Patent Application
20120317682
Humans grow corn for food and energy applications. Humans also grow many other crops, including soybeans and cotton. Insects eat and damage plants and thereby undermine these human efforts. Billions of dollars are spent each year to control insect pests and additional billions are lost to the damage they inflict. Synthetic organic chemical insecticides have been the primary tools used to control insect pests but biological insecticides, such as the insecticidal proteins derived from Bacillus thuringiensis (Bt), have played an important role in some areas. The ability to produce insect-resistant plants through transformation with Bt insecticidal protein genes has revolutionized modern agriculture and heightened the importance and value of insecticidal proteins and their genes.
Several Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include Cry1Ab, Cry1Ac, Cry1F and Cry3Bb in corn, Cry1Ac and Cry2Ab in cotton, and Cry3A in potato.
The commercial products expressing these proteins express a single protein except in cases where the combined insecticidal spectrum of 2 proteins is desired (e.g., Cry1Ab and Cry3Bb in corn combined to provide resistance to lepidopteran pests and rootworm, respectively) or where the independent action of the proteins makes them useful as a tool for delaying the development of resistance in susceptible insect populations (e.g., Cry1Ac and Cry2Ab in cotton combined to provide resistance management for tobacco budworm). See also US 2009 0313717, which relates to a Cry2 protein plus a Vip3Aa, Cry1F, or Cry1A for control of Helicoverpa zea or armigerain. WO 2009 132850 relates to Cry1F or Cry1A and Vip3Aa for controlling Spodoptera frugiperda. US 2008 0311096 relates in part to Cry1Ab for controlling Cry1F-resistant ECB.
That is, some of the qualities of insect-resistant transgenic plants that have led to rapid and widespread adoption of this technology also give rise to the concern that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been suggested for preserving the utility of Bt-based insect resistance traits which include deploying proteins at a high dose in combination with a refuge, and alternation with, or co-deployment of, different toxins (McGaughey et al. (1998), “B.t. Resistance Management,” Nature Biotechnol. 16:144-146).
The proteins selected for use in an IRM stack need to exert their insecticidal effect independently so that resistance developed to one protein does not confer resistance to the second protein (i.e., there is not cross resistance to the proteins). If, for example, a pest population selected for resistance to “Protein A” is sensitive to “Protein B”, one would conclude that there is not cross resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A alone.
In the absence of resistant insect populations, assessments can be made based on other characteristics presumed to be related to mechanism of action and cross-resistance potential. The utility of receptor-mediated binding in identifying insecticidal proteins likely to not exhibit cross resistance has been suggested (van Mellaert et al. 1999). The key predictor of lack of cross resistance inherent in this approach is that the insecticidal proteins do not compete for receptors in a sensitive insect species.
In the event that two Bt toxins compete for the same receptor, then if that receptor mutates in that insect so that one of the toxins no longer binds to that receptor and thus is no longer insecticidal against the insect, it might be the case that the insect will also be resistant to the second toxin (which competitively bound to the same receptor). That is, the insect is said to be cross-resistant to both Bt toxins. However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to those two toxins.
Cry1Fa is useful in controlling many lepidopteran pests species including the European corn borer (ECB; Ostrinia nubilalis (Hubner)) and the fall armyworm (FAW; Spodoptera frugiperda), and is active against the sugarcane borer (SCB; Diatraea saccharalis). The Cry1Fa protein, as produced in corn plants containing event TC1507, is responsible for an industry-leading insect resistance trait for FAW control. Cry1Fa is further deployed in the Herculex®, SmartStax™, and WideStrike™ products.
The ability to conduct (competitive or homologous) receptor binding studies using Cry1Fa protein is limited because the most common technique available for labeling proteins for detection in receptor binding assays inactivates the insecticidal activity of the Cry1Fa protein.
Additional Cry toxins are listed at the website of the official B. t. nomenclature committee (Crickmore et al.; lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). There are currently nearly 60 main groups of “Cry” toxins (Cry1-Cry59), with additional Cyt toxins and VIP toxins and the like. Many of each numeric group have capital-letter subgroups, and the capital letter subgroups have lower-cased letter sub-subgroups. (Cry1 has A-L, and Cry1A has a-i, for example).
The subject invention relates in part to the surprising discovery that a fall armyworm (Spodoptera frugiperda; FAW) population resistant to the insecticidal activity of the Cry1Fa protein is not resistant to the insecticidal activity of the Vip3Ab protein. The subject pair of toxins provides non-cross-resistant action against FAW.
As one skilled in the art will recognize with the benefit of this disclosure, plants expressing Vip3Ab and Cry1Fa, or insecticidal portions thereof, will be useful in delaying or preventing the development of resistance to either of these insecticidal proteins alone.
The subject invention is also supported by the discovery that Vip3Ab and Cry1Fa do not compete with each other for binding sites in the gut of FAW.
Thus, the subject invention relates in part to the use of a Vip3Ab protein in combination with a Cry1Fa protein. Plants (and acreage planted with such plants) that produce Vip3Ab plus Cry1Fa are included within the scope of the subject invention.
The subject invention also relates in part to triple stacks or “pyramids” of three toxins, or more, with Vip3Ab and Cry1Fa being the base pair. In some preferred pyramid embodiments, the selected toxin(s) have non-cross-resistant action against FAW. Some preferred proteins for these triple-stack pyramid combinations are Cry1Fa plus Vip3Ab plus Cry1C, Cry1D, Cry1Be, or Cry1E. These particular triple stacks would, according to the subject invention, advantageously and surprisingly provide non-cross-resistant action against FAW. This can help to reduce or eliminate the requirement for refuge acreage.
With Cry1Fa being active against both FAW and European cornborer (ECB), and in light of the data presented herein, a quad (four-way) stack could also be selected to provide four proteins, wherein three of the four have non-cross-resistant activity against ECB, and three of the four have non-cross-resistant activity against FAW. This could be obtained by using Cry1Be (active against both ECB and FAW) together with the subject pair of proteins, plus one additional protein that is active against ECB. Such quad stacks, according to the subject invention, are:
As reported herein, a Vip3Ab toxin produced in transgenic corn (and other plants; cotton and soybeans, for example) can be very effective in controlling fall armyworm (FAW; Spodoptera frugiperda) that have developed resistance to Cry1Fa activity. Thus, the subject invention relates in part to the surprising discovery that fall armyworm resistant to Cry1Fa are susceptible (i.e., are not cross-resistant) to Vip3Ab. Stated another way, the subject invention also relates in part to the surprising discovery that Vip3Ab toxin is effective at protecting plants (such as maize plants) from damage by Cry1Fa-resistant fall armyworm. For a discussion of this pest, see e.g. Tabashnik, PNAS (2008), vol. 105 no. 49, 19029-19030.
The subject invention includes the use of Vip3Ab toxin to protect corn and other economically important plant species (such as soybeans) from damage and yield loss caused by fall armyworm feeding or to fall armyworm populations that have developed resistance to Cry1Fa.
The subject invention thus teaches an IRM stack comprising Vip3Ab to prevent or mitigate the development of resistance by fall armyworm to Cry1Fa.
The present invention provides compositions for controlling lepidopteran pests comprising cells that produce a Cry1Fa core toxin-containing protein and a Vip3Ab core toxin-containing protein.
The invention further comprises a host transformed to produce both a Cry1Fa insecticidal protein and a Vip3Ab insecticidal protein, wherein said host is a microorganism or a plant cell. The subject polynucleotide(s) are preferably in a genetic construct under control of (operably linked to/comprising) a non-Bacillus-thuringiensis promoter(s). The subject polynucleotides can comprise codon usage for enhanced expression in a plant.
It is additionally intended that the invention provides a method of controlling lepidopteran pests comprising contacting said pests or the environment of said pests with an effective amount of a composition that contains a Cry1Fa core toxin-containing protein and further contains a Vip3Ab core toxin-containing protein.
An embodiment of the invention comprises a maize plant comprising a plant-expressible gene encoding a Vip3Ab core toxin-containing protein and a plant-expressible gene encoding a Cry1Fa core toxin-containing protein, and seed of such a plant.
A further embodiment of the invention comprises a maize plant wherein a plant-expressible gene encoding a Vip3Ab core toxin-containing protein and a plant-expressible gene encoding a Cry1Fa core toxin-containing protein have been introgressed into said maize plant, and seed of such a plant.
As described in the Examples, competitive binding studies using radiolabeled Vip3Ab core toxin protein show that the Cry1Fa core toxin protein does not compete for binding in FAW insect tissues to which Vip3Ab binds. These results also indicate that the combination of Cry1Fa and Vip3Ab proteins is an effective means to mitigate the development of resistance in FAW populations to Cry1Fa (and likewise, the development of resistance to Vip3Ab), and would likely increase the level of resistance to this pest in corn plants expressing both proteins. Thus, based in part on the data described herein, it is thought that co-production (stacking) of the Vip3Ab and Cry1Fa proteins can be used to produce a high dose IRM stack for FAW. With Cry1Fa being active against both FAW and European cornborer (ECB), the subject pair of toxins provides non-competitive action against the FAW.
Other proteins can be added to this pair to expand insect-control spectrum. Another deployment option would be to use Cry1Fa and Vip3Ab proteins in combination with another, third toxin/gene, and to use this triple stack to mitigate the development of resistance in FAW to any of these toxins. Thus, another deployment option of the subject invention would be to use two, three, or more proteins in crop-growing regions where FAW can develop resistant populations.
Accordingly, the subject invention also relates in part to triple stacks or “pyramids” of three (or more) toxins, with Cry1Fa and Vip3Ab toxins being the base pair.
In some preferred pyramid embodiments, the three selected proteins provide non-cross-resistant action against FAW. Some preferred “triple action” pyramid combinations are Cry1Fa plus Vip3Ab plus either Cry1C or Cry1D. See U.S. Ser. No. 61/284,281 (filed Dec. 16, 2009), which shows that Cry1C is active against Cry1F-resistant FAW, and U.S. Ser. No. 61/284,252 (filed Dec. 16, 2009), which shows that Cry1D is active against Cry1F-resistant FAW. These two applications also show that Cry1C does not compete with Cry1F for binding in FAW membrane preparations, and that Cry1D does not compete with Cry1F for binding in FAW membrane preparations. In some embodiments, Cry1Be or Cry1E could be combined with Vip3A and Cry1F as the third anti-FAW protein. For use of Cry1Be with Cry1F, see U.S. Ser. No. 61/284,290 (filed Dec. 16, 2009). For use of Cry1E with Cry1F, see U.S. Ser. No. 61/284,278 (filed Dec. 16, 2009). These particular triple stacks would, according to the subject invention, advantageously and surprisingly provide three proteins providing non-cross-resistant action against FAW. This can help to reduce or eliminate the requirement for refuge acreage.
In light of the data presented herein, a quad (four-way) stack could also be selected to provide three proteins with non-cross-resistant action against ECB and three proteins with non-cross-resistantaction against FAW. This could be obtained by using Cry1Be (active against both ECB and FAW) together with Cry1Fa (active against both ECB and FAW) together with the subject Vip3Ab (active against FAW) and a fourth protein—having ECB-toxicity (See U.S. Ser. No. 61/284,290, filed Dec. 16, 2009, which relates to combinations of Cry1Fa and Cry1Be.) Examples of quad stacks, according to the subject invention, are:
Plants (and acreage planted with such plants) that produce any of the subject combinations of proteins are included within the scope of the subject invention. Additional toxins/genes can also be added, but the particular stacks discussed above advantageously and surprisingly provide multiple modes of action against FAW and/or ECB. This can help to reduce or eliminate the requirement for refuge acreage. A field thus planted of over 10 acres is thus included within the subject invention.
GENBANK can also be used to obtain the sequences for any of the genes and proteins disclosed or mentioned herein. See Appendix A, below.
U.S. Pat. No. 5,188,960 and U.S. Pat. No. 5,827,514 describe Cry1Fa core toxin containing proteins suitable for use in carrying out the present invention. U.S. Pat. No. 6,218,188 describes plant-optimized DNA sequences encoding Cry1Fa core toxin-containing proteins that are suitable for use in the present invention.
Cry1Fa is in the Herculex®, SmartStax™, and WidesStrike™ products. A vip3Ab gene could be combined into, for example, a Cry1Fa product such as Herculex®, SmartStax™, and WideStrike™. Accordingly, use of Vip3Ab could be significant in reducing the selection pressure on these and other commercialized proteins. Vip3Ab could thus be used as in the 3 gene combination for corn and other plants (cotton and soybeans, for example).
Combinations of proteins described herein can be used to control lepidopteran pests. Adult lepidopterans, for example, butterflies and moths, primarily feed on flower nectar and are a significant effector of pollination. Nearly all lepidopteran larvae, i.e., caterpillars, feed on plants, and many are serious pests. Caterpillars feed on or inside foliage or on the roots or stem of a plant, depriving the plant of nutrients and often destroying the plant's physical support structure. Additionally, caterpillars feed on fruit, fabrics, and stored grains and flours, ruining these products for sale or severely diminishing their value. As used herein, reference to lepidopteran pests refers to various life stages of the pest, including larval stages.
Some chimeric toxins of the subject invention comprise a full N-terminal core toxin portion of a Bt toxin and, at some point past the end of the core toxin portion, the protein has a transition to a heterologous protoxin sequence. The N-terminal, insecticidally active, toxin portion of a Bt toxin is referred to as the “core” toxin. The transition from the core toxin segment to the heterologous protoxin segment can occur at approximately the toxin/protoxin junction or, in the alternative, a portion of the native protoxin (extending past the core toxin portion) can be retained, with the transition to the heterologous protoxin portion occurring downstream.
As an example, one chimeric toxin of the subject invention, is a full core toxin portion of Cry1Fa (roughly the first 600 amino acids) and a heterologous protoxin (the remainder of the protein to the C-terminus). In one preferred embodiment, the portion of a chimeric toxin comprising the protoxin is derived from a Cry1Ab protein toxin. In a preferred embodiment, the portion of a chimeric toxin comprising the protoxin is derived from a Cry1Ab protein toxin.
A person skilled in this art will appreciate that Bt toxins, even within a certain class such as Cry1F, will vary to some extent in length and the precise location of the transition from core toxin portion to protoxin portion. Typically, the Cry1Fa toxins are about 1150 to about 1200 amino acids in length. The transition from core toxin portion to protoxin portion will typically occur at between about 50% to about 60% of the full length toxin. The chimeric toxin of the subject invention will include the full expanse of this N-terminal core toxin portion. Thus, the chimeric toxin will comprise at least about 50% of the full length of the Cry1Fa Bt toxin protein. This will typically be at least about 590 amino acids. With regard to the protoxin portion, the full expanse of the Cry1Ab protoxin portion extends from the end of the core toxin portion to the C-terminus of the molecule.
Genes and Toxins
The genes and toxins useful according to the subject invention include not only the full length sequences disclosed but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein. As used herein, the terms “variants” or “variations” of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term “equivalent toxins” refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins.
As used herein, the boundaries represent approximately 95% (Cry1Fa's and Vip3Ab's), 78% (Cry1F's and Vip3A's), and 45% (Cry1's and Vip3's) sequence identity, per “Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins,” N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean. Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813. These cut offs can also be applied to the core toxins only (for Cry1Fa, for example).
It should be apparent to a person skilled in this art that genes encoding active toxins can be identified and obtained through several means. The specific genes or gene portions exemplified herein may be obtained from the isolates deposited at a culture depository. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these 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. Genes that encode active fragments may also be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these protein toxins.
Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the 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 these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to “essentially the same” sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments of genes encoding proteins that retain pesticidal activity are also included in this definition.
A further method for identifying the genes encoding the toxins and gene portions useful according to the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO93/16094. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. Preferably, hybridization is conducted under stringent conditions by techniques well-known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170. Some examples of salt concentrations and temperature combinations are as follows (in order of increasing stringency): 2×SSPE or SSC at room temperature; 1×SSPE or SSC at 42° C.; 0.1×SSPE or SSC at 42° C.; 0.1×SSPE or SSC at 65° C. Detection of the probe provides a means for determining in a known manner whether hybridization has occurred. 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.
Variant Toxins
Certain toxins of the subject invention have been specifically exemplified herein. Since 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 homology with an exemplified toxin. This amino acid homology will typically be greater than 75%, preferably be greater than 90%, and most preferably be greater than 95%. The amino acid homology 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 ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may 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 does not materially alter the biological activity of the compound. Below is 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 biological activity of the toxin.
Recombinant Hosts.
The genes encoding the toxins of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. Conjugal transfer and recombinant transfer can be used to create a Bt strain that expresses both toxins of the subject invention. Other host organisms may also be transformed with one or both of the toxin genes then used to accomplish the synergistic effect. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is control of the pest. Alternatively, 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, then can be applied to the environment of the target pest.
Where the Bt toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes 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, Agrobactenum, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobactenium 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, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.
A wide variety of methods is available for introducing a Bt gene encoding a toxin into a microorganism host under conditions which 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, which is incorporated herein by reference.
Treatment of Cells.
Bacillus thuringiensis or recombinant cells expressing the Bt toxins can be treated to prolong the toxin activity and stabilize the cell. The pesticide microcapsule that is formed comprises the Bt toxin or toxins within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Suitable host cells may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi.
The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.
Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin gene or genes, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, various acids and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host environment. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.
The cells generally will have enhanced structural stability which will enhance resistance to environmental conditions. Where the pesticide is in a proform, the method of cell treatment should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen. For example, formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide. The method of treatment should retain at least a substantial portion of the bio-availability or bioactivity of the toxin.
Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene or genes into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; survival in aqueous environments; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
Growth of Cells.
The cellular host containing the B.t. insecticidal gene or genes may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
The B.t. cells producing the toxins of the invention can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art.
Formulations.
Formulated bait granules containing an attractant and spores, crystals, and toxins of the B.t. isolates, or recombinant microbes comprising the genes obtainable from the B.t. isolates 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 B.t. 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 rheological 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 lepidopteran pest, e.g., foliage or soil, by spraying, dusting, sprinkling, or the like.
Plant Transformation.
A preferred recombinant host for production of the insecticidal proteins of the subject invention is a transformed plant. Genes encoding Bt toxin proteins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in Escherichia 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, M13mp series, pACYC184, inter alia. Accordingly, the DNA fragment having the sequence encoding the Bt toxin protein 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 sufficiently described in EP 120 516, Lee and Gelvin (2008), Hoekema (1985), Fraley et al., (1986), and An et al., (1985), and is well established in the art.
Once the inserted DNA has been integrated in the plant genome, it is relatively stable. The transformation vector normally contains a selectable marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as Bialaphos, Kanamycin, G418, Bleomycin, or Hygromycin, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.
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 in Agrobacteria. 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 et al., 1978). 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 a preferred embodiment of the subject invention, plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Pat. No. 5,380,831, which is hereby incorporated by reference. While some truncated toxins are exemplified herein, it is well-known in the Bt art that 130 kDa-type (full-length) toxins have an N-terminal half that is the core toxin, and a C-terminal half that is the protoxin “tail.” Thus, appropriate “tails” can be used with truncated/core toxins of the subject invention. See e.g. U.S. Pat. No. 6,218,188 and U.S. Pat. No. 6,673,990. In addition, methods for creating synthetic Bt genes for use in plants are known in the art (Stewart and Burgin, 2007). One non-limiting example of a preferred transformed plant is a fertile maize plant comprising a plant expressible gene encoding a Cry1Fa protein, and further comprising a second plant expressible gene encoding a Vip3Ab protein.
Transfer (or introgression) of the Cry1Fa- and Vip3Ab-determined trait(s) into inbred maize lines can be achieved by recurrent selection breeding, for example by backcrossing. In this case, a desired recurrent parent is first crossed to a donor inbred (the non-recurrent parent) that carries the appropriate gene(s) for the Cry1F- and Vip3Ab-determined traits. The progeny of this cross is then mated back to the recurrent parent followed by selection in the resultant progeny for the desired trait(s) to be transferred from the non-recurrent parent. After three, preferably four, more preferably five or more generations of backcrosses with the recurrent parent with selection for the desired trait(s), the progeny will be heterozygous for loci controlling the trait(s) being transferred, but will be like the recurrent parent for most or almost all other genes (see, for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles of Cultivar Development, Vol. 1: Theory and Technique, 360-376).
Insect Resistance Management (IRM) Strategies.
Roush et al., for example, outlines two-toxin strategies, also called “pyramiding” or “stacking,” for management of insecticidal transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353, 1777-1786).
On their website, the United States Environmental Protection Agency (epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge—2006.htm) publishes the following requirements for providing non-transgenic (i.e., non-B.t.) refuges (a section of non-Bt crops/corn) for use with transgenic crops producing a single Bt protein active against target pests.
In addition, the National Corn Growers Association, on their website: (ncga.com/insect-resistance-management-fact-sheet-bt-corn)
also provides similar guidance regarding the refuge requirements. For example:
As stated by Roush et al. (on pages 1780 and 1784 right column, for example), stacking or pyramiding of two different proteins each effective against the target pests and with little or no cross-resistance can allow for use of a smaller refuge. Roush suggests that for a successful stack, a refuge size of less than 10% refuge, can provide comparable resistance management to about 50% refuge for a single (non-pyramided) trait. For currently available pyramided Bt corn products, the U.S. Environmental Protection Agency requires significantly less (generally 5%) structured refuge of non-Bt corn be planted than for single trait products (generally 20%).
There are various ways of providing the IRM effects of a refuge, including various geometric planting patterns in the fields (as mentioned above) and in-bag seed mixtures, as discussed further by Roush et al. (supra), and U.S. Pat. No. 6,551,962.
The above percentages, or similar refuge ratios, can be used for the subject double or triple stacks or pyramids. For triple stacks with three modes of action against a single target pest, a goal would be zero refuge (or less than 5% refuge, for example). This is particularly true for commercial acreage—of over 10 acres for example.
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.
Unless specifically indicated or implied, the terms “a”, “an”, and “the” signify “at least one” as used herein.
Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.
Examples are given showing that Vip3Ab1 is active against Spodoptera frugiperda (fall armyworm) wild type larvae, and against a field collected strain of Spodoptera frugiperda found in Puerto Rico that is resistant to the Bacillus thuringiensis crystal toxin Cry1Fa. This biological data supports the utility of Vip3Ab1 to be used to combat the development of Cry1 resistance in insects, since insects developing resistance to the Cry1Fa toxins would continue to be susceptible to the toxicity of Vip3Ab1.
Similarly, in Spodoptera frugiperda, 125I radiolabeled Cry1Fa binds to receptor proteins and the binding can be displaced using non-radiolabeled Cry1Fa. However, Vip3Ab1 cannot displace the binding of 125I Cry1Fa from its receptor in these experiments. These results indicate that Vip3Ab1 has a unique binding site as compared to Cry1Fa. The ability of Vip3Ab1 to exert toxicity against insects that are resistant to Cry1Fa stems from its demonstrated non-interaction at the site where these toxins bind. Further data is presented that shows the nature of Cry1Fa resistance in Spodoptera frugiperda is due to the inability of Cry1Fa to bind to BBMV's prepared from this insect. The biological activity of Vip3Ab1 against Cry1Fa resistant S. frugiperda larvae that lost their ability to bind Cry1Fa, further supports the non-interacting target site of Vip3Ab1 as compared to Cry1Fa.
The genes encoding the Cry1Fa and Vip3Ab1 pro toxins were expressed in Pseudomonas fluorescens expression strains and the full length proteins isolated as insoluble inclusion bodies. The washed inclusion bodies were solubilized by stirring at 37° C. in buffer containing 20 mM CAPS buffer, pH 11, +10 mM DDT, +0.1% 2-mercaptoethanol, for 2 hrs. The solution was centrifuged at 27,000×g for 10 min. at 37° C. and the supernatant treated with 0.5% (w/v) TCPK treated trypsin (Sigma). This solution was incubated with mixing for an additional 1 hr. at room temperature, filtered, then loaded onto a Pharmacia Mono Q 1010 column equilibrated with 20 mM CAPS pH 10.5. After washing the loaded column with 2 column volumes of buffer, the truncated toxin was eluted using a linear gradient of 0 to 0.5 M NaCl in 20 mM CAPS in 15 column volumes at a flow rate of 1.0 ml/min. Purified trypsin truncated Cry proteins eluted at about 0.2-0.3 M NaCl. The purity of the proteins was checked by SDS PAGE and with visualization using Coomassie brilliant blue dye. In some cases, the combined fractions of the purified toxin were concentrated and loaded onto a Superose 6 column (1.6 cm dia., 60 cm long), and further purified by size exclusion chromatography. Fractions comprising a single peak of the monomeric molecular weight were combined, and concentrated, resulting in a preparation more than 95% homogeneous for a protein having a molecular weight of about 60,000 kDa.
Processing of Vip3Ab1 was achieved in a similar manner starting with the purified full length 85 kDa protein (DIG-307) provided by Monte Badger. The protein (12 mg) was dialyzed into 50 mM sodium phosphate buffer, pH 8.4, then processed by adding 1 mg of solid trypsin and incubating for 1 hrs. at room temperature. The solution was loaded onto a MonoQ anion exchange column (1 cm dia., 10 cm. long), and eluted with a linear gradient of NaCl from 0 to 500 mM in 20 mM sodium phosphate buffer, pH 8.4 over 7 column volumes. Elution of the protein was monitored by SDS-PAGE. The major processed band had a molecular weight of 65 kDa, as determined by SDS-PAGE using molecular weight standards for comparison.
Purified proteins were tested for insecticidal activity in bioassays conducted with neonate Spodoptera frugiperda (J. E. Smith) larvae on artificial insect diet. The Cry1F-resistant FAW were collected from fields of Herculex I (Cry1Fa) corn in Puerto Rico, and brought into the Dow AgroSciences Insectary for continuous rearing. Characterization of this strain of resistant-FAW is outlined in the internal report by Schlenz, et al (Schlenz et al., 2008).
Insect bioassays were conducted in 128-well plastic bioassay trays (C-D International, Pitman, N.J.). Each well contained 0.5 mL of multi-species lepidoptera diet (Southland Products, Lake Village, Ark.). A 40 μL aliquot of the purified Cry or Vip3Ab1 protein diluted to various concentrations in 10 mM CAPS, pH 10.5, or control solution was delivered by pipette onto the 1.5 cm2 diet surface of each well (26.7 μL/cm2). Sixteen wells were tested per sample. The negative control was a buffer solution blank containing no protein. Positive controls included preparations of Cry1F. The treated trays were held in a fume hood until the liquid on the diet surface had evaporated or was absorbed into the diet.
Within a few hours of eclosion, individual larvae were picked up with a moistened camelhair brush and deposited on the treated diet, one larva per well. The infested wells were then sealed with adhesive sheets of clear plastic that are vented to allow gas exchange (C-D International, Pitman, N.J.). The bioassay trays were held under controlled environmental conditions (28° C., ˜40% RH, 16:8 [L:D] photoperiod). After 5 days, the total number of insects exposed to each protein sample, the number of dead insects, and the weight of surviving insects were recorded.
Iodination of Cry1F has been reported to destroy both the toxicity and the binding capacity of this protein when tested against tobacco budworm larvae and BBMV's prepared from these insects (Luo et al., 1999; Sheets and Storer, 2001). The inactivation is presumably due to the need for unmodified tyrosine residues near its binding site. When Cry1F was iodinated using the Iodo-bead method, the protein lost all of its ability to exhibit specific binding characteristics using BBMV's from H. virescens. Using non-radiolabeled NaI to iodinate Cry1F employing the Iodo-bead method, the iodinated Cry1F also lost its insecticidal activity against H. virescens.
Earlier studies in our laboratories demonstrated that Cry1Fa could be fluorescently labeled using maleimide conjugated labeling reagents that specifically alkylate proteins at cysteine residues. Since the Cry1Fa trypsin core toxin contains a single cysteine residue at position 205, labeling the protein with such a reagent would result in alkylation of the protein at a single specific site. It was determined that Cry1Fa could be fluorescently labeled with fluorescein-5-maleimide and that the labeled protein retained insecticidal activity. Based upon the retention of biological activity of the cysteine fluorescein labeled Cry1Fa, it was determined that we could also radioiodinate the fluorescein portion of the label by the method of Palmer et al., (Palmer et al., 1997), and attach it to the cysteine of Cry1Fa and have a radiolabeled Cry1Fa that retains biological activity.
Fluorescein-5-maleimide was dissolved to 10 mM (4.27 mg/ml) in DMSO, then diluted to 1 mM in PBS as determined by its molar extinction coefficient of 68,000 M−1cm−1. To a 70 μl solution of PBS containing two Iodobeads, 0.5 mCi of Na125I was added behind lead shielding. The solution was allowed to mix at room temperature for 5 min., then 10 μl of the 1 mM fluorescein-5-maleimide was added. The reactants were allowed to react for 10 min., and then removed from the iodobeads. To the reacted solution was added 2 μg of highly purified trypsin truncated Cry1Fa core toxin in PBS. The protein was incubated with the iodinated fluorescein-5-maleimide solution for 48 hrs at 4° C. The reaction was stopped by adding 2-mercapto ethanol to 14 mM. The reaction mixture was then added to a Zebra spin column equilibrated in 20 mM CAPS, 150 mM KCl, pH 9, and centrifuged at 1,500×g for 2 min. to separate non-reacted iodinated dye from the protein. The 125I radiolabeled fluorescein-Cry1Fa was counted in a gamma counter to determine its specific activity determined based upon an assumed 80% recovery of the input toxin. The protein was also characterized by SDS-PAGE and visualized by phosphor imaging to assure that the radioactivity measured was covalently associated with the Cry1Fa protein.
Standard methods of protein quantification and SDS-polyacrylamide gel electrophoresis were employed as taught, for example, in Sambrook et al. (Sambrook and Russell, 2001) and updates thereof. Last instar S. frugiperda larvae were fasted overnight and then dissected after chilling on ice for 15 minutes. The midgut tissue was removed from the body cavity, leaving behind the hindgut attached to the integument. The midgut was placed in a 9× volume of ice cold homogenization buffer (300 mM mannitol, 5 mM EGTA, 17 mM Tris base, pH7.5), supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich P-2714) diluted as recommended by the supplier. The tissue was homogenized with 15 strokes of a glass tissue homogenizer. BBMV's were prepared by the MgCl2 precipitation method of Wolfersberger (Wolfersberger, 1993). Briefly, an equal volume of a 24 mM MgCl2 solution in 300 mM mannitol was mixed with the midgut homogenate, stirred for 5 minutes and allowed to stand on ice for 15 min. The solution was centrifuged at 2,500×g for 15 min at 4° C. The supernatant was saved and the pellet suspended into the original volume of 0.5× diluted homogenization buffer and centrifuged again. The two supernatants were combined and centrifuged at 27,000×g for 30 min at 4° C. to form the BBMV fraction. The pellet was suspended into BBMV Storage Buffer (10 mM HEPES, 130 mM KCl, 10% glycerol, pH 7.4) to a concentration of about 3 mg/ml protein. Protein concentration was determined using BSA as the standard.
L-leucine-p-nitroanilide aminopeptidase activity (a marker enzyme for the BBMV fraction) was determined prior to freezing the samples. Briefly, 50 μl of L-leucine-p-nitroanilide (1 mg/ml in PBS) was added to 940 ml 50 mM Tris HCl in a standard cuvette. The cuvette was placed in a Cary 50 Bio spectrophotometer, zeroed for absorbance reading at 405 nm, and the reaction initiated by adding 10 μl of either insect midgut homogenate or insect BBMV preparation. The increase in absorbance at 405 nm was monitored for 5 minutes at room temperature. The specific activity of the homogenate and BBMV preparations was determined based upon the kinetics of the absorbance increase over time during a linear increase in absorbance per unit total protein added to the assay based upon the following equation:
ΔOD/(min*mg)=Aminopeptidase Rate(ΔOD/ml*min)/[protein](mg/ml)
The specific activity of this enzyme typically increased 7-fold compared to that found in the starting midgut homogenate fraction. The BBMV's were aliquoted into 250 μl samples, flash frozen in liquid N2 and stored at −80° C.
Analysis of proteins by SDS-PAGE was conducted under reducing (i.e. in 5% β-mercaptoethanol, BME) and denaturing (i.e. heated 5 minutes at 90° in the presence of 4% SDS) conditions. Proteins were loaded into wells of a 4% to 20% tris-glycine polyacrylamide gel (BioRad; Hercules, Calif.) and separated at 200 volts for 60 minutes. Protein bands were detected by staining with Coomassie Brilliant Blue R-250 (BioRad) for one hour, and destained with a solution of 5% methanol in 7% acetic acid. The gels were imaged and analyzed using a BioRad Fluoro-S Multi Imager™. Relative molecular weights of the protein bands were determined by comparison to the mobilities of known molecular weight proteins observed in a sample of BenchMark™ Protein Ladder (Invitrogen, Carlsbad, Calif.) loaded into one well of the gel.
Radio-purity of the iodinated Cry proteins and measurement of radioactive Cry1Fa in pull down assays was determined by SDS-PAGE and phosphorimaging. Briefly, SDS-PAGE gels were imaged by wrapping the gels in Mylar film (12 μm thick), after separation and fixation of the protein, then exposing the gel under a Molecular Dynamics storage phosphor screen (35 cm×43 cm) for at least overnight, and up to 4 days. The plates were developed using a Molecular Dynamics Storm 820 phosphor-imager and the image was analyzed using ImageQuant™ software.
Mortality results from bioassays of the full length Vip3Ab1 protein tested at a variety of doses against wild type and Cry1Fa resistant S. frugiperda larvae are shown in
A bioassay was also conducted to compare the biological activity of Vip3Ab1 against wild type S. frugiperda versus Cry1Fa resistant S. frugiperda (
Additional bioassay replications were conducted to generate median lethal concentrations (LC50), median growth inhibition concentrations. Table 2 shows (GI50) and 95% confidence intervals of Cry1F-suseptible Spodoptera frugiperda and Cry1F-resistant Spodoptera frugiperda to Vip3Ab1 compared to controls.
Radiolabeled competition binding assays were conducted to determine if Vip3Ab1 interacts at the same site that Cry1Fa binds in FAW. A competition assay was developed to measure the ability of Vip3Ab to compete with the binding of 125I radiolabeled Cry1Fa.
Insects can develop resistance to the toxicity of Cry proteins through a number of different biochemical mechanisms, but the most common mechanism is due to a reduction in the ability of the Cry toxin protein to bind to its specific receptor in the gut of the insect (Heckel et al., 2007; Tabashnik et al., 2000; Xu et al., 2005). This can be brought about thought small point mutations, large gene deletions, or though other genetic or biochemical mechanisms. When we investigated the BBMV proteins from Cry1Fa resistant S. frugiperda to understand the nature of their resistance to Cry1Fa, we discovered that BBMV's prepared from Cry1Fa resistant insects were much less able to bind 125I radiolabeled Cry1Fa as compared to BBMV's prepared from the wild type insects (
Paenibacillus
popilliae
Paenibacillus
popilliae
Paenibacillus
popilliae
P. lentimorbus
P. popilliae
P. lentimorbus
P. lentimorbus
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/60810 | 12/16/2010 | WO | 00 | 8/30/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61284290 | Dec 2009 | US | |
| 61284252 | Dec 2009 | US | |
| 61284281 | Dec 2009 | US | |
| 61284278 | Dec 2009 | US |
