cells were thawed and re-suspended in 50 mM sodium acetate buffer, pH 5 (buffer A) containing protease inhibitor cocktail V from Calbiochem™. A crude cleared lysate was obtained by passing the cells through a homogenizer at 30,000 psi, followed by centrifugation at 13,800 g for 20 min. The supernatant was adjusted to less than 5 mSiemens/cm conductivity by dilution with buffer A. This material was loaded onto a SP-HP HiTrap™ column (cation exchange, GE Healthcare) and eluted with a linear gradient to 1 M NaCl in Buffer A. Fractions were desalted and subjected for identification of insecticidal activity. Active fractions were pooled, buffer exchanged into 50 mM Tris, pH9 (buffer B) and applied to a Mono Q™ column (anion exchange, GE Healthcare). Protein elution was achieved with a salt gradient form 0 to 1 M NaCl in buffer B. Active fractions were identified in artificial diet insect feeding assays. Further purification was achieved by size exclusion chromatography. Highly enriched, active fractions were analyzed by SDS-PAGE. The candidate protein band was excised, digested with trypsin and analyzed by nano-liquid chromatography/electrospray tandem mass spectrometry (nano-LC/ESI-MS/MS) on a Thermo Q Exactive Orbitrap™ mass spectrometer (Thermo Fisher Scientific) interfaced with an Eksigent® NanoLC 1-D™ Plus nano-LC system (AB Sciex). Ten product ion spectra were collected in a data dependent acquisition mode after a MS1 survey scan.

Protein identification was done by database searches using Mascot (Matrix Science). The search against a DuPont Pioneer in-house database, which combines in-house bacterial protein sequences, and the SWISS-PROT protein database identified the protein of SEQ ID NO: 1, which was designated PIP-47Aa, encoded by the polynucleotide of SEQ ID NO: 2 from strain SS62E1.

Example 2—Identification of Homologous Proteins of PIP-47Aa

Gene identities may be determined by conducting BLAST (Basic Local Alignment 20 Search Tool; Altschul, et al., (1993) J. Mol. Biol. 215:403-410; see also ncbi.nlm.nih.gov/BLAST/, which can be accessed using the www prefix) searches under default parameters for similarity to sequences contained in the publically available BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the 25 SWISS-PROT protein sequence database, EMBL, and DDBJ databases. In addition to public databases internal DuPont Pioneer databases were searched. The polypeptide sequence of SEQ ID NO: 1 was analyzed.

The search identified several homologs of PIP-47Aa (SEQ ID NO: 1) having varying percent identity to PIP-47Aa (SEQ ID NO: 1). PIP-47Aa homologs designated as PIP-47Ba (SEQ ID NO: 4), PIP-47Bb (SEQ ID NO: 6) and PIP-47Fa (SEQ ID NO: 8) were identified from a company internal bacterial genome database from Pseudomonas putida, Pseudomonas fulva, and Pseudomonas chlororaphis respectively. PIP-47Ba (SEQ ID NO: 4), PIP-47Bb (SEQ ID NO: 6) and PIP-47Fa (SEQ ID NO: 8) are encoded by the DNA sequence of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively. The more distant PIP-47Aa (SEQ ID NO: 2) homologs; hypothetical protein Plu2048 (Accession # NP_929309); hypothetical protein plu2046 (Accession # NP_929307); the conserved hypothetical protein (Accession # YP_003041397); the conserved hypothetical protein (Accession # YP_003041380); hypothetical protein HMPREF9690_02266 (Accession # ZP_17107944); and the hypothetical protein (Accession # ZP_05881874) were identified from the NCBI public database, and designated herein as: Plu2048 (SEQ ID NO: 10 encoded by polynucleotide of SEQ ID NO: 9); Plu2046 (SEQ ID NO: 12 encoded by SEQ ID NO: 11); YP003041397 (SEQ ID NO: 14 encode by SEQ ID NO: 13); YP003041380 (SEQ ID NO: 16 encode by SEQ ID NO: 15); ZP17107944 (SEQ ID NO: 16 encode by SEQ ID NO: 15); and ZP05881874 (SEQ ID NO: 18 encode by SEQ ID NO: 17), respectively. FIG. 1A-1B shows an alignment of the amino acid sequences of PIP-47Aa (SEQ ID NO: 1), PIP-47Ba (SEQ ID NO: 4), PIP-47Bb (SEQ ID NO: 6), PIP-47Fa (SEQ ID NO: 8) and the distant homologs: Plu2048 (SEQ ID NO: 10); Plu2046 (SEQ ID NO: 12); YP003041397 (SEQ ID NO: 14); YP003041380 (SEQ ID NO: 16); ZP17107944 (SEQ ID NO: 18); and ZP05881874 (SEQ ID NO: 20). FIG. 3 shows an alignment of the amino acid sequences of PIP-47Aa (SEQ ID NO: 2), PIP-47Ba (SEQ ID NO: 4), PIP-47Bb (SEQ ID NO: 6), and PIP-47Fa (SEQ ID NO: 8). The percent identities of the PIP-47 homologs are shown in Table 5.

TABLE 5

PIP-47Aa
PIP-47Bb
PIP-47Ba
PIP-47Fa
ZP05881874
ZP17107944
YP003041380
Plu2048
YP003041397
Plu2046

(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID

NO: 2)
NO: 4)
NO: 6)
NO: 8)
NO: 20)
NO: 18)
NO: 16)
NO: 10)
NO: 14)
NO: 12)

PIP-47Aa

89
84
45
43
41
41
41
40
36

(SEQ ID

NO: 2)

PIP-47Bb

83
45
45
43
43
43
42
38

(SEQ ID

NO: 4)

PIP-47Ba

43
46
42
43
42
44
39

(SEQ ID

NO: 6)

PIP-47Fa

40
41
37
35
33
32

(SEQ ID

NO: 8)

ZP05881874

46
40
39
37
34

(SEQ ID

NO: 20)

ZP17107944

38
38
39
37

(SEQ ID

NO: 18)

YP003041380

88
50
47

(SEQ ID

NO: 16)

Plu2048

52
49

(SEQ ID

NO: 10)

YP003041397

74

(SEQ ID

NO: 14)

Plu2046

(SEQ ID

NO: 12)

Example 3—E. coli Expression of PIP-47Aa and Homologous Proteins

The PIP-47Aa gene was amplified by PCR using genomic DNA isolated from strain SS62E5 using the PIP-47Aa forward primer (SEQ ID NO: 423) and PIP-47Aa reverse primer (SEQ ID NO: 424). The resulting PCR product was DNA sequence verified and subcloned into pCOLD™-1 (Takara Bio Inc., Seta 3-4-1, Otsu, Shiga, Japan 520-2193) in frame with an N-terminal His-6 tag followed by a Factor Xa cleavage site. Nucleic acid sequences encoding the homologs PIP-47Ba (SEQ ID NO: 3), PIP-47Bb (SEQ ID NO: 5), and PIP-47Fa (SEQ ID NO: 7) were cloned in a similar manner, using their respective genomic DNA preparation as the template for PCR gene amplification and the primer pairs: SEQ ID NO: 425/SEQ ID NO: 426; SEQ ID NO: 427/SEQ ID NO: 428; and SEQ ID NO: 429/SEQ ID NO: 430; respectively.

The coding sequence (SEQ ID NO: 9) for the homolog Plu2046 (SEQ ID NO: 10) was obtained through gene synthesis with compatible 5′ and 3′ ends for downstream cloning into pCOLD™-1.

pCOLD™1 plasmid DNA, containing the respective PIP-47 gene insert, was transformed into competent BL21-DE3 E. coli cells for recombinant protein expression. E. coli cells were grown overnight at 37° C. with carbenicillin selection and then inoculated to a fresh 2×YT medium (1:25) and further grown to an optical density of about 0.8. At that point cells were chilled in the presence of 1 mM ITPG and further grown at 16° C. for 16 hours to induce protein expression. The E. coli expressed His-tagged proteins were purified by immobilized metal ion chromatography using Ni-NTA agarose (Qiagen, Germany) according to the manufacturer's protocols.

Example 4—Insecticidal Activity of PIP-47Aa and Homologous Proteins

A series of concentrations of the purified PIP-47Aa protein (SEQ ID NO: 2) and homologs PIP-47Ba (SEQ ID NO: 4), PIP-47Bb (SEQ ID NO: 6), PIP-47Fa (SEQ ID NO: 8) and Plu2048 (SEQ ID NO: 10) were assayed against coleopteran, lepidopteran and hemipteran species. Concentrations for 50% mortality (LC50) or inhibition of 50% of the individuals (IC50) were calculated in two independent experiments.

To measure insecticidal activities against WCRW (Diabrotica virgifera) bioassays were conducted using 20 ul of the purified protein samples applied topically over 75 ul artificial WCRW diet (Bio-Sery F9800B based) in each of a 96 well bioassay plate (BD Falcon 353910) then air dried. A variable number of neonate Diabrotica virgifera neonates (3 to 9) were placed into each well of the 96 well plate. The assay was run for four days at 25° C. with no light and then scored for mortality and stunting.

Southern Corn rootworm SCRW (Diabrotica undecimpunctata howardi), Northern Corn rootworm (NCRW, Diabrotica barberi) San Antonio beetle (Diabrotica speciosa) sensitivities were assessed in similar fashion. 20 ul of the purified protein samples applied topically over 75 ul artificial SCRW diet (Bio-Sery F9800B based) in each of a 96 well bioassay plate (BD Falcon 353910) then air dried. A variable number of neonates (3 to 5) were placed into each well of the 96 well plate. The assay was run for 4 days at 25° C. with no light and then scored for mortality and stunting.

Crucifer flea beetle, Phyllotreta crucierae assays were conducted with a field collected wild population. The beetles were starved for 48 hours prior for use in assays. Purified protein was tested at various doses using droplet feeding bioassay techniques. A portion of the protein sample was mixed with sucrose suspension supplemented with canola seedling juice and delivered to test insect daily for three days. After three days all surviving beetles were provided with canola leaf disc. Mortality of the beetles was monitored after 10 days.

Lepidoptera feeding assays were conducted on an artificial diet in a 96 well plate set up. The purified protein was incorporated with the Lepidopteran-specific artificial diet in a ratio of 10 ul protein and 40 ul of diet mixture. Two to five neonate larvae were placed in each well to feed ad libitum for 5 days. Results were expressed as positive for larvae reactions such as stunting and or mortality. Results were expressed as negative if the larvae were similar to the negative control that is feeding diet to which the above buffer only has been applied.

PIP-47Aa, PIP-47Ba and PIP-47Bb proteins were assayed on corn earworm (Helicoverpa zea), black cutworm (Agrotis fall armyworm (Spodoptera frugiperda) and soybean looper (Pseudoplusia includens). The insecticidal activity is shown in Table 6.

TABLE 6

protein
insect
highest dose
IC50
effect

PIP-47Aa
SCRW
68
ug/cm2
~20-40
ug/cm
death

SEQ ID NO: 2

PIP-47Aa
Lygus
200
ppm

Inactive at dose tested

SEQ ID NO: 2

PIP-47Aa
SBL
600
ppm

Inactive at dose tested

SEQ ID NO: 2

PIP-47Aa
FAW
68
ug/cm2

Inactive at dose tested

SEQ ID NO: 2

PIP-47Aa
BCW
68
ug/cm2

Inactive at dose tested

SEQ ID NO: 2

PIP-47Aa
CEW
68
ug/cm2

Inactive at dose tested

SEQ ID NO: 2

PIP-47Aa
WCRW
1167
ppm
10-30
ppm
death

SEQ ID NO: 2

PIP-47Aa
NCRW
75
ug/cm2

severe stunting, single

SEQ ID NO: 2

dose only

PIP-47Aa

D. speciosa

1167
ppm
163
ppm
death

SEQ ID NO: 2

PIP-47Aa
Crucifer flea
500
ppm
260
ppm
death

SEQ ID NO: 2
beetle

PIP-47Ba
WCRW
1167
ppm
>400
ppm
death

SEQ ID NO: 4

PIP-47Bb
WCRW
600
ppm
20-40
ppm
death

SEQ ID NO: 6

PIP-47Bb
SCRW
750
ppm
~440
ppm
severe stunting

SEQ ID NO: 6

PIP-47Bb
CEW
580
ppm

Inactive at dose tested

SEQ ID NO: 6

PIP-47Bb
FAW
580
ppm

Inactive at dose tested

SEQ ID NO: 6

PIP-47Bb
ECB
580
ppm

Inactive at dose tested

SEQ ID NO: 6

PIP-47Bb
BCW
580
ppm

Inactive at dose tested

SEQ ID NO: 6

Plu2048
WCRW
1000
ppm

Inactive at dose tested

SEQ ID NO: 10

Example 5—Lack of Cross Resistance of PIP-47Aa in mCry3A Resistant Strain of WCRW

A WCRW strain resistant to mCry3A was developed by selections of WCRW on mCry3A transgenic maize plants with T0 expression level of mCry3A at >10,000 ppm of total proteins in roots (U.S. Ser. No. 13/800,233). Seven selections were made on F3, F6, F7, F8, F10, F12, F14 larvae. F16 eggs of the mCry3A-resistant insects had a resistance ratio (RR) of >46-fold to mCry3A compared with the susceptible laboratory colony, and were used for cross resistance testing of PIP-47Aa. Standardized WCRW diet incorporation bioassays were utilized to evaluate the effects of PIP-47Aa on WCRW larvae. WCRW neonate larvae were placed on the plates containing the bioassay diet and insecticidal protein with 4 replicates for each concentration treatment for 3 days after initiation of each bioassay. Insect mortality and severe stunting was scored and used to calculate inhibitory concentrations (1050 and LC50) based on probit analysis. The resistance ratio (RR) was calculated as follows: RR=(LC/IC50 of resistant WCRW)/(LC/IC50 of susceptible WCRW). As shown in Table 7 mCry3A-resistant WCRW insects were sensitive to PIP-47Aa (SEQ ID NO: 2).

TABLE 7

WCRW

strain
LC/IC
sensitivity, ppm
range, ppm
resistance ratio

mCry3A
LC50
63
44-87
1

sensitive
IC50
14
11-18
1

mCry3A
LC50
46
35-59
0.74

resistant
IC50
12
9-15
0.89

Example 6—Agrobacterium-Mediated Stable Transformation of Maize

For Agrobacterium-mediated maize transformation, the method of Zhao was employed (U.S. Pat. No. 5,981,840 and International Patent Publication Number WO 1998/32326, the contents of which are hereby incorporated by reference). Briefly, immature embryos were isolated from maize and the embryos contacted with an Agrobacterium suspension, where the bacteria were capable of transferring the PIP-47Aa coding sequence (SEQ ID NO: 1) to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos were immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos were co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos were cultured on solid medium with antibiotic, but without a selecting agent, for Agrobacterium elimination and for a resting phase for the infected cells. Next, inoculated embryos were cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). The immature embryos were cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus was then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium were cultured on solid medium to regenerate the plants.

For detection of the PIP-47Aa protein (SEQ ID NO: 2) in leaf tissue 4 lyophilized leaf punches/sample were pulverized and resuspended in 1004 PBS containing 0.1% Tween 20 (PBST), 1% beta-mercaoptoethanol containing 1 tablet/7 mL complete Mini proteinase inhibitor (Roche 1183615301). The suspension was sonicated for 2 min and then centrifuged at 4° C., 20,000 g for 15 min. To a supernatant aliquot 1/3 volume of 3× NuPAGE® LDS Sample Buffer (Invitrogen™ (CA, USA), 1% B-ME containing 1 tablet/7 mL complete Mini proteinase inhibitor was added. The reaction was heated at 80° C. for 10 min and then centrifuged. A supernatant sample was loaded on 4-12% Bis-Tris Midi gels with MES running buffer as per manufacturer's (Invitrogen™) instructions and transferred onto a nitrocellulose membrane using an iBlot® apparatus (Invitrogen™). The nitrocellulose membrane was incubated in PBST containing 5% skim milk powder for 2 hours before overnight incubation in affinity-purified rabbit anti-PIP-47Aa in PBST overnight. The membrane was rinsed three times with PBST and then incubated in PBST for 15 min and then two times 5 min before incubating for 2 hours in PBST with goat anti-rabbit-HRP for 3 hours. The detected proteins were visualized using ECL Western Blotting Reagents (GE Healthcare cat # RPN2106) and Kodak® Biomax® MR film. For detection of the PIP-47Aa protein in roots the roots were lyophilized and 2 mg powder per sample was resuspended in LDS, 1% beta-mercaoptoethanol containing 1 tablet/7 mL Complete Mini proteinase inhibitor was added. The reaction was heated at 80° C. for 10 min and then centrifuged at 4° C., 20,000 g for 15 min. A supernatant sample was loaded on 4-12% Bis-Tris Midi gels with MES running buffer as per manufacturer's (Invitrogen™) instructions and transferred onto a nitrocellulose membrane using an iBlot® apparatus (Invitrogen™). The nitrocellulose membrane was incubated in PBST containing 5% skim milk powder for 2 hours before overnight incubation in affinity-purified polyclonal rabbit anti-PIP-47Aa antibody in PBST overnight. The membrane was rinsed three times with PBST and then incubated in PBST for 15 min and then two times 5 min before incubating for 2 hours in PBST with goat anti-rabbit-HRP for 3 hrs. The antibody bound insecticidal proteins were detected using ECL™ Western Blotting Reagents (GE Healthcare cat # RPN2106) and Kodak® Biomax® MR film.

Transgenic maize plants positive for expression of the insecticidal proteins are tested for pesticidal activity using standard bioassays known in the art. Such methods include, for example, root excision bioassays and whole plant bioassays. See, e.g., US Patent Application Publication Number US 2003/0120054 and International Publication Number WO 2003/018810.

Example 7—Expression Vector Construct for Expression of PIP-47Aa in Plants

The plant expression vector, PHP61246, was constructed to include a transgene cassette containing the PIP-47Aa gene (SEQ ID NO: 2) under control of the Sb-RCc3 promoter (U.S. patent application Ser. No. 13/371,526) in combination with an enhancer element. The PIP-47Aa gene was fused at the N-terminus to a chloroplast transit peptide with the purpose of directing and accumulating the insecticidal protein into plastids within the root. Another vector, PHP61185, under the same enhancer-promoter combination but without chloroplast targeting was also constructed and tested in transgenic maize events for efficacy against corn rootworm.

T0 GH efficacy results for events generated from the PHP61246 and PHP61185 constructs are shown in FIG. 4. Efficacy for events derived both constructs was observed relative to negative control events as measured by root protection from Western corn rootworm. Root protection was measured according to the number of nodes of roots injured (CRWNIS=corn rootworm node injury score) using the method developed by Oleson, et al. (2005) [J. Econ Entomol. 98(1):1-8]. The root injury score is measured from “0” to “3” with “0” indicating no visible root injury, “1” indicating 1 node of root damage, “2” indicating 2 nodes or root damage, and “3” indicating a maximum score of 3 nodes of root damage. Intermediate scores (eg. 1.5) indicate additional fractions of nodes of damage (eg. one and a half nodes injured).

FIG. 4 shows that the majority of events (each star represents a single event) from PHP61246 and PHP61185 perform better than the negative control and have rootworm injury scores of <1.0. A proportion of events from PHP61246 showed excellent CRWNIS scores of <0.5.

Example 8—Creation of Variants of PIP-47Aa with Multiple Amino Acid Substitutions

To create variants of PIP-47Aa (SEQ ID NO: 2) with multiple amino acid changes, variant libraries were generated by family shuffling (Chia-Chun J. Chang et al, 1999, Nature Biotechnology 17, 793-797) of the genes encoding the PIP-47 homologs: PIP-47Aa (SEQ ID NO: 2); PIP-47Ba (SEQ ID NO: 4); PIP-47Bb (SEQ ID NO: 6); and PIP-47Fa (SEQ ID NO: 8). To facilitate homologous recombination among genes, the codon usage of PIP-47Ba and PIP-47Bb were normalized according to that of PIP-47Aa. The mutation rate of the libraries was controlled by mixing different portion of fragmented DNA from PIP-47Ba (SEQ ID NO: 3), PIP-47Bb (SEQ ID NO: 5) with that of PIP-47Aa (SEQ ID NO: 1). Four sub-libraries with expected different mutation rates were generated. The resulting variants from this family of shuffled libraries of PIP-47-Aa, PIP-47Ba and PIP-47Bb were named as “lib-XXX”, where “XXX” is a unique designation for each clone and its sub-library origin.

To create additional PIP-47 polypeptide variants another type of library was generated by semi-synthetic DNA shuffling (Stutzman-Engwall et al, 2005, Metabolic Engineer, 7:27-37) using the PIP-47Bb coding sequence (SEQ ID NO: 5) as the backbone and the library was generated by spiking DNA oligos carrying diversity from the PIP-47Fa coding sequence (SEQ ID NO: 7). The library mutation rate was controlled by varying amount of spiked oligos to the fragmented PIP-047Fa DNA. 5 sub-libraries with different mutation rates were generated. The variants from this library were named as “Fablib2-XXX”, “Fablib3-XXX” or “Fablib4-XXX”, where “XXX” is a unique designation for each clone and its sub-library origin.

Another sequence diversity of PIP-047Aa and PIP-047Bb was generated by replacement of N-terminal 52 amino acids with that of PIP-047Fa. The resulting active variants were named as Fa-Aa-Chim 2 and Fa-Bb-Chim-1.

After transforming the resulting variants from the libraries into E. coli cells, the colonies were picked and cultured in 96-well plates for protein expression. Cell lysates were generated by B-PER® Protein Extraction Reagent from Thermo Fisher Scientific (3747 N Meridian Rd, Rockford, Ill. USA 61101) and screened for WCRW insecticidal activity. The total number of variants screened from all the libraries was 1128. The WCRW active PIP-47 variants were DNA sequenced and the amino acid sequenced deduced.

The resulting “lib-XXX” series WCRW active PIP-47 polypeptide variant clone designations, corresponding coding sequence identifier, and the corresponding protein sequence identifier are shown in Table 8. The resulting “Fablib2-XXX”, “Fablib3-XXX” or “Fablib4-XXX” series WCRW active PIP-47 polypeptide variant clone designations, corresponding coding sequence identifier, and the corresponding protein sequence identifier are shown in Table 8.

TABLE 8

Polynucleotide
Polypeptide SEQ

Clone
SEQ ID NO
ID NO

Falib4-1E1
SEQ ID NO: 21
SEQ ID NO: 221

Falib4-1G1
SEQ ID NO: 22
SEQ ID NO: 222

Falib4-1F2
SEQ ID NO: 23
SEQ ID NO: 223

Falib4-1H2
SEQ ID NO: 24
SEQ ID NO: 224

Falib4-1B3
SEQ ID NO: 25
SEQ ID NO: 225

Falib4-1H7
SEQ ID NO: 26
SEQ ID NO: 226

Falib4-1F9
SEQ ID NO: 27
SEQ ID NO: 227

Falib4-2G1
SEQ ID NO: 28
SEQ ID NO: 228

Falib4-2H1
SEQ ID NO: 29
SEQ ID NO: 229

Falib4-2A4
SEQ ID NO: 30
SEQ ID NO: 230

Falib4-2G5
SEQ ID NO: 31
SEQ ID NO: 231

Falib4-2H5
SEQ ID NO: 32
SEQ ID NO: 232

Falib4-2G7
SEQ ID NO: 33
SEQ ID NO: 233

Falib4-2F9
SEQ ID NO: 34
SEQ ID NO: 234

Falib4-2D10
SEQ ID NO: 35
SEQ ID NO: 235

Falib4-2B11
SEQ ID NO: 36
SEQ ID NO: 236

Falib4-2H11
SEQ ID NO: 37
SEQ ID NO: 237

Falib4-3D2
SEQ ID NO: 38
SEQ ID NO: 238

Falib4-3B5
SEQ ID NO: 39
SEQ ID NO: 239

Falib4-3F6
SEQ ID NO: 40
SEQ ID NO: 240

Falib4-3A7
SEQ ID NO: 41
SEQ ID NO: 241

Falib4-3E7
SEQ ID NO: 42
SEQ ID NO: 242

Falib4-3B9
SEQ ID NO: 43
SEQ ID NO: 243

Falib4-3G9
SEQ ID NO: 44
SEQ ID NO: 244

Falib4-3A12
SEQ ID NO: 45
SEQ ID NO: 245

Falib4-4B1
SEQ ID NO: 46
SEQ ID NO: 246

Falib4-4A2
SEQ ID NO: 47
SEQ ID NO: 247

Falib4-4E2
SEQ ID NO: 48
SEQ ID NO: 248

Falib4-4G2
SEQ ID NO: 49
SEQ ID NO: 249

Falib4-4C3
SEQ ID NO: 50
SEQ ID NO: 250

Falib4-4A4
SEQ ID NO: 51
SEQ ID NO: 251

Falib4-4B4
SEQ ID NO: 52
SEQ ID NO: 252

Falib4-4H4
SEQ ID NO: 53
SEQ ID NO: 253

Falib4-4A5
SEQ ID NO: 54
SEQ ID NO: 254

Falib4-4B5
SEQ ID NO: 55
SEQ ID NO: 255

Falib4-4C5
SEQ ID NO: 56
SEQ ID NO: 256

Falib4-4G5
SEQ ID NO: 57
SEQ ID NO: 257

Falib4-4A6
SEQ ID NO: 58
SEQ ID NO: 258

Falib4-4B6
SEQ ID NO: 59
SEQ ID NO: 259

Falib4-4H6
SEQ ID NO: 60
SEQ ID NO: 260

Falib4-4A7
SEQ ID NO: 61
SEQ ID NO: 261

Falib4-4B7
SEQ ID NO: 62
SEQ ID NO: 262

Falib4-4H7
SEQ ID NO: 63
SEQ ID NO: 263

Falib4-4B8
SEQ ID NO: 64
SEQ ID NO: 264

Falib4-4C9
SEQ ID NO: 65
SEQ ID NO: 265

Falib4-4G9
SEQ ID NO: 66
SEQ ID NO: 266

Falib4-4A10
SEQ ID NO: 67
SEQ ID NO: 267

Falib4-4B10
SEQ ID NO: 68
SEQ ID NO: 268

Falib4-4C10
SEQ ID NO: 69
SEQ ID NO: 269

Falib4-4E10
SEQ ID NO: 70
SEQ ID NO: 270

Falib4-4A12
SEQ ID NO: 71
SEQ ID NO: 271

Falib4-4C12
SEQ ID NO: 72
SEQ ID NO: 272

Fa-Aa-chim 2
SEQ ID NO: 73
SEQ ID NO: 273

Fa-Bb-chim 1
SEQ ID NO: 74
SEQ ID NO: 274

lib-1B1
SEQ ID NO: 75
SEQ ID NO: 275

lib-1D1
SEQ ID NO: 76
SEQ ID NO: 276

lib-1H1
SEQ ID NO: 77
SEQ ID NO: 277

lib-1B2
SEQ ID NO: 78
SEQ ID NO: 278

lib-1D2
SEQ ID NO: 79
SEQ ID NO: 279

lib-1E2
SEQ ID NO: 80
SEQ ID NO: 280

lib-1G2
SEQ ID NO: 81
SEQ ID NO: 281

lib-1H2
SEQ ID NO: 82
SEQ ID NO: 282

lib-1B3
SEQ ID NO: 83
SEQ ID NO: 283

lib-1H3
SEQ ID NO: 84
SEQ ID NO: 284

lib-1B4
SEQ ID NO: 85
SEQ ID NO: 285

lib-1G4
SEQ ID NO: 86
SEQ ID NO: 286

lib-1H4
SEQ ID NO: 87
SEQ ID NO: 287

lib-1A5
SEQ ID NO: 88
SEQ ID NO: 288

lib-1C5
SEQ ID NO: 89
SEQ ID NO: 289

lib-1B6
SEQ ID NO: 90
SEQ ID NO: 290

lib-1G6
SEQ ID NO: 91
SEQ ID NO: 291

lib-1B7
SEQ ID NO: 92
SEQ ID NO: 292

lib-1G7
SEQ ID NO: 93
SEQ ID NO: 293

lib-1H7
SEQ ID NO: 94
SEQ ID NO: 294

lib-1A8
SEQ ID NO: 95
SEQ ID NO: 295

lib-1B8
SEQ ID NO: 96
SEQ ID NO: 296

lib-1F8
SEQ ID NO: 97
SEQ ID NO: 297

lib-1H8
SEQ ID NO: 98
SEQ ID NO: 298

lib-1A9
SEQ ID NO: 99
SEQ ID NO: 299

lib-1C9
SEQ ID NO: 100
SEQ ID NO: 300

lib-1A10
SEQ ID NO: 101
SEQ ID NO: 301

lib-1G10
SEQ ID NO: 102
SEQ ID NO: 302

lib-1B11
SEQ ID NO: 103
SEQ ID NO: 303

lib-1E12
SEQ ID NO: 104
SEQ ID NO: 304

lib-2B1
SEQ ID NO: 105
SEQ ID NO: 305

lib-2A3
SEQ ID NO: 106
SEQ ID NO: 306

lib-2C4
SEQ ID NO: 107
SEQ ID NO: 307

lib-2H4
SEQ ID NO: 108
SEQ ID NO: 308

lib-2A5
SEQ ID NO: 109
SEQ ID NO: 309

lib-2C5
SEQ ID NO: 110
SEQ ID NO: 310

lib-2E5
SEQ ID NO: 111
SEQ ID NO: 311

lib-2D6
SEQ ID NO: 112
SEQ ID NO: 312

lib-2A7
SEQ ID NO: 113
SEQ ID NO: 313

lib-2B7
SEQ ID NO: 114
SEQ ID NO: 314

lib-2E8
SEQ ID NO: 115
SEQ ID NO: 315

lib-2H8
SEQ ID NO: 116
SEQ ID NO: 316

lib-2H9
SEQ ID NO: 117
SEQ ID NO: 317

lib-2D11
SEQ ID NO: 118
SEQ ID NO: 318

lib-2A12
SEQ ID NO: 119
SEQ ID NO: 319

lib-2B12
SEQ ID NO: 120
SEQ ID NO: 320

lib-2D12
SEQ ID NO: 121
SEQ ID NO: 321

lib-2F12
SEQ ID NO: 122
SEQ ID NO: 322

lib-3B1
SEQ ID NO: 123
SEQ ID NO: 323

lib-3E1
SEQ ID NO: 124
SEQ ID NO: 324

lib-3D2
SEQ ID NO: 125
SEQ ID NO: 325

lib-3G2
SEQ ID NO: 126
SEQ ID NO: 326

lib-3E3
SEQ ID NO: 127
SEQ ID NO: 327

lib-3H3
SEQ ID NO: 128
SEQ ID NO: 328

lib-3A4
SEQ ID NO: 129
SEQ ID NO: 329

lib-3D4
SEQ ID NO: 130
SEQ ID NO: 330

lib-3H4
SEQ ID NO: 131
SEQ ID NO: 331

lib-3G6
SEQ ID NO: 132
SEQ ID NO: 332

lib-3F8
SEQ ID NO: 133
SEQ ID NO: 333

lib-3B9
SEQ ID NO: 134
SEQ ID NO: 334

lib-3B10
SEQ ID NO: 135
SEQ ID NO: 335

lib-3E10
SEQ ID NO: 136
SEQ ID NO: 336

lib-3H10
SEQ ID NO: 137
SEQ ID NO: 337

lib-3B11
SEQ ID NO: 138
SEQ ID NO: 338

lib-3F11
SEQ ID NO: 139
SEQ ID NO: 339

lib-4C1
SEQ ID NO: 140
SEQ ID NO: 340

lib-4D1
SEQ ID NO: 141
SEQ ID NO: 341

lib-4B2
SEQ ID NO: 142
SEQ ID NO: 342

lib-4G2
SEQ ID NO: 143
SEQ ID NO: 343

lib-4A3
SEQ ID NO: 144
SEQ ID NO: 344

lib-4B3
SEQ ID NO: 145
SEQ ID NO: 345

lib-4A4
SEQ ID NO: 146
SEQ ID NO: 346

lib-4G4
SEQ ID NO: 147
SEQ ID NO: 347

lib-4H4
SEQ ID NO: 148
SEQ ID NO: 348

lib-4D5
SEQ ID NO: 149
SEQ ID NO: 349

lib-4H5
SEQ ID NO: 150
SEQ ID NO: 350

lib-4G8
SEQ ID NO: 151
SEQ ID NO: 351

lib-4C9
SEQ ID NO: 152
SEQ ID NO: 352

lib-4F9
SEQ ID NO: 153
SEQ ID NO: 353

lib-4A10
SEQ ID NO: 154
SEQ ID NO: 354

lib-4A11
SEQ ID NO: 155
SEQ ID NO: 355

lib-4B11
SEQ ID NO: 156
SEQ ID NO: 356

lib-4A12
SEQ ID NO: 157
SEQ ID NO: 357

lib-4C12
SEQ ID NO: 158
SEQ ID NO: 358

lib-4E12
SEQ ID NO: 159
SEQ ID NO: 359

Falib5-1G1
SEQ ID NO: 160
SEQ ID NO: 360

Falib5-1A2
SEQ ID NO: 161
SEQ ID NO: 361

Falib5-1E2
SEQ ID NO: 162
SEQ ID NO: 362

Falib5-1G2
SEQ ID NO: 163
SEQ ID NO: 363

Falib5-1H2
SEQ ID NO: 164
SEQ ID NO: 364

Falib5-1A3
SEQ ID NO: 165
SEQ ID NO: 365

Falib5-1E3
SEQ ID NO: 166
SEQ ID NO: 366

Falib5-1G4
SEQ ID NO: 167
SEQ ID NO: 367

Falib5-1C5
SEQ ID NO: 168
SEQ ID NO: 368

Falib5-1B6
SEQ ID NO: 169
SEQ ID NO: 369

Falib5-1F6
SEQ ID NO: 170
SEQ ID NO: 370

Falib5-1G6
SEQ ID NO: 171
SEQ ID NO: 371

Falib5-1B7
SEQ ID NO: 172
SEQ ID NO: 372

Falib5-1G7
SEQ ID NO: 173
SEQ ID NO: 373

Falib5-1F8
SEQ ID NO: 174
SEQ ID NO: 374

Falib5-1G9
SEQ ID NO: 175
SEQ ID NO: 375

Falib5-1C10
SEQ ID NO: 176
SEQ ID NO: 376

Falib5-1G10
SEQ ID NO: 177
SEQ ID NO: 377

Falib5-1E11
SEQ ID NO: 178
SEQ ID NO: 378

Falib5-1F12
SEQ ID NO: 179
SEQ ID NO: 379

Falib5-1G12
SEQ ID NO: 180
SEQ ID NO: 380

Falib5-2D1
SEQ ID NO: 181
SEQ ID NO: 381

Falib5-2H1
SEQ ID NO: 182
SEQ ID NO: 382

Falib5-2B2
SEQ ID NO: 183
SEQ ID NO: 383

Falib5-2D2
SEQ ID NO: 184
SEQ ID NO: 384

Falib5-2E2
SEQ ID NO: 185
SEQ ID NO: 385

Falib5-2F2
SEQ ID NO: 186
SEQ ID NO: 386

Falib5-2C3
SEQ ID NO: 187
SEQ ID NO: 387

Falib5-2G3
SEQ ID NO: 188
SEQ ID NO: 388

Falib5-2H3
SEQ ID NO: 189
SEQ ID NO: 389

Falib5-2H4
SEQ ID NO: 190
SEQ ID NO: 390

Falib5-2G5
SEQ ID NO: 191
SEQ ID NO: 391

Falib5-2A6
SEQ ID NO: 192
SEQ ID NO: 392

Falib5-2D7
SEQ ID NO: 193
SEQ ID NO: 393

Falib5-2D8
SEQ ID NO: 194
SEQ ID NO: 394

Falib5-2E8
SEQ ID NO: 195
SEQ ID NO: 395

Falib5-2A9
SEQ ID NO: 196
SEQ ID NO: 396

Falib5-2B9
SEQ ID NO: 197
SEQ ID NO: 397

Falib5-2E9
SEQ ID NO: 198
SEQ ID NO: 398

Falib5-2H9
SEQ ID NO: 199
SEQ ID NO: 399

Falib5-2D11
SEQ ID NO: 200
SEQ ID NO: 400

Falib5-2F11
SEQ ID NO: 201
SEQ ID NO: 401

Falib5-2G11
SEQ ID NO: 202
SEQ ID NO: 402

Falib5-2A12
SEQ ID NO: 203
SEQ ID NO: 403

Falib5-3A2
SEQ ID NO: 204
SEQ ID NO: 404

Falib5-3E2
SEQ ID NO: 205
SEQ ID NO: 405

Falib5-3D3
SEQ ID NO: 206
SEQ ID NO: 406

Falib5-3G4
SEQ ID NO: 207
SEQ ID NO: 407

Falib5-3C5
SEQ ID NO: 208
SEQ ID NO: 408

Falib5-3D5
SEQ ID NO: 209
SEQ ID NO: 409

Falib5-3D10
SEQ ID NO: 210
SEQ ID NO: 410

Falib5-4F1
SEQ ID NO: 211
SEQ ID NO: 411

Falib5-4F3
SEQ ID NO: 212
SEQ ID NO: 412

Falib5-4C4
SEQ ID NO: 213
SEQ ID NO: 413

Falib5-4D4
SEQ ID NO: 214
SEQ ID NO: 414

Falib5-4H4
SEQ ID NO: 215
SEQ ID NO: 415

Falib5-4B5
SEQ ID NO: 216
SEQ ID NO: 416

Falib5-4C5
SEQ ID NO: 217
SEQ ID NO: 417

Falib5-4H5
SEQ ID NO: 218
SEQ ID NO: 418

Falib5-4D7
SEQ ID NO: 219
SEQ ID NO: 419

Falib5-4F12
SEQ ID NO: 220
SEQ ID NO: 420

The percent identity of the PIP-47 polypeptide variants compared to PIP-47Aa (SEQ ID NO: 2) and the number of amino acid changes compared to PIP-47Aa (SEQ ID NO: 2) are shown in Table 9. The diversity of the resulting amino acid substitutions at each position of the WCRW active PIP-47 polypeptide variants compared to PIP-41Aa (SEQ ID NO: 2) is shown in Table 10. The amino acid substitutions of the WCRW active PIP-47 polypeptide variants compared to PIP-47Aa (SEQ ID NO: 2) are shown are shown in Table 11.

TABLE 9

# of a.a.

% Identity to
substitutions

PIP-47Aa (SEQ
compared to

ID NO: 2)
SEQ ID NO: 2
Variants designation

75%
76
Falib5-1A3

80%
61-63
Falib4-2H11, Falib5-1B6, Falib5-1G4, Falib5-2A12,

81%
58-60
Falib4-4B1, Falib4-4C5, Falib4-4H7, Fa-Bb-Chim 1, Falib5-1F8, Falib5-2A9,

Falib5-2F2, Falib5-2H3

82%
55-57
Falib4-4C9, Falib5-1G2, Falib5-2B2, Falib5-2D1, Falib5-2G3, Falib5-4D4

83%
53-54
Falib4-3B5, Falib4-4A2, Falib4-4B7, Falib4-4G2, Falib5-1B7, Falib5-1E3,

Falib5-2A6, Falib5-4H4

84%
49-53
Falib4-2A4, Falib4-3E7, Falib4-4B6, Falib4-4E10, Falib4-4E2, Falib4-4G5,

Falib5-1A2, Falib5-1C5, Falib5-1F12, Falib5-1G10, Falib5-1G12, Falib5-2D11,

Falib5-2D2, Falib5-2E8, Falib5-2G11, Falib5-2H1, Falib5-2H4, Falib5-3C5,

Falib5-3D5, Falib5-4B5, Falib5-4D7, Falib5-4F12

85%
47-48
Falib4-2F9, Falib4-2G7, Falib4-3F6, Falib4-3G9, Falib4-4A12, Falib4-4C10,

Falib4-4C12, Falib4-4C3, Falib4-4H4, Falib5-1F6, Falib5-1G1, Falib5-2B9,

Falib5-2E2, Falib5-2F11, Falib5-4C4, Falib5-4F3

86%
44-46
Falib4-1B3, Falib4-1H2, Falib4-2G1, Falib4-2H1, Falib4-3B9, Falib4-4A10,

Falib4-4A4, Falib4-4A7, Falib4-4B10, Falib4-4B4, Falib4-4G9, Falib4-4H6, Fa-

Aa-Chim-2, Falib5-1E11, Falib5-1E2, Falib5-1G6, Falib5-1G7, Falib5-1G9,

Falib5-2C3, Falib5-2D7, Falib5-2D8, Falib5-2E9, Falib5-2G5, Falib5-3D10,

Falib5-3E2, Falib5-3G4, Falib5-4H5

87%
40-41
lib-2A12, Falib4-1G1, Falib4-1H7, Falib4-3A7, Falib4-4B5, Falib5-3D3

88%
38-41
lib-1D1, lib-2A7, lib-1G4, lib-3H3, lib-3G6, lib-3D2, lib-3E3, lib-4A11, Falib4-

1E1, Falib4-1F2, Falib4-1F9, Falib4-2B11, Falib4-2D10, Falib4-3A12, Falib4-

3D2, Falib4-4A5, Falib4-4A6, Falib5-1C10, Falib5-1H2, Falib5-2H9, Falib5-4C5

89%
34-38
lib-1G2, lib-1B4, lib-4D1, lib-1H4, lib-4B11, lib-1A9, lib-3B1, lib-1H2, lib-3E1,

lib-2B1, lib-4C9, Falib4-2G5, Falib4-2H5, Falib4-4B8, Falib5-3A2, Falib5-4F1,

lib-1B2

90%
31-34
lib-4C12, lib-4A12, lib-1G7, lib-4H4, lib-1B8, lib-2C5, lib1E12

91%
27-31
lib-2B12, lib-2F12, lib-1G10, lib-1A5, lib-3A4, lib-4E12, lib-3B9, lib-2H4, lib-

1B6, lib-1B7, lib-3D4, lib-1D2, lib-2D11, lib-2H8, lib-1F8, lib-1B1, lib-2A3, lib-

3H10, lib-1C5, lib-2H9

92%
22-25
lib-3H4, lib-2C4, lib-4A4, lib-2D12, lib-4F9, lib-1A10, lib-1E2, lib-4B3, lib-2D6,

lib-4A3, lib-2E8, lib-1B11, lib-4D5, lib-1H3, lib-3E10, lib-4H5

93%
20-21
lib-2A5, lib-3B10, lib-4B2

94%
16-19
lib-2E5, lib-4A10, lib-1A8, lib-4G4, lib-1C9, lib-1H1, lib-1H7, lib-4G2

95%
15-16
lib-1G6, lib-1B3, lib-4G8

96%
12-14
lib-3F8, lib-3G2, lib-3B11

97%
9
lib-4C1

98%
6
lib-3F11

TABLE 10

Position
PIP-47Aa
Derivative

2
H
S,

3
A
T,

4
P
Q,

5
G
N, Deleted

6
A
H, Deleted

7
I
A, K,

Deleted

8
P
H, Deleted

9
S
T

10
E
V, T

11
K
E

12
E
K,

13
S
G, V, L,

14
A
L,

15
H
A

16
A
W

17
W
L,

18
L
N

19
T
E,

20
E
T, G, S,

21
T
A, E, K, H,

Deleted

22
K
E, T, A

23
A
S, R

24
N
R, K, V

25
A
V, K, E

26
K
S, L, N

27
S
F

28
T
A

29
A
T, Q, I

30
L
T, V, A

31
R
N, H,

32
G
P

34
I
F

35
F
Y

36
A
V, S

37
Q
R, H

38
D
E

39
Y
R

40
N
S, H

41
R
H

41/42
—
Insert-P

42
Q
S, H

43
L
Q, S

44
L
F, V

45
T
S

46
A
E, P, S

47
T
S

48
G
L,

49
Q
R, L

50
S
T, A

51
M
P

52
R
G

53
S
T

57
A
I

58
I
D, A

62
F
Y

63
S
G, A

65
A
T

66
K
G, S

67
G
S

68
T
G, A, L

71
G
D, A,

72
S
G, R, N

74
A
L

75
K
Q, N

76
D
A

78
D
N

79
A
N, K, S

80
N
D

81
V
A

84
G
S

85
S
T, K

86
A
V

88
V
E, T

90
I
F, L

91
Y
Q, C, H

92
E
D, K

96
T
S

97
A
S, C

98
I
V

99
D
N

100
I
T, M

103
R
T

106
G
E

107
Y
W

110
L
N

112
Q
P

115
D
G, A

116
Q
A, R

117
K
R, N

119
K
H

120
S
T, A

121
A
T, K

124
R
Y

130
F
L,

132
Q
K,

133
Q
E

135
A
S

136
E
S, R, N

137
I
N

141
S
T

142
L
P

143
L
E

144
A
S

146
Q
S

147
V
S

148
T
S

149
Q
L,

150
I
V

151
Q
R, M

153
K
Q

156
D
G

158
N
D

161
V
I

164
F
C

165
V
A

168
F
S

169
I
H

170
G
D

172
K
E, A

173
N
T, A

174
Q
A, K

176
V
I

178
Q
E

179
I
T

181
Q
R, K

182
S
F, G

184
S
K,

185
S
E, N,

188
N
S, K

193
Y
E,

194
S
F, C

196
Q
K,

198
E
N

200
Q
E

202
N
F

203
F
L,

206
N
H

207
I
T

208
L
M

212
D
N, I

213
S
G, A

222
A
P

226
T
S

228
K
V

230
S
T, A

231
S
D, N

232
H
Q, K

235
T
A

236
I
L,

237
T
N

243
T
S, L

245
S
T

246
Q
P

247
A
S

248
I
V, K

250
Q
T

253
V
G

254
E
G

255
S
G

257
N
E

260
K
R

262
V
T,

264
S
A, Y

265
K
D, E, R, N

267
Q
L,

272
Q
E, N

273
Q
T, K

275
L
P,

276
G
D

277
D
N

281
Q
P

282
V
G,

283
R
K

284
E
A, P

287
K
S

295
S
G

TABLE 11

Clone

designation
Mutation List (vs PIP-47Aa)

Falib4-1E1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, I100M,

R103T, S120A, E136N, D212N, S213G, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-1G1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, D212N, S213G, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-1F2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, A074V, D078N, A079N, V081A, A097S, S120A,

R124Y, E136N, D212N, S213G, T237N, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-1H2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

A097S, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, D212N, S213G, V262T, Q272N, Q273T,

G276D, Q281P, R283K, S295G

Falib4-1B3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071D, D078N, A079N, A097S,

S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272N, Q273T, G276D,

Q281P, R283K, S295G

Falib4-1H7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, S063A, K066G, G067S, T068L, G071D, D078N, A079N, A097S, S120A, E136N,

D212N, S213G, T243L, S245T, A247S, I248K, Q250T, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-1F9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, I100M,

R103T, S120A, E136N, D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-2G1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066S, T068A, G071D, D078N, A079N, A097S, S120A,

E136N, N188K, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272N, Q273T, G276D,

Q281P, R283K, S295G

Falib4-2H1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, Q132K, Q133E, A135S, E136R, D212N, S213G, T237N, V262T, Q272N, Q273T,

G276D, Q281P, R283K, S295G

Falib4-2A4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071A, S072R, A074L, K075Q,

D076A, D078N, A079N, V081A, A097S, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q,

T237N, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-2G5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D212N,

S213G, T237N, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-2H5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, R124Y, E136N,

D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-2G7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A,

D078N, A079N, A097C, I098V, D099N, I100M, R103T, S120A, R124Y, E136N, D212N, S213G, T237N, V262T,

Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-2F9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066G, G067S, T068L, G071A, S072R, A074L, K075Q, D076A, D078N,

A079N, A097S, D115A, Q116A, K117N, K119H, S120T, A121T, Q132K, Q133E, A135S, E136R, D212N, S213G,

V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

2D10
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V081A, A097S, S120A, E136N,

D212N, S213G, T237N, V262T, S264Y, K265D, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

2B11
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, S085T, I090F, A097S, S120A,

E136N, D212N, S213G, E254G, V262T, Q272N, Q273K, G276D, Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

2H11
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D076A, D078N, A079N, A097C, I098V, D099N,

I100M, R103T, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, S141T, L143E, A144S, Q146S,

V147S, T148S, Q149L, I150V, Q151M, K153Q, N188K, D212N, S213G, T226S, K228V, S230T, S231N, H232Q,

T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-3D2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, S063A, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D212N,

S213G, T226S, K228V, S230T, S231D, H232Q, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-3B5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

A097S, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, V176I, Q178E, I179T, Q181R, S182G,

S184K, S185E, N188K, D212N, S213G, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-3F6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

A097S, S120A, E136N, N188K, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, V262T, S264Y, K265D,

Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-3A7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D212N,

S213G, T237N, T243L, S245T, A247S, I248K, Q250T, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-3E7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, S063A, K066S, T068A, G071D, D078N, A079N, V088T, I090F, Y091H,

E092D, A097C, I098V, I100M, S120A, Q133E, A135S, E136S, N202F, N206H, D212N, S213G, S231N, T237N,

T243S, A247S, I248K, V262T, K265N, Q272E, G276D, Q281P, R283K, S295G

Falib4-3B9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A097S, Y107W, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T,

Q272E, G276D, Q281P, R283K, S295G

Falib4-3G9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A097C, I098V, D099N, I100M, S120A, E136N, I169H, K172E, N173T, Q174A, V176I, D212N, S213G,

V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

3A12
D038E, R041H, A046P, Q049L, S063A, K066G, G067S, T068L, G071D, D078N, A079N, A097C, I098V, D099N,

I100M, R103T, S120A, E136N, D212N, S213G, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-4B1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, S063A, K066G, G067S, T068L, G071A, S072R, A074L, K075Q, D076A, D078N,

A079N, V081A, A097S, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, I169H, K172E, N173T,

Q174A, V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, Y193C, D212N, S213G, V262T, Q272E,

G276D, Q281P, R283K, S295G

Falib4-4A2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, I100M,

R103T, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, S185N, N188K, Y193E, S194C, Q196K,

E198N, Q200E, N202F, N206H, D212N, S213G, V262T, S264P, Q272E, G276D, Q281P, R283K, S295G

Falib4-4E2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V081A, A097C, I098V, D099N,

I100M, R103T, S120A, E136N, Y193E, S194C, Q196K, E198N, Q200E, N202F, D212N, S213G, T226S, K228V,

S230T, S231D, H232Q, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-4G2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, V088T, I090L, Y091Q, E092D, A097S, S120A, Q132K, Q133E, A135S, E136R, L208M, D212N, S213G,

T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-4C3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, I100M,

R103T, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, S264Y, K265D,

Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-4A4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068G, G071D, D078N, A079N, A097S, I100M, R103T, Y107W,

L110N, Q112P, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, V262T, Q272N, Q273T,

G276D, Q281P, R283K, S295G

Falib4-4B4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, I100M,

R103T, S120A, E136N, V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, D212N, S213G, V262T,

Q272E, G276D, Q281P, R283K, S295G

Falib4-4H4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T,

Q272E, G276D, D277N, Q281P, R283K, S295G

Falib4-4A5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A097S, S120A, E136N, D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-4B5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D212N,

S213G, T226S, T237N, T243L, S245T, A247S, I248K, Q250T, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-4C5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037R,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071A, S072R, A074L, K075Q,

D076A, D078N, A079N, V081A, A097S, S120A, E136N, Q196K, E198N, Q200E, N202F, D212N, S213G, T226S,

K228V, S230T, S231D, H232Q, T237N, T243L, S245T, A247S, I248K, Q250T, V262T, Q272N, Q273T, G276D,

Q281P, R283K, S295G

Falib4-4G5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A086V, A097S, S120A, E136N, V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, Y193E, S194C,

Q196K, D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-4A6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, S063A, K066G, G067S, T068L, G071D, D078N, A079N, V081A, A097S, S120A,

Q132K, Q133E, A135S, E136R, D212N, S213G, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-4B6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, F168S, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, D212N,

S213G, V262T, S264Y, K265D, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-4H6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A097S, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272E,

G276D, Q281P, R283K, S295G

Falib4-4A7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V081A, A097C, I098V, D099N,

I100M, R103T, S120A, E136N, D212N, S213G, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D, Q281P,

R283K, S295G

Falib4-4B7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, Y091C, A097C, I098V, D099N, I100M, R103T, S120A, Q132K, Q133E, A135S, E136R, D212N, S213G,

T237N, T243L, S245T, A247S, I248K, Q250T, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-4H7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, I034F,

Q037H, D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q,

E092D, A097C, I098V, D099N, I100M, R103T, S120A, R124Y, E136N, Y193E, S194C, Q196K, E198N, Q200E,

N202F, N206H, I207T, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272N, Q273T,

G276D, Q281P, R283K, S295G

Falib4-4B8
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D212N,

S213G, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-4C9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, Y193E,

S194C, Q196K, E198N, Q200E, N202F, D212N, S213G, T226S, K228V, V262T, Q272E, G276D, Q281P, R283K,

S295G

Falib4-4G9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A097C, I098V, D099N, I100M, R103T, S120A, E136N, D212N, S213G, T237N, V262T, Q272E, G276D,

Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

4A10
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

A097S, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, V262T, Q272E, G276D, Q281P,

R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

4B10
D038E, R041H, A046P, Q049L, S063A, K066G, G067S, T068L, G071D, D078N, A079N, A097S, R103T, S120A,

R124Y, E136N, Y193E, S194C, Q196K, E198N, Q200E, N202F, D212N, S213G, V262T, Q272N, Q273T, G276D,

Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

4C10
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, I100M,

R103T, S120A, E136N, V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, D212N, S213G, V262T,

Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

4E10
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, Y107W, L110N, Q112P,

D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, F130L, E136N, D212N, S213G, T243L, S245T, A247S,

I248K, Q250T, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

4A12
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, S120A,

E136N, Q196K, E198N, Q200E, N202F, N206H, I207T, D212N, S213G, A222P, T226S, K228V, S230T, S231D,

H232Q, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib4-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

4C12
D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071A, S072R, A074L, K075Q,

D076A, D078N, A079N, V081A, A097C, I098V, D099N, I100M, R103T, S120A, R124Y, E136N, D212N, S213G,

T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Fa-Aa-chim 2
H002S, A003T, P004Q, G005N, A006H, I007K, P008H, S009I, E010T, K011E, E012K, S013T, A014L, H015A,

A016W, W017L, L018N, E020T, T021H, K022E, A023S, A025K, K026L, A029Q, L030T, R031N, G032P, A036V,

Q037L, Y039R, N040S, Q042S, L043S, L044F, T045S, A046E, T047S, G048L, Q049L, S050T, M051P, R052G

Fa-Bb-chim 1
H002S, A003T, P004Q, G005N, A006H, I007K, P008H, S009I, E010T, K011E, E012K, S013T, A014L, H015A,

A016W, W017L, L018N, E020T, T021H, K022E, A023S, A025K, K026L, A029Q, L030T, R031N, G032P, A036V,

Q037L, Y039R, N040S, Q042S, L043S, L044F, T045S, A046E, T047S, G048L, Q049L, S050T, M051P, R052G,

F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D212N, S213G, V262T, Q272E, G276D,

Q281P, R283K, S295G

lib-1B1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, A046P,

Q049L, G071D, D078N, A079N, A097S, K117R, S120A, S230T, H232K, N257E, K260R, S264A, G276D

lib-1D1
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, K066A, T068A, G071D, K075N,

D078N, A079K, S120A, F203L, S230T, H232K, V253G, N257E, K260R, S264A, Q281P, R283K, E284P, K287S

lib-1H1
G005Del, A006Del, I007Del, P008Del, N024K, K026N, A029I, L030V, Q037H, S053T, D212N, S213G, S264A,

G276D, Q281P, R283K, E284P, K287S

lib-1B2
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, D038E, R041H, A046P, Q049L, K066A, T068A, G071D, K075N, D078N, A079K, S085K, I248V, N257E,

K260R, S264A, G276D, R283K, E284P, K287S, S295G

lib-1D2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, R041H,

A046P, Q049L, K066A, T068A, D078N, A079N, S085K, A097S, E136N, D212I, S213A, Q281P, R283K, S295G

lib-1E2
I007A, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H, Q037H,

Q049R, S050A, S053T, T068A, S085K, K117R, S120A, A121K, Q181K, I248V, N257E, K260R, S264A

lib-1G2
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049L, F062Y, K066A, T068A, G071D, K075N, D078N,

A079K, E136N, D212N, S213G, I248V, N257E, K260R, Q281P, R283K, E284P, K287S

lib-1H2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, K066A, T068A, G071D, K075N, D078N, A079K, S085K, G106E, D212I, S213A,

Q272E, G276D, Q281P, R283K, E284P, K287S, S295G

lib-1B3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, Q049R, S050A, S053T, F062Y, S230T, H232K,

S264A, Q272E, K287S

lib-1H3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, G106E, S230T, H232K

lib-1B4
S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H, Q037H,

R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, K066A, T068A, G071D, K075N, D078N,

A079K, S085K, A097S, S120A, V161I, D212N, S213G, I248V, N257E, K260R, S264A

lib-1G4
I007A, S013V, T019E, E020G, T021E, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, F062Y, K066S, T068A, G071D,

K075N, D078N, A079K, S085K, A097S, G106E, S120A, D212I, S213A, I248V, N257E, K260R, S264A, S295G

lib-1H4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, T019E, E020G, T021K, K022A, A023R, N024V,

A025K, K026S, S027F, A029T, L030A, R031H, Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R,

S050A, S053T, K066A, T068A, G071D, D078N, A079N, D212I, S213A, V262T, Q281P, R283K, E284P, K287S

lib-1A5
I007A, S013V, T019E, E020G, T021Del, N024R, A025V, A029T, L030A, R031H, K066A, T068A, G071D, D078N,

A079K, S085K, A097S, K117R, S120A, A121K, D212I, S213A, K260R, S264A, Q281P, R283K, E284P, K287S

lib-1C5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

R041H, Ins41/42P, Q042H, L043Q, K075N, D078N, A079K, A097S, G106E, K117R, N257E, K260R, S264A,

G276D

lib-1B6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, S053T,

K066A, T068A, G071D, D078N, A079K, S085K, D212N, S213G, N257E, K260R, S264A, Q281P, R283K, E284P,

K287S

lib-1G6
S013L, A097S, K117R, S120A, A121K, D212I, S213A, S230T, H232K, N257E, K260R, G276D, Q281P, R283K,

S295G

lib-1B7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, A046P,

Q049L, A097S, K117R, S120A, A121K, D212I, S213A, I248V, N257E, K260R, Q281P, R283K, E284P, K287S

lib-1G7
I007A, S013G, T019E, E020S, T021E, K022A, A023R, N024V, A025E, K026S, S027F, A029T, L030A, R031H,

F035Y, A036S, Q037H, D038E, N040H, R041H, F062Y, K066A, T068A, G071D, D078N, A079N, G106E, D212N,

S213G, S230T, H232K

lib-1H7
G005Del, A006Del, I007Del, P008Del, S009T, S013L, K022T, N024K, K026N, A029I, L030V, D078N, A079N,

A097S, Q181K, D212I, S213A, K260R

lib-1A8
I007A, K066S, T068A, G071D, D078N, A079N, A097S, K117R, S120A, A121K, D212I, S213A, V262T, Q272E,

G276D, Q281P, R283K

lib-1B8
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049R, S050A, S053T, S120A, E136N, D212N, S213G, I248V, S264A, G276D, Q281P,

R283K, S295G

lib-1F8
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, G106E, D212I, S213A, S230T, H232K, N257E, K260R, S264A, Q281P, R283K,

S295G

lib-1H8
H002Del, A003Del, P004Del, G005Del, A006Del, I007Del, P008Del, S009Del, E010Del, K011Del, E012Del,

S013Del, A014Del, H015Del, A016Del, W017Del, L018Del, T019Del, E020Del, T021Del, K022Del, A023Del,

N024Del, A025Del, K026Del, S027Del, T028Del, A029Del, L030Del, R031Del, G032Del, N033Del, I034Del,

A036Del, Q037Del, D038Del, Y039Del, N040Del, R041Del, Q042Del, L043Del, L044Del, T045Del, A046Del,

T047Del, G048Del, Q049Del, S050Del, K066S, T068A, G071D, D078N, A079N, G106E, D212N, S213G, S230T,

N257E, Q281P, R283K, E284A, K287S, S295G

lib-1A9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, A029T, L030A, R031H, Q037H, R041H,

Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, F062Y, K066S, T068A, G071D, D078N, A079N,

S120A, E136N, D212N, S213G, S230T, Q281P, R283K, E284P, K287S, S295G

lib-1C9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K066A, T068A, G071D, S085K, A097S, G106E,

D212I, S213A, I248V, V262T

lib-1A10
I007A, S013V, T019E, E020G, T021Del, N024R, A025V, A029T, L030A, R031H, Q037H, D038E, R041H, A046P,

K066A, T068A, G071D, G106E, V165A, I248V, N257E, K260R, G276D, Q281P

lib-1G10
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, S120A, D212I, S213A,

G276D, Q281P, R283K

lib-1B11
I007A, K022T, N024K, K026N, A029I, L030V, Q037H, D038E, R041H, A046P, Q049L, K066A, T068A, G071D,

A097S, K117R, E136N, V161I, D212I, S213A, N257E, K260R, S264A, Q281P, R283K

lib-1E12
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, F062Y, K066S, T068A, G071D, D078N, A079K, K117R, S120A, A121K, Q181K, D212I, S213A,

I248V, N257E, K260R, S264A

lib-2B1
G005Del, A006Del, I007Del, P008Del, S009T, S013L, K022T, N024K, K026N, A029I, L030V, Q037H, R041H,

Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, D078N, A079N, V161I, D212N, S213G, S230T, I236L,

N257E, K260R, S264A, Q281P, R283K, E284P, K287S

lib-2A3
I007A, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H, R041H, Ins41/42P, Q042H,

L043Q, A046S, Q049R, S050A, S053T, K066A, T068A, G071D, K075N, D078N, A079K, G106E, D212N, S213G,

S295G

lib-2C4
I007A, S013V, A046P, Q049L, F062Y, K066A, T068A, G071D, K075N, D078N, A079K, A097S, K172A, N173A,

Q174K, D212N, S213G, T235A, N257E, K260R, S264A, Q281P, R283K, E284P, K287S

lib-2H4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, K066S,

T068A, G071D, D078N, A079N, G106E, S120A, D212N, S213G, V262T, G276D, Q281P, R283K, S295G

lib-2A5
S013V, Q037H, D038E, R041H, T068A, G071D, K075N, D078N, A079N, G106E, K117R, S120A, A121K, D212N,

S213G, I248V, S264A, Q281P, R283K, S295G

lib-2C5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, K066A, D078N, A079N, V081A, S120A,

D212N, S213G, N257E, K260R, S264A, S295G

lib-2E5
I007A, A046P, Q049L, K066A, T068A, G071D, D078N, A079N, S120A, E136N, D212I, S213A, S230T, N257E,

K260R, S264A, Q281P, R283K, S295G

lib-2D6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, S053T,

F062Y, K066S, T068A, G071D, D212N, S213G, S230T, H232K, N257E, K260R, S264A

lib-2A7
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, K066A, T068A, G071D, D078N, A079N,

S120A, D212N, S213G, S230T, H232K, N257E, K260R, S264A, G276D, Q281P, R283K, E284P, K287S, S295G

lib-2B7
I007A, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H, D038E, R041H, A046P, Q049L,

K066S, T068A, G071D, K075N, D078N, A079K, S085K, D212I, S213A, I248V, N257E, K260R, S264A, G276D,

Q281P, R283K, E284P, K287S

lib-2E8
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, K066A,

T068A, G071D, D078N, A079N, N257E, K260R, S264A, Q281P, R283K, E284P, K287S

lib-2H8
I007A, S013V, K022T, N024K, K026N, A029I, L030V, Q037H, D038E, R041H, A046P, Q049L, K066A, T068A,

G071D, N080D, S120A, E136N, V161I, D212N, S213G, N257E, K260R, Q281P, R283K, S295G

lib-2H9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A046P, Q049L, F062Y,

K066S, T068A, G071D, D078N, A079N, A097S, S120A, N158D, D212I, S213A, I248V, N257E, K260R, S264A

lib-2D11
I007A, S013L, K022T, N024K, K026N, A029I, L030V, Q037H, D038E, R041H, A046P, Q049L, F062Y, K066S,

T068A, G071D, D078N, A079N, D212I, S213A, I248V, N257E, K260R, Q281P, R283K, E284P

lib-2A12
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

R041H, Ins41/42P, Q042H, L043Q, A046P, Q049L, S053T, K066A, T068A, G071D, K075N, D078N, A079K,

S085K, A097S, K117R, S120A, A121K, K172A, N173A, Q174K, D212N, S213G, N257E, K260R, S264A, S295G

lib-2B12
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

R041H, Ins41/42P, Q042H, L043Q, A046P, Q049L, S053T, K066A, T068A, G071D, K075N, D078N, A079K,

S085K, A097S, K117R, S120A, A121K, K172A, N173A, Q174K, D212N, S213G, N257E, K260R, S264A, S295G

lib-2D12
R041H, A046S, Q049R, S050A, K066A, T068A, G071D, K075N, D078N, A079K, S085K, G106E, S120A, A121K,

D212N, S213G, S230T, H232K, Q281P, R283K, E284P, K287S

lib-2F12
Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, K066A, T068A, G071D, K075N,

D078N, A079K, S085K, G106E, S120A, S230T, H232K, I248V, N257E, Q281P, R283K, E284P, K287S, S295G

lib-3B1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, K066A,

T068A, G071D, S085K, K117R, S120A, A121K, D212N, S213G, S230T, I248V, N257E, K260R, S264A, Q281P,

V282G, R283K, E284P, K287S, S295G

lib-3E1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, K022T, N024K, K026N, A029I, L030V, A046S, S053T,

G071D, K075N, D078N, A079K, A097S, K117R, S120A, A121K, Q151R, F203L, S230T, H232K, N257E, K260R,

S264A, G276D, Q281P, R283K, E284P, K287S, S295G

lib-3D2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, T019E, E020G, T021Del, N024R, A025V, A029T,

L030A, R031H, Q037H, D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, D078N, A079N, A097S, G106E,

D212N, S213G, I248V, K260R, V262T, Q272E, Q281P, R283K, E284P, K287S

lib-3G2
I007A, K066S, T068A, G071D, G106E, S120A, S230T, H232K, Q281P, R283K, E284P, K287S, S295G

lib-3E3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, K022T, N024K, K026N, A029I, L030V, Q037H, D038E,

R041H, Ins41/42P, Q042H, L043Q, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S,

K117R, S120A, N257E, K260R, S264A, Q281P, R283K, E284P, K287S, S295G

lib-3H3
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, T028A, A029T, L030A,

R031H, Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, F062Y, K066S, T068A,

G071D, D078N, A079N, A097S, K117R, S120A, A121K, D212I, S213A, Q267L, Q272E, Q281P, R283K, E284P,

K287S

lib-3A4
I007A, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H, Q037H,

R041H, Ins41/42P, Q042H, L043Q, A097S, S120A, V161I, Q181K, D212N, S213G, I248V, V262T, Q272E,

G276D, Q281P, R283K, E284P, K287S

lib-3D4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, A046P,

Q049L, K066A, T068A, G071D, K075N, A097S, D212N, S213G, N257E, Q281P, R283K, E284P, K287S, S295G

lib-3H4
Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, G071D, S085K, A097S, K117R,

S120A, A121K, D212I, S213A, N257E, K260R, S264A, Q281P, R283K, E284P, K287S, S295G

lib-3G6
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, K075N, D078N, A079K, S085K, A097S, G106E,

K117R, S120A, A121K, K172A, N173A, Q174K, D212N, S213G, N257E, K260R, S264A, Q281P, R283K

lib-3F8
I007A, Q037H, R041H, Ins41/42P, Q042H, L043Q, S085K, K117R, D212N, S213G, S230T, H232K, N257E,

K260R

lib-3B9
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, T068A, S085K, A097S, D212I, S213A, I248V, N257E,

K260R, S264A, G276D, Q281P, R283K, K287S

lib-3B10
T021A, L044V, Q049L, F062Y, S063G, S072N, N080D, V088E, E092K, A097S, G106E, S120A, D212N, S213G,

I248V, Q272E, G276D, Q281P, R283K, E284A, S295G

lib-3E10
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, A046P,

K066S, T068A, G071D, K075N, D078N, A079K, A097S, V161I, D212I, S213A

lib-3H10
K022T, N024K, K026N, A029I, L030V, Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A,

S053T, K066A, T068A, G071D, D078N, A079N, A097S, S120A, D212N, S213G, S230T, H232K, V262T, S295G

lib-3B11
S085K, G106E, S120A, S230T, N257E, K260R, S264A, Q272E, G276D, Q281P, R283K, S295G

lib-3F11
A046P, Q181K, D212N, S213G, K260R, S264A

lib-4C1
I007A, S053T, S120A, D212N, S213G, N257E, K260R, S264A, S295G

lib-4D1
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, K066S, T068A, G071D, D078N, A079N,

S085K, A097S, G106E, K117R, S120A, A121K, D212N, S213G, N257E, K260R, S264A, S295G

lib-4B2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K066A, T068A, G071D, K075N, D078N, A079K,

G106E, K117R, S120A, A121K, D212N, S213G, I248V, Q281P

lib-4G2
K022T, N024K, K026N, A029I, L030V, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, G106E,

V161I, I248V

lib-4A3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, S053T,

K066A, T068A, G071D, E136N, V161I, D212N, S213G, N257E, K260R, S264A, Q281P, R283K

lib-4B3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, Q037H, D038E, R041H, A046P, Q049L, S053T, K117R,

Q181K, D212N, S213G, N257E, K260R, S264A, Q281P, R283K, E284P, K287S

lib-4A4
Q037H, D038E, R041H, A046P, Q049L, K066A, T068A, G071D, D078N, A079K, S085K, A097S, S120A, D212N,

S213G, N257E, K260R, S264A, G276D, Q281P, R283K, S295G

lib-4G4
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, Q037H, R041H,

Ins41/42P, Q042H, L043Q, G106E, N257E, K260R, S264A, G276D, Q281P, R283K

lib-4H4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, K066A,

T068A, G071D, D078N, A079K, K117R, S120A, A121K, N188S, D212I, S213A, I248V, K260R, Q272E, G276D,

Q281P, R283K

lib-4D5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K026N, A029I, L030V, Q049L, K066S, T068A,

G071D, D078N, A079N, A097S, S120A, S230T, N257E, K260R, S264A, Q281P, R283K, S295G

lib-4H5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, F062Y, K066S, T068A, G071D, A097S, I100T, D212I, S213A

lib-4G8
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, S085K, S120A, Q272E, G276D, Q281P,

R283K, E284P, K287S

lib-4C9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, Q037H, R041H, Ins41/42P, Q042H,

L043Q, A046S, Q049R, S050A, S053T, K066A, T068A, G071D, K075N, D078N, A079K, A097S, S120A, E136N,

S230T, H232K, N257E, K260R, S264A, Q281P, R283K, S295G

lib-4F9
T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H, Q037H, R041H,

Ins41/42P, Q042H, L043Q, A046S, S053T, K066S, T068A, G071D, D078N, A079N, N257E, K260R, Q281P,

R283K, E284P, K287S

lib-4A10
F062Y, K066A, T068A, G071D, D078N, A079N, Q116R, S120A, E136N, S230A, H232K, V262T, Q281P, R283K,

E284P, K287S

lib-4A11
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N,

G106E, E136N, D212I, S213A, S230T, H232K, I248V, N257E, K260R, S264A

lib-4B11
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, T019E, E020G, T021K, K022A, A023R, N024V,

A025K, K026S, S027F, A029T, L030A, R031H, Q037H, R041H, Ins41/42P, Q042H, L043Q, A046P, S053T,

K066A, T068A, G071D, S085K, S120A, D212N, S213G, N257E, Q272E, G276D, Q281P, R283K, E284P, K287S

lib-4A12
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, F062Y, K066S, T068A, G071D,

S120A, A121K, N257E, K260R, S264A, Q281P, R283K, S295G

lib-4C12
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, Q049R, S050A, S053T, K066A, T068A, K075N, D078N,

A079K, S085K, D212I, S213A, N257E, Q272E, G276D

lib-4E12
I007A, S013V, T019E, E020G, T021K, K022A, A023R, N024V, A025K, K026S, S027F, A029T, L030A, R031H,

Q037H, R041H, Ins41/42P, Q042H, L043Q, A046S, S072G, A097S, G106E, K117R, S120A, A121K, Q181K,

D212N, S213G, S230T, H232K, S295G

Falib5-1G1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T,

K265R, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-1A2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, R124Y, Q132K,

Q133E, A135S, E136R, V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, D212N, S213G, A247S,

I248K, Q250T, V262T, Q272E, L275P, G276D, Q281P, R283K, S295G

Falib5-1E2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, T096S, A097S, S120A, Q132K,

Q133E, A135S, E136R, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, S264Y, K265D,

Q272E, G276D, Q281P, R283K, S295G

Falib5-1G2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, S120A, Q132K, Q133E, A135S, E136R, Y193E, S194C, Q196K, E198N, Q200E, N202F, D212N, S213G,

T243L, S245T, A247S, I248K, Q250T, V262T, S264Y, K265D, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-1H2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D212N,

S213G, T237N, V262T, S264Y, K265D, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-1A3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026S, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A097C, I098V, D099N, I100M, R103T, Y107W, L110N, D115A, Q116A, K117N, K119H, S120T, A121T,

R124Y, E136N, S141T, L143E, A144S, Q146S, V147S, T148S, Q149L, I150V, Q151M, K153Q, N188K, Y193E,

S194C, Q196K, E198N, Q200E, N202F, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, I248T,

V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-1E3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V081A, A097C, I098V, D099N,

I100M, R103T, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, F164C, D212N, S213G, T226S,

K228V, S230T, S231D, H232Q, T237N, T243L, S245T, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-1G4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071D, D078N, A079N, V088T,

I090L, Y091Q, E092D, A097C, I098V, D099N, I100M, R103T, Y107W, L110N, S120A, E136N, S141T, L143E,

A144S, Q146S, V147S, T148S, N188K, N206H, I207T, D212N, S213G, T243L, S245T, A247S, I248K, Q250T,

V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-1C5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, S120A, E136N, I137N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, T243L, S245T,

A247S, I248K, Q250T, V262T, S264Y, K265D, Q272E, G276D, Q281P, R283K, S295G

Falib5-1B6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097C, I098V, D099N, L110N, Q112P, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, Q132K, Q133E,

A135S, E136R, Q196K, E198N, Q200E, N202F, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N,

V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-1F6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

A097S, R103T, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, S264Y,

K265D, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-1G6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V081A, A097S, Y107W, L110N,

Q112P, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, T243L, V262T, Q272E,

G276D, Q281P, R283K, S295G

Falib5-1B7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A097S, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, V176I, Q178E, I179T, Q181R,

S182G, S184K, S185E, N188K, D212N, S213G, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-1G7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071N, D078N, A079N, A097S, Y107W, L110N, Q112P,

S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272N, Q273T, G276D,

Q281P, R283K, S295G

Falib5-1F8
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, V088T, I090L, Y091Q, E092D, A097C, I098V, D099N, I100M, R103T, D115A, Q116A, K117N, K119H,

S120T, A121T, R124Y, E136N, N188K, D212N, S213G, T243L, S245T, A247S, I248K, Q250T, V262T, Q272E,

G276D, Q281P, R283K, S295G

Falib5-1G9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272N, Q273T,

G276D, Q281P, R283K, S295G

Falib5-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

1C10
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

1G10
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, Y193E, S194C, Q196K, E198N, Q200E, N202F, N206H, I207T, D212N,

S213G, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

1E11
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D,

Q281P, R283K, S295G

Falib5-1F12
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V,

D099N, I100M, R103T, S120A, R124Y, E136N, S194F, D212N, S213G, T226S, K228V, S230T, S231D, H232Q,

V262T, S264Y, K265E, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

1G12
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, V176I,

Q178E, I179T, Q181R, S182G, S184K, S185E, Q196K, E198N, Q200E, N202F, N206H, I207T, D212N, S213G,

T226S, K228V, T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-2D1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, S063A, K066G, G067S, T068L, G071D, D078N, A079N, V088T, I090L, Y091Q,

E092D, A097C, I098V, D099N, I100M, R103T, S120A, E136N, Y193E, S194C, Q196K, E198N, Q200E, N202F,

N206H, I207T, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, V262T, Q272E, G276D, Q281P, R283K,

S295G

Falib5-2H1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D156G,

V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, N206H, I207T, D212N, S213G, T226S, K228V,

S230T, S231D, H232Q, T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-2B2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, A065T, K066S, T068A, G071D, D078N, A079N, V081A, V088T, I090L,

Y091Q, E092D, A097C, I098V, D099N, I100M, R103T, Y107W, L110N, S120A, E136N, Y193E, S194C, Q196K,

E198N, Q200E, N202F, N206H, I207T, D212N, S213G, T237N, T243S, V262T, Q272N, Q273T, Q281P, R283K,

S295G

Falib5-2D2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097C, I098V, D099N, I100M, R103T, S120A, E136N, Y193E, S194C, Q196K, E198N, Q200E, N202F, N206H,

I207T, D212N, S213G, T237N, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-2E2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, F062Y, K066S, T068A, G071D, D078N, A079N, V081A, V088T,

I090L, Y091Q, E092D, A097S, S120A, E136N, D212N, S213G, T237N, T243L, S245T, A247S, I248K, Q250T,

V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-2F2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V081A, V088T, I090L, Y091Q,

E092D, A097C, I098V, D099N, I100M, R103T, Y107W, L110N, S120A, E136N, D212N, S213G, T226S, K228V,

S230T, S231D, H232Q, T237N, T243L, S245T, A247S, I248K, Q250T, V262T, S264Y, K265D, Q272N, Q273T,

G276D, Q281P, R283K, S295G

Falib5-2C3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071A, S072R, A074L, K075Q,

D078N, A079N, A097S, Y107W, S120A, E136N, D212N, S213G, T243L, S245T, V262T, Q272E, G276D, Q281P,

R283K, S295G

Falib5-2G3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A,

D078N, A079N, A097S, S120A, E136N, V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, Y193E,

S194C, Q196K, E198N, Q200E, N202F, N206H, I207T, D212N, S213G, T237N, V262T, Q272E, G276D, Q281P,

R283K, S295G

Falib5-2H3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071D, D078N, A079N, A097C,

I098V, D099N, I100M, S120A, Q132K, Q133E, A135S, E136R, I169H, K172E, N173T, Q174A, V176I, Q178E,

I179T, Q181R, S182G, S184K, S185E, N188K, D212N, S213G, Q246P, V262T, S264Y, K265D, Q272N, Q273T,

G276D, Q281P, R283K, S295G

Falib5-2H4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L,

Y091Q, E092D, A097C, I098V, D099N, I100M, R103T, Y107W, L110N, Q112P, S120A, E136N, D212N, S213G,

V262T, S264Y, K265D, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-2G5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, D212N, S213G, V262T, S264Y, K265D, Q272N, Q273T, G276D, Q281P,

R283K, S295G

Falib5-2A6
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, I100M,

R103T, Y107W, L110N, Q112P, S120A, E136N, Y193E, S194C, Q196K, E198N, Q200E, N202F, D212N, S213G,

T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-2D7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058D, S063A, K066G, G067S, T068L, G071D, D078N, A079N, A097C,

I098V, D099N, I100M, R103T, S120A, E136N, D212N, S213G, V262T, S264Y, K265D, Q272N, Q273T, G276D,

Q281P, R283K, S295G

Falib5-2D8
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, V176I,

Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, D212N, S213G, S255G, V262T, Q272N, Q273T, G276D,

Q281P, R283K, S295G

Falib5-2E8
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, Y107W, L110N, Q112P, S120A, Q132K, Q133E, A135S, E136R, L142P, D212N, S213G, T243L, S245T,

A247S, I248K, Q250T, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-2A9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, Y107W, L110N, Q112P,

D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, Q132K, Q133E, A135S, E136R, Y193E, S194C, Q196K,

E198N, Q200E, N202F, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D,

Q281P, R283K, S295G

Falib5-2B9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, I090F, A097C, I098V, D099N,

I100M, R103T, S120A, E136N, V176I, Q178E, I179T, Q181R, S182G, S184K, S185E, N188K, D212N, S213G,

V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-2E9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, I100M,

R103T, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272N, Q273T,

G276D, Q281P, R283K, S295G

Falib5-2H9
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066G, G067S, T068L, G071D, D078N, A079N, V081A, A097C, I098V,

D099N, I100M, R103T, S120A, E136N, D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

2D11
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, I100M, R103T, S120A, E136N, G170D, N188K, I207T, D212N, S213G, T226S, K228V, S230T, S231D,

H232Q, T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-2F11
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T,

Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

2G11
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, S120A, E136N, S182F, I207T, D212N, S213G, S230T, S231D, H232Q, T243L, S245T, A247S, I248K,

Q250T, V262T, S264Y, K265D, Q272E, G276D, Q281P, R283K, S295G

Falib5-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

2A12
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, Q132K, Q133E, A135S, E136R, Y193E, S194C, Q196K, E198N, Q200E, D212N,

S213G, T226S, K228V, S230T, S231D, H232Q, T237N, T243L, S245T, A247S, I248K, Q250T, V262T, S264Y,

K265D, Q272E, G276D, Q281P, R283K, S295G

Falib5-3A2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, V176I,

D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-3E2
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, D115A, Q116A, K117N,

K119H, S120T, A121T, R124Y, E136N, D212N, S213G, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D,

Q281P, R283K, S295G

Falib5-3D3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066G, G067S, T068L, G071D, D078N, A079N, A097S, S120A, E136N,

D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-3G4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V081A, A097S, S120A, E136N,

D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, T243L, S245T, V262T, Q272N, Q273T, G276D,

Q281P, R283K, S295G

Falib5-3C5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

A097C, I098V, D099N, I100M, R103T, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q,

T237N, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-3D5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D078N, A079N, V088T,

I090L, Y091Q, E092D, A097C, I098V, D099N, I100M, R103T, S120A, E136N, N188K, N202F, N206H, I207T,

D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

3D10
D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071A, S072R, A074L, K075Q, D076A, D078N, A079N,

V081A, A097S, S120A, R124Y, E136N, V176I, D212N, S213G, T237N, V262T, Q272N, Q273T, G276D, Q281P,

R283K, S295G

Falib5-4F1
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097S, S120A, E136N, D212N,

S213G, V262T, S264Y, K265D, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-4F3
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071A, S072R, A074L, K075Q,

D076A, D078N, A079N, V081A, A097S, S120A, E136N, N188K, D212N, S213G, T243L, S245T, A247S, I248K,

Q250T, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-4C4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V, D099N, R103T,

D115G, S120A, E136N, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, T243L, S245T, A247S,

V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-4D4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L,

Y091Q, E092D, A097C, I098V, D099N, I100M, R103T, S120A, E136N, Y193E, S194C, Q196K, E198N, Q200E,

N202F, N206H, D212N, S213G, T226S, K228V, S230T, S231D, H232Q, T237N, V262T, Q272E, G276D, Q281P,

R283K, S295G

Falib5-4H4
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, S063A, K066G, G067S, T068L, G071A, S072R, A074L, K075Q, D076A, D078N,

A079N, A097S, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, V176I, Q178E, I179T, Q181R,

S182G, S184K, S185E, N188K, D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-4B5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, D115A, Q116A, K117N, K119H, S120T, A121T, R124Y, E136N, D212N, S213G, T226S, K228V, S230T,

S231D, H232Q, T237N, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-4C5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, F062Y, K066S, T068A, G071D, D078N, A079N, A097C, I098V,

D099N, I100M, R103T, S120A, E136N, D212N, S213G, V262T, Q272E, G276D, Q281P, R283K, S295G

Falib5-4H5
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, F062Y, K066S, T068A, G071D, D078N, A079N, V088T, I090L, Y091Q, E092D,

A097S, D115A, Q116A, K117N, K119H, S120T, A121T, E136N, Y193E, S194C, D212N, S213G, V262T, Q272E,

G276D, Q281P, R283K, S295G

Falib5-4D7
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071A, S072R, A074L, K075Q,

D076A, D078N, A079N, V081A, A097C, I098V, D099N, I100M, R103T, S120A, E136N, D212N, S213G, T237N,

V262T, S264Y, K265D, Q272N, Q273T, G276D, Q281P, R283K, S295G

Falib5-4F12
G005Del, A006Del, I007Del, P008Del, S009T, E010V, S013L, K022T, N024K, K026N, A029I, L030V, Q037H,

D038E, R041H, A046P, Q049L, A057I, I058A, S063A, K066G, G067S, T068L, G071D, D078N, A079N, V088T,

I090L, Y091Q, E092D, A097C, I098V, D099N, I100M, R103T, Y107W, L110N, Q112P, S120A, E136N, D212N,

S213G, T237N, V262T, Q272N, Q273T, G276D, Q281P, R283K, S295G

Example 9—Identification of Amino Acid Positions Affecting the Protein Stability and Function of PIP-47Aa

The protein sequence alignment of: PIP-47Aa (SEQ ID NO:2); the active closely related PIP-47Ba (SEQ ID NO:4), PIP-47Bb (SEQ ID NO:6); the distant related active homolog PIP-47Fa (SEQ ID NO: 8); and inactive homologs: Plu2048 (SEQ ID NO: 10); Plu2046 (SEQ ID NO: 12); YP003041397 (SEQ ID NO: 14); YP003041380 (SEQ ID NO: 16); ZP17107944 (SEQ ID NO: 18); and ZP05881874 (SEQ ID NO: 20) is shown in FIG. 1A-1B. To understand the role of some of the conserved positions between the PIP-47 polypeptides in insecticidal activity and protein stability, amino acids T47, Y73, Q95, P138, L142, G170, Q205, Y221, W256, and T279 relative to PIP-47Aa (SEQ ID NO: 2) were selected for saturation mutagenesis. Saturated mutagenesis was designed for selected positions using the mutagenesis oligonucleotides as shown in Table 12. Mutants were generated using degenerate oligos for each site using sewing and rescuing PCR strategy of two overlapping fragments of N-terminus (no mutation) and C-terminus (with mutations) for each site using sewing and rescuing PCR strategy of two overlapping fragments of N-terminus (no mutation) and C-terminus (with mutations) gene). As an alternative method to perform saturation mutagenesis, a QuikChange™ Lightning kit from Agilent™ Technologies was used. Table 12 shows the amino acid substitutions identified at each selected positions that were mutagenized, the amino acid substitutions that expressed a soluble protein in the cell lysate, and sequence variations that allow retention of insecticidal activity that were identified.

TABLE 12

Oligo

Identified

Residue
name

mutations
Active mutants
Soluble expressed

T47
047SM-
SEQ ID NO: 431
G, A, L, M, P, S,
G, A, L, M, P,
G, A, L, M, P, S, C,

T47F

C, N, D, E, K, R,
S, C, N, D, E,
N, D*, E, K, R, W,

047SM-
SEQ ID NO: 432
Y, I, W, F
K, R, W, I, Y
I, Y

T47R

Y73
047SM-
SEQ ID NO: 433
G, A, V, L, W, S,
G, C, E
F, G*, C*, E*

Y73F

T, C, Q, E, K, R,

047SM-
SEQ ID NO: 434
N, H, F, I, D, P, M

Y73R

Q95
047SM-
SEQ ID NO: 435
G, A, V, L, M, W,
A, V, M, S, T,
A, V, M*, S, T, N*,

Q95F

F, P, S, T, N, E,
N, H
L*, P*, K*, H*

047SM-
SEQ ID NO: 436
K, R, H

Q95R

P138
047SM-
SEQ ID NO: 437
G, A, V, L, I, W,
none
none

P138F

C, N, Q, E, K, R,

047SM-
SEQ ID NO: 438
F, D, M, H, S, T, Y

P138R

L142
047SM-
SEQ ID NO: 439
G, V, M, F, P, S,
G, V, M, S, T,
G, V, M, S, T, C, N,

L142F

T, C, N, Q, D, E,
C, N, Q, D, E, H
Q, D, E, H, K*, P*, R

047SM-
SEQ ID NO: 440
K, R, H

L142R

G170
047SM-
SEQ ID NO: 441
A, V, L, I, M, W,
A, V, L, I, M,
A, M, S*, C*, Y, N,

G170F

S, T, C, Y, N, Q,
S, C, Y, N, E,
V*, K*, R*, L*, Q*,

047SM-
SEQ ID NO: 442
E, K, R, P
K, R
I*, E*, K*

G170R

Q205
047SM-
SEQ ID NO: 443
G, A, V, L, I, M,
A, V, P, S, T,
A, V, S, T, C, N, D,

Q204F

W, P, S, T, C, Y,
C, Y, N, D, R
L*, P*, Y*, R*

047SM-
SEQ ID NO: 444
N, D, E, K, R

Q204R

Y221
047SM-
SEQ ID NO: 445
G, A, V, L, P, S,
A, V, L, I, M,
A, V, L, I, M, S*,

Y221F

T, Q, D, E, K, R,
S, Q, H, G
Q*, H, G*, K*, W, C

047SM-
SEQ ID NO: 446
M, W, I, F, H, C

Y221R

W256
047SM-
SEQ ID NO: 447
G, A, V, L, I, M,
none
none

W256F

P, S, C, Y, N, Q,

047SM-
SEQ ID NO: 448
D, R, H

W256R

T279
047SM-
SEQ ID NO: 449
G, A, V, M, W, P,
S, C, N, E
S, C, N, E*

T279F

S, C, Y, N, Q, D,

047SM-
SEQ ID NO: 450
E, K, R, H

T279R

*significantly reduced soluble expression

Example 10—Identification of Motifs Affecting the Protein Stability and Function of PIP-47Aa

To further identify sequence space affecting protein structural stability and insecticidal function, three motifs (amino acids A77-P87 of SEQ ID NO: 2 (motif 1), amino acids Y107-S120 of SEQ ID NO: 2 (motif 2), amino acids K233-A247 of SEQ ID NO: 2 (motif 3), of PIP-47Aa (SEQ ID NO: 2) shared by the four PIP-47 polypeptides: PIP-47Aa (SEQ ID NO: 2); PIP-47Ba (SEQ ID NO: 4); PIP-47Bb (SEQ ID NO: 6); and PIP-47Fa) were identified (FIG. 3), and selected for saturation mutagenesis. Saturated mutagenesis was designed for position A77-P87, Y107-S120 and K233-A247 of SEQ ID NO: 2 using the mutagenesis oligonucleotides as shown in Table 13, Table 14 and Table 15, respectively. Mutants were generated as described in Example 9. Table 13, Table 14 and Table 15 show the amino acid substitutions identified at each position that were mutagenized of Motif 1, Motif 2 and Motif 3, respectively, the amino acid substitutions that expressed a soluble protein in the cell lysate (Motif 2 & Motif 3), and sequence variations that allow retention of insecticidal activity.

TABLE 13

Oligo

sequence

Position
Oligo name
identifier
Identified mutations
Active mutants

A77
047SM-A77F
SEQ ID
G, Y, V, L, W, S, T, C,
S, C, Q, P, N

NO: 489
Q, E, K, R, N, H, F, I, D, P, M

D78
047SM-D78F
SEQ ID
G, A, V, L, W, S, T, C, Q, E, K, R,
G, A, V, L, W, S, C, Q, E, R, F, I,

NO: 490
N, H, F, I, Y, P, M
Y, M, N, H, T, K,

A79
047SM-A79F
SEQ ID
G, Y, V, L, W, S, T, C, Q, E, K, R,
G, Y, V, L, W, Q, E, K, R, N, D, M,

NO: 491
N, F, I, D, P, M
F, I, C, S, T,

N80
047SM-N80F
SEQ ID
G, A, V, L, W, S, T, C, Q, E, K, R,
G, A, V, L, W, S, T, C, Q, E, K, R,

NO: 492
Y, F, D, P, M
Y, F, D, M

V81
047SM-V81F
SEQ ID
G, A, Y, L, W, S, T, C, Q, E, K, R,
G, A, L, W, S, T, C,

NO: 493
N, F, I, D, P, M
Q, E, K, R, N, I, D, P, M

S82
047SM-S82F
SEQ ID
G, A, V, L, W, Y, T, C,
G, A, V, W, T, C, Q, E, K, N, H, F,

NO: 494
Q, E, K, R, N, H, F, I, D, P, M
I, D, M

P83
047SM-P83F
SEQ ID
G, A, V, L, W, S, T, C, Q, E, K, R,
G, A, V, L, W, S, T, C, Q, E, K, R,

NO: 495
N, F, I, D,
N, F, I, D,

G84
047SM-G84F
SEQ ID
Y, A, V, L, W, S, T, C, Q, E, R, N,
Y, A, V, L, W, S, T, C, Q, E, R, N,

NO: 496
F, I, D, P, M
F, I, D, P, M

S85
047SM-S85F
SEQ ID
G, A, V, L, W, Y, T, C, Q, E, R, F,
G, A, V, L, W, Y, T, C, Q, E, R, F,

NO: 497
D, P, M
D, P, M

A86
047SM-A86F
SEQ ID
G, Y, V, L, W, S, T, C, Q, E, K, R,
G, Y, V, L, W, S, T, C, Q, E, K, R,

NO: 498
N, H, F, I, D, P, M
N, H, F, I, D, P, M

P87
047SM-P87F
SEQ ID
G, A, V, L, W, S, T, C, Q, E, K, R,
None

NO: 499
N, H, F, I, D, Y, M

TABLE 14

Oligo

Identified

Soluble

Position
name
Sequence
mutations
Active mutants
expressed

Y107
047SM-Y107F
SEQ ID NO: 500
A, C, D, E, F, G,
C, F, G, H, L
C, E*, F, H, L,

047SM-Y107R
SEQ ID NO: 501
H, I, K, L, P, Q, S*

R, S, T, V,

N108
047SM-N108F
SEQ ID NO: 502
A, C, E, F, G, I,
A, C, E, F, G,
A, C, E, F, G,

047SM-N108R
SEQ ID NO: 503
K, L, M, P, Q, R,
I, K, L, M, Q,
I, K, L, M, Q,

S, T, W
R, S, T, W
R, S, T, W

P109
047SM-P109F
SEQ ID NO: 504
G, A, I, V, L, M,
G, A, L, M, C,

047SM-P109R
SEQ ID NO: 505
F, S, T, C, Y, Q,
Y, E, K

D, E, K, R, H

L110
047SM-L110F
SEQ ID NO: 506
A, C, E, F, G, K,
A, E, F, M, N,
A, C, E, F, G,

047SM-L110R
SEQ ID NO: 507

M, N, P, R, S, T,
P, S, T, V, W
K, M, N, P, R,

V, W

S, T, V, W

D111
047SM-D111F
SEQ ID NO: 508
A, C, E, F, G, H,
A, E, F, G, H,
A, C, E, F, G,

047SM-D111R
SEQ ID NO: 509
L, N, P, Q, S, T,
N, Q, S, V, Y
H, L, N, Q, S,

V, W, Y

T, V, Y

Q112
047SM-Q112F
SEQ ID NO: 510
A, C, G, H, K, L,
C, P, V,
A*, C$*, H*, P*,

047SM-Q112R
SEQ ID NO: 511
M, P, R, S, T, V, W

S*, V*

P113
047SM-P113F
SEQ ID NO: 512
C, F, G, L, Q, R,
C, G
C, G, S*, W*, Y*

047SM-P113R
SEQ ID NO: 513
S, T, W, Y

T114
047SM-T114F
SEQ ID NO: 514
C, E, F, G, H, I,
C, E, F, G, H,
C, E, F, G, H,

047SM-T114R
SEQ ID NO: 515
K, L, N, P, Q, R,
I, K, L, N, Q,
I, K, L, N, Q,

S, V, W, Y,
R, S, V, W, Y,
R, S, V, W, Y,

D115
047SM-D115F
SEQ ID NO: 516
A, E, F, G, H, I,
A, E, F, G, H,
A, E, F, G, H,

047SM-D115R
SEQ ID NO: 517
L, P, Q, R, S, T, Y
I, L, P, Q, R,
I, L, P, Q, R,

S, T, Y
S, T, Y

Q116
047SM-Q116F
SEQ ID NO: 518
A, C, F, H, I, L,
A, C, F, H, I,
A, C, F, H, I,

047SM-Q116R
SEQ ID NO: 519
N, P, R, S, T, V,
L, N, P, R, S,
L, N, P, R, S,

W,
T, V, W,
T, V, W,

K117
047SM-K117F
SEQ ID NO: 520
A, D, E, F, G, H,
A, D, E, F, G,
A, D, E, F, G,

047SM-K117R
SEQ ID NO: 521
I, L, N, P, Q, R,
H, I, L, N, P,
H, I, L, N, P,

S, T, V, Y
Q, R, S, T, V, Y
Q, R, S, T, V, Y

P118
047SM-P118F
SEQ ID NO: 522
A, C, D, E, G, H,
A, C, D, E, G,
A, C, D, E, G,

047SM-P118R
SEQ ID NO: 523
I, K, L, M, N, Q,
H, I, K, L, M,
H, I, K, L, M,

R, S, T, V, W
N, Q, R, S, T,
N, Q, R, S, T,

V, W
V, W

K119
047SM-K119F
SEQ ID NO: 524
A, C, D, E, F, C,
A, C, D, E, F,
A, C, D, E, F,

047SM-K119R
SEQ ID NO: 525
H, I, L, M, N, P,
G, H, I, L, M,
G, H, I, L, M,

Q, R, S, T, V, W,
N, P, Q, R, S,
N, P, Q, R, S,

T, V, W
T, V, W

S120
047SM-S120F
SEQ ID NO: 526
A, D, E, F, G, H,
A, D, E, F, G,
A, D, E, F, G,

047SM-S120R
SEQ ID NO: 527
I, L, M, P, Q, R,
H, I, L, M, P,
H, I, L, M, P,

T, V, Y
Q, R, T, V, Y
Q, R, T, V, Y

TABLE 15

Oligo

Identified

Soluble

Position
name
Sequence
mutations
Active mutants
expressed

K233
047SM-K233F
SEQ ID NO: 451
G, A, V, L, I, M,

M, N, Q, D, W*,

047SM-K233R
SEQ ID NO: 452
W, F, P, S, T, C,

I, C, L, F

Y, N, Q, D, E, R, H

G234
047SM-G234F
SEQ ID NO: 453
A, C, F, H, I, K,
A, C, F, H, K,
A, C*, F*, H, K,

047SM-G234R
SEQ ID NO: 454
L, M, P, R, S, V,
L, M, P, R, S,
L*, M, P, R, S,

W, Y
V, Y
V*, Y*

T235
047SM-T235F
SEQ ID NO: 455
A, C, E, F, G, K,
A, C, E, F, G,
A, C, E, F, G,

047SM-T235R
SEQ ID NO: 456
L, M, N, P, R, S,
K, L, M, N, R,
K, L, M, N, P*,

V, W
S, V, W
Q, R, S, V, W

I236
047SM-I236F
SEQ ID NO: 457
A, C, D, E, F, G,
A, C, F, L, P,
A, C, F, L, P,

047SM-I236R
SEQ ID NO: 458
K, L, N, P, Q, R,
T, V
T, V, W*

S, T, V, W, Y

T237
047SM-T237F
SEQ ID NO: 459
A, C, D, E, F, G,
A, C, D, E, F,
A, C, D, E, F,

047SM-T237R
SEQ ID NO: 460
H, I, K, L, P, Q,
G, H, I, K, L,
G, H, I, K, L,

R, S, V, W,
Q, R, S, V, W,
P*, Q, R, S, V, W

F238
047SM-F238F
SEQ ID NO: 461
A, C, D, E, G, K,
A, D, L, P, T,
A, C, D, E, G,

047SM-F238R
SEQ ID NO: 462
L, N, P, R, S, T,
V, W, Y
K, L, N, P, R,

V, W, Y

S, T, V, W, Y

Q239
047SM-Q239F
SEQ ID NO: 463
C, G, R, Y, S, P,
C, G, R, Y, S,
C, G, R, S, W,

047SM-Q239R
SEQ ID NO: 464
W, E, T, M, A, K,
P, W, E, T, M,
E, T, M, A, K,

H, I, V, L, D
A, K, H, I, V,
P*, H, I, V, L,

L, D
Y*, D

S240
047SM-S240F
SEQ ID NO: 465
A, E, G, H, K, L,
A, E, G, H, L,
A, E, G, H, K,

047SM-S240R
SEQ ID NO: 466
M, N, P, Q, R, T,
M, N, P, Q, T, V
L, M, N, P, Q,

W, V

R, T, W, V

S241
047SM-S241F
SEQ ID NO: 467
A, C, D, F, G, H,
A, C, D, F, G,
A, C, D, F, G,

047SM-S241R
SEQ ID NO: 468
I, K, L, M, N, P,
H, I, K, L, M,
H*, I, K, L, M,

Q, R, T, V, W, Y,
N, Q, R, T, V,
N, P*, Q, R, T,

W, Y,
V, W, Y,

Y242
047SM-Y242F
SEQ ID NO: 469
A, C, D, E, F, G,
C, F, G, H, I,
C*, F, G*, H, I,

047SM-Y242R
SEQ ID NO: 470
H, I, K, L, M, N,
L, M, P, Q, R,
L*, M, N, P, Q,

P, Q, R, S, T, V, W
S, T, V, W
R, S*, T*, V, W

T243
047SM-T243F
SEQ ID NO: 471
A, D, E, G, H, I,
D, E, H, I, K,
A, D, E, G, H,

047SM-T243R
SEQ ID NO: 472
K, L, M, N, P, Q,
L, M, N, P, Q,
I, K, L, M, N,

R, S, V, Y
R, S, V, Y
P, Q, R, S, V, Y

L244
047SM-L244F
SEQ ID NO: 473
A, C, D, F, G, K,
A, C, F, G, K,
A, C, F, G*, K*,

047SM-L244R
SEQ ID NO: 474
P, Q, R, S, T, V,
P, Q, R, S, T,
P*, Q, R*, S, T,

W, Y
V, W, Y
V, W, Y

S245
047SM-S245F
SEQ ID NO: 475
A, C, D, E, G, H,
A, C, D, G, L,
A, C, D, E, G,

047SM-S245R
SEQ ID NO: 476
L, M, N, P, Q, R,
M, N, Q, R, T,
H, L, M, N, P*,

T, V, W, Y
V, W, Y
Q, R, T, V, W, Y

Q246
047SM-Q246F
SEQ ID NO: 477
A, C, E, F, G, H,
A, C, E, F, G,
A, C, E*, F*, G,

047SM-Q246R
SEQ ID NO: 478
I, K, L, M, P, R,
H, I, K, L, M,
H, I, K*, L, M,

S, T, W, V
P, R
P*, R, S, T, W*,

V*

A247
047SM-A247F
SEQ ID NO: 479
C, D, F, G, H, I,
D, F, K, M, S, V
C, D*, F, G, I,

047SM-A247R
SEQ ID NO: 480
K, L, M, N, P, Q,

K, L, M, N, P*,

R, S, V, W,

Q, S, V, W

*Significantly reduced soluble expression

Example 11—Combinatorial Mutagenesis of Flexible Positions to Generate Active Variants with Multiple Mutations

Amino acids positions A79-S85 within motif 1 and T114-S120 within motif 2 of PIP-47Aa (SEQ ID NO:2, FIG. 1) were selected to perform combinatorial mutagenesis. The library variants were generated using QuikChange™ Lightning kit from Agilent™ Technologies according to manufacturer's instruction and mutagenesis oligos Mot-1-comb-F: 5′-CAGCTATGCCAAGGATGCGGACKSCVAKMTKKSCNNKKSCKSCGCACCAGTGAGCATCT ACGAAG-3′ (SEQ ID NO: 528) for Motif 1 (positions A79-S85) and Mot-2-comb-F: 5′-CTACAACCCACTGGACCAGCCGKSCVAKVAKNNKNNKNNKKSCGCCGGTGACCGTGAG CACTTC-3′ (SEQ ID NO: 529) for Motif 2 (positions) T114-S120. The mutant variants were expressed and screened for insecticidal activity as described in example 8. After screening 282 isolates from the Motif 1 (positions A79-S85) combinatorial mutagenesis library, 99 active variants were identified. Table 16 shows the resulting PIP-47 variant designations, PIP-47 variant gene sequences (SEQ ID NOs), the Motif 1 amino acid sequences of the PIP-47 variants (SEQ ID NOs), and the number of amino acid substitutions (mutations) compared to PIP-47Aa (SEQ ID NO: 2).

TABLE 16

Variants
DNA sequence
Motif 1 AA sequence
Mutations

PIP-47Aa
SEQ ID NO: 1
ANVSPGS (amino acids 79-85 of
0

SEQ ID NO: 2)

cmbM1-01
SEQ ID NO: 530

SHI
SCSS (amino acids 79-85 of
5

SEQ ID NO: 629)

cmbM1-02
SEQ ID NO: 531

GQL
SLCS (amino acids 79-85 of
5

SEQ ID NO: 630)

cmbM1-03
SEQ ID NO: 532

CHLA
PSS (amino acids 79-85 of
5

SEQ ID NO: 631)

cmbM1-04
SEQ ID NO: 533
AHISLSC (amino acids 79-85 of
5

SEQ ID NO: 632)

cmbM1-05
SEQ ID NO: 534

GHLALS
S (amino acids 79-85 of
6

SEQ ID NO: 633)

cmbM1-06
SEQ ID NO: 535
AQISLSC (amino acids 79-85 of
5

SEQ ID NO: 634)

cmbM1-07
SEQ ID NO: 536

GQI
SLSS (amino acids 79-85 of
5

SEQ ID NO: 635)

cmbM1-08
SEQ ID NO: 537

S
NVTSGS (amino acids 79-85 of
3

SEQ ID NO: 636)

cmbM1-09
SEQ ID NO: 538

GHIG
PGS (amino acids 79-85 of
4

SEQ ID NO: 637)

cmbM1-10
SEQ ID NO: 539
AHLSKSS (amino acids 79-85 of
4

SEQ ID NO: 638)

cmbM1-11
SEQ ID NO: 540

GHM
SPSC (amino acids 79-85 of
5

SEQ ID NO: 639)

cmbM1-12
SEQ ID NO: 541

G
NMGLCC (amino acids 79-85 of
6

SEQ ID NO: 640)

cmbM1-13
SEQ ID NO: 542

SHL
SNSC (amino acids 79-85 of
6

SEQ ID NO: 641)

cmbM1-14
SEQ ID NO: 543
AHVSSCS (amino acids 79-85 of
3

SEQ ID NO: 642)

cmbM1-15
SEQ ID NO: 544
ANLSLGC (amino acids 79-85 of
3

SEQ ID NO: 643)

cmbM1-16
SEQ ID NO: 545

GH
VSPGC (amino acids 79-85 of
3

SEQ ID NO: 644)

cmbM1-17
SEQ ID NO: 546

SHL
SSCA (amino acids 79-85 of
6

SEQ ID NO: 645)

cmbM1-18
SEQ ID NO: 547

C
NICVSS (amino acids 79-85 of
5

SEQ ID NO: 646)

cmbM1-19
SEQ ID NO: 548

SHIASSC
(amino acids 79-85 of
7

SEQ ID NO: 647)

cmbM1-20
SEQ ID NO: 549

SHMGSSC
(amino acids 79-85 of
7

SEQ ID NO: 648)

cmbM1-21
SEQ ID NO: 550

S
NLAPSS (amino acids 79-85 of
4

SEQ ID NO: 649)

cmbM1-22
SEQ ID NO: 551
AHISVSS (amino acids 79-85 of
4

SEQ ID NO: 650)

cmbM1-23
SEQ ID NO: 552
ANISCSG (amino acids 79-85 of
4

SEQ ID NO: 651)

cmbM1-24
SEQ ID NO: 553
AKIAICS (amino acids 79-85 of
5

SEQ ID NO: 652)

cmbM1-25
SEQ ID NO: 554
ANISLCA (amino acids 79-85 of
4

SEQ ID NO: 653)

cmbM1-26
SEQ ID NO: 555

SDL
SYSG (amino acids 79-85 of
6

SEQ ID NO: 654)

cmbM1-27
SEQ ID NO: 556

G
NVSPGS (amino acids 79-85 of
1

SEQ ID NO: 655)

cmbM1-28
SEQ ID NO: 557

C
NMSHCC (amino acids 79-85 of
5

SEQ ID NO: 656)

cmbM1-29
SEQ ID NO: 558

S
NVGHGA (amino acids 79-85 of
4

SEQ ID NO: 657)

cmbM1-30
SEQ ID NO: 559

S
NICTAS (amino acids 79-85 of
5

SEQ ID NO: 658)

cmbM1-31
SEQ ID NO: 560

CHI
SVSS (amino acids 79-85 of
5

SEQ ID NO: 659)

cmbM1-32
SEQ ID NO: 561
AHICSCC (amino acids 79-85 of
6

SEQ ID NO: 660)

cmbM1-33
SEQ ID NO: 562

C
NISLSS (amino acids 79-85 of
4

SEQ ID NO: 661)

cmbM1-34
SEQ ID NO: 563

S
NLSSSS (amino acids 79-85 of
4

SEQ ID NO: 662)

cmbM1-35
SEQ ID NO: 564

S
NIGSAC (amino acids 79-85 of
6

SEQ ID NO: 663)

cmbM1-36
SEQ ID NO: 565

SHLCKSC
(amino acids 79-85 of
7

SEQ ID NO: 664)

cmbM1-37
SEQ ID NO: 566

GHICSCG
(amino acids 79-85 of
7

SEQ ID NO: 665)

cmbM1-38
SEQ ID NO: 567

SDI
SSCC (amino acids 79-85 of
6

SEQ ID NO: 666)

cmbM1-39
SEQ ID NO: 568

C
NMSPAC (amino acids 79-85 of
4

SEQ ID NO: 667)

cmbM1-40
SEQ ID NO: 569

S
NICLGC (amino acids 79-85 of
5

SEQ ID NO: 668)

cmbM1-41
SEQ ID NO: 570

C
NMAASC (amino acids 79-85 of
6

SEQ ID NO: 669)

cmbM1-42
SEQ ID NO: 571

S
NVSSCS (amino acids 79-85 of
3

SEQ ID NO: 670)

cmbM1-43
SEQ ID NO: 572
ANISPSC (amino acids 79-85 of
3

SEQ ID NO: 671)

cmbM1-44
SEQ ID NO: 573

CHI
SRSC (amino acids 79-85 of
6

SEQ ID NO: 672)

cmbM1-45
SEQ ID NO: 574

S
NISLSC (amino acids 79-85 of
5

SEQ ID NO: 673)

cmbM1-46
SEQ ID NO: 575
ANISFGS (amino acids 79-85 of
2

SEQ ID NO: 674)

cmbM1-47
SEQ ID NO: 576

CHL
SLCS (amino acids 79-85 of
5

SEQ ID NO: 675)

cmbM1-48
SEQ ID NO: 577

GHL
SHGC (amino acids 79-85 of
5

SEQ ID NO: 676)

cmbM1-49
SEQ ID NO: 578

C
NMSLCC (amino acids 79-85 of
5

SEQ ID NO: 677)

cmbM1-50
SEQ ID NO: 579

C
NLANCC (amino acids 79-85 of
6

SEQ ID NO: 678)

cmbM1-51
SEQ ID NO: 580
ANLCMSS (amino acids 79-85 of
4

SEQ ID NO: 679)

cmbM1-52
SEQ ID NO: 581
AHISLGC (amino acids 79-85 of
4

SEQ ID NO: 680)

cmbM1-53
SEQ ID NO: 582

SHM
SSGC (amino acids 79-85 of
5

SEQ ID NO: 681)

cmbM1-54
SEQ ID NO: 583

GHL
STSS (amino acids 79-85 of
5

SEQ ID NO: 682)

cmbM1-55
SEQ ID NO: 584

SQI
SLCA (amino acids 79-85 of
6

SEQ ID NO: 683)

cmbM1-56
SEQ ID NO: 585

S
NVSPGC (amino acids 79-85 of
2

SEQ ID NO: 684)

cmbM1-57
SEQ ID NO: 586
AHISRSS (amino acids 79-85 of
4

SEQ ID NO: 685)

cmbM1-58
SEQ ID NO: 587

CHI
SPAA (amino acids 79-85 of
5

SEQ ID NO: 686)

cmbM1-59
SEQ ID NO: 588
ADLCSGS (amino acids 79-85 of
4

SEQ ID NO: 687)

cmbM1-60
SEQ ID NO: 589
ANISLSS (amino acids 79-85 of
3

SEQ ID NO: 688)

cmbM1-61
SEQ ID NO: 590

S
NISPCG (amino acids 79-85 of
4

SEQ ID NO: 689)

cmbM1-62
SEQ ID NO: 591

CHMGGSC
(amino acids 79-85 of
7

SEQ ID NO: 690)

cmbM1-63
SEQ ID NO: 592
AHLALGC (amino acids 79-85 of
5

SEQ ID NO: 691)

cmbM1-64
SEQ ID NO: 593

S
NVSPGS (amino acids 79-85 of
1

SEQ ID NO: 692)

cmbM1-65
SEQ ID NO: 594

SEI
SFGS (amino acids 79-85 of
4

SEQ ID NO: 693)

cmbM1-66
SEQ ID NO: 595

CHLC
PGA (amino acids 79-85 of
5

SEQ ID NO: 694)

cmbM1-67
SEQ ID NO: 596

SHL
SVAS (amino acids 79-85 of
5

SEQ ID NO: 695)

cmbM1-68
SEQ ID NO: 597

S
NVSPGC (amino acids 79-85 of
2

SEQ ID NO: 696)

cmbM1-69
SEQ ID NO: 598
ANVSPGC (amino acids 79-85 of
1

SEQ ID NO: 697)

cmbM1-70
SEQ ID NO: 599

S
NLSPGS (amino acids 79-85 of
2

SEQ ID NO: 698)

cmbM1-71
SEQ ID NO: 600
ADVSFSC (amino acids 79-85 of
4

SEQ ID NO: 699)

cmbM1-72
SEQ ID NO: 601
ANLSLCC (amino acids 79-85 of
4

SEQ ID NO: 700)

cmbM1-73
SEQ ID NO: 602

SHL
SVSC (amino acids 79-85 of
6

SEQ ID NO: 701)

cmbM1-74
SEQ ID NO: 603
ANISFSG (amino acids 79-85 of
4

SEQ ID NO: 702)

cmbM1-75
SEQ ID NO: 604

S
NICFSS (amino acids 79-85 of
5

SEQ ID NO: 703)

cmbM1-76
SEQ ID NO: 605

SD
VCLGC (amino acids 79-85 of
5

SEQ ID NO: 704)

cmbM1-77
SEQ ID NO: 606

S
NMCSSS (amino acids 79-85 of
5

SEQ ID NO: 705)

cmbM1-78
SEQ ID NO: 607
ADVCPGC (amino acids 79-85 of
3

SEQ ID NO: 706)

cmbM1-79
SEQ ID NO: 608

CDMAVA
S (amino acids 79-85 of
6

SEQ ID NO: 707)

cmbM1-80
SEQ ID NO: 609
AEISFGC (amino acids 79-85 of
4

SEQ ID NO: 708)

cmbM1-81
SEQ ID NO: 610

G
NLSFCS (amino acids 79-85 of
4

SEQ ID NO: 709)

cmbM1-82
SEQ ID NO: 611

CKL
SQCG (amino acids 79-85 of
6

SEQ ID NO: 710)

cmbM1-83
SEQ ID NO: 612

C
NMSRGS (amino acids 79-85 of
3

SEQ ID NO: 711)

cmbM1-84
SEQ ID NO: 613

SHI
STCA (amino acids 79-85 of
6

SEQ ID NO: 712)

cmbM1-85
SEQ ID NO: 614

CHI
SLGC (amino acids 79-85 of
5

SEQ ID NO: 713)

cmbM1-86
SEQ ID NO: 615

SDICD
GA (amino acids 79-85 of
6

SEQ ID NO: 714)

cmbM1-87
SEQ ID NO: 616
AHIGHAA (amino acids 79-85 of
6

SEQ ID NO: 715)

cmbM1-88
SEQ ID NO: 617

S
NVCPGG (amino acids 79-85 of
3

SEQ ID NO: 716)

cmbM1-89
SEQ ID NO: 618
ANVGPGS (amino acids 79-85 of
1

SEQ ID NO: 717)

cmbM1-90
SEQ ID NO: 619
ANVGPGC (amino acids 79-85 of
2

SEQ ID NO: 718)

cmbM1-91
SEQ ID NO: 620

SHICH
GS (amino acids 79-85 of
5

SEQ ID NO: 719)

cmbM1-92
SEQ ID NO: 621
ADVSLCC (amino acids 79-85 of
4

SEQ ID NO: 720)

cmbM1-93
SEQ ID NO: 622

C
NLSPSC (amino acids 79-85 of
4

SEQ ID NO: 721)

cmbM1-94
SEQ ID NO: 623

GELCVS
S (amino acids 79-85 of
6

SEQ ID NO: 722)

cmbM1-95
SEQ ID NO: 624

CDL
SLSC (amino acids 79-85 of
6

SEQ ID NO: 723)

cmbM1-96
SEQ ID NO: 625

SHL
SFCA (amino acids 79-85 of
6

SEQ ID NO: 724)

cmbM1-97
SEQ ID NO: 626

SHI
SSCS (amino acids 79-85 of
5

SEQ ID NO: 725)

cmbM1-98
SEQ ID NO: 627

CKLGESC
(amino acids 79-85 of
7

SEQ ID NO: 726)

cmbM1-99
SEQ ID NO: 628
ANVCHGC (amino acids 79-85 of
3

SEQ ID NO: 727)

After screening 188 isolates from the Motif 2 (positions T114-S120) combinatorial mutagenesis library, 69 active variants were recovered. Table 17 shows the resulting PIP-47 variant designations, PIP-47 variant gene sequences (SEQ ID NOs), the amino acid sequences of the Motif 2 region of the PIP-47 variants (SEQ ID NOs), and the number of amino acid substitutions (mutations) compared to PIP-47Aa (SEQ ID NO: 2).

TABLE 17

Variants
DNA sequence
Motif 2 AA sequence
Mutations

PIP-47Aa
SEQ ID NO: 1
TDQKPKSAGD (amino acids 114-123 of
0

SEQ ID NO: 2)

cmbM2-01
SEQ ID NO: 728

GEHHLF
SAGD (amino acids 114-123 of
6

SEQ ID NO: 797)

cmbM2-02
SEQ ID NO: 729

G
DDDAGAAGD (amino acids 114-123 of
6

SEQ ID NO: 798)

cmbM2-03
SEQ ID NO: 730

CNNY
PASAGD (amino acids 114-123 of
5

SEQ ID NO: 799)

cmbM2-04
SEQ ID NO: 731

S
DQLNKGAGD (amino acids 114-123 of
4

SEQ ID NO: 800)

cmbM2-05
SEQ ID NO: 732

S
DKESVAAGD (amino acids 114-123 of
6

SEQ ID NO: 801)

cmbM2-06
SEQ ID NO: 733

GKHVST
SAGD (amino acids 114-123 of
6

SEQ ID NO: 802)

cmbM2-07
SEQ ID NO: 734

SQKVCFC
AGD (amino acids 114-123 of
7

SEQ ID NO: 803)

cmbM2-08
SEQ ID NO: 735
TDQKQKSAGD (amino acids 114-123 of
1

SEQ ID NO: 804)

cmbM2-09
SEQ ID NO: 736

GNELCS
SAGD (amino acids 114-123 of
6

SEQ ID NO: 805)

cmbM2-10
SEQ ID NO: 737

G
DHAFLSAGD (amino acids 114-123 of
5

SEQ ID NO: 806)

cmbM2-11
SEQ ID NO: 738

G
DDFLSCAGD (amino acids 114-123 of
6

SEQ ID NO: 807)

cmbM2-12
SEQ ID NO: 739

A
DQKPKSAGD (amino acids 114-123 of
1

SEQ ID NO: 808)

cmbM2-13
SEQ ID NO: 740

CKDSLVA
AGD (amino acids 114-123 of
7

SEQ ID NO: 809)

cmbM2-14
SEQ ID NO: 741

SHNLAT
SAGD (amino acids 114-123 of
6

SEQ ID NO: 810)

cmbM2-15
SEQ ID NO: 742

G
DKGNISAGD (amino acids 114-123 of
5

SEQ ID NO: 811)

cmbM2-16
SEQ ID NO: 743

SNHSFGA
AGD (amino acids 114-123 of
7

SEQ ID NO: 812)

cmbM2-17
SEQ ID NO: 744

CNNNWL
SAGD (amino acids 114-123 of
6

SEQ ID NO: 813)

cmbM2-18
SEQ ID NO: 745

GHEVQQ
SAGD (amino acids 114-123 of
6

SEQ ID NO: 814)

cmbM2-19
SEQ ID NO: 746

GHNAATA
AGD (amino acids 114-123 of
7

SEQ ID NO: 815)

cmbM2-20
SEQ ID NO: 747

GNKHLL
SAGD (amino acids 114-123 of
6

SEQ ID NO: 816)

cmbM2-21
SEQ ID NO: 748

GQNPLW
SAGD (amino acids 114-123 of
6

SEQ ID NO: 817)

cmbM2-22
SEQ ID NO: 749

CQEFSLA
AGD (amino acids 114-123 of
7

SEQ ID NO: 818)

cmbM2-23
SEQ ID NO: 750

SHDMGSA
AGD (amino acids 114-123 of
7

SEQ ID NO: 819)

cmbM2-24
SEQ ID NO: 751

GHNNAI
SAGD (amino acids 114-123 of
6

SEQ ID NO: 820)

cmbM2-25
SEQ ID NO: 752

C
DQDSQAAGD (amino acids 114-123 of
5

SEQ ID NO: 821)

cmbM2-26
SEQ ID NO: 753

S
DQCFSSAGD (amino acids 114-123 of
4

SEQ ID NO: 822)

cmbM2-27
SEQ ID NO: 754

A
DEISIAAGD (amino acids 114-123 of
6

SEQ ID NO: 823)

cmbM2-28
SEQ ID NO: 755

CHNVNF
SAGD (amino acids 114-123 of
6

SEQ ID NO: 824)

cmbM2-29
SEQ ID NO: 756

GQHLMV
SAGD (amino acids 114-123 of
6

SEQ ID NO: 825)

cmbM2-30
SEQ ID NO: 757

S
DNVFFAAGD (amino acids 114-123 of
6

SEQ ID NO: 826)

cmbM2-31
SEQ ID NO: 758

CKK
KLLSAGD (amino acids 114-123 of
5

SEQ ID NO: 827)

cmbM2-32
SEQ ID NO: 759

SHNRWN
SAGD (amino acids 114-123 of
6

SEQ ID NO: 828)

cmbM2-33
SEQ ID NO: 760

G
DQSLSAAGD (amino acids 114-123 of
5

SEQ ID NO: 829)

cmbM2-34
SEQ ID NO: 761

SENDSRA
AGD (amino acids 114-123 of
7

SEQ ID NO: 830)

cmbM2-35
SEQ ID NO: 762

G
DHSYESAGD (amino acids 114-123 of
5

SEQ ID NO: 831)

cmbM2-36
SEQ ID NO: 763
TDDNSKSAGD (amino acids 114-123 of
3

SEQ ID NO: 832)

cmbM2-37
SEQ ID NO: 764

GQNLGL
SAGD (amino acids 114-123 of
6

SEQ ID NO: 833)

cmbM2-38
SEQ ID NO: 765

C
DNFLLAAGD (amino acids 114-123 of
6

SEQ ID NO: 834)

cmbM2-39
SEQ ID NO: 766

C
DNWALCAGD (amino acids 114-123 of
6

SEQ ID NO: 835)

cmbM2-40
SEQ ID NO: 767

SH
QCVWSAGD (amino acids 114-123 of
5

SEQ ID NO: 836)

cmbM2-41
SEQ ID NO: 768

GNDQSR
SAGD (amino acids 114-123 of
6

SEQ ID NO: 837)

cmbM2-42
SEQ ID NO: 769

SKNDFFC
AGE* (amino acids 114-123
8

of SEQ ID NO: 838)

cmbM2-43
SEQ ID NO: 770

GKHCTF
SAGD (amino acids 114-123 of
6

SEQ ID NO: 839)

cmbM2-44
SEQ ID NO: 771

CQNAVFC
AGD (amino acids 114-123 of
7

SEQ ID NO: 840)

cmbM2-45
SEQ ID NO: 772

SNNQL
KSAGD (amino acids 114-123 of
5

SEQ ID NO: 841)

cmbM2-46
SEQ ID NO: 773

SHELIPA
AGD (amino acids 114-123 of
7

SEQ ID NO: 842)

cmbM2-47
SEQ ID NO: 774

C
DQGALSAGD (amino acids 114-123 of
4

SEQ ID NO: 843)

cmbM2-48
SEQ ID NO: 775

C
DDLFRSAGD (amino acids 114-123 of
5

SEQ ID NO: 844)

cmbM2-49
SEQ ID NO: 776

GQDPCLA
AGD (amino acids 114-123 of
7

SEQ ID NO: 845)

cmbM2-50
SEQ ID NO: 777

SEESWI
SAGD (amino acids 114-123 of
6

SEQ ID NO: 846)

cmbM2-51
SEQ ID NO: 778

SEDNRV
SAGD (amino acids 114-123 of
6

SEQ ID NO: 847)

cmbM2-52
SEQ ID NO: 779
TDQISNAAGD (amino acids 114-123 of
4

SEQ ID NO: 848)

cmbM2-53
SEQ ID NO: 780

SHELQS
SAGD (amino acids 114-123 of
6

SEQ ID NO: 849)

cmbM2-54
SEQ ID NO: 781

SEHSCS
SAGD (amino acids 114-123 of
6

SEQ ID NO: 850)

cmbM2-55
SEQ ID NO: 782

S
DQKTKSAGD (amino acids 114-123 of
2

SEQ ID NO: 851)

cmbM2-56
SEQ ID NO: 783

S
DELVLCAGD (amino acids 114-123 of
6

SEQ ID NO: 852)

cmbM2-57
SEQ ID NO: 784

SQHAVL
SAGD (amino acids 114-123 of
6

SEQ ID NO: 853)

cmbM2-58
SEQ ID NO: 785
TEHITCSAGD (amino acids 114-123 of
5

SEQ ID NO: 854)

cmbM2-59
SEQ ID NO: 786

G
DQLSKAAGD (amino acids 114-123 of
4

SEQ ID NO: 855)

cmbM2-60
SEQ ID NO: 787

GNDDRTA
AGD (amino acids 114-123 of
7

SEQ ID NO: 856)

cmbM2-61
SEQ ID NO: 788
TDQKPMSAGD (amino acids 114-123 of
1

SEQ ID NO: 857)

cmbM2-62
SEQ ID NO: 789
TDHKPRSAGD (amino acids 114-123 of
2

SEQ ID NO: 858)

cmbM2-63
SEQ ID NO: 790

SHHSSM
SAGD (amino acids 114-123 of
6

SEQ ID NO: 859)

cmbM2-64
SEQ ID NO: 791
TDDKPNSAGD (amino acids 114-123 of
2

SEQ ID NO: 860)

cmbM2-65
SEQ ID NO: 792
TDNTVISAGD (amino acids 114-123 of
4

SEQ ID NO: 861)

cmbM2-66
SEQ ID NO: 793
TNNMNVAAGD (amino acids 114-123 of
6

SEQ ID NO: 862)

cmbM2-67
SEQ ID NO: 794

GNKTVS
SAGD (amino acids 114-123 of
6

SEQ ID NO: 863)

cmbM2-68
SEQ ID NO: 795

S
DKIVLCAGD (amino acids 114-123 of
6

SEQ ID NO: 864)

cmbM2-69
SEQ ID NO: 796

CQ
QCSFAAGD (amino acids 114-123 of
5

SEQ ID NO: 865)

*random mutation

A third combinatorial mutagenesis library that covered both Motif 1 (positions A79-S85) and Motif 2 (positions T114-S120) was also generated using the same mutagenesis oligos described above. After screening 188 isolates, 22 active variants were identified. Table 18 shows the resulting PIP-47 variant designations, the PIP-47 variant gene sequences (SEQ ID NO:), the amino acid sequences of the Motif 1 region and Motif 2 region of the PIP-47 variants (SEQ ID NO:), and the number of amino acid substitutions (mutations) compared to PIP-47Aa (SEQ ID NO: 2).

TABLE 18

Motif 1 region and Motif 2 region

Variants
DNA sequence
AA sequence
Mutations

PIP-47Aa
SEQ ID NO: 1
YAKDADANVSPGSAP--YNPLDQPTDQKPKS
0

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 2)

cmbM1 + M2-01
SEQ ID NO: 866
YAKDADANMASSAAP--YNPLDQPADHKFQS
5 + 4

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 888)

cmbM1 + M2-02
SEQ ID NO: 867
YAKDADSNVSPGSAP--YNPLDQPCQHCLIS
1 + 6

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 889)

cmbM1 + M2-03
SEQ ID NO: 868
YAKDGDSELALSGAP-YNPLDQPSQQNSIC
8 + 6

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 890)

cmbM1 + M2-04
SEQ ID NO: 869
YAKDADGEICSACAP--YNPLDQPSHEFIPC
7 + 7

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 891)

cmbM1 + M2-05
SEQ ID NO: 870

S
AKDADSQMSASSAP-YNPLDQPSHDNAIA
6 + 7

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 892)

cmbM1 + M2-06
SEQ ID NO: 871
YAKDADSNIGPAGAP-YNPLDQPGHHKTKC
5 + 5

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 893)

cmbM1 + M2-07
SEQ ID NO: 872
YAKDADCHICPGSAP-YNPLDQPTDENAKS
4 + 3

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 894)

cmbM1 + M2-08
SEQ ID NO: 873
YAKDADAHMSFCCAP-YNPLDQPSDKNCKA
5 + 5

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 895)

cmbM1 + M2-09
SEQ ID NO: 874
YAKDADSHISSGCAP-YNPLDQPTDQNPNS
5 + 2

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 896)

cmbM1 + M2-10
SEQ ID NO: 875
YAKDADSHISLAAAP-YNPLDQPTHQSINS
6 + 4

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 897)

cmbM1 + M2-11
SEQ ID NO: 876
YAKDADSNISLCGAP-YNPLDQPTDQKTKS
5 + 1

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 898)

cmbM1 + M2-12
SEQ ID NO: 877
YAKDADGHICPGSAP-YNPLDQPTDKKPNS
4 + 2

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 899)

cmbM1 + M2-13
SEQ ID NO: 878
YAKDADSNLACCSAP-YNPLDQPTDENPKS
5 + 2

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 900)

cmbM1 + M2-14
SEQ ID NO: 879
YAKDADANISPGCAP-YNPLDQPTDNNANS
2 + 4

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 901)

cmbM1 + M2-15
SEQ ID NO: 880
YAKDADADLCPSSAP-YNPLDQPSHHTLSA
4 + 7

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 902)

cmbM1 + M2-16
SEQ ID NO: 881
YAKDADSHIGPSCAP-YNPLDQPTDHKTSS
6 + 3

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 903)

cmbM1 + M2-17
SEQ ID NO: 882
YAKDADSHISYCSAP-YNPLDQPSDDNPTC
5 + 5

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 904)

cmbM1 + M2-18
SEQ ID NO: 883
YAKDADSHIGLGCAP-YNPLDQPTDQKRES
6 + 2

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 905)

cmbM1 + M2-19
SEQ ID NO: 884
YAKDADSDLSPGAAP-YNPLDQPSDDLIAS
4 + 5

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 906)

cmbM1 + M2-20
SEQ ID NO: 885
YAKDADCDICYASAP-YNPLDQPSHDGFPA
6 + 7

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 907)

cmbM1 + M2-21
SEQ ID NO: 886
YAKDADSNISPGGAP-YNPLDQPTDDNPNA
3 + 4

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 908)

cmbM1 + M2-22
SEQ ID NO: 887
YAKDADANVCRGSAP-YNPLDQPTDQKTKS
2 + 1

(amino acids 73-87 & amino acids

107-120 of SEQ ID NO: 909)

Example 12—Characterization of PIP-47Aa and PIP-47Bb Processing by Extract of Corn Tissues

Based on western analysis using polyclonal antibodies raised against PIP-47Aa transgenic corn leaves and roots expressing PIP-47Aa accumulate the expected full-length PIP-47Aa at about 35 kDa but also an approximately 28 kDa processing product. Incubation of purified PIP-47Aa (SEQ ID NO: 2) with nontransgenic root extract showed processing pattern similar to that of PIP-47Aa (SEQ ID NO: 2) containing transgenic tissue. To determine the processing site(s), the ˜28 kDa corn root processing products of PIP-47Aa (SEQ ID NO: 2) and PIP-47Bb (SEQ ID NO: 6) were generated by incubations of purified PIP-47Aa (SEQ ID NO: 2) and PIP-47Bb (SEQ ID NO: 6) with root extract from nontransgenic corn in a PBS-buffer at 37 C for 2 hr. The incubated samples were run on a 4-12% Bis-Tris gels in MOPS buffer (Invitrogen™) and blotted onto a PVDF membrane. The membrane was stained with Coomassie™ Blue R250, destained with 50% methanol, processing bands excised and subjected to Edman sequencing. For PIP-47Aa (SEQ ID NO: 2) the results indicated an approximately equal mixture of proteins with the N-terminal amino acids ATGQSMR (amino acids 46-52 of SEQ ID NO: 2) and SGADAIN (amino acids 53-59 of SEQ ID NO: 2). The results for PIP-47Bb (SEQ ID NO: 6) indicated a protein with the N-terminal amino acids GLSM (amino acids 44-47 of SEQ ID NO: 6).

Example 13—Generating Processing-Resistant Variants of PIP-47Aa by Modification of Processing Sites

To modify the first proteolytic processing site, ATGQSMR (amino acids 46-52 of SEQ ID NO: 2), the N-terminal 52 amino acids of PIP-47Aa was replaced by that of PIP-47Fa (SEQ ID NO: 8) to generate a PIP-47Fa-Aa chimera (SEQ ID NO: 73), which partially prevents processing by corn tissue extract and retains full insecticidal activity. The second processing site was mutagenized by saturation mutagenesis on SGADA (amino acids 53-56) of PIP-47Fa-Aa chimera using mutagenesis oligo 5′-GTTTCTCAGAAAGTTTACTCACACCTGGANNKNNKNNKNNKNNKATCAACCCCTTCTTCA GCCCGGCC-3′ (SEQ ID NO: 922). After screening 192 isolates from the library, 142 active variants were identified. Among them, 47 variants were tested for protein processing by incubating with corn tissue extract for 2 hours at 37° C. followed by a western analysis and 4 processing-resistant variants were identified. Table 19 shows the resulting PIP-47 variant designations, the PIP-47 variant gene sequences (SEQ ID NOs), the amino acid sequences of PIP-47 variants (SEQ ID NOs), and the amino acid substitutions in the two proteolytic processing sites compared to PIP-47Aa (SEQ ID NO: 2). In a further experiment, when wild type protein (PIP-47Aa (SEQ ID NO: 2) was incubated with corn tissue extract for 0, 1 and 5 hours at 37° C., significant protein truncation was observed at 1 hour incubation and >90 protein was processed at 5 hour incubation (data not shown). In contrast, the processing resistant variant proteins B4* (SEQ ID NO: 916) and D4* (SEQ ID NO: 918) showed <10% proteins were processed after 5 hours incubation (data not shown).

TABLE 19

Variant
NA sequence
AA sequence
Proteolytic cleavage site

PIP-47Aa
SEQ ID NO: 1
SEQ ID NO: 1
T A T G Q S M R S G A D A (amino

acids 45-58 of SEQ ID NO: 2)

B4 mutant^†
SEQ ID NO: 910
SEQ ID NO: 916
S E S L L T P G S R P C V (amino

acids 45-58 of SEQ ID NO: 916)

B4 mutant^††
SEQ ID NO: 911
SEQ ID NO: 917
S E S L L T P G S R P C V (amino

acids 45-58 of SEQ ID NO: 917)

D4 mutant^††
SEQ ID NO: 912
SEQ ID NO: 918
S E S L L T P G C G K M P (amino

acids 45-58 of SEQ ID NO: 918)

H8-1
SEQ ID NO: 913
SEQ ID NO: 919
S E S L L T P G D V C E K (amino

mutant^†

acids 45-58 of SEQ ID NO: 919)

H8-2
SEQ ID NO: 914
SEQ ID NO: 920
S E S L L T P G C E S M T (amino

mutant^†

acids 45-58 of SEQ ID NO: 920)

H1 mutant^††
SEQ ID NO: 915
SEQ ID NO: 921
S E S L L T P G D V S M T (amino

acids 45-58 of SEQ ID NO: 921)

^†in PIP-47Fa-Aa chimera backbone (SEQ ID NO: 73);

^††in PIP-47Aa backbone (SEQ ID NO: 2).

The polynucleotides encoding PIP-47Aa (SEQ ID NO: 1) and the PIP-47 variants B4* (SEQ ID NO: 910), B4** (SEQ ID NO: 911), H8-2* (SEQ ID NO: 914), H1** (SEQ ID NO: 915) were cloned into a plant expression vector and transiently expressed in bush bean (Royal Burgundy) leaf disks by Agrobacterium infection. Soluble proteins were extracted from the infected leaf disks and subjected to western analysis using polyclonal antibodies raised against PIP-47Aa to detect in vivo processing of PIP-47Aa and the mutant variants. The results showed that ˜50% wild type PIP-47Aa protein was processed, but the processing products from all 4 mutant proteins were significantly reduced to ˜0 for H1**—SEQ ID NO: 921) and ˜10% for B4* (SEQ ID NO: 916), B4** (SEQ ID NO: 917), and H8-2* (SEQ ID NO: 920) (data not shown).

Example 14—Particle Bombardment Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing a nucleotide sequence encoding the insecticidal protein. The ears are husked and surface sterilized in 30% Clorox® bleach plus 0.5% Micro detergent for 20 minutes and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment. A plasmid vector DNA comprising the nucleotide sequence encoding the insecticidal protein operably linked to a promoter is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl₂precipitation procedure as follows: 100 μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCl₂and 10 μl 0.1 M spermidine.

Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment. The sample plates are bombarded at level #4 in a particle gun. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA

Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5″ pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for expression of PIP-47Aa by assays known in the art, such as, for example, immunoassays and Western blotting.

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D and 2.88 g/l L-proline (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite™ (added after bringing to volume with D-I H₂O) and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose and 2.0 mg/l 2,4-D (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after bringing to volume with D-I H₂O) and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H₂O) (Murashige and Skoog, (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite™ (added after bringing to volume with D-I H₂O) and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/l glycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositol and 40.0 g/l sucrose (brought to volume with polished D-I H₂O after adjusting pH to 5.6) and 6 g/l bacto-agar (added after bringing to volume with polished D-I H₂O), sterilized and cooled to 60° C.

The above description of various illustrated embodiments of the disclosure is not intended to be exhaustive or to limit the invention to the precise form disclosed. While specific embodiments of, and examples for, the invention are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the invention, as those skilled in the relevant art will recognize. The teachings provided herein of the invention can be applied to other purposes, other than the examples described above. The invention may be practiced in ways other than those particularly described in the foregoing description and examples. Numerous modifications and variations of the invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

These and other changes may be made to the invention in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the invention to the specific embodiments disclosed in the specification and the claims.

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, manuals, books or other disclosures) in the Background of the Invention, Detailed Description, and Examples is herein incorporated by reference in their entireties.

Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight; temperature is in degrees centigrade; and pressure is at or near atmospheric.

	Number	Date	Country
Parent	14912356	Feb 2016	US
Child	16001133		US

INSECTICIDAL PROTEINS AND METHODS FOR THEIR USE

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