Patent Grant
7566818
The present invention relates to a novel toxin complex (TC) protein obtainable from Xenorhabdus and to novel insecticidal combinations comprising this protein and other TC proteins.
There is a great need for developing genes that can be expressed in plants in order to provide transgenic insecticidal plants that effectively control various insects. Recently, considerable attention has been given to toxin complex (TC) genes, obtainable from a variety of organisms, including Photorhabdus, Xenorhabdus, Paenibacillus, Serratia, and Pseudomonas.
There has been substantial progress in the cloning of genes encoding insecticidal toxins from both Photorhabdus luminescens and Xenorhabdus nematophilus. Toxin-complex encoding genes from P. luminescens were examined first. See WO 98/08932. Parallel genes were more recently cloned from X. nematophilus. Morgan et al., Applied and Environmental Microbiology 2001, 67:20062-69; WO 95/00647 relates to the use of Xenorhabdus protein toxin to control insects, but it does not recognize orally active toxins. WO 98/08388 relates to orally administered pesticidal agents from Xenorhabdus. U.S. Pat. No. 6,048,838 relates to protein toxins/toxin complexes, having oral activity, obtainable from Xenorhabdus species and strains.
Four different toxin complexes (TCs)—Tca, Tcb, Tcc and Tcd—have been identified in Photorhabdus spp. Each of these toxin complexes resolves as either a single or dimeric species on a native agarose gel but resolution on a denaturing gel reveals that each complex consists of a range of species between 25-280 kDa. See WO 97/17432, WO 98/08932, and R. H. ffrench-Constant and Bowen, 57 Cell. Mol. Life Sci. 828-833 (2000).
Genomic libraries of P. luminescens were screened with DNA probes and with monoclonal and/or polyclonal antibodies raised against the toxins. Four tc loci were cloned: tca, tcb, tcc and tcd. The tca locus is a putative operon of three open reading frames (ORFs), tcaA, tcaB, and tcaC, transcribed from the same DNA strand, with a smaller terminal ORF (tcaZ) transcribed in the opposite direction. The tcc locus also is comprised of three ORFs putatively transcribed in the same direction (tccA, tccB, and tccC). The tcb locus is a single large ORF (tcbA), and the tcd locus is composed of two ORFs (tcdA and tcdB) ; tcbA and tcdA, each about 7.5 kb, encode large insect toxins. TcdB has some level of homology to TcaC. It was determined that many of these gene products were cleaved by proteases. For example, both TcbA and TcdA are cleaved into three fragments termed i, ii and iii (e.g. TcbAi, TcbAii and TcbAiii). Products of the tca and tcc ORFs are also cleaved. See WO 98/08932 and R. H. french-Constant and D. J. Bowen, Current Opinions in Microbiology, 1999, 12:284-288.
Bioassays of the Tca toxin complexes revealed them to be highly toxic to first instar tomato hornworms (Manduca sexta) when given orally (LD50 of 875 ng per square centimeter of artificial diet). R. H. french-Constant and Bowen 1999. Feeding was inhibited at Tca doses as low as 40 ng/cm2. Given the high predicted molecular weight of Tca, on a molar basis, P. luminescens toxins are highly active and relatively few molecules appear to be necessary to exert a toxic effect. R. H. ffrench-Constant and Bowen, Current Opinions in Microbiology, 1999, 12:284-288.
WO 99/42589 and U.S. Pat. No. 6,281,413 disclose TC ORFs from Photorhabdus luminescens. WO 00/30453 and WO 00/42855 disclose TC proteins from Xenorhabdus.
While the exact molecular interactions of the TCs with each other, and their mechanism(s) of action, are not currently understood, it is known, for example, that the Tca toxin complex (comprised of TcaA, TcaB, and TcaC) of Photorhabdus is toxic to Manduca sexta. In addition, some TC proteins are known to have “stand alone” insecticidal activity, while other TC proteins are known to potentiate or enhance the activity of the stand-alone toxins. It is known that the TcdA protein is active, alone, against Manduca sexta, but that TcdB and TccC, together, can be used (in conjunction with TcdA) to greatly enhance the activity of TcdA. TcbA is the other main, stand-alone toxin from Photorhabdus. The activity of this toxin (TcbA) can also be greatly enhanced by TcdB- together with TccC-like proteins.
TcaA, TcaB, TccA, and TccB are referred to as “small toxins”, as distinguished from the “large toxins” TcbA and TcdA. The pair TcaA+TcaB in Toxin C is analogous to the large toxin TcdA in Toxin A; and in the pair TccA+TccB in Toxin D is likewise analogous to the large toxin TcdA. N. R. Waterfield, et al., “The tc genes of Photorhabdus: a growing family,” T
Some Photorhabdus TC proteins have some level of sequence homology with other Photorhabdus TC proteins. As indicated above, TccA has some level of homology with the N terminus of TcdA, and TccB has some level of homology with the C terminus of TcdA. Furthermore, TcdA is about 280 kDa, and TccA together with TccB are of about the same size, if combined, as that of TcdA. Though TccA and TccB are much less active on SCR than TcdA, TccA and TccB from Photorhabdus strain W14 are called “Toxin D.” “Toxin A” (TcdA), “Toxin B” (Tcb or TcbA), and “Toxin C” (TcaA and TcaB) are also indicated above.
Furthermore, TcaA has some level of homology with TccA and likewise with the N terminus of TcdA. Still further, TcaB has some level of homology with TccB and likewise with the N terminus of TcdA. TcdB has a significant level of similarity to TcaC.
Relatively recent cloning efforts in Xenorhabdus nematophilus also appear to have identified novel insecticidal toxin genes with homology to the P. luminescens tc loci. See, e.g., WO 98/08388 and Morgan et al, Applied and Environmental Microbiology 2001, 67:20062-69. In R. H. ffrench-Constant and D. J. Bowen Current Opinions in Microbiology, 1999, 12:284-288, cosmid clones were screened directly for oral toxicity to another lepidopteran, Pieris brassicae. One orally toxic cosmid clone was sequenced. Analysis of the sequence in that cosmid suggested that there are five different ORF's with similarity to Photorhabdus tc genes; orf2 and orf5 both have some level of sequence relatedness to both tcbA and tcdA, whereas orf1 is similar to tccB, orf3 is similar to tccC and orf4 is similar to tcaC. Importantly, a number of these predicted ORFs also share the putative cleavage site documented in P. luminescens, suggesting that active toxins may also be proteolytically processed.
Five typical TC proteins from Xenorhabdus have heretofore been identified: XptA1, XptA2, XptB1, XptC1, and XptD1. XptA1 and XptA2 were known to have stand-alone toxin activity. The XptA2 protein was known to have some degree of similarity to the TcdA protein. XptB1 and XptC1 are Xenorhabdus potentiators that were known to enhance the activity of either (or both) of the XptA toxins. XptD1 was known to have some level of homology with TccB, and XptC1 was known to have some level of similarity to TcaC. XptB1 has some level of similarity to TccC.
United States Patent Application 20040194164 of Scott B. Bintrim et al. on “Xenorhabdus TC proteins and genes for pest control” discloses a set of novel Xenorhabdus TC proteins and genes obtainable from the Xwi strain of Xenorhabdus nematophilus. It also provides an exochitinase obtainable from from the Xwi strain of Xenorhabdus nematophilus.
TC proteins and genes have more recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen. Waterfield et al., TRENDS in Microbiology, Vol. 9, No. 4, April 2001.
TC proteins and lepidopteran-toxic Cry proteins have very recently been discovered in Paenibacillus. See U.S. Ser. No. 60/392,633 (Bintrim et al.), filed Jun. 28, 2002. Bacteria of the genus Paenibacillus are distinguishable from other bacteria by distinctive rRNA and phenotypic characteristics (C. Ash et al. (1993), “Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test: Proposal for the creation of a new genus Paenibacillus,” Antonie Van Leeuwenhoek 64:253-260). Some species in this genus are known to be pathogenic to honeybees (Paenibacillus larvae) and to scarab beetle grubs (P. popilliae and P. lentimorbus.) P. larvae, P. popilliae, and P. lentimorbus are considered obligate insect pathogens involved with milky disease of scarab beetles (D. P. Stahly et al. (1992), “The genus Bacillus: insect pathogens,” p. 1697-1745, In A. Balows et al., ed., The Procaryotes, 2nd Ed., Vol. 2, Springer-Verlag, New York, N.Y.).
Although some Xenorhabdus TC proteins have been found to “correspond” (have a similar function and some level of sequence homology) to some of the Photorhabdus TC proteins, the “corresponding” proteins share only about 40% (approximately) sequence identity with each other. This is also true for the more recently discovered TC proteins from Paenibacillus (those proteins and that discovery are the subject of co-pending U.S. Ser. No. 60/392,633).
Some TC proteins have stand alone insecticidal activity, but it is known that this activity can be enhanced when such proteins are used in combination with other TC proteins. In this regard, three relevant classes of TC proteins have heretofore been identified: Class A proteins, Class B proteins, and Class C proteins. A protein belonging to one of these classes is referred to hereinafter as a “Protein A”, a “Protein B”, or a “Protein C”. Proteins are assigned to Class A, Class B, or Class C based on sequence similarity, as discussed in greater detail hereinafter. Class A proteins have stand alone insecticidal activity. Typical examples of Class A proteins are the “large toxins” TcdA, TcdA2, TcdA4, and TcBA from Photorhabdus luminescens, XptA1Xwi and XptA2Xwi from Xenorhabdus nematophilus, and SepA from Serratia entomophila. Class A proteins should also be understood to include the small toxin pairs that correspond to the large toxins, e.g. TcaA+TcaB in Toxin C and TccA+TccB in Toxin D. Class B and Class C proteins lack significant stand alone insecticidal activity, and have been referred to as potentiators. Typical Class B proteins are TcdB1, TcdB2, TcaC from Photorhabdus luminescens, XptC1Xwi Xenorhabdus nematophilus, PptB11529 from Paenibacilluss spp., and SepB from Serratia entomophila. Typical Class C proteins are TccC2 from Photorhabdus luminescens, XptB1Xwi from Xenorhabdus nematophilus, and XptC1Xb from Xenorhabdus bovienii. It has repeatedly been found that use of a Protein A in combination with a Protein B and a Protein C substantially enhances insecticidal activity over that obtained with the Protein A alone.
United States Patent Application Publication 2004/0208907 demonstrates that the activity of a Protein A can be potentiated by a Protein B and Protein C even if the Protein B and/or Protein C originates from an entirely distinct species from the one that produces the Protein A.
In light of concerns about insects developing resistance to a given pesticidal toxin, and in light of other concerns—some of which are discussed above, there is a continuing need for the discovery of new insecticidal toxins and other proteins that can be used to control insects.
The present invention provides the novel toxin complex (TC) protein complex XTC-2 comprising XptB1 (SEQ ID NO:11), XptC1 (SEQ ID NO:10), XptD1 (SEQ ID NO:2), XptE1 (SEQ ID NO:4), and an exochitinase of SEQ ID NO:6.
The invention also provides a novel toxin complex (TC) protein, XptE1Xwi and DNA sequences encoding it. XptE1Xwi complements the previously known XptD1Xwi in a similar fashion to the pair TcaA+TcaB. That is, the XptD1Xwi+XptE1Xwi pair provides the equivalent of a “large toxin” lass A protein.
The invention also provides insecticidal compositions comprising XptE1Xwi, and methods of controlling insects using it.
More specifically, the invention provides an isolated protein that has toxin activity against an insect, wherein said protein has at least 50% sequence identity with the amino acid sequence of SEQ ID NO:4(XptE1Xwi).
The invention also provides insectidical compositions comprising, in combination, a protein having the amino acid sequence of SEQ ID NO:2 (XptD1Xwi), a protein having the amino acid sequence of SEQ ID NO:4 (XptE1Xwi), a Protein B, and a Protein C, wherein
said Protein B is a 130-180 kda toxin complex potentiator having an amino acid sequence at least 40% identical to a sequence selected from the group consisting of:
SEQ ID NO:7 (TcdB1), SEQ ID NO:8 (TcdB2), SEQ ID NO:9 (TcaC), SEQ ID NO:10 (XptC1Xwi), SEQ ID NO:11 (XptB1Xb), SEQ ID NO:12 (PptB1(orf5)), and SEQ ID NO:13 (SepB); and
said Protein C is a 90-120 kDa toxin complex potentiator having an amino acid sequence at least 35% identical to a sequence selected from the group consisting of:
SEQ ID NO:14 (TccC1), SEQ ID NO:15 (TccC2), SEQ ID NO:16 (TccC3), SEQ ID NO:17 (TccC4), SEQ ID NO:18 (TccC5), SEQ ID NO:19 (XptB1Xwi), SEQ ID NO:20 (XptC1Xb), SEQ ID NO:21 (PptC1 (orf 6 long)), SEQ ID NO:22 (PptC1 (orf 6 short)), SEQ ID NO:23 (SepC).
Isolated polynucleotides and isolated proteins. As used herein, reference to “isolated” polynucleotides and/or “purified” toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature. Thus, reference to “isolated” and/or “purified” signifies the involvement of the “hand of man” as described herein. For example, a bacterial toxin “gene” of the subject invention put into a plant for expression is an “isolated polynucleotide.” Likewise, a Xenorhabdus protein, exemplified herein, produced by a plant is an “isolated protein.”
Toxin activity against an insect. By toxin activity against an insect it is meant that a protein function as an orally active insect control agent (alone or in combination with other proteins), as demonstrated by its ability to disrupt or deter insect activity, growth, and/or feeding. Toxin activity may or may not cause death of the insect. When an insect comes into contact with an effective amount of a “toxin” of the subject invention delivered via transgenic plant expression, formulated protein composition(s), sprayable protein composition(s), a bait matrix or other delivery system, the results are typically death of the insect, inhibition of the growth and/or proliferation of the insect, and/or prevention of the insects from feeding upon the source (preferably a transgenic plant) that makes the toxins available to the insects. Complete lethality to feeding insects is preferred but is not required to achieve functional activity. If an insect avoids the toxin or ceases feeding, that avoidance will be useful in some applications, even if the effects are sublethal or lethality is delayed or indirect. For example, if insect resistant transgenic plants are desired, the reluctance of insects to feed on the plants is as useful as lethal toxicity to the insects because the ultimate objective is avoiding insect-induced plant damage.
Sequence identity. Unless otherwise specified, as used herein percent sequence identity and/or similarity of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990), J. Mol. Biol. 215:402-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al (1997), Nucl. Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See NCBI/NIH website. The scores can also be calculated using the methods and algorithms of Crickmore et al. as described in the Background section, above.
Hybridizes under stringent conditions. As used herein “stringent” conditions for hybridization refers to conditions which achieve the same, or about the same, degree of specificity of hybridization as the conditions employed by the current applicants. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed by standard methods (see, e.g., Maniatis, T., E. F. Fritsch, J. Sambrook [1982] Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). In general, hybridization and subsequent washes were carried out under conditions that allowed for detection of target sequences. For double-stranded DNA gene probes, hybridization was carried out overnight at 20-25E C below the melting temperature (Tm) of the DNA hybrid in 6× SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285):
Tm=81.5E C+16.6 Log[Na+]+0.41 (% G+C)−0.61(% formamide)−600/length of duplex in base pairs.
Washes are typically carried out as follows:
(1) Twice at room temperature for 15 minutes in 1× SSPE, 0.1% SDS (low stringency wash).
(2) Once at Tm-20EC for 15 minutes in 0.2× SSPE, 0.1% SDS (moderate stringency wash).
For oligonucleotide probes, hybridization was carried out overnight at 10-20EC below the melting temperature (Tm) of the hybrid in 6× SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula:
Tm (E C)=2(number T/A base pairs)+4(number G/C base pairs)
(Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown [ed.], Academic Press, New York, 23:683-693).
Washes were typically carried out as follows:
(1) Twice at room temperature for 15 minutes 1× SSPE, 0.1% SDS (low stringency wash).
(2) Once at the hybridization temperature for 15 minutes in 1× SSPE, 0.1% SDS (moderate stringency wash).
In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment>70 or so bases in length, the following conditions can be used:
Protein B. Protein B refers to a 130-180 kDa toxin complex potentiator having an amino acid sequence at least 40% identical to a sequence selected from the group consisting of:
Protein C. Protein C refers to a 90-120 kDa toxin complex potentiator having an amino acid sequence at least 35% identical to a sequence selected from the group consisting of:
Potentiator. Potentiator protein refers to a TC “Protein B” or “Protein C.”
Associating a toxic protein with a material to be protected can be achieved by, e.g. applying the toxic protein to the material, admixing it with the material, and, in the case of transgenic plant material, by causing the plant cells to express the toxic protein.
Effective amount of a toxic protein means an amount that provides toxic activity against an insect.
We describe here the purification and characterization of a novel lepidopertan-active protein toxin complex termed Xenorhabdus Toxin Complex 2 (XTC-2) isolated from Xenorhabdus nematophilus (Xwi strain). We determined XTC-2 to be comprised of five different proteins, XptC1Xwi (a 170 kDa “B” type protein), XptD1Xwi (a 140 kDa protein), XptE1Xwi (a 130 kDa protein), XptB1Xwi (112 kDa “C” type protein) and an exochitinase. The molecular weight of XTC-2 was estimated to be between 1,150-1,300 kDa based upon migration on native polyacrylamide gel electrophoresis (PAGE) and elution volume from size exclusion chromatography. Based upon its estimated molecular weight and the size of the individual protein subunits, we determined that multiple copies of the subunits are most likely present in the complete native protein. When fed to insects in a top load diet bioassay, XTC-2 results in inhibition of larval growth, with fifty percent inhibition of growth (GI50) occurring with approximately 1 ng/cm2 of applied material when tested against corn earworm larvae (CEW, Helicoverpa zea,) and 10-70 ng/cm2 vs tobacco budworm larvae (TBW, Heliothis virescens). The toxin complex has weak activity against beet army worm (BAW, Spodoptera exigua), and did not inhibit growth of southern corn rootworm larvae (SCR, Diahrotica undecimpunctata howardi) at protein concentrations as high as 1,000 ng/cm2. This limited spectrum of insect bioassays demonstrates that XTC-2 has activity against lepidopteran insects, but further bioassays would be needed to fully characterize its spectrum of insect activity.
Methods
Materials. Aqueous solutions were prepared using Milli-Q purified water. All solvents used were HPLC grade. All other chemicals were analytical grade from commercial supply houses. Bovine serum albumin (BSA) Fraction V (cat. #A7906), and the general protease inhibitors for bacterial cell lysates (cat. #P8465) were both from Sigma Chemical, (St. Louis, Mo.). Native molecular weight protein markers were from Amersham (Piscataway, N.J., cat. #17-0445-01). Protein liquid chromatography was performed on an AKTA purifier 900 chromatography system.
Protein Purification. Cell pellets obtained from a 2 liter culture after overnight incubation of the Xenhorabdus nematophihis bacteria were provided by Scott Bintrim, and typically stored frozen at −20° C. for less than two weeks. Frozen cells were suspended in 250 ml of 50 mM Tris-HCl pH 8.0, 0.10 M NaCl, 1 mM DTT, 10% glycerol and lysozyme (0.6 mg/ml). A small amount of glass beads (0.5 mm), was added and the solution gently shaken to facilitate suspension. The cells were then disrupted in approx. 50 ml batches by sonicating them at maximum output power two times for 30 seconds, keeping the lysate cold using an ice bath. The broken cells were then centrifuged at 48,000×g for 60 min. at 4° C. The supernatant was collected, a bacterial protease inhibitor cocktail added (2.0 ml), and the solution dialyzed against 25 mM Tris-HCl, pH 8.0 overnight. The protein was then loaded onto a Q sepharose XL (1.6×10 cm) anion exchange column. Bound proteins were eluted using a linear 0 to 1M NaCl gradient in 10 column volumes at a flow rate of 5 ml/min and collecting in 5 ml fractions. The high molecular weight toxin complexes eluted in the early fractions. These fractions were combined and concentrated to 2 ml using a 100 kDa filter concentrator. The concentrated proteins were then loaded onto a superose 200 size exclusion column (1.6×60 cm), using 50 mM Tris-HCl, 100 mM NaCl, 5% glycerol, 0.05% Tween-20, pH 8.0 at a flow rate of 1.0 m/min. The large molecular weight proteins eluting from the column, were combined, brought to 1.5 M ammonium sulfate concentration and loaded onto a phenyl superose (0.5×5 cm) hydrophobic-interaction column. Proteins were eluted using a decreasing linear gradient of 1.5 to 0 M ammonium sulfate in 25 mM Tris-HCl, pH 8.0 over 20 column volumes at a flow rate of 1 ml/min. The toxin complexes eluted together as a broad peak at low salt concentration. The protein was dialyzed overnight against 25 mM Tris-HCl, and loaded onto a high resolution MonoQ (0.5×5 cm) anion exchange column. The two separate toxin complexes were resolved with baseline resolution using a linear gradient of 0 to IM NaCl in 25 mM Tris-HCl obtained in 20 column volumes. The proteins were identified by N-terminal amino acid sequencing and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry analysis as described below.
Gel electrophoresis. Purification was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and denaturing conditions by method of Laemmli (Laemmli, 1970). Various quantities of protein were loaded into wells of a 4-20% tris-glycine polyacrylamide gel (Zaxis, Hudson, Ohio) and separated by applying 170 volts of electricity for 90 minutes. Detection of the separated protein bands was achieved by staining the gel with Coomassie brilliant blue R-250 (BioRad, Hercules, Calif.) for one hour, then destaining first with a solution of 45% methanol and 10% acetic acid, then with a solution of 5% methanol and 7% acetic acid. The gels were imaged and analyzed using a BioRad Fluro-S Multi Imager™. Relative molecular weight of the protein bands was determined by including a sample of BenchMark™ Protein Ladder (Life Technologies, Rockville, Md.) in one well of the gel.
Proteolytic digestion. SDS gel slices containing Coomassie blue stained bands were removed from the gel and placed into a siliconized Eppendorf microcentrifuge tubes. The gel slices were destained with 50% acetonitrile in 25 mM NH4HCO3 until the slices became clear and the solvent removed using a Speed-Vac (Savant Instruments, Holbrook, N.Y.). The dried gel slices were crushed and enough sequencing grade trypsin at 12.5 μg/ml (Roche Diagnostics, Indianapolis, Ind.) was added to cover the gel pieces. After 30 min incubation at room temperature the excess trypsin was removed and replaced with 25 mM NH4HCO3. The digestion was allowed to proceed for 4 hr at 37° C. The digested peptides were then extracted by shaking for one hour with a solution of 50% acetonitrile in 0.5% trifluoro acetic acid (TFA, Sigma). After brief centrifugation to pellet the gel pieces, the supernatant containing the peptides was transferred to a fresh tube and dried in a Speed-Vac. The peptides were then suspended in 6 μL of 0.1% TFA, absorbed to a C18 ZipTip resin (Millipore, Bedford, Mass.) and eluted with 75% acetonitrile/0.1% TFA. The eluent was stored at −20° C. freezer until MALDI-TOF mass spectral analysis.
Fragment derivatization. To facilitate MALDI-Post Source Decay (PSD) sequencing the peptides were modified using Ettan™ CAF™ Sequencing Kit (Amersham). This modification improves fragmentation efficiency at peptide bonds and results in the formation of only positively charged y ions which become separated in the MALDI reflection. Briefly the peptides were absorbed onto a C18 Ziptip resin. The lysine residues at C-terminus were protected by converting to homoarginine using O-methylisourea-hydrogen sulfate and incubating overnight. After washing the ziptip with water to remove the quanidination reagent, the N-terminal of the peptides were sulfonated with provided CAF reagent solution for at least three min. The ziptip was washed and the derivatized peptides were eluted with 80% acetonitrile for MALDI-PSD sequencing.
Mass Spectrometry Analysis. The extracted peptides were analyzed using MALDI-TOF mass spectrometry. The instrument used was a Voyager DE-STR MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, Mass.). The instrument utilizes a 337 nm nitrogen laser for the desorption/ionization event and a 3.0 meter reflectron time-of-flight tube.
To generate peptide mass fingerprints the samples derived above were spotted onto a MALDI stainless steel plate in a 1:1 ratio of 0.5 μL of sample with 0.5 μL of matrix mixed on the plate using the dried droplet spotting technique (air dried). The matrix was a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile with 0.1% TFA. External calibration was performed by using a solution of Calibration Mix 2 (PE Biosystems, Foster City, Calif.). Internal calibration was performed using, if detected, the autolytic trypsin peak at m/z 842.50, 1045.56, or 2211.10. Acquired spectra were de-isotoped and peptide mass fingerprints (PMF) tables were exported for database searching.
Chemically modified peptides were used for MALDI-PSD sequencing. The instrument was first calibrated with an angiotensin peptide at 1296 Da. Calibration for PSD fragments was performed using the fragmented angiotensin peptide as described by the instrument manual. MALDI-MS was first performed on the samples in both positive and negative modes to generate mass fingerprints. From the fingerprints at least five peptides with high intensities were selected for MALDI-PSD analysis. PSD fragmentation and detection was performed by incrementally increasing laser intensity by 50 V and tilting the reflectron mirror ratio from 1.0 to 0.2. The fragment spectra from different mirror ratios were stitched together by instrument control software. The mass differences between neighboring peaks were calculated and their corresponding amino acid was determined from an amino acid mass table.
Edman N-terminal amino acid sequencing. After SDS PAGE separation, proteins were transferred to PVDF membrane (Bio-Rad) using standard procedure (100V for one hr). The membrane was stained briefly with Coomassie blue, the protein bands removed, dried and sent for N-terminal sequencing. N-terminal sequencing was performed with a Procise 494 (Applied Biosystem, Freemont, Mass.) using a standard protocol provided by the manufacture. Sequence was determined by accompanied software Sequence Pro Ver. 1.0 and then manually corrected for erroneous sequence assignments.
Database searching and protein identification. Proteomics search engine Mascot (London, UK) was used for database searching and protein identification. A proprietary toxin database was constructed by compiling all the known (public and private) Photorhabdus and Xenorhabdus toxin related protein and DNA translation sequences. PMF tables were pasted in the Mascot search form and the search performed against both the toxin database and NCBI non-redundant protein database. No molecular weight and pI restrictions were applied during the search, nor during the numerous modifications. The mass error tolerance was set at 0.1 Da and missed cleavage was set at 1.
Insect bioassays. Corn earworm (CEW, Helicoverpa zea) used in these studies were supplied as eggs by the insectary at North Carolina State University. Tobacco budworm (TBW, Heliothis virescens) and beet armyworm (BAW, Spodoptera exigua) eggs were obtained from the insectary of Dow AgroSciences. Southern corn rootworm eggs (SCR, Diabrotica undecimpunctata howardi) were supplied by FrenchAg Research, Lamberton, Minn., or Crop Characteristics, Inc., Farmington, Minn. The eggs were washed and held at 24° C. and 50% RH until they hatched. The artificial diet consists of 2-4% powdery solids such as soy flour, yeast, wheat germ, casein, sugar, vitamins, and cholesterol suspended in a 1.0-2.0% dissolved agar in water matrix. For bioassay, toxin protein complexes were diluted in ten-fold increments into 10 mM sodium phosphate buffer, pH 7.0 to concentrations ranging from 1 to 1000 ng toxin per cm2 then applied to the surface of the artificial diet. Each concentration was assayed separately with 8 replications by placing newly emerged neonates onto the treated diet and holding the test at 28° C. for five days. The weights of the larvae were measured at the end of the time period in addition to recording mortality or stunting of the insects. Dead larvae were scored as zero weight. GI50 was determined using the measured weights of the insects and the Trimmed Spearman Karber statistical method (Hamilton et al., 1977).
Results
Toxin Complex Purification and Characterization. Initial anion chromatography of the cell lysate from Xenorhabdus nematophilus resulted in poor resolution but captured the majority of the high molecular weight proteins, which eluted between 100-200 mM salt concentrations. Subsequent size exclusion chromatography isolated a well-resolved high molecular weight fraction eluting at 40 ml very near the void volume of the column. This fraction was further resolved into two broad and symmetrical peaks in an approximate 7:1 ratio by hydrophobic-interaction chromatography. The high molecular weight proteins are located in the larger peak eluting at approximately 0.4 M ammonium sulfate and were resolved into two separate peaks using a high resolution MonoQ anion exchange column. The two peaks elute between 35-45 mS/cm conductance. When analyzed by SDS-PAGE, the first peak was determined to consist of three proteins. Using a calibration curve generated from separation of the Benchmark Ladder, these three proteins have apparent molecular weights of 280, 172, and 85 kDa. The molecular weight of these proteins correspond to those found in Xeno Toxin Complex 1 (XTC-1) previously identified (Morgan et al., 2001; Sergeant et al., 2002) composed of proteins XptA2Xwi, XptC1Xwi and XptB1Xwi, respectively. Confirmation of the identity of these proteins was done by MALDI-TOF analysis of each of the protein bands.
The second peak from the MonoQ anion exchange column termed as Xeno Toxin Complex 2 (XTC-2), is composed of at least 4 proteins as determined by SDS-PAGE. Based upon migration on SDS PAGE under reducing conditions, the apparent molecular weights of the four protein bands correspond to 172, 153, 131, and 85 kDa. When analyzed by native PAGE using a 3-8% tris acetate gel, a single high molecular weight protein band is obtained, showing that the individual proteins resolved by SDS-PAGE form a single protein complex under native conditions. Using four molecular weight standards in the native gel, we generated a standard curve for migration of proteins under native conditions. Based upon the relative migration of XTC-2 by PAGE under native conditions and compared to known standards, it has an apparent molecular weight of about 1,300 kDa. Note that due to the very high molecular weight of XTC-2, this value was obtained by extrapolation from the calibration curve. The high molecular weight of XTC-2 indicates that the complex is most likely not only composed of at least the four different proteins resolved by SDS-PAGE, but that multiple copies of the different proteins are most likely included as part of the complex.
When loaded on to a calibrated Superose 200 1×30 cm size exclusion column, XTC-2 eluted as a single symmetrical peak, very close to the void volume of the column. Three separate injections of XTC-2 were made using the calibrated superose 200 1×30 cm column, resulting in elution of the toxin complex at 8.13, 8.19 and 8.02 ml (average=8.11 ml). Using a calibration curve for this size exclusion column, this elution volume corresponded to an apparent molecular weight of 1,150 kDa (range 1,100-1,200 kDa). This value is close to the value obtained by native PAGE analysis (1,300 kDa), and further demonstrates that XTC-2 is most likely composed of multiple copies of the different subunits that can be separated by SDS-PAGE under denaturing conditions.
Densitometric analysis of the SDS-PAGE gel indicates that the different protein bands comprising the XTC-2 are not present at equal concentrations or molar ratios (Table 1). XptC1Xwi is present in the lowest amount, based upon density of staining by coomassie blue dye. Given that this is the largest protein (172 kDa) of the five proteins comprising XTC-2, on a molar basis the content of this protein is very low relative to the molar amount of the other proteins subunits comprising the total toxin. Since the exochintinase and XptB1Xwi was poorly resolved on this gel, we did not determine the relative proportions of these two different proteins comprising the entire Toxin Complex.
Protein Identification. The proteins in the complexes were resolved by SDS-PAGE. From previous work it is known that XTC-1 is composed of four 280 kDa toxin subunits (named XptA2Xwi) and one XptC1Xwi and one XptB1Xwi protein. The components of XTC-1 were confirmed as expected as XptA2Xwi, XptC1Xwi and XptB1Xwi,. Although the particular preparation of XTC-2 illustrated here contains a high molecular weight protein (˜280 kDa) possibly corresponding to XptA2Xwi, in subsequent steps this protein was removed and thus is not part of XTC-2. Two very faint proteins at about 32 kDa were evaluated by MALDI and appear to be breakdown products of the XptD1Xwi and XptB1Xwi proteins (data not shown). These data suggest that toxin complex 2 is composed of at least 4 peptides.
To identify these peptides the protein bands were either blotted onto PVDF membrane for N-terminal sequencing, or analyzed with MALDI-TOF. Initial attempt to identify the peptides in lane 3 using MALDI peptide mass fingerprints (PMF) failed to produce positive hits due to lack of protein sequences in our proprietary toxin database. We subsequently searched for all the Photorhabdus and Xenorhabdus sequences from our previous sequencing efforts and from publicly available database (NCBI non-redundant database and Swiss-Prot database). Obtained DNA sequences were translated into amino acid sequences. This new set of amino acid sequences was then updated to our toxin database. The database searching was repeated against the new toxin database and positive hits with high confidences were returned for all bands in XTC-2. The top band (MW ˜172 kDa) was found to be highly similar to (>95%), if not identical to XptC1Xwi, a protein also found in XTC-1. Forty matching tryptic fragments cover 35% of amino acids in the sequence, yielding a highly significant Mowse score of 290, (a score greater than 40 is significant at the 0.05 level). The second band (MW ˜153) was highly similar to, if not identical to, XptD1Xwi. Forty four matching fragments cover 44% of amino acids in the sequence, producing a Mowse score of 270. The significant hit (Mowse score 121) for the third band (˜131 kDa) corresponds to Xwi XptE1Xwi. However, calculated MW from the XptE1Xwi sequence at hand is only 57 kD, significantly smaller than 131 kD as indicated by SDS PAGE, indicating the sequence in the toxin database is incomplete. The fourth band (˜85 kDa) was found to contain at least two peptides. This was confirmed upon close inspection of the gel that shows two very slightly resolved bands (not very obvious on the digital image). Database searching using mix of the PMFs revealed that one protein is highly similar to, if not identical to Xeno XptB1Xwi (Mowse score 111, coverage 24% and matching peptides 18), the other matches with a Xwi exochitinase (Mowse score 195, coverage 52%, and matching peptides 21). The detail database search results are attached as appendices.
To confirm the identities of these proteins, they were also subjected to N-terminal and internal sequencing. The N-terminal sequence of band 1, determined by Edman degradation, corresponded to N-terminus of XptC1Xwi. The N-terminal sequence of band 2 was identical to N-terminus of XptD1Xwi.
At least five fragments from each of bands corresponding to XptE1Xwi, XptD1Xwi and XptB1Xwi were selected for MALDI-PSD analysis. All the internal fragment sequences matched with their corresponding protein sequences as predicted by MALDI PMF data, indicating positive identification of these 5 proteins.
It is clear that this toxin complex 2 is formed from 5 proteins, namely XptC1Xwi XptE1Xwi, XptD1Xwi, XptB1Xwiand a putative exochitinase. By inspecting Xwi cosmids that were previously characterized, we found that all or at least part of the genes encoding these 5 proteins were located on cosmid 8C3 where the toxin genes (XptA2Xwi, and XptA1Xwi) of XTC-1 are also located (
It is not unusual to find toxin complex and auxiliary genes clustered together in the genome. In cosmid 8c3, there are two large operons composed of toxin-like genes. The first one contains a truncated xptE1Xwi gene, a xptD1Xwi gene (which together would form a toxin A like complex), an exochitanase gene and a gene for xptA1Xwi. The structure of the DNA sequence of the upstream region of the exochitanase region is revealing. Its ribosome binding site is sequestered in a large stem loop structure that would prevent this gene from being transcribed except as part of the operon. Of course, in the genomic sequence xptE1Xwi would not be truncated. We do not know if xptE1Xwi is the first gene in that operon or whether there are more toxin like genes upstream from it. The second operon which is transcribed in the opposite orientation contains a toxin A gene (xptA2Xwi), a xptC1Xwi gene and a xptB1Xwi gene. These two operons are in extremely close proximity, the two ends being within a few hundred bases of each other. While the second operon contains all the genes necessary for toxicity, the first operon does not. Toxin gene clustering, as seen in this and other cases, would ensure that genetic recombination would not separate the first toxin gene operon from the second since the second contains genes essential for the generation of an active complex from the first.
Bioactivity. Highly purified preparations of XTC-1 and XTC-2 were bioassayed against CEW, TBW, BAW and SCR larvae. Both complexes exhibited a similar spectrum of activity against these four insects. Neither complex was active against SCR larvae when tested at a diet concentration of up to 1 μg/cm2. (data not shown). Both complexes, however, were very active against CEW and TBW, but principally against CEW larvae. The primary effect of these toxin complexes against these two insects was stunting, but at the highest concentrations tested (1 μg/cm2), some death of the larvae was observed. LC-50 values were >1 μg/cm2 for both of these toxins against these insects. Bioassay data are presented in the bar graphs shown in
Discussion
This experiment demonstrates the first discovery and characterization of a second complete toxin complex (XTC-2) found in the Xwi strain of Xenorhabdus nematophihis. Using an active cosmid 8C3 prepared by Dow AgroSciences scientists, and from data provided by researchers at HRI using a different cosmid (cHRIM1), scientists at both institutions were able to identify the original toxin complex (XTC-1) that consisted of three proteins, XptA2Xwi, XptC1Xwi and XptB1Xwi. The 8C3 cosmid contains additional genes with unknown function. Some of these genes encode for proteins that we have purified and identified in XTC-2. These include genes encoding XptC1Xwi, XptB1Xwi and the exochitinase. However, parts of the genes encoding the XptD1Xwi and XptE1Xwi proteins were either truncated or abscent in the cHRIM1 cosmid. Another protein belonging to XTC-2 (XptE1Xwi) was missing from the cHRIM1 cosmid. The cHRIM1 cosmid also contained an exochitinase gene directly downstream of the xptD1Xwi gene that was considered not to contribute to bioactivity based on gene inactivation studies. The lack of the complete set of proteins comprising XTC-2 in the cHRIM1 cosmid probably resulted in the poor level of biological activity seen for this cosmid and thus most likely prevented these researchers from identifying these proteins as part of an active protein complex. We now have a more complete picture of the genes in cosmid 8C3 and a better understanding of their function.
The XTC-2 is a very large protein complex, estimated at between 1,150-1,300 kDa, depending on the method utilized to determine protein size. It is composed of at least five different protein subunits. Some of these subunits must be present in multiple copies in the complete toxin complex, since the sum of the molecular weights of the individual components (626 kDa) equals only about half of the molecular weight of XTC-2. The stochiometry of XTC-1 is 4:1:1, for the XptA2Xwi, XptC1Xwi and XptB1Xwi proteins respectively 1. This toxin complex does not have an exochitinase associated with it. Given the homology between XptE1Xwi and XptD1Xwi proteins with XptA2Xwi, it is reasonable to assume that these two proteins represent gene cleavage products, that when put together, organize into a protein of similar structure as XptA2Xwi. Thus, XptE1Xwi+XptD1Xwi together could be equivalent to one XptA2Xwi protein. Thus, if XTC-1 contains four XptA2Xwi subunits, then by inference we could assume that XTC-2 contains four XptE1Xwi and four XptD1Xwi subunits. This would add up to a combined molecular weight of 1,136 kDa, which is still lower than the estimated molecular weight of the entire XTC-2. The addition of a single XptC1Xwi, XptB1Xwi, and exochitinase protein would add 342 kDa to the weight of the complex, resulting in a overall size of 1,478 kDa, which is about 13% larger than the estimated size of the complete toxin complex. These estimates assume that there is no significant truncation or other processing of the protein subunits upon forming the toxin complex. What ever the true stochiometry of the toxin complex turns out to be, it is clearly composed of multiple copies of identical subunits.
The exochitinase was identified by MALDI from what first appeared to be a homogeneous protein band migrating at a molecular mass consistent with that of a truncated XptB1Xwi (85 kDa). While the truncated XptB1Xwi in XTC-2 was positively identified, careful MALDI analysis enabled the discovery that the signal actually was derived from two co-migrating proteins, XptB1Xwi and an exochitinase.
This is the first demonstration of bioactivity from a Xenorhabdus toxin complex containing co-purified XptD1Xwi and exochitinase proteins, and might indicate an important role for this exochitinase in toxin complex mode of action. The precise role of the exochitinase protein in producing insecticidal activity is not known, nor do we know if the exochitinase being bound in the toxin complex has any biological activity. Toxicity bioassay studies using Xeno Toxin Complex 1, recombinant XptA2Xwi and other accessory proteins from Xenorhabdus and Photoharabdus, clearly demonstrate that an exochitinase enzyme is not needed for expression of high levels of oral insecticidal activity with these proteins. What is not known is if the chitinase acts in some manner to increase insecticidal activity beyond what is expressed in its absence. Expression of exo and endochitinase genes in plants have been shown to increase the activity of Bt toxins. (Ding et al., 1998; Regev et al., 1996). Such enzymatic activity might assist in breaking down chitin barriers in the peritrophic membrane separating the toxin from its site of action in the gut of the insect. Being able to express these proteins individually in heterologous systems and obtaining them in purified form would allow more careful biochemical studies aimed at determining the individual role of each protein in producing insecticidal activity.
XptD1Xwi and XptE1Xwi are two proteins very similar to XptA2Xwi. They may represent unique domains of XptA2Xwi that evolutionarily have been separated into two separate gene products. Having these proteins in purified form so that individual bioassays and binding studies may be conducted may give clues to how these proteins function in vivo. Such studies may also provide insight how the XptA2Xwi protein might be mutated or tuncated and still retain biological activity. The discovery of Xeno Toxin Complex 2 is an important finding and we demonstrate here the activity of these proteins with utility for insect control. It should be noted that XptD1Xwi and XptE1Xwi are about half the size of XptA2Xwi, and thus might represent effective alternatives transgenes should they individually possess the biological potency and spectrum required for insect resistance product goal. They also provide information that can be used to engineer XptA2Xwi to make the protein smaller yet still retain activity.
Proteins and toxins. The present invention provides easily administered, functional proteins. The invention also provides a method for delivering insecticidal toxins that are functionally active and effective against many orders of insects, preferably lepidopteran insects.
The subject invention provides new classes of toxins having advantageous pesticidal activities. One way to characterize these classes of toxins and the polynucleotides that encode them is by defining a polynucleotide by its ability to hybridize, under a range of specified conditions, with an exemplified nucleotide sequence (the complement thereof and/or a probe or probes derived from either strand) and/or by their ability to be amplified by PCR using primers derived from the exemplified sequences.
There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or western blotting. Antibodies to the toxins disclosed herein, or to equivalent toxins, or to fragments of these toxins, can be readily prepared using standard procedures. Toxins of the subject invention can be obtained from a variety of sources/source microorganisms.
One skilled in the art would readily recognize that toxins (and genes) of the subject invention can be obtained from a variety of sources. A toxin “from” or “obtainable from” the subject Xwi isolate means that the toxin (or a similar toxin) can be obtained from Xwi or some other source, such as another bacterial strain or a plant. For example, one skilled in the art will readily recognize that, given the disclosure of a bacterial gene and toxin, a plant can be engineered to produce the toxin. Antibody preparations, nucleic acid probes (DNA and RNA), and the like may be prepared using the polynucleotide and/or amino acid sequences disclosed herein and used to screen and recover other toxin genes from other (natural) sources.
Delivery of toxins. There are many other ways in which toxins can be incorporated into an insect's diet. For example, it is possible to adulterate the larval food source with the toxic protein by spraying the food with a protein solution, as disclosed herein. Alternatively, the purified protein could be genetically engineered into an otherwise harmless bacterium, which could then be grown in culture, and either applied to the food source or allowed to reside in the soil in an area in which insect eradication was desirable. Also, the protein could be genetically engineered directly into an insect food source. For instance, the major food source for many insect larvae is plant material. Therefore the genes encoding toxins can be transferred to plant material so that said plant material expresses the toxin of interest.
Transfer of the functional activity to plant or bacterial systems typically requires nucleic acid sequences, encoding the amino acid sequences for the toxins, integrated into a protein expression vector appropriate to the host in which the vector will reside. One way to obtain a nucleic acid sequence encoding a protein with functional activity is to isolate the native genetic material from the bacterial species which produce the toxins, using information deduced from the toxin's amino acid sequence, as disclosed herein. The native sequences can be optimized for expression in plants, for example, as discussed in more detail below. Optimized polynucleotide can also be designed based on the protein sequence.
Polynucleotides and probes. The subject invention further provides nucleotide sequences that encode the toxins of the subject invention. The subject invention further provides methods of identifying and characterizing genes that encode pesticidal toxins. In one embodiment, the subject invention provides unique nucleotide sequences that are useful as hybridization probes and/or primers for PCR techniques. The primers produce characteristic gene fragments that can be used in the identification, characterization, and/or isolation of specific toxin genes. The nucleotide sequences of the subject invention encode toxins that are distinct from previously described toxins.
The polynucleotides of the subject invention can be used to form complete “genes” to encode proteins or peptides in a desired host cell. For example, as the skilled artisan would readily recognize, the subject polynucleotides can be appropriately placed under the control of a promoter in a host of interest, as is readily known in the art.
As the skilled artisan knows, DNA typically exists in a double-stranded form. In this arrangement, one strand is complementary to the other strand and vice versa. As DNA is replicated in a plant (for example), additional complementary strands of DNA are produced. The “coding strand” is often used in the art to refer to the strand that binds with the anti-sense strand. The mRNA is transcribed from the “anti-sense” strand of DNA. The “sense” or “coding” strand has a series of codons (a codon is three nucleotides that can be read as a three-residue unit to specify a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest. In order to produce a protein in vivo, a strand of DNA is typically transcribed into a complementary strand of mRNA which is used as the template for the protein. Thus, the subject invention includes the use of the exemplified polynucleotides shown in the attached sequence listing and/or equivalents including the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the exemplified DNA are included in the subject invention.
In one embodiment of the subject invention, bacterial isolates can be cultivated under conditions resulting in high multiplication of the microbe. After treating the microbe to provide single-stranded genomic nucleic acid, the DNA can be contacted with the primers of the invention and subjected to PCR amplification. Characteristic fragments of toxin-encoding genes will be amplified by the procedure, thus identifying the presence of the toxin-encoding gene(s).
Further aspects of the subject invention include genes and isolates identified using the methods and nucleotide sequences disclosed herein. The genes thus identified encode toxins active against pests.
Toxins and genes of the subject invention can be identified and obtained by using oligonucleotide probes, for example. These probes are detectable nucleotide sequences which may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO 93/16094. The probes (and the polynucleotides of the subject invention) may be DNA, RNA, or PNA. In addition to adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U; for RNA molecules), synthetic probes (and polynucleotides) of the subject invention can also have inosine (a neutral base capable of pairing with all four bases; sometimes used in place of a mixture of all four bases in synthetic probes). Thus, where a synthetic, degenerate oligonucleotide is referred to herein, and “n” is used generically, “n” can be G, A, T, C, or inosine. Ambiguity codes as used herein are in accordance with standard IUPAC naming conventions as of the filing of the subject application (for example, R means A or G, Y means C or T, etc.).
As is well known in the art, if a probe molecule hybridizes with a nucleic acid sample, it can be reasonably assumed that the probe and sample have substantial homology/similarity/identity. Preferably, hybridization of the polynucleotide is first conducted followed by washes under conditions of low, moderate, or high stringency by techniques well-known in the art, as described in, for example, Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170. For example, as stated therein, low stringency conditions can be achieved by first washing with 2×SSC (Standard Saline Citrate)/0.1% SDS (Sodium Dodecyl Sulfate) for 15 minutes at room temperature. Two washes are typically performed. Higher stringency can then be achieved by lowering the salt concentration and/or by raising the temperature. For example, the wash described above can be followed by two washings with 0.1×SSC/0.1% SDS for 15 minutes each at room temperature followed by subsequent washes with 0.1×SSC/0.1% SDS for 30 minutes each at 55E C. These temperatures can be used with other hybridization and wash protocols set forth herein and as would be known to one skilled in the art (SSPE can be used as the salt instead of SSC, for example). The 2×SSC/0.1% SDS can be prepared by adding 50 ml of 20×SSC and 5 ml of 10% SDS to 445 ml of water. 20×SSC can be prepared by combining NaCl (175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), and water to 1 liter, followed by adjusting pH to 7.0 with 10 N NaOH. 10% SDS can be prepared by dissolving 10 g of SDS in 50 ml of autoclaved water, diluting to 100 ml, and aliquotting.
Detection of the probe provides a means for determining in a known manner whether hybridization has been maintained. Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the subject invention.
Hybridization characteristics of a molecule can be used to define polynucleotides of the subject invention. Thus the subject invention includes polynucleotides (and/or their complements, preferably their full complements) that hybridize with a polynucleotide exemplified herein.
Duplex formation and stability depend on substantial complementarily between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.
PCR technology. Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki, Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim [1985] “Enzymatic Amplification of P-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia,” Science 230:1350-1354). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3′ ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5′ ends of the PCR primers. The extension product of each primer can serve as a template for the other primer, so each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.
The DNA sequences of the subject invention can be used as primers for PCR amplification. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5N end) of the exemplified primers fall within the scope of the subject invention. Mutations, insertions, and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan.
Modification of genes and toxins. The genes and toxins useful according to the subject invention include not only the specifically exemplified full-length sequences, but also portions, segments and/or fragments (including internal and/or terminal deletions compared to the full-length molecules) of these sequences, variants, mutants, chimerics, and fusions thereof Proteins of the subject invention can have substituted amino acids so long as they retain the characteristic pesticidal/ functional activity of the proteins specifically exemplified herein. “Variant” genes have nucleotide sequences that encode the same toxins or equivalent toxins having pesticidal activity equivalent to an exemplified protein. The terms “variant proteins” and “equivalent toxins” refer to toxins having the same or essentially the same biological/functional activity against the target pests and equivalent sequences as the exemplified toxins. As used herein, reference to an “equivalent” sequence refers to sequences having amino acid substitutions, deletions, additions, or insertions which improve or do not adversely affect pesticidal activity. Fragments retaining pesticidal activity are also included in this definition. Fragments and other equivalents that retain the same or similar function, or “toxin activity,” as a corresponding fragment of an exemplified toxin are within the scope of the subject invention. Changes, such as amino acid substitutions or additions, can be made for a variety of purposes, such as increasing (or decreasing) protease stability of the protein (without materially/substantially decreasing the functional activity of the toxin).
Equivalent toxins and/or genes encoding these equivalent toxins can be obtained/derived from wild-type or recombinant bacteria and/or from other wild-type or recombinant organisms using the teachings provided herein. Other Bacillus, Paenibacillus, Photorhabdus, and Xenorhabdus species, for example, can be used as source isolates.
Variations of genes may be readily constructed using standard techniques for making point mutations, for example. In addition, U.S. Pat. No. 5,605,793, for example, describes methods for generating additional molecular diversity by using DNA reassembly after random fragmentation. Variant genes can be used to produce variant proteins; recombinant hosts can be used to produce the variant proteins. Using these “gene shuffling” techniques, equivalent genes and proteins can be constructed that comprise any 5, 10, or 20 contiguous residues (amino acid or nucleotide) of any sequence exemplified herein. As one skilled in the art knows, the gene shuffling techniques can be adjusted to obtain equivalents having, for example, 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, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 contiguous residues (amino acid or nucleotide), corresponding to a segment (of the same size) in any of the exemplified sequences (or the complements (full complements) thereof). Similarly sized segments, especially those for conserved regions, can also be used as probes and/or primers.
Fragments of full-length genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.
It is within the scope of the invention as disclosed herein that toxins may be truncated and still retain functional activity. By “truncated toxin” is meant that a portion of a toxin protein may be cleaved and yet still exhibit activity after cleavage. Cleavage can be achieved by proteases inside or outside of the insect gut. Furthermore, effectively cleaved proteins can be produced using molecular biology techniques wherein the DNA bases encoding said toxin are removed either through digestion with restriction endonucleases or other techniques available to the skilled artisan. After truncation, said proteins can be expressed in heterologous systems such as E coli, baculoviruses, plant-based viral systems, yeast and the like and then placed in insect assays as disclosed herein to determine activity. It is well-known in the art that truncated toxins can be successfully produced so that they retain functional activity while having less than the entire, full-length sequence. It is well known in the art that B. t. toxins can be used in a truncated (core toxin) form. See, e.g., Adang el al, Gene 36:289-300 (1985), “Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp kurstaki HD-73 and their toxicity to Manduca sexta.” There are other examples of truncated proteins that retain insecticidal activity, including the insect juvenile hormone esterase (U.S. Pat. No. 5,674,485 to the Regents of the University of California). As used herein, the term “toxin” is also meant to include functionally active truncations.
Certain toxins of the subject invention have been specifically exemplified herein. As these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin. Equivalent toxins will have amino acid similarity (and/or homology) with an exemplified toxin. The amino acid identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. Preferred polynucleotides and proteins of the subject invention can also be defined in terms of more particular identity and/or similarity ranges. For example, the identity and/or similarity can be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.
The amino acid homology/similarity/identity will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which is ultimately responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected to be tolerated. For example, these substitutions can be in regions of the protein that are not critical to activity. Analyzing the crystal structure of a protein, and software-based protein structure modeling, can be used to identify regions of a protein that can be modified (using site-directed mutagenesis, shuffling, etc.) to actually change the properties and/or increase the functionality of the protein.
Various properties and three-dimensional features of the protein can also be changed without adversely affecting the toxin activity/functionality of the protein. Conservative amino acid substitutions can be expected to be tolerated/to not adversely affect the three-dimensional configuration of the molecule. Amino acids can be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution is not adverse to the biological activity of the compound. Table 1 provides a listing of examples of amino acids belonging to each class.
In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the functional/biological activity of the toxin.
Because of the degeneracy/redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create alternative DNA sequences that encode the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention.
Optimization of sequence for expression in plants. To obtain high expression of heterologous genes in plants it may be preferred to reengineer said genes so that they are more efficiently expressed in (the cytoplasm of) plant cells. Maize is one such plant where it may be preferred to re-design the heterologous gene(s) prior to transformation to increase the expression level thereof in said plant. Therefore, an additional step in the design of genes encoding a bacterial toxin is reengineering of a heterologous gene for optimal expression.
One reason for the reengineering of a bacterial toxin for expression in maize is due to the non-optimal G+C content of the native gene. For example, the very low G+C content of many native bacterial gene(s) (and consequent skewing towards high A+T content) results in the generation of sequences mimicking or duplicating plant gene control sequences that are known to be highly A+T rich. The presence of some A+T-rich sequences within the DNA of gene(s) introduced into plants (e.g., TATA box regions normally found in gene promoters) may result in aberrant transcription of the gene(s). On the other hand, the presence of other regulatory sequences residing in the transcribed MRNA (e.g., polyadenylation signal sequences (AAUAAA), or sequences complementary to small nuclear RNAs involved in pre-mRNA splicing) may lead to RNA instability. Therefore, one goal in the design of genes encoding a bacterial toxin for maize expression, more preferably referred to as plant optimized gene(s), is to generate a DNA sequence having a higher G+C content, and preferably one close to that of maize genes coding for metabolic enzymes. Another goal in the design of the plant optimized gene(s) encoding a bacterial toxin is to generate a DNA sequence in which the sequence modifications do not hinder translation.
The table below (Table 2) illustrates how high the G+C content is in maize. For the data in Table 2, coding regions of the genes were extracted from GenBank (Release 71) entries, and base compositions were calculated using the MacVector.TM. program (IBI, New Haven, Conn.). Intron sequences were ignored in the calculations.
Due to the plasticity afforded by the redundancy/degeneracy of the genetic code (i.e., some amino acids are specified by more than one codon), evolution of the genomes in different organisms or classes of organisms has resulted in differential usage of redundant codons. This “codon bias” is reflected in the mean base composition of protein coding regions. For example, organisms with relatively low G+C contents utilize codons having A or T in the third position of redundant codons, whereas those having higher G+C contents utilize codons having G or C in the third position. It is thought that the presence of “minor” codons within a mRNA may reduce the absolute translation rate of that mRNA, especially when the relative abundance of the charged tRNA corresponding to the minor codon is low. An extension of this is that the diminution of translation rate by individual minor codons would be at least additive for multiple minor codons. Therefore, mRNAs having high relative contents of minor codons would have correspondingly low translation rates. This rate would be reflected by subsequent low levels of the encoded protein.
In engineering genes encoding a bacterial toxin for maize (or other plant, such as cotton or soybean) expression, the codon bias of the plant has been determined. The codon bias for maize is the statistical codon distribution that the plant uses for coding its proteins and the preferred codon usage is shown in Table 3. After determining the bias, the percent frequency of the codons in the gene(s) of interest is determined. The primary codons preferred by the plant should be determined as well as the second and third choice of preferred codons. Afterwards, the amino acid sequence of the bacterial toxin of interest is reverse translated so that the resulting nucleic acid sequence codes for exactly the same protein as the native gene wanting to be heterologously expressed. The new DNA sequence is designed using codon bias information so that it corresponds to the most preferred codons of the desired plant. The new sequence is then analyzed for restriction enzyme sites that might have been created by the modification. The identified sites are further modified by replacing the codons with second or third choice preferred codons. Other sites in the sequence which could affect transcription or translation of the gene of interest are the exon:intron junctions (5′ or 3′), poly A addition signals, or RNA polymerase termination signals. The sequence is further analyzed and modified to reduce the frequency of TA or GC doublets. In addition to the doublets, G or C sequence blocks that have more than about four residues that are the same can affect transcription of the sequence. Therefore, these blocks are also modified by replacing the codons of first or second choice, etc. with the next preferred codon of choice.
It is preferred that the plant optimized gene(s) encoding a bacterial toxin contain about 63% of first choice codons, between about 22% to about 37% second choice codons, and between about 15% to about 0% third choice codons, wherein the total percentage is 100%. Most preferred the plant optimized gene(s) contains about 63% of first choice codons, at least about 22% second choice codons, about 7.5% third choice codons, and about 7.5% fourth choice codons, wherein the total percentage is 100%. The preferred codon usage for engineering genes for maize expression are shown in Table 3. The method described above enables one skilled in the art to modify gene(s) that are foreign to a particular plant so that the genes are optimally expressed in plants. The method is further illustrated in PCT application WO 97/13402.
In order to design plant optimized genes encoding a bacterial toxin, the amino acid sequence of said protein is reverse translated into a DNA sequence utilizing a non-redundant genetic code established from a codon bias table compiled for the gene sequences for the particular plant, as shown in Table 2. The resulting DNA sequence, which is completely homogeneous in codon usage, is further modified to establish a DNA sequence that, besides having a higher degree of codon diversity, also contains strategically placed restriction enzyme recognition sites, desirable base composition, and a lack of sequences that might interfere with transcription of the gene, or translation of the product mRNA.
Thus, synthetic genes that are functionally equivalent to the toxins/genes of the subject invention can be used to transform hosts, including plants. Additional guidance regarding the production of synthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.
In some cases, especially for expression in plants, it can be advantageous to use truncated genes that express truncated proteins. Höfte et al. 1989, for example, discussed in the Background Section above, discussed protoxin and core toxin segments of B. t. toxins. Preferred truncated genes will typically encode 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the full-length toxin.
Transgenic hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. In preferred embodiments, transgenic plant cells and plants are used. Preferred plants (and plant cells) are corn, maize, and cotton.
In preferred embodiments, expression of the toxin gene results, directly or indirectly, in the intracellular production (and maintenance) of the pesticide proteins. Plants can be rendered insect-resistant in this manner. When transgenic/recombinant/transformed/transfected host cells (or contents thereof) are ingested by the pests, the pests will ingest the toxin. This is the preferred manner in which to cause contact of the pest with the toxin. The result is control (killing or making sick) of the pest. Sucking pests can also be controlled in a similar manner. Alternatively, suitable microbial hosts, e.g., Pseudomonas such as P. fluorescens, can be applied where target pests are present; the microbes can proliferate there, and are ingested by the target pests. The microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, can then be applied to the environment of the target pest.
Where the toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, certain host microbes should be used. Microorganism hosts are selected which are known to occupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetohacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharornyces, Cryptococcus, Kluyvetomyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; 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, Sporobolomnyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Also of interest are pigmented microorganisms.
Insertion of genes to form transgenic hosts. One aspect of the subject invention is the transformation/transfection of plants, plant cells, and other host cells with polynucleotides of the subject invention that express proteins of the subject invention. Plants transformed in this manner can be rendered resistant to attack by the target pest(s).
A wide variety of methods are available for introducing a gene encoding a pesticidal protein into the target host under conditions that allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,135,867.
For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13 mp series, pACYC184, etc. Accordingly, the sequence encoding the toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and described in EP 120 516; Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley el al., Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J. 4:277-287.
A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and inAgrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters el al. [1978] Mol. Gen. Genet. 163:181-187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.
The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
In some preferred embodiments of the invention, genes encoding the bacterial toxin are expressed from transcriptional units inserted into the plant genome. Preferably, said transcriptional units are recombinant vectors capable of stable integration into the plant genome and enable selection of transformed plant lines expressing mRNA encoding the proteins.
Once the inserted DNA has been integrated in the genome, it is relatively stable there (and does not come out again). It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA. The gene(s) of interest are preferably expressed either by constitutive or inducible promoters in the plant cell. Once expressed, the mRNA is translated into proteins, thereby incorporating amino acids of interest into protein. The genes encoding a toxin expressed in the plant cells can be under the control of a constitutive promoter; a tissue-specific promoter, or an inducible promoter.
Several techniques exist for introducing foreign recombinant vectors into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include the introduction of genetic material coated onto microparticles directly into cells (U.S. Pat. Nos. 4,945,050 to Cornell and 5,141,131 to DowElanco, now Dow AgroSciences, LLC). In addition, plants may be transformed using Agrobacterium technology, see U.S. Pat. No. 5,177,010 to University of Toledo; 5,104,310 to Texas A&M; European Patent Application 0131624B1; European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot; U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot; European Patent Applications 116718, 290799, 320500 all to Max Planck; European Patent Applications 604662 and 627752, and U.S. Pat. No. 5,591,616, to Japan Tobacco; European Patent Applications 0267159 and 0292435, and U.S. Pat. No. 5,231,019, all to Ciba Geigy, now Novartis; U.S. Pat. Nos. 5,463,174 and 4,762,785, both to Calgene; and U.S. Pat. Nos. 5,004,863 and 5,159,135, both to Agracetus. Other transformation technology includes whiskers technology. See U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca. Electroporation technology has also been used to transform plants. See WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and 5,384,253, both to Dekalb; and WO 92/09696 and WO 93/21335, both to Plant Genetic Systems. Furthermore, viral vectors can also be used to produce transgenic plants expressing the protein of interest. For example, monocotyledonous plant can be transformed with a viral vector using the methods described in U.S. Pat. No. 5,569,597 to Mycogen Plant Science and Ciba-Giegy, now Novartis, as well as U.S. Pat. Nos. 5,589,367 and 5,316,931, both to Biosource.
As mentioned previously, the manner in which the DNA construct is introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation may be employed. For example, various methods for plant cell transformation are described herein and include the use of Ti or Ri-plasmids and the like to perform Agrobacterium mediated transformation. In many instances, it will be desirable to have the construct used for transformation bordered on one or both sides by T-DNA borders, more specifically the right border. This is particularly useful when the construct uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode for transformation, although T-DNA borders may find use with other modes of transformation. Where Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the host for homologous recombination with T-DNA or the Ti or Ri plasmid present in the host. Introduction of the vector may be performed via electroporation, tri-parental mating and other techniques for transforming gram-negative bacteria which are known to those skilled in the art. The manner of vector transformation into the Agrobacterium host is not critical to this invention. The Ti or Ri plasmid containing the T-DNA for recombination may be capable or incapable of causing gall formation, and is not critical to said invention so long as the vir genes are present in said host.
In some cases where Agrobacterium is used for transformation, the expression construct being within the T-DNA borders will be inserted into a broad spectrum vector such as pRK2 or derivatives thereof as described in Ditta et al., (PNAS USA(1980) 77:7347-7351 and EPO 0 120 515, which are incorporated herein by reference. Included within the expression construct and the T-DNA will be one or more markers as described herein which allow for selection of transformed Agrobacteriumand transformed plant cells. The particular marker employed is not essential to this invention, with the preferred marker depending on the host and construction used.
For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time to allow transformation thereof. After transformation, the Agrobacteria are killed by selection with the appropriate antibiotic and plant cells are cultured with the appropriate selective medium. Once calli are formed, shoot formation can be encouraged by employing the appropriate plant hormones according to methods well known in the art of plant tissue culturing and plant regeneration. However, a callus intermediate stage is not always necessary. After shoot formation, said plant cells can be transferred to medium which encourages root formation thereby completing plant regeneration. The plants may then be grown to seed and said seed can be used to establish future generations. Regardless of transformation technique, the gene encoding a bacterial toxin is preferably incorporated into a gene transfer vector adapted to express said gene in a plant cell by including in the vector a plant promoter regulatory element, as well as 3′ non-translated transcriptional termination regions such as Nos and the like.
In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue types I, II, and III, hypocotyl, meristem, root tissue, tissues for expression in phloem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques described herein.
As mentioned above, a variety of selectable markers can be used, if desired. Preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which encode for resistance or tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos); imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorsulfuron; bromoxynil, dalapon and the like.
In addition to a selectable marker, it may be desirous to use a reporter gene. In some instances a reporter gene may be used with or without a selectable marker. Reporter genes are genes which are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Wising et al. Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes include the beta-glucuronidase (GUS) of the uidA locus of E. coli, the chloramphenicol acetyl transferase gene from Tn9 of E. coli, the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria, and the luciferase genes from firefly Photinus pyralis. An assay for detecting reporter gene expression may then be performed at a suitable time after said gene has been introduced into recipient cells. A preferred such assay entails the use of the gene encoding beta-glucuronidase (GUS) of the uidA locus of E. coli as described by Jefferson el al., (1987 Biochem. Soc. Trans. 15, 17-19) to identify transformed cells.
In addition to plant promoter regulatory elements, promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoter regulatory elements of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No. 6,166,302, especially Example 7E) and the like may be used. Plant promoter regulatory elements include but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat-shock promoters, and tissue specific promoters. Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences and the like may be present and thus may improve the transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA by affecting transcription, mRNA stability, and the like. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include but are not limited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coat protein leader sequence, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan. Constitutive promoter regulatory elements may also be used thereby directing continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoter regulatory elements are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and these may also be used.
Promoter regulatory elements may also be active during a certain stage of the plant's development as well as active in plant tissues and organs. Examples of such include but are not limited to pollen-specific, embryo-specific, corn-silk-specific, cotton-fiber-specific, root-specific, seed-endosperm-specific promoter regulatory elements and the like. Under certain circumstances it may be desirable to use an inducible promoter regulatory element, which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes), light (RUBP carboxylase), hormone (Em), metabolites, chemical, and stress. Other desirable transcription and translation elements that function in plants may be used. Numerous plant-specific gene transfer vectors are known in the art.
Standard molecular biology techniques may be used to clone and sequence the toxins described herein. Additional information may be found in Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, which is incorporated herein by reference.
Resistance Management. With increasing commercial use of insecticidal proteins in transgenic plants, one consideration is resistance management. That is, there are numerous companies using Bacillus thuringiensis toxins in their products, and there is concern about insects developing resistance to B. t. toxins. One strategy for insect resistance management would be to combine the TC toxins produced by Xenorhabdus, Photorhabdus, and the like with toxins such as B. t. crystal toxins, soluble insecticidal proteins from Bacillus stains (see, e.g., WO 98/18932 and WO 99/57282), or other insect toxins. The combinations could be formulated for a sprayable application or could be molecular combinations. Plants could be transformed with bacterial genes that produce two or more different insect toxins (see, e.g., Gould, 38 Bioscience 26-33 (1988) and U.S. Pat. No. 5,500,365; likewise, European Patent Application 0 400 246 A1 and U.S. Pats. 5,866,784; 5,908,970; and 6,172,281 also describe transformation of a plant with two B. t. crystal toxins). Another method of producing a transgenic plant that contains more than one insect resistant gene would be to first produce two plants, with each plant containing an insect resistance gene. These plants could then be crossed using traditional plant breeding techniques to produce a plant containing more than one insect resistance gene. Thus, it should be apparent that the phrase “comprising a polynucleotide” as used herein means at least one polynucleotide (and possibly more, contiguous or not) unless specifically indicated otherwise.
Formulations and Other Delivery Systems. Formulated bait granules containing spores and/or crystals of the subject Paenibacillus isolate, or recombinant microbes comprising the genes obtainable from the isolate disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of cells may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include Theological agents, surfactants, emulsifiers, dispersants, or polymers.
As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about 102 to about 104 cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the pest, e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.
Another delivery scheme is the incorporation of the genetic material of toxins into a baculovirus vector. Baculoviruses infect particular insect hosts, including those desirably targeted with the toxins. Infectious baculovirus harboring an expression construct for the toxins could be introduced into areas of insect infestation to thereby intoxicate or poison infected insects.
Insect viruses, or baculoviruses, are known to infect and adversely affect certain insects. The affect of the viruses on insects is slow, and viruses do not immediately stop the feeding of insects. Thus, viruses are not viewed as being optimal as insect pest control agents. However, combining the toxin genes into a baculovirus vector could provide an efficient way of transmitting the toxins. In addition, since different baculoviruses are specific to different insects, it may be possible to use a particular toxin to selectively target particularly damaging insect pests. A particularly useful vector for the toxins genes is the nuclear polyhedrosis virus. Transfer vectors using this virus have been described and are now the vectors of choice for transferring foreign genes into insects. The virus-toxin gene recombinant may be constructed in an orally transmissible form. Baculoviruses normally infect insect victims through the mid-gut intestinal mucosa. The toxin gene inserted behind a strong viral coat protein promoter would be expressed and should rapidly kill the infected insect.
In addition to an insect virus or baculovirus or transgenic plant delivery system for the protein toxins of the present invention, the proteins may be encapsulated using Bacillus thuringiensis encapsulation technology such as but not limited to U.S. Pat. Nos. 4,695,455; 4,695,462; 4,861,595 which are all incorporated herein by reference. Another delivery system for the protein toxins of the present invention is formulation of the protein into a bait matrix, which could then be used in above and below ground insect bait stations. Examples of such technology include but are not limited to PCT Patent Application WO 93/23998, which is incorporated herein by reference.
Plant RNA viral based systems can also be used to express bacterial toxin. In so doing, the gene encoding a toxin can be inserted into the coat promoter region of a suitable plant virus which will infect the host plant of interest. The toxin can then be expressed thus providing protection of the plant from insect damage. Plant RNA viral based systems are described in U.S. Pat. No. 5,500,360 to Mycogen Plant Sciences, Inc. and U.S. Pat. Nos. 5,316,931 and 5,589,367 to Biosource Genetics Corp.
In addition to producing a transformed plant, there are other delivery systems where it may be desirable to reengineer the bacterial gene(s). For example, a protein toxin can be constructed by fusing together a molecule attractive to insects as a food source with a toxin. After purification in the laboratory such a toxic agent with “built-in” bait could be packaged inside standard insect trap housings.
Mutants. Mutants of the Xenorhabdus Xwi isolate of the invention can be made by procedures that are well known in the art. For example, asporogenous mutants can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.
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