WO2011084631A1 - Use of cry1ab in combination with cry1be for management of resistant insects - Google Patents

Abstract

The subject invention includes methods and plants for controlling European corn borer and/or fall armyworm insects, said plants comprising a Cry1Ab insecticidal protein and a Cry1Be insecticidal protein, and various combinations of other proteins comprising this pair of proteins, to delay or prevent development of resistance by the insects.

Description

DAS-P0199

USE OF Cry l Ab IN COMBINATION WITH Cry l Be FOR MANAGEMENT OF RESISTANT INSECTS

Background of the Invention

|0001] 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. 100021 Several Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include Cry l Ab, Cry l Ac, Cry I F and Cry3Bb in com, Cry l Ac and Cry2Ab in cotton, and Cry3A in potato.

[0003] 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. , Cry l Ab 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., Cry l Ac and Cry2Ab in cotton combined to provide resistance management for tobacco budworm). See also U.S. Patent Application Publication No. 2009/0313717, which relates to a Cry2 protein plus a Vip3 Aa, Cry 1 F, or Cry 1 A for control of Helicoverpa zea or armigerain. WO 2009/1 32850 relates to Cry I F or Cry l A and Vip3Aa for controlling Spodoptera frugiperda. U.S. Patent Application Publication No. 2008/031 1096 relates in part to Cry l Ab for controlling Cry l F-resistant European corn borer (ECB; Ostrinia nubilalis (Hiibner)).

[0004| 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 5/-based insect resistance traits which include deploying proteins at a high dose in combination with a

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refuge, and alternation with, or co-deployment of, different toxins (McGaughey et al.

( 1998), "B.t. Resistance Management," Nature Biotechnol. 16: 144- 146).

[0005] The proteins selected for use in an insect resistant management (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.

[0006] 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.

|0007] In the event that two Bt toxins compete for the same receptor in an insect, 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 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.

|0008] For example, Cry 1 Fa protein is useful in controlling many lepidopteran pests species including ECB and the fall armyworm (FAW; Spodoptera frugiperda), and is active against the sugarcane borer (SCB; Diatraea saccharalis) . The Cry lFa protein, as produced in transgenic corn plants containing event TCI 507, is responsible for an industry-leading insect resistance trait for FAW control. Cry 1 Fa is further deployed in the Herculex®, SmartStax™, and WideStrike™ products.

[0009] The ability to conduct (competitive or homologous) receptor binding studies using Cry 1 Fa protein has been limited because a common technique available for labeling proteins for detection in receptor binding assays tends to inactivate the insecticidal activity of the Cry 1 Fa protein.

10010] 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

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currently nearly 60 main groups of "Cry" toxins (Cryl-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. (Cry 1 has A-L, and Cry l A has a-i, for example).

Brief Summary of the Invention

100111 The subject invention relates in part to the surprising discovery that Cry 1 Ab and Cry 1 Be do not compete for binding to sites in European corn borer (ECB; Ostrinia nubilalis (Hiibner)) or fall armyworm (FAW; Spodoptera frugiperda) gut cell membrane preparations. As one skilled in the art will recognize with the benefit of this disclosure, plants that produce both of these proteins (including insecticidal portions of the full-length proteins) can delay or prevent the development of resistance to any of these insecticidal proteins alone. Corn and soybean are some preferred plants. ECB is the preferred target insect for the subject pair of toxins.

[0012] Thus, the subject invention relates in part to the use of a Cry 1 Ab protein in combination with a Cry l Be protein. Plants (and acreage planted with such plants) that produce both of these proteins are included within the scope of the subject invention.

[0013| The subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, with Cry l Ab and Cry l Be being the base pair. In some preferred pyramid embodiments, the combination of the selected toxins provides three sites of action against ECB. Some preferred "three sites of action" pyramid combinations include the subject base pair of proteins plus Cry2A, Cry 1 1, and DIG-3 as the third protein for targeting ECB. These particular triple stacks would, according to the subject invention, advantageously and surprisingly provide three sites of action against ECB. This can help to reduce or eliminate the requirement for refuge acreage.

(0014) Although the subject invention is disclosed herein as a base pair of toxins, Cry l Ab and Cry 1 Be, which, either alone or in a "pyramid" of three or more toxins, provides for insect-resistance against ECB in corn, it should be understood that the combinations of Cry 1 Ab and Cry 1 Be, described herein, can be also used with additional proteins for targeting FAW in both soybean or corn.

[0015| Additional toxins/genes can also be added according to the subject invention. For example, if Cry 1 Fa is stacked with the subject pair of proteins (Cry 1 Fa and Cry 1 Be are both

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active against both FAW and ECB), adding one additional protein to this triple stack wherein the fourth added protein targets ECB, would provide three sites of action against FAW, and three sites of action against ECB. This added protein (the fourth protein for targeting FAW) could be selected from the group consisting of Cry 1 Fa, Vip3Ab, or Cry I E. This would result in a four-protein stack having three sites of action against two insects (ECB and FAW).

BRIEF DESCRIPTION OF THE TABLES

Table 1 : provides examples of Amino Acids within the Four Classes of Amino

Acids.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : is a graph illustrating the percent binding of labeled Cry l Ab versus Cry 1 Be for ECB BBMVs.

Figure 2: is a graph illustrating the percent binding of labeled Cry l Ab versus Cry 1 Be with FAW BBMVs.

Figure 3: is a graph illustrating the percent binding of labeled Cry 1 Be versus Cry 1 Ab with FAW BBMVs.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The subject invention relates in part to the surprising discovery that Cry 1 Ab and Cry l Be do not compete with each other for binding sites in the gut of the European corn borer (ECB; Ostrinia nubilalis (Hiibner)) or the fall armyworms (FAW; Spodoptera frugiperda). Thus, a Cry 1 Ab protein can be used in combination with a Cry l Be protein in transgenic corn (and other plants; e.g., cotton and soybeans, for example) to delay or prevent ECB from developing resistance to either of these proteins alone. The subject pair of proteins can be effective at protecting plants (such as maize plants) from damage by Cry- resistant ECB.. That is, one use of the subject invention is to protect corn and other economically important plant species from damage and yield loss caused by ECB populations that could develop resistance to Cry l Ab or Cry l Be.

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|0017| The subject invention thus teaches an insect resistant management (1RM) stack comprising Cry 1 Ab and Cry 1 Be to prevent or mitigate the development of resistance by ECB to either or both of these proteins.

|0018| Further, although the subject invention, disclosed herein, teaches an IRM stack comprising Cry l Ab and Cry l Be for preventing resistance by ECB to either or both of these proteins, it is within the scope of the invention disclosed herein that one or both of Cry l Ab and Cry 1 Be may be adapted, either alone or in combination, to prevent resistance by FAW to either or both of these proteins.

[0019] The present invention provides compositions for controlling lepidopteran pests comprising cells that produce a CrylAb core toxin-containing protein and a Cry l Be core toxin-containing protein.

|0020) The invention further comprises a host transformed to produce both a Cry 1 Ab insecticidal protein and a Cry l Be 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 a non-Bacillus-thuringiensis promoters). The subject polynucleotides can comprise codon usage for enhanced expression in a plant.

100211 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 Cry l Ab insecticidal protein and further contains a Cry l Be insecticidal protein.

|0022) An embodiment of the invention comprises a maize plant comprising a plant- expressible gene encoding a Cry 1 Be core toxin-containing protein and a plant-expressible gene encoding a Cry 1 Ab core toxin-containing protein, and seed of such a plant.

[0023] A further embodiment of the invention comprises a maize plant wherein a plant- expressible gene encoding a Cry 1 Be insecticidal protein and a plant-expressible gene encoding a Cry l Ab insecticidal protein have been introgressed into said maize plant, and seed of such a plant.

[0024] As described in the Examples, competitive receptor binding studies using radiolabeled Cry 1 Ab and Cry 1 Be proteins show that the Cry 1 Be protein does not compete for binding in ECB or FAW tissues to which Cry l Ab binds. These results also indicate that the combination of Cry 1 Ab and Cry 1 Be proteins can be an effective means to mitigate the development of resistance in ECB and FAW populations to either of these proteins.

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Thus, based in part on the data described herein, it is thought that co-production (stacking) of the Cry 1 Be and Cry 1 Ab proteins can be used to produce a high dose IRM stack for ECB. |0025| Other proteins can be added to this pair. For example, the subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, with Cry 1 Ab and Cry 1 Be being the base pair. In some preferred pyramid embodiments, the selected toxins have three separate sites of action against FAW. Some preferred "three sites of action" pyramid combinations include the subject base pair of proteins plus Cry 1 Fa, Vip3Ab, Cry l C, Cry l D, or Cry I E as the third protein for targeting FAW. These particular triple stacks would, according to the subject invention, advantageously and surprisingly provide three sites of action against FAW. This can help to reduce or eliminate the requirement for refuge acreage. By "separate sites of action," it is meant any of the given proteins do not cause cross-resistance with each other.

100261 Additional toxins/genes can also be added according to the subject invention. For example, if Cry 1 Fa is stacked with the subject pair of proteins (both Cry 1 Fa and Cry 1 Be are both active against both FAW and European cornborer (ECB)), adding one additional protein to this triple stack wherein the fourth added protein targets ECB, would provide three sites of action against FAW, and three sites of action against ECB. This added protein (the fourth protein) could be selected from the group consisting of Cry2A, Cry l I, and DIG- 3 (see U.S. Patent Application Serial No. 61 /284,278 (filed December 16, 2009) and US 2010 00269223). This would result in a four-protein stack having three sites of action against two insects (ECB and FAW).

|0027] Thus, one deployment option is to use the subject pair of proteins in combination with a third toxin/gene, and to use this triple stack to mitigate the development of resistance in ECB and/or FAW to any of these toxins. Accordingly, the subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins. In some preferred pyramid embodiments, the selected toxins have three separate sites of action against ECB and/or FAW.

|0028) Included among deployment options of the subject invention would be to use two, three, or more proteins of the subject proteins in crop-growing regions where ECB and/or FAW can develop resistant populations.

(0029) For guidance regarding the use of Cry 1 Fa and Cry 1 Be (for controlling ECB and/or FAW), see U.S. Patent Application Serial No. 61/284,290 (filed December 16, 2009). With Cry l Fa being active against ECB (and FAW), Cry l Ab plus Cry l Be plus Cry l Fa would,

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according to the subject invention, advantageously and surprisingly provide three sites of action against ECB. This can help to reduce or eliminate the requirement for refuge acreage.

[0030] Cry 1 Fa is deployed in the Herculex®, SmartStax™, and WidesStrike™ products. The subject pair of genes (Cry l Ab and Cry 1 Be) could be combined into, for example, a Cry 1 Fa product such as Herculex®, SmartStax™, and WideStrike™. Accordingly, the subject pair of proteins could be significant in reducing the selection pressure on these and other proteins. The subject pair of proteins could thus be used as in the three gene combinations for corn and other plants (cotton and soybeans, for example).

[0031 ] As discussed above, additional toxins/genes can also be added according to the subject invention. For targeting ECB, Cry2A, Cry 1 1, and/or D1G-3 can be used. See U.S. Patent Application Serial No. 61 /284,278 (filed December 16, 2009) and US 2010

00269223. For the combination of Cry I F and Cry l Ab (for controlling ECB), see U.S. Patent Application Publication No. 2008/031 1096.

|0032] 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 sites of action against ECB and/ or FAW. This can help to reduce or eliminate the requirement for refuge acreage. A field thus planted of over ten acres is thus included within the subject invention.

[0033] GENBANK can also be used to obtain the sequences for any of the genes and proteins discussed herein. See Appendix A, below. Patents can also be used. For example, U.S. Patent No. 5, 188,960 and U.S. Patent No. 5,827,514 describe Cry 1 Fa core toxin containing proteins suitable for use in carrying out the present invention. U.S. Patent No. 6,218, 188 describes plant-optimized DNA sequences encoding Cry l Fa core toxin- containing proteins that are suitable for use in the present invention.

[0034| 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,

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reference to lepidopteran pests refers to various life stages of the pest, including larval stages.

|0035) 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.

|0036] As an example, one chimeric toxin of the subject invention, is a full core toxin portion of Cry 1 Ab (approximately amino acids 1 to 601 ) and/or a heterologous protoxin (approximately amino acids 602 to the C-terminus). In one preferred embodiment, the portion of a chimeric toxin comprising the protoxin is derived from a Cry 1 Ab protein toxin. In a preferred embodiment, the portion of a chimeric toxin comprising the protoxin is derived from a Cry l Ab protein toxin.

|0037] A person skilled in this art will appreciate that Bt toxins, even within a certain class such as Cry 1 Be, will vary to some extent in length and the precise location of the transition from core toxin portion to protoxin portion. Typically, the Cry l Be toxins are about 1 150 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 Cry 1 Be protein. This will typically be at least about 590 amino acids. With regard to the protoxin portion, the full expanse of the Cry 1 Ab protoxin portion extends from the end of the core toxin portion to the C-terminus of the molecule.

|0038] 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

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toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins.

[0039] As used herein, the boundaries represent approximately 95% (Cry 1 Ab's and Cry l Be's), 78% (Cryl A's and Cry l B's), and 45% (Cry l '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.

|0040| 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.

|0041 | 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 D A 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.

|0042] 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

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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): 2X SSPE or SSC at room temperature; I X SSPE or SSC at 42° C; 0.1 X SSPE or SSC at 42° C; 0.1 X 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.

|0043| 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.

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Table 1 : Exam les of Amino Acids within the Four Classes of Amino Acids

|0044] 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.

[0045] 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.

100461 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.

[0047] 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,

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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.

|0048] 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. Patent No. 5,135,867, which is incorporated herein by reference.

100491 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.

|0050] 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.

[0051] Treatment of the microbial cell, e.g., a microbe containing the Bt 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

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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.

|0052) 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.

[0053] Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the Bt 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.

|0054] Growth of cells. The cellular host containing the Bt 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

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the Bt gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

|0055| The Bt 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 Bt spores and crystals from the fermentation broth by means well known in the art. The recovered Bt 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.

[0056] Formulations. Formulated bait granules containing an attractant and spores, crystals, and toxins of the Bt isolates, or recombinant microbes comprising the genes obtainable from the Bt 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 Bt 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.

|0057) 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.

|0058] The formulations can be applied to the environment of the lepidopteran pest, e.g., foliage or soil, by spraying, dusting, sprinkling, or the like.

BDDB01 641 1412v l DAS-P0199

[0059] 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, M 13mp series, pACYCl 84, 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.

|0060] 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.

|0061| 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

BDDB01 641 1412v l DAS-P0199

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 DN A 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.

|0062] 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.

|0063| 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. Patent 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. Patent No. 6,218, 188 and U.S. Patent 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).

BDDB01 641 1412vl DAS-P0199

One non-limiting example of a preferred transformed plant is a fertile maize plant comprising a plant expressible gene encoding a Cry 1 Ab protein, and further comprising a second plant expressible gene encoding a Cry 1 Be protein.

|0064] Transfer (or introgression) of the Cryl Ab- and Cry l Be-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 Cry 1 A- and Cry 1 Be- 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 nonrecurrent 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 Cuitivar Development, Vol. 1 : Theory and Technique, 360-376).

[0065| 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).

[0066] 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.

"The specific structured requirements for corn borer-protected Bt (Cryl Ab or Cry I F) corn products are as follows:

Structured refuges: 20% non-Lepidopteran Bt corn refuge in Corn Belt;

50% non-Lepidopteran Bt refuge in Cotton Belt

Blocks

Internal (i.e. , within the Bt field)

External (i.e., separate fields within ½ mile (¼ mile if possible) of the

Bt field to maximize random mating)

In-field Strips

Strips must be at least 4 rows wide (preferably 6 rows) to reduce

the effects of larval movement"

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|0067) In addition, the National Corn Growers Association, on their website:

(ncga.com/insect-resistance-management-fact-sheet-bt-corn) |0068] also provides similar guidance regarding the refuge requirements. For example:

"Requirements of the Corn Borer IRM:

-Plant at least 20% of your corn acres to refuge hybrids

-In cotton producing regions, refuge must be 50%

-Must be planted within 1 /2 mile of the refuge hybrids

-Refuge can be planted as strips within the Bt field; the refuge strips must be at least 4 rows wide

-Refuge may be treated with conventional pesticides only if economic thresholds are reached for target insect

-Bt-based sprayable insecticides cannot be used on the refuge corn

-Appropriate refuge must be planted on every farm with Bt corn"

|0069| 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-Z?r corn be planted than for single trait products (generally 20%).

|0070| 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. Patent No. 6,551 ,962.

|00711 The above percentages, or similar refuge ratios, can be used for the subject double or triple stacks or pyramids. For triple stacks with three sites 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.

[0072| 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.

[0073| Unless specifically indicated or implied, the terms "a", "an", and "the" signify "at least one" as used herein.

|0074] 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

BDDB0 I 641 1412v l DAS-P0199

solvent mixture proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.

EXAMPLES

Example 1 - ¹²⁵I Labeling of Cry Proteins

Iodination of Cry toxins. Purified truncated Cry toxins were was iodinated using lodo-Beads or Iodo-gen (Pierce). Briefly, two lodo-Beads were washed twice with 500 μΙ of phosphate buffered saline, PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 7.5), and placed into a 1 .5 ml centrifuge tube behind lead shielding. To this was added 100 μΐ of PBS. In a hood and through the use of proper radioactive handling techniques, 0.5 mCi Na¹²⁵l (17.4 Ci/mg, Lot 01 14, Amersham) was added to the PBS solution with the Iodo-Bead. The components were allowed to react for 5 minutes at room temperature, then 2-25 μg of highly pure truncated Cry protein was added to the solution and allowed to react for an additional 3-5 minutes. The reaction was terminated by removing the solution from the iodo-beads and applying it to a 0.5 ml desalting Zeba spin column (InVitrogen) equilibrated in PBS. The iodo-bead was washed twice with 10 μΐ of PBS each and the wash solution also applied to the desalting column. The radioactive solution was eluted through the desalting column by centrifugation at 1 ,000 x g for 2 min. In the case of Cry 1 Da, the Iodo- gen method was used to conduct the radiolabeling procedure. Using this procedure, the cry toxin in 100 mM phosphate buffer (pH 8) was first cleaned of lipopolysaccharides (LPS) by passing it through a small 0.5 ml polymyxin column multiple times. To the iodo-gen tube (Pierce Chem. Co.) was added 20 μg of the LPS-free Cry 1 Da toxin, then 0.5 mCi of Na^{l 25}l. The reaction mixture was shaken for 15 min at 25 °C. The solution was removed from the tube, and 50 μΐ of 0.2M non-radiolabeled Nal added to quench the reaction. The protein was dialyzed vs PBS with 3 changes of buffer to remove any unbound ^l25I.

Radio-purity of the iodinated Cry proteins was determined by SDS-PAGE, phosphorimaging and gamma counting. Briefly, 2 μΐ of the radioactive protein was separated by SDS-PAGE. After separation, the gels were dried using a BioRad gel drying apparatus following the manufacturer's instructions. The dried gels were imaged by wrapping them in Mylar film (12 μπι thick), and exposing them under a Molecular Dynamics storage phosphor screen (35 cm x 43 cm), for 1 hour. The plates were developed

BDDB01 641 1412vl DAS-P0199

using a Molecular Dynamics Storm 820 phosphorimager and the imaged analyzed using ImageQuant™ software. The radioactive band along with areas immediately above and below the band were cut from the gel using a razor blade and counted in a gamma counter. Radioactivity was only detected in the Cry protein band and in areas below the band. No radioactivity was detected above the band, indicating that all radioactive contaminants consisted of smaller protein components than the truncated Cry protein. These components most probably represent degradation products.

Example 2 - BBMV Preparation Protocol

Preparation and Fractionation of Solubilized BBMV's. Last instar Spodoptera frugiperda, Ostrinia nubilalis, or Heleothis. zea larvae were fasted overnight and then dissected in the morning 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 9X volume of ice cold homogenization buffer (300 mM mannitol, 5 mM EGTA, 17 mM tris. base, pH 7.5), supplemented with Protease Inhibitor Cocktail ¹ (Sigma P-2714) diluted as recommended by the supplier. The tissue was homogenized with 1 5 strokes of a glass tissue homogenizer. BBMV's were prepared by the gCh precipitation method of Wolfersberger ( 1993). Briefly, an equal volume of a 24 mM MgC 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 x g for 15 min at 4° C. The supernatant was saved and the pellet suspended into the original volume of 0.5-X diluted homogenization buffer and centrifuged again. The two supernatants were combined, centrifuged at 27,000 x g for 30 min at 4° C to form the BBMV fraction. The pellet was suspended into 10 ml homogienization buffer and supplemented to protease inhibitiors and centrifuged again at 27,000 x g of r30 min at 4 °C to wash the BBMV's. The resulting pellet was suspended into BBMV Storage Buffer ( 10 mM HEPES, 130 mM C1, 10% glycerol, pH 7.4) to a concentration of about 3 mg/ml protein. Protein concentration was determined by using the Bradford method ( 1976) with bovine serum albumin (BSA) as the standard. Alkaline phosphatase determination was made prior to freezing the samples using the Sigma assay following manufacturer's instructions. The specific activity of this marker enzyme in the BBMV fraction typically increased 7-fold compared to that found in

¹ Final concentration of cocktail components (in μ ) are AEBSF (500), EDTA (250 mM), Bestatin (32), E-64 (0.35), Leupeptin (0.25), and Aprotinin (0.075).

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the midgut homogenate fraction. The BBMV's were aliquoted into 250 μΐ samples, flash frozen in liquid 2 and stored at -80°C.

Example 3 - Method to Measure Binding of ^{l 23}l Cry Proteins to BBMV Proteins

Binding of ^l25I Cry Proteins to BBMV's. To determine the optimal amount of BBMV protein to use in the binding assays, a saturation curve was generated. ^{I 25}I radiolabeled Cry protein (0.5 nM) was incubated for 1 hr. at 28 °C with various amounts of BBMV protein, ranging from 0-500 μg/ml in binding buffer (8 mM NaHPO^, 2 mM K.H2PO4, 150 mM NaCl, 0.1 % bovine serum albumin, pH 7.4). Total volume was 0.5 ml. Bound ¹²⁵I Cry protein was separated from unbound by sampling 150 μΐ of the reaction mixture in triplicate from a 1.5 ml centrifuge tube into a 500 μΐ centrifuge tube and centrifuging the samples at 14,000 x g for 6 minutes at room temperature. The supernatant was gently removed, and the pellet gently washed three times with ice cold binding buffer. The bottom of the centrifuge containing the pellet was cut out and placed into a 13 x 75-mm glass culture tube. The samples were counted for 5 minutes each in the gamma counter. The counts contained in the sample were subtracted from background counts (reaction with out any protein) and was plotted versus BBMV protein concentration. The optimal amount of protein to use was determined to be 0.15 mg/ml of BBMV protein.

To determine the binding kinetics, a saturation curve was generated. Briefly, BBMV's (1 50 μg/ml) were incubated for 1 hr. at 28 °C with increasing concentrations of ^{l 25}I Cry toxin, ranging from 0.01 to 10 nM. Total binding was determined by sampling 150 μΐ of each concentration in triplicate, centrifugation of the sample and counting as described above. Non-specific binding was determined in the same manner, with the addition of 1 ,000 nM of the homologous trypsinized non-radioactive Cry toxin added to the reaction mixture to saturate all non-specific receptor binding sites. Specific binding was calculated as the difference between total binding and non-specific binding.

Homologous and heterologous competition binding assays were conducted using 150 μg ml BBMV protein and 0.5 nM of the ¹²⁵I radiolabeled Cry protein. The concentration of the competitive non-radiolabeled Cry toxin added to the reaction mixture ranged from 0.045 to 1 ,000 nM and were added at the same time as the radioactive ligand, to assure true binding competition. Incubations were carried out for 1 hr. at 28 °C and the amount of ^{l 25}I Cry protein bound to its receptor toxin measured as described above with

BDDB01 641 1412v l DAS-P0199

non-specific binding subtracted. One hundred percent total binding was determined in the absence of any competitor ligand. Results were plotted on a semi-logarithmic plot as percent total specific binding versus concentration of competitive ligand added.

Example 4 - Summary of Results

Figure 1 shows percent specific binding of ^I25I Cry 1 Ab (0.5 nM) in BBMV's from ECB versus competition by unlabeled homologous Cry l Ab (♦) and heterologous Cryl Be (·). The displacement curve for homologous competition by Cry l Ab results in a sigmoidal shaped curve showing 50% displacement of the radioligand at about 0.5 nM of Cry l Ab. Cry 1 Be also displaces ^l25l Cry 1 Be from its binding site but requires approximately 40 nM concentration (80-fold higher than required by Cry l Ab), to displace 50% of the ^{l 25}I Cry 1 Ab from its binding site.

Figure 2 shows percent specific binding of ^{l 25}I Cry l Ab (0.5 nM) in BBMV's from FAW versus competition by unlabeled homologous Cry lAb (♦) and heterologous Cryl Be (·). The displacement curve for homologous competition by CrylAb results in a sigmoidal shaped curve showing 50% displacement of the radioligand at about 0.3 nM of Cryl Ab. Cry 1 Be displaces ¹²⁵I CrylAb by 50% at a concentration of approximately 300 nM, or about 1 ,000-fold greater than required by CrylAb. Error bars represent the range of values obtained from duplicate determinations.

Figure 3 shows percent specific binding of ^l25I CrylBe (0.5 nM) in BBMV's from FAW versus competition by unlabeled homologous Cryl Be (·) and heterologous Cry l Ab (♦). The displacement curve for homologous competition by Cry 1 Be results in a sigmoidal shaped curve showing 50% displacement of the radioligand at about 2 nM of Cry 1 Be. Cry l Ab at a concentration of 1 ,000 nM (2,000-fold greater than ¹²⁵I Cry l Be being displaced) results approximately 50% displacement. There is an approximate 500-fold lower affinity of Cry 1 Ab to compete for the binding of Cry 1 Be. Error bars represent the range of values obtained from triplicate determinations.

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Reference List

Heckel,D.G., Gahan.L.J., Baxter,S.W., Zhao.J.Z., Shelton,A. ., Gould,F., and

Tabashnik,B.E. (2007). The diversity of Bt resistance genes in species of Lepidoptera. J Invertebr Pathol 95, 192- 197.

Luo.K., Banks,D., and Adang.M.J. (1999). Toxicity, binding, and permeability analyses of four bacillus thuringiensis cry l delta-endotoxins using brush border membrane vesicles of spodoptera exigua and spodoptera frugiperda. Appl. Environ. Microbiol. 65, 457-464.

Palmer, M., Buchkremer, M, Valeva, A, and Bhakdi, S. Cysteine-specific radioiodination of proteins with fluorescein maleimide. Analytical Biochemistry 253, 175-179. 1997.

Ref Type: Journal (Full)

Sambrook_jJ. and Russell,D.W. (2001 ). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory).

Schlenz, M. L., Babcock, J. M., and Storer, N. P. Response of Cry 1 F-resistant and

Susceptible European Corn Borer and Fall Armyworm Colonies to Cry 1 A.105 and

Cry l 2Ab2. DAI 0830, 2008. Indianapolis, Dow AgroSciences. Derbi Report.

Sheets, J. J. and Storer, N. P. Analysis of Cry l Ac Binding to Proteins in Brush Border Membrane Vesicles of Corn Earworm Larvae (Heleothis zed). Interactions with Cry 1 F Proteins and Its Implication for Resistance in the Field. DA1-0417, 1 -26. 2001. Indianapolis, Dow AgroSciences.

Tabashnik.B.E., Liu,Y.B., Finson.N., Masson,L., and Heckel,D.G. ( 1997). One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins. Proc. Natl. Acad. Sci. U. S. A 94, 1640-1644.

Tabashnik,B.E., Malvar,T., Liu,Y.B., Finson.N., Borthakur,D., Shin,B.S., Park,S.H., Masson,L., de Maagd,R.A., and Bosch,D. (1996). Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins. Appl. Environ. Microbiol. 62, 2839-2844.

Tabashnik,B.E., Roush,R.T., Earle,E.D., and Shelton,A.M. (2000). Resistance to Bt toxins. Science 287, 42.

Wolfersberger,M.G. ( 1993). Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the gypsy moth (Lymantria dispar). Arch. Insect Biochem. Physiol 24, 139- 147.

Xu,X., Yu,L., and Wu,Y. (2005). Disruption of a cadherin gene associated with resistance to Cry 1 Ac {delta}-endotoxin of Bacillus thuringiensis in Helicoverpa armigera. Appl Environ Microbiol 71, 948-954.

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Appendix A

List of delta-endotoxins - from Crickmore et al. website (cited in application)

Accession Number is to NCBI entry

Name Acc No. Authors Year Source Strain Comment

CrylAal AAA22353 Schnepf et al 1985 Bt kurstaki HD1

CrylAa2 AAA22552 Shibano et al 1985 Bt sotto

CrylAa3 BAA00257 Shimizu et al 1988 Bt aizawai IPL7

CrylAa4 CAA31886 Masson et al 1989 Bt entomocidus

CrylAa5 BAA04468 Udayasuriyan et al 1994 Bt Fu-2-7

Bt kurstaki NRD-

CrylAa6 AAA86265 Masson et al 1994

12

CrylAa7 AAD46139 Osman et al 1999 BtC12

CrylAa8 126149 Liu 1996 DNA sequence only

Bt dendrolimus

!Aa9 BAA77213 Nagamatsu et al

T84A1

Bt kurstaki HD-1-

CrylAalO AAD55382 Hou and Chen 1999

02

CrylAal 1 CAA70856 Tounsi et al 1999 Bt kurstaki

CrylAal2 AAP80146 Yao et al 2001 Bt Ly30

CrylAal3 AAM44305 Zhong et al 2002. Bt sotto

CrylAal4 AAP40639 Ren et al 2002 unpublished

CrylAal5 AAY66993 Sauka et al 2005 Bt INTA Mol-12

CrylAbl AAA22330 Wabiko et al 1986 Bt berliner 1715

CrylAb2 AAA22613 Thorne et al 1986 Bt kurstaki

CrylAb3 AAA22561 Geiser et al 1986 Bt kurstaki HD1

CrylAb4 BAA00071 ondo et al 1987 Bt kurstaki HD1

CrylAb5 CAA28405 Hofte et al 1986 Bt berliner 1715

Bt kurstaki NRD-

Crv1Ab6 AAA22420 Hefford et al 1987

12

CrylAb7 CAA31620 Haider & Ellar 1988 Bt aizawai IC1

CrylAb8 AAA22551 Oeda et al 1987 Bt aizawai IPL7

CrylAb9 CAA38701 Chak & Jen 1993 Bt aizawai HD133

CrylAblO A29125 Fischhoff et al 1987 Bt kurstaki HD1

CrylAbl 1 112419 Ely & Tippett 1995 Bt A20 DNA sequence only

CrylAbl2 AAC64003 Silva-Werneck et al 1998 Bt kurstaki S93

CrylAbl 3 AAN76494 Tan et al 2002 Bt c005

Meza- Basso &

CrylAbl4 AAG 16877 2000 Native Chilean Bt

Theoduloz

BDDB016411412vl DAS-P01 9

CrylAbl5 ΑΑΟΪ3302 Li et al 2001 Bt B-Hm-16

CrylAbl6 AAK55546 Yu et al 2002 Bt AC- 11

CrylAbl7 AAT46415 Huang et al 2004 Bt WB9

CrylAblS AAQ88259 Stobdan et al 2004 Bt

CrylAbl9 AAW31761 Zhong et al 2005 Bt X-2

CrylAb2Q ABB72460 Liu et al 2006 BtC008

CrylAb21 ABS 18384 Swiecicka et al 2007 Bt IS5056

CrylAb22 ABW87320 Wu and Feng 2008 BtS2491Ab

CrylAb- like AA 14336 Nagarathinam et al

Bt kunthala RX24 uncertain sequence

CrylAb- like AA 14337 Nagarathinam et al Bt kunthala RX28 uncertain sequence

CrylAb- like AAK 14338 Nagarathinam et al 2001 Bt kunthala RX27 uncertain sequence

CrylAb- like ABG88858 Lin et a! 2006 Bt Iy4a3 insufficient sequence

CrylAcl AAA22331 Adang et al 1985 Bt kurstaki HD73

CrylAc2 AAA22338 Von Tersch et al 1991 Bt kenyae

CrylAc3 CAA38098 Dardenne et al 1990 Bt BTS89A

CrvlAc4 AAA73077 Feitelson Bt kurstaki

1991

PS85A1

CrvlAc5 AAA22339 Feitelson Bt kurstaki

1992

PS81GG

CrvlAc6 AAA86266 Masson et al Bt kurstaki NRD-

1994

12

CrylAc7 AAB46989 Herrera et al 1994 Bt kurstaki HD73

CrylAc8 AAC44841 Omolo et al 1997 Bt kurstaki HD73

CrylAc9 AAB49768 G leave et al 1992 Bt DSIR732

CrylAclO CAA05505 Sun Bt kurstaki YBT-

1997

1520

CrylAcl 1 CAA 10270 akhdoom &

Riazuddin 1998

CrylAcl2 II24I8 Ely & Tippett 1995 Bt A20 DNA sequence only Crv1Acl3 AAD38701 Qiao et al 1999 Bt kurstaki HD1

CrylAcl4 AAQ06607 Yao et al 2002 Bt Ly30

CrylAcIS AAN07788 Tzeng et al 2001 Bt from Taiwan

CrylAcl6 AAU87037 Zhao et al 2005 Bt H3

CrylAcl7 A AX 18704 Hire et al 2005 Bt kenyae HD549

CrylAcIS AAY88347 Kaur & Allam 2005 BtSK-729

CrylAcl9 ABD37053 Gao et al 2005 Bt C-33

CrylAc20 ABB89046 Tan et al 2005

CrylAc21 AAY66992 Sauka et al 2005 INTAMol-12

CrylAc22 ABZ01836 Zhang & Fang 2008 Bt W015-1

CrylAc23 CAQ30431 Kashyap et al 2008 Bt

CrylAc24 ABL01535 Arango et al 2008 Bt 146-158-01

BDDB016411412vl DAS-P0199

Cr lAc25 FJ513324 Guan Peng et al 2008 Bt Tm37-6 No NCBI link July 09 CrylAc26 FJ617446 Guan Peng et al 2009 Bt Tm41-4 No NCBI link July 09 CrylAc27 FJ617447 Guan Peng et al 2009 Bt Tm44-1B No NCBI link July 09 CrylAc28 AC 90319 Li et al 2009 Bt Q-12

CrylAdl AAA22340 Feitelson 1993 Bt aizawai PS81I

Crv1Ad2 CAA01880 Anonymous 1995 Bt PS81 R1

CrylAel AAA22410 Lee & Aronson 1991 Bt alesti

CrylAfl AAB82749 Kang et al 1997 B NT0423

CrylAel AAD46137 Mustafa 1999

CrylAhl AAQ 14326 Tan et al 2000

CrylAh2 ABB76664 Qi et al 2005 Bt alesti

CrylAil AA039719 Wang et al 2002

CrylA- like AAK14339 Nagarathinam et al 2001 Bt kunthala nags3 uncertain sequence

CrylBal CAA29898 Brizzard & Whiteley 1988 ^{Bt thurin}g>ensis

⁷ HD2

CrvlBa2 CAA65003 Soetaert 1996 Bt entomocidus

HD110

CrvlBa3 AA 63251 Zhang et al 2001

CrvlBa4 AA 51084 Nathan et al 2001 Bt entomocidus

HD9

CrvlBa5 ABO20894 Song et al 2007 Btsfw-12

Cry 1 Ba6 ABL60921 Martins et al 2006 Bt S60I

CrvlBbl AAA22344 Donovan et al 1994 Bt EG 5847

CrvlBcl CAA86568 Bishop et al 1994 Bt morrisoni

CrylBdl AAD 10292 uo et al 2000 Bt wuhanensis

HD525

CrvlBd2 AAM93496 Isakova et al 2002 Bt 834

CrvlBel AAC32850 Payne et al 1998 Bt PS158C2

CrvlBe2 AAQ52387 Baum et al 2003

CrylBe3 FJ7I6I02 Xiaodong Sun et al 2009 Bt NCBI link July

CrvlBfl CAC50778 Arnaut et al 2001

CrvlBf2 AAQ52380 Baum et al 2003

CrvlBel AAO39720 Wang et al 2002

CrvlCal CAA30396 Honee et al 1988 Bt entomocidus

60.5

CrvlCa2 CAA31951 Sanchis et al 1989 Bt aizawai 7.29

CrvICa3 AAA22343 Feitelson 1993 Bt aizawai PS811

CrvlCa4 CAA01886 Van Mellaert et al 1990 Bt entomocidus

HD110

CrvICa5 CAA65457 Strizhov 1996 Bt aizawai 7.29

CrvlCa6 AAF37224 Yu et al 2000 Bt AF-2

CrvlCa7 AAG50438 Aixing et al 2000 Bt J8

CrvlCa8 AAM00264 Chen et al 2001 Bt c002

CrvICa9 AAL79362 ao et al 2003 BtGlO-OlA

BDDB016411412vl DAS-P0199

CrvlCalO AAN 16462 Lin et al 2003 Bt E05-20a

CrvlCall AAX53094 Cai et al 2005 Bt C-33

CrvlCbl 97880 alman et al 1993 Bt galleriae HD29

CrvlCb2 AAG35409 Song et al 2000 Bt cboi

CrvlCb3 ACD50894 Huang et al 2008 Bt 087

CrvlCb- Thammasittirong et

AAX63901 2005 Bt TA476-1 insufficient sequence like al

CrvlDal CAA38099 Hofte et al 1990 Bt aizawai HD68

Cry 1 Da2 176415 Payne & Sick 1997 DNA sequence only

CrvlDbl CAA80234 Lambert 1993 Bt BTS00349A

Crv1Db2 AA 48937 Li et al 2001 Bt B-Pr-88

CrvlDcl AB 35074 Lertwiriyawong et al 2006 Bt JC291

CrvlEal CAA37933 Visser et al 1990 Bt kenyae 4F1

CrvlEa2 CAA39609 Bosse et al 1990 Bt kenyae

CrvlEa3 AAA22345 Feitelson 1991 Bt kenyae PS8 IF

Barboza-Corona et Bt kenyae LBIT-

CrvlEa4 AAD04732 1998

al 147

CrvlEa5 A15535 Botterman et al 1994 DNA sequence only

CrvlEa6 AAL50330 Sun et al 1999 Bt YBT-032

CrvlEa7 AAW72936 Huehne et al 2005 Bt JC190

CrvlEa8 ABX11258 Huang et al 2007 Bt HZM2

Bt aizawai

CrvlEbl AAA22346 Feitelson 1993

PS81A2

Bt aizawai

CrvlFal AAA22348 Chambers et al 1991

EG6346

CrvlFa2 AAA22347 Feitelson 1993 Bt aizawai PS81I

CrvlFbl CAA80235 Lambert 1993 Bt BTS00349A

Bt morrisoni

CrvlFb2 BAA25298 asuda & Asano 1998

ΓΝΑ67

CrvlFb3 AAF21767 Song et al 1998 Bt morrisoni

CrvlFb4 A AC 10641 Payne et al 1997

CrvlFb5 AAO 13295 Li et al 2001 Bt B-Pr-88

CrvlFb6 ACD50892 Huang et al 2008 Bt012

CrvlFb7 ACD50893 Huang et al 2008 Bt 087

CrvlGal CAA80233 Lambert 1993 Bt BTS0349A

CrvlGa2 CAA70506 Shevelev et al 1997 Bt wuhanensis

Bt wuhanensis

CrvlGbl AAD 10291 uo & Chak 1999

HD525

CrvlGb2 AA013756 Li et al 2000 Bt B-Pr-88

CrvlGc AAQ52381 Baum et al 2003

CrvlHal CAA80236 Lambert 1993 Bt BTS02069AA

Bt morrisoni

CrvlHbl AAA79694 Koo et al 1995

BF190

CrvlH-

AAF01213 Srifah et al 1999 Bt JC291 insufficient sequence like

BDDB0164ll412vl DAS-P01 9

Cry Hal CAA44633 Tailor et al 1992 Bt kurstaki

Crvlla2 AAA22354 Gleave et al 1993 Bt kurstaki

Crvlla3 AAC36999 Shin et al 1995 Bt kurstaki HD1

Crvlla4 AAB00958 Kostichka et al 1996 Bt AB88

Crvlla5 CAA70124 Selvapandiyan 1996 Bt 61

Crvlla6 AAC26910 Zhong et al 1998 Bt kurstaki S101

Crvlla7 AAM73516 Porcar et al 2000 Bt

Crvlla8 AA 66742 Song et al 2001

Crvlla9 AAQ08616 Yao et al 2002 Bt Ly30

CrvlIalO AAP86782 Espindola et al 2003 Bt thuringiensis

Crvllall CAC85964 Tounsi et al 2003 Bt kurstaki BNS3

Crvllal2 AAV53390 Grossi de Sa et al 2005 Bt

Crvllal3 ABF83202 Martins et al 2006 Bt

Crvllal4 ACG6387I Liu & Guo 2008 Btll

Cry Hal 5 FJ617445 GuanPengetal 2009 BtE-lB No NCB1 link July

2009

Cry Hal 6 FJ617448 GuanPengetal 2009 BtE-lA No NCBI link July

2009

Cryllbl AAA82114 Shin et al 1995 Bt entomocidus

BP465

Cryllb2 ABW88019 Guan et al 2007 Bt PP61

Cryllb3 ACD75515 Liu & Guo 2008 Bt GS8

Cryllcl AAC62933 Osman et al 1998 BtC18

Cryllc2 AAE71691 Osman et al 2001

Crylldl AAD44366 Choi 2000

Cryllel AAG43526 Song et al 2000 Bt BTC007

Cryllfl AAQ52382 Baum et al 2003

Cryll-like AAC31094 Payne et al 1998 insufficient sequence

Cryll-like ABG88859 Lin & Fang 2006 Bt Iy4a3 insufficient sequence

CrylJal AAA22341 Donovan 1994 Bt EG5847

CrylJbl AAA98959 Von Tersch &

Gonzalez 1994 BtEG5092

CrylJcl AAC31092 Payne et al 1998

CrylJc2 AAQ52372 Baum et al 2003

CrylJdl CAC50779 Arnaut et al 2001 Bt

Crvl al AAB00376 oo et al Bt morrisoni

1995

BF190

Cry 1 Lai AAS60191 Je et al 2004 Bt kurstaki 1

Cry 1 -like AAC31091 Payne et al 1998 insufficient sequence

Crv2Aal AAA22335 Donovan et al 1989 Bt kurstaki

Crv2Aa2 AAA83516 Widner & Whiteley 1989 Bt kurstaki HD1

Crv2Aa3 D86064 Sasaki et al 1997 Bt sotto DNA sequence only

Crv2Aa4 AAC04867 Misra et al 1998 Bt kenyae HD549

Crv2Aa5 CAA 10671 Yu & Pang 1999 Bt SL39

BDDB016411412v 1 DAS-P0I99

Crv2Aa6 CAA 10672 Yu & Pang 1999 Bt YZ71

Crv2Aa7 CAA 10670 Yu & Pang 1999 Bt CY29

Crv2Aa8 AA013734 Wei et al 2000 Bt Dongbei 66

Crv2Aa9 AAO13750 Zhang et al 2000

Crv2Aal0 AAQ04263 Yao et al 2001

Crv2Aal 1 AAQ52384 Baum et al 2003

Crv2Aal2 ABI83671 Tan et al 2006 Bt Rpp39

Crv2Aal ABL01536 Arango et al 2008 Bt 146-158-01

Crv2Aal4 ACF04939 Hire et al 2008 Bt HD-550

Crv2Abl AAA22342 Widner & Whiteley 1989 Bt kurstaki HD1

Crv2Ab2 CAA39075 Dankocsik et al 1990 Bt kurstaki HD1

Crv2Ab3 AAG36762 Chen et al 1999 Bt BTC002

Crv2Ab4 AAO 13296 Li et al 2001 Bt B-Pr-88

Crv2Ab5 AAQ04609 Yaoetal 2001 Bt Iy30

Crv2Ab6 AAP59457 Wang et al 2003 Bt WZ-7

Crv2Ab7 AAZ66347 Udayasuriyan et al 2005 Bt 14-1

Crv2Ab8 ABC95996 Huang et al 2006 Bt WB2

Crv2Ab9 ABC74968 Zhang et al 2005 Bt LLB6

Crv2Abl0 EF157306 Lin et al 2006 Bt LyD

Crv2Abll CAM84575 Saleem et al 2007 Bt C BL-BT1

Cry2Abl2 AB 21764 Lin et al 2007 Bt LyD

Crv2Abl3 ACG76120 Zhu et al 2008 Bt ywc5-4

Cry2Abl4 ACG76121 Zhu et al 2008 Bt Bts

Cry2Acl CAA40536 Aronson 1991 Bt shanghai S 1

Cry2Ac2 AAG35410 Song et al 2000

Cry2Ac3 AAQ52385 Baum et al 2003

Cry2Ac4 ABC95997 Huang et al 2006 Bt WB9

Cry2Ac5 ABC74969 Zhang et al 2005

Crv2Ac6 ABC74793 Xia et al 2006 Bt wuhanensis

Crv2Ac7 CAL 18690 Saleem et al 2008 Bt SBSBT-1

Cry2Ac8 CAM09325 Saleem et al 2007 Bt CMBL-BT1

Crv2Ac9 CAM09326 Saleem etal 2007 Bt CMBL-BT2

Crv2Acl0 ABN15104 Bai et al 2007 Bt QCL-1

Cry2Acl 1 CAM83895 Saleem et al 2007 Bt HD29

Crv2Acl2 CAM83896 Saleem et al 2007 Bt CMBL-BT3

Crv2Adl AAF09583 Choi et al 1999 Bt BR30

Cry2Ad2 ABC86927 Huang et al 2006 Bt WB10

Crv2Ad3 CAK29504 Saleem et al 2006 Bt5_2AcT(l)

Cry2Ad4 CAM32331 Saleem et al 2007 Bt C BL-BT2

Cry2Ad5 CA078739 Saleem et al 2007 Bt HD29

Cry2Ael AAQ52362 Baum et al 2003

Crv2Afl ABO30519 Beard et al 2007 Bt C8I

Crv2Ag ACH9I610 Zhu et al 2008 Bt JF19-2

Cry2Ah EU939453 Zhang et al 2008 Bt No NCB1 link July 09

BDDB016411412v 1 DAS-P0199

Crv2Ah2 ACL80665 Zhang et al 2009 Bt BRC-ZQL3

Cry2Ai FJ788388 Udayasuriyan et al 2009 Bt No NCBI link July 09

Crv3Aa1 AAA22336 Herrnstadt et al 1987 Bt san diego

Crv3Aa2 AAA2254 I Sekar et al 1987 Bt tenebrionis

Crv3Aa3 CAA68482 Hofte et al 1987

Crv3Aa4 AAA22542 McPherson et al 1988 Bt tenebrionis

Bt morrisoni

Crv3Aa5 AAA50255 Donovan et al 1988

EG2158

Crv3Aa6 AAC43266 Adams et al 1994 Bt tenebrionis

Crv3Aa7 CAB4141 1 Zhang et al 1999 Bt 22

Crv3Aa8 AAS79487 Gao and Cai 2004 Bt YM-03

Crv3Aa9 AAW05659 Bulla and Candas 2004 Bt UTD-001

Crv3Aal O AAU2941 1 Chen et al 2004 Bt 886

Crv3Aal 1 AAW82872 Kurt et al Bt tenebrionis

2005

Mm2

Crv3Aal 2 ABY49136 Sezen et al 2008 Bt tenebrionis

Crv3Bal CAA34983 Sick et al 1990 Bt tolworthi 43F

Crv3Ba2 CAA00645 Peferoen et al 1990 Bt PGSI208

Crv3Bb1 AAA22334 Donovan et al 1992 Bt EG4961

Crv3Bb2 AAA74198 Donovan et al 1995 Bt EG5144

Crv3Bb3 115475 Peferoen et al 1995 DNA sequence only

Bt kurstaki

Crv3Ca1 CAA42469 Lambert et al 1992

BtI 109P

Crv4Aa l CAA68485 Ward & Ellar 1987 Bt israelensis

Crv4Aa2 BAA001 79 Sen et al Bt israelensis

1988

HD522

Crv4Aa3 CAD30148 Berry et al 2002 Bt israelensis

Crv4A-

AAY96321

like ahalakshmi et al 2005 Bt LDC-9 insufficient sequence

Chungjatpornchai et

Crv4Bal CAA303 I 2 Bt israelensis

1988

al 4Q2-72

Crv4Ba2 CAA301 14 Tungpradubkul et al 1988 Bt israelensis

Crv4Ba3 AAA22337 Yamamoto et al 1988 Bt israelensis

Crv4Ba4 BAA00178 Bt israelensis

Sen et al 1988

HD522

Crv4Ba5 CAD30095 Berry et al 2002 Bt israelensis

Crv4Ba-

ABC47686 Mahalakshmi et al

like 2005 Bt LDC-9 insufficient sequence

Cry4Cal EU646202 Shu et al 2008 No NCBI link July 09 Cry4Cbl FJ403208 Jun & Furong 2008 Bt HS 18- 1 No NCBI link July 09 Cry4Cb2 FJ597622 Jun & Furong 2008 Bt Ywc2-8 No NCBI link July 09 Cry4Cc l FJ403207 Jun & Furong 2008 Bt MC28 No NCBI link July 09

Crv5Aal AAA67694 Bt darmstadiensis

Narva et al 1994

PS 17

Cry5Abl AAA67693 Narva et al 1 991 Bt darmstadiensis

BDDB01 641 14 12v l DAS-P0199

PS 17

Crv5Acl 134543 Payne et al 1997 DNA sequence only

Crv5Adl ABQ82087 Lenane et al 2007 Bt L366

Crv5Bal AAA68598 Foncerrada & Narva 1997 Bt PS86Q3

Crv5Ba2 ABW88932 Guo et al 2008 YBT 1518

Crv6Aal AAA22357 Narva et al 1993 Bt PS52A1

Crv6Aa2 AAM46849 Bai et al 2001 YBT 1518

Crv6Aa3 ABH03377 Jia et al 2006 Bt 96418

Crv6Bal AAA22358 Narva et al 1991 Bt S69D1

Crv7Aal AAA22351 Lambert et al Bt galleriae

1992

PGSI245

Crv7Abl AAA21 120 Narva & Fu 1994 Bt dakota HD51 1

Crv7Ab2 AAA21 121 Narva & Fu 1994 Bt kumamotoensis

867

Crv7Ab3 ABX24522 Song et al 2008 Bt WZ-9

Cr 7Ab4 EU380678 Shu et al 2008 Bt No NCBI link July 09

Crv7Ab5 ABX79555 Aguirre-Arzola et al 2008 Bt monterrey GM- 33

Crv7Ab6 ACI44005 Deng et al 2008 Bt HQ 122

Cry7Ab7 FJ940776 Wang et al 2009 No NCBI link Sept 09

Cry7Ab8 GUI 45299 Feng Jing 2009 No NCBI link Nov 09

Crv7Bal ABB70817 Zhang et al 2006 Bt huazhongensis

Crv7Cal ABR67863 Gao et al 2007 Bt BTH-13

Crv7Dal ACQ99547 Yi et al 2009 Bt LH-2

Crv8Aal AAA21 1 17 Narva & Fu 1992 Bt kumamotoensis

Cry8Abl EU044830 Cheng et al 2007 Bt B-JJX No NCBI link July 09

Crv8Bal AAA21 1 18 Narva & Fu 1993 Bt kumamotoensis

Crv8Bb l CAD57542 Abad et al 2002

Crv8Bcl CAD57543 Abad et al 2002

Crv8Cal AAA21 I I 9 Sato et al. Bt japonensis

1995

Buibui

Crv8Ca2 AAR98783 Shu et al 2004 Bt HBF- 1

Cry8Ca3 EU625349 Du et al 2008 Bt FTL-23 No NCBI link July 09

Crv8Dal BAC07226 Asano et al 2002 Bt galleriae

Crv8Da2 BD 133574 Asano et al 2002 Bt DNA sequence only

Crv8Da3 BD 133575 Asano et al 2002 Bt DNA sequence only

Crv8Dbl BAF93483 Yamaguchi et al 2007 Bt BBT2-5

Crv8Eal AAQ73470 Fuping et al 2003 Bt 185

Cry8Ea2 EU047597 Liu et al 2007 Bt B-DLL No NCBI link July 09

Crv8Fal AAT48690 Shu et al 2004 Bt 185 also AAW81032

Crv8Gal AAT46073 Shu et al 2004 Bt HBF-18

Crv8Ga2 ABC42043 Yan et al 2008 Bt 145

Cry8Ga3 FJ 198072 Xiaodong et al 2008 Bt FCD1 14 No NCBI link July 09

Cr 8Hal EF465532 Fuping et al 2006 Bt 185 No NCBI link July 09

BDDB01 641 1412v l DAS-P0199

Cr 8Ial EU381044 Yan et al 2008 Bt su4 No NCBI link July 09

Cry8Ja l EU625348 Du et al 2008 Bt FPT-2 No NCBI link July 09

Cry8 al FJ422558 Quezado et al 2008 No CBI link July 09

Crv8 a2 ACN87262 Noguera & Ibarra 2009 Bt kenyae

Crv8-like FJ770571 Noguera & Ibarra 2009 Bt canadensis DNA sequence

Crv8-like ABS53003 Mangena et al 2007 Bt

Crv9Aal CAA41 122 Shevelev et al 1991 Bt galleriae

Crv9Aa2 CAA41425 Gleave et al 1992 Bt DSIR517

Cry9Aa3 GQ249293 Su et al 2009 Bt SC5(D2) No NCBI link July 09

Cry9Aa4 GQ249294 Su et al 2009 Bt T03C001 No NCBI link July 09

Crv9Aa

AAQ52376 Baum et al 2003 incomplete sequence like

Crv9Bal CAA52927 Shevelev et al 1993 Bt galleriae

Crv9Bbl AAV28716 Silva-Werneck et al 2004 Bt japonensis

Crv9Cal CAA85764 Lambert et al 1996 Bt tolworthi

Cry9Ca2 AAQ52375 Baum et al 2003

Bt japonensis

Crv9Dal BAA 19948 Asano 1997

N 141

Crv9Da2 AAB97923 Wasano & Ohba 1998 Bt japonensis

Cry9Da3 GQ249295 Su et al 2009 Bt T03B001 No NCBI link July 09 Cry9Da4 GQ249297 Su et al 2009 Bt T03B001 No NCBI link July 09

Bt kurstaki

Crv9Dbl AAX78439 Flannagan & Abad 2005

DP 1019

Bt aizawai SS - Crv9Eal BAA34908 Midoh & Oyama 1998

10

Cry9Ea2 AAO 12908 Li et al 2001 Bt B-Hm- 16

Cry9Ea3 ABM21765 Lin et al 2006 Bt lyA

Cry9Ea4 ACE88267 Zhu et al 2008 Bt ywc5-4

Crv9Ea5 ACF04743 Zhu et al 2008 Bts

Cry9Ea6 ACG63872 Liu & Guo 2008 Bt 1 1

Cry9Ea7 FJ380927 Sun et al 2008 No NCBI link July 09 Cry9Ea8 GQ249292 Su et al 2009 GQ249292 No NCBI link July 09 Crv9Ebl CAC50780 Arnaut et al 2001

Cry9Eb2 GQ249298 Su et al 2009 Bt T03B001 No NCBI link July 09 Crv9Ec l AAC63366 Wasano et al 2003 Bt galleriae

Bt kurstaki

Crv9Ed l AAX78440 Flannagan & Abad

DP 1019

Cry9Ee l GQ249296 Su et al 2009 Bt T03B001 No NCBI link Aug 09 Crv9-like AAC63366 Wasano et al 1998 Bt galleriae insufficient sequence Crv l OAal AAA22614 Thorne et al 1986 Bt israelensis

Bt israelensis

Crv l OAa2 E00614 Aran & Toomasu 1996 DNA sequence only

ONR-60A

Crv l 0Aa3 CAD30098 Berry et al 2002 Bt israelensis

Cry 1 OA

DQ 167578 Mahalakshmi et al 2006 Bt LDC-9 incomplete sequence like

BDDB01 641 1412v 1 DAS-P0199

CrvllAal AAA22352 Donovan et al 1988 Bt israelensis

CrvllAa2 AAA22611 Adams et al 1989 Bt israelensis

CrvllAa3 CAD30081 Berry et al 2002 Bt israelensis

DQ166531 Mahalakshmi et al 2007 Bt LDC-9 incomplete sequence like

CrvllBal CAA60504 Delecluse et al 1995 Bt jegathesan 367

CrvllBbl AAC97162 Orduz et al 1998 Bt medellin

Crvl2Aal AAA22355 Narva et al 1991 Bt PS33F2

Crvl3Aa] AAA22356 Narva et al 1992 Bt PS63B

CrvMAal AAA21516 Narva et al 1994 Bt sotto PS80JJ1

Crvl5Aal AAA22333 Brown & Whiteley 1992 Bt thompsoni

Crvl6Aal CAA63860 Barloy et al 1996 Cb malaysia CHI 8

Crvl7Aal CAA67841 Barloy et al 1998 Cb malaysia CHI 8

Paeni bacillus

Crvl8Aal CAA67506 Zhang et al 1997

popilliae

Paenibacillus

Crvl8Bal AAF89667 Patel et al 1999

popilliae

Paenibacillus

Crvl8Cal AAF89668 Patel et al 1999

popilliae

Crvl9Aal CAA68875 Rosso & Delecluse 1996 Bt jegathesan 367

Crvl9Bal BAA32397 Hwang et al 1998 Bt higo

Crv20Aal AAB93476 Lee & Gill 1997 Bt fukuokaensis

Crv20Bal ACS93601 Noguera & Ibarra 2009 Bt higo LBIT-976

Crv20-like G0144333 Yi et al 2009 BtY-5 DNA sequence only

Crv21Aal 132932 Payne et al 1996 DNA sequence only

Crv21Aa2166477 Feitelson 1997 DNA sequence only

Crv21Bal BAC06484 Sato & Asano 2002 Bt roskildiensis

Crv22Aal 134547 Payne et al 1997 DNA sequence only

Crv22Aa2 CAD43579 Isaac et al 2002 Bt

Crv22Aa3 ACD93211 Du et al 2008 Bt FZ-4

Crv22Abl AA 50456 Baum et al 2000 Bt EG4140

Crv22Ab2 CAD43577 Isaac et al 2002 Bt

Crv22Bal CAD43578 Isaac et al 2002 Bt

Crv23Aal AAF76375 Donovan et al 2000 Bt Binary with Cry37Aal

Crv24Aal AAC61891 Kawalek and Gill 1998 Bt jegathesan

Crv24Bal BAD32657 Ohgushi et al 2004 Bt sotto

Crv24Cal CAJ43600 Beron & Salerno 2005 Bt FCC-41

Crv25Aal AAC61892 Kawalek and Gill 1998 Bt jegathesan

Wojciechowska et Bt finitimus B-

Crv26Aal AAD25075 1999

al 1166

Crv27Aal BAA82796 Saitoh 1999 Bt higo

Bt finitimus B-

Crv28Aal AAD24189 Wojciechowska et al 1999

1161

Crv28Aa2 AAG00235 Moore and Debro 2000 Bt finitimus

Crv29Aal CAC80985 Delecluse et al 2000 Bt medellin

BDDB016411412vl DAS-P0199

Crv30Aal CAC80986 Delecluse et al 2000 Bt medellin

Crv30Ba1 BAD00052 Ito et al 2003 Bt entomocidus

Crv30Cal BAD67157 Ohgushi et al 2004 Bt sotto

Crv30Ca2 ACU24781 Sun and Park 2009 Bt jegathesan 367

Cry30Dal EF095955 Shu et al 2006 Bt Y41 No NCBI link July09

Crv30Db1 BAE80088 Kishida et a) Bt aizawai BUN1-

2006

14

Crv30Eal ACC95445 Fang eta] 2007 Bt S2160-1

Cry30Ea2 FJ499389 Jun et al 2008 Bt Ywc2-8 No NCBI link July09

Crv30Fal ACI22625 Tan et al 2008 Bt MC28

Crv30Gal ACG60020 Zhu et al 2008 Bt HS18-1

Crv31Aal BAB 11757 Saitoh & Mizuki 2000 Bt 84-HS-l-ll

Crv31Aa2 AAL87458 Jung and Cote 2000 Bt M15

Crv31Aa3 BAE79808 Uemori et al 2006 BtB0195

Crv31Aa4 B AF32571 Yasutake et al 2006 Bt 79-25

Crv31Aa5 BAF32572 Yasutake et al 2006 Bt 92-10

Crv31Abl BAE79809 Uemori et al 2006 Bt B0195

Crv31Ab2 BAF32570 Yasutake et al 2006 Bt 31 -5

Crv31Acl BAF34368 Yasutake et al 2006 Bt 87-29

Crv32Aal AAG36711 Balasubramanian et

al 2001 Bt yunnanensis

Crv32Bal BAB78601 Takebe et al 2001 Bt

Crv32Cal BAB78602 Takebe et al 2001 Bt

Crv32Dal BAB78603 Takebe et al 2001 Bt

Crv33Aal AAL26871 Kim et al 2001 Bt dakota

Crv34Aal AAG50341 Ellis et al 2001 Bt PS80JJ1 Binary with Cry35Aal

Crv34Aa2 AAK64560 Rupar et al 2001 Bt EG5899 Binary with Cry35Aa2

Cry34Aa3 AAT29032 Schnepfetal 2004 Bt PS69Q Binary with Cry35Aa3

Crv34Aa4 AAT29030 Schnepfetal 2004 Bt PS185GG Binary with Cry35Aa4 Crv34Abl A AG41671 Moel lenbeck et al 2001 Bt PS149B1 Binary with Cry35Abl

Cry34Acl AAG50118 Ellis et al 2001 Bt PS167H2 Binary with Cry35Acl

Crv34Ac2 AAK64562 Rupar et al 2001 Bt EG9444 Binary with Cry35Ab2

Cry34Ac3 AAT29029 Schnepfetal 2004 Bt R1369 Binary with Cry35Ab3

Crv34Bal AAK64565 Rupar et al 2001 Bt EG4851 Binary with Cry35Bal

Cry34Ba2 AAT29033 Schnepfetal 2004 Bt PS20IL3 Binary with Cry35Ba2

Crv34Ba3 AAT29031 Schnepfetal 2004

Bt PS201HH2 Binary with Cry35Ba3

Cry35Aal AAG50342 Ellis et al 2001 BIPS80JJ1 Binary with Cry34Aal

Crv35Aa2 AAK64561 Rupar et al 2001 Bt EG5899 Binary with Cry34Aa2

Crv35Aa3 AAT29028 Schnepfetal 2004 Bt PS69Q Binary with Cry34Aa3

Cry35Aa4 AAT29025 Schnepfetal 2004 Bt PS185GG Binary with Cry34Aa4 Crv35Abl AAG41672 Moel lenbeck et al 2001 Bt PS149B1 Binary with Cry34Abl

Crv35Ab2 AAK64563 Rupar et al 2001 Bt EG9444 Binary with Cry34Ac2

Crv35Ab3 AY536891 AAT29024 2004 Bt KR1369 Binary with Cry34Ab3

Crv35Acl AAG50117 Ellis et al 2001 Bt PS167H2 Binary with Cry34Acl

BDDB016411412vl DAS-P0199

Crv35Bal AA 64566 upar et al 2001 Bt EG4851 Binary with Cry34Bal

Crv35Ba2 AAT29027 Sc nepf et al 2004 Bt PS201 L3 Binary with Cry34Ba2

Crv35Ba3 AAT29026 Schnepf et al 2004 Bt PS201 HH2 Binary with Cry34Ba3

Crv36Aa) AAK64558 Rupar et al 2001 Bt

Crv37Aal AAF76376 Donovan et al 2000 Bt Binary with Cry23Aa

Crv38Aal AA 64559 Rupar et al 2000 Bt

Crv39Aal BAB72016 Ito et al 2001 Bt aizawai

Crv40Aal BAB72018 Ito et al 2001 Bt aizawai

Crv40Bal BAC77648 Ito et al 2003 Bunl-14

Cry40Cal EU381045 Shu et al 2008 Bt Y41 No NCBI link July09

Crv40Da1 ACF15199 Zhang et al 2008 Bt S2096-2

Crv41Aal BAD35157 Yamashita et al 2003 Bt A 1462

Crv41 Abl BAD35163 Yamashita et al 2003 Bt A 1462

Crv42Aal BAD35166 Yamashita et al 2003 Bt A 1462

Crv43Aal BAD 15301 Yokoyama and

Tanaka 2003 P. lentimorbus

semadara

Crv43Aa2 BAD95474 Nozawa 2004 P. popilliae

popilliae

CQ_ 3Bai BAD 15303 ^Yok° ama and^.

Tanaka 2003 P. lentimorbus

semadara

Crv43-like BAD 15305 ^Yok°y^arr>a and

2 P. lentimorb

Tanaka 003 us

semadara

Cry44Aa BAD08532 Ito et al 2004 Bt entomocidus

I A288

Cry45Aa BAD22577 Okumura et al 2004 Bt 89-T-34-22

Crv46Aa BAC79010 Ito et al 2004 Bt dakota

Crv46Aa2 BAG68906 Ishikawa et al 2008 Bt A 1470

Crv46Ab BAD35170 Yamagiwa et al 2004 Bt

Crv47Aa AAY24695 Kongsuwan et al 2005 Bt CAA890

Crv48Aa CAJ18351 Jones and Berry 2005 Bs IAB59 binary with 49Aa Crv48Aa2 CAJ86545 Jones and Berry 2006 Bs 47-6B binary with 49Aa2 Crv48Aa3 CAJ86546 Jones and Berry 2006 Bs NHA15b binary with 49Aa3 Crv48Ab CAJ86548 Jones and Berry 2006 Bs LP1 G binary with 49Abl Crv48Ab2 CAJ86549 Jones and Berry 2006 Bs 2173 binary with 49Aa4 Cry49Aa CAH56541 Jones and Berry 2005 Bs IAB59 binary with 48Aa

Cry49Aa2 CAJ86541 Jones and Berry 2006 Bs 47-6B binary with 48Aa2

Cry49Aa3 CAJ86543 Jones and Berry 2006 BsNHA15b binary with 48Aa3 Cry49Aa4 CAJ86544 Jones and Berry 2006 Bs 2173 binary with 48Ab2 Cry49Abl CAJ86542 Jones and Berry 2006 Bs LP1 G binary with 48Ab l Crv50Aal BAE86999 Ohgushi et al 2006 Bt sotto

Cry51 Aal ΑΒΠ 4444 Meng et al 2006 Bt F 14- I

Cry52Aal EF613489 Song et al 2007 Bt Y41 No NCBI link July09 Cry52Bal FJ361760 Jun et al 2008 Bt BM59-2 No NCBI link July09 Cry53Aal EF633476 Song et al 2007 Bt Y41 No NCBI link July09

BDDB0I 641 J4l2vl DAS-P0199

Cry53Abl FJ361759 Jun et al 2008 Bt MC28 No NCBI link July09

Crv54Aal ACA52194 Tan et al 2009 Bt MC28

Crv55Aal ABW8893 1 Guo et al 2008 YBT 1 51 8

Crv55Aa2 AAE33526 Bradfisch et al 2000 BT Y41

Cry56Aal FJ597621 Jun & Furong 2008 Bt Ywc2-8 No NCBI link July09

Cry56Aa2 GQ483512 Guan Peng et al 2009 Bt G7- l No NCBI link Aug09

Crv57Aal ANC87261 Noguera & Ibarra 2009 Bt kim

Crv58Aal ANC87260 Noguera & Ibarra 2009 Bt entomocidus

Crv59Aal AC 43758 Noguera & Ibarra 2009 Bt kim LBIT-980

BDDB0I 641 1412v l DAS.P0199

BDDBOl 641 1412vl DAS-P0199

BDDBOl 641 1412vl

Claims

We claim:

1. A transgenic plant comprising DNA encoding a Cry lAb insecticidal protein and DNA encoding a Cry 1 Be insecticidal protein.

2. The transgenic plant of claim 1, said plant further comprising DNA encoding a third insecticidal protein, said third protein being selected from the group consisting of Cry2A, Cry II, and DIG-3.

3. The transgenic plant of claim 3, said plant further comprising DNA encoding an insecticidal CrylFa protein, and DNA encoding a fourth insecticidal protein selected from the group consisting of CrylFa, Vip3Ab, CrylCa, and CrylE.

4. Seed of a plant of any of claims 1 -3.

5. A field of plants comprising non-5t refuge plants and a plurality of plants of any of claims 1-3, wherein said refuge plants comprise less than 40% of all crop plants in said field.

6. The field of plants of claim 5, wherein said refuge plants comprise less than 30% of all the crop plants in said field.

7. The field of plants of claim 5, wherein said refuge plants comprise less than 20% of all the crop plants in said field.

8. The field of plants of claim 5, wherein said refuge plants comprise less than 10% of all the crop plants in said field.

9. The field of plants of claim 5, wherein said refuge plants comprise less than 5% of all the crop plants in said field.

10. The field of plants of claim 5, wherein said refuge plants are in blocks or strips.

1 1. A mixture of seeds comprising refuge seeds from non-5? refuge plants, and a

plurality of seeds of claim 4, wherein said refuge seeds comprise less than 40% of all the seeds in the mixture.

12. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 30% of all the seeds in the mixture.

13. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 20% of all the seeds in the mixture.

14. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 10% of all the seeds in the mixture.

15. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 5% of all the seeds in the mixture.

16. A method of managing development of resistance to a Cry protein by an insect, said method comprising planting seeds to produce a field of plants of any of claims 5-10.

17. A field of any of claims 5-10, wherein said plants occupy more than 10 acres.

18. A plant of any of claims 1-3, wherein said plant is selected from the group

consisting of corn, soybeans, and cotton.

19. The plant of claim 18, wherein said plant is a maize plant.

20. A plant cell of a plant of any of claims 1-3, wherein said plant cell comprises said DNA encoding said CrylBe insecticidal protein and said DNA encoding said CrylAb insecticidal protein, wherein said CrylBe insecticidal protein is at least 99% identical with SEQ ID NO: l, and said CrylAb insecticidal protein is at least 99% identical with SEQ ID NO:2.

21. A plant of any of claims 1-3, wherein said CrylBe insecticidal protein comprises SEQ ID NO: l, and said CrylAb insecticidal protein comprises SEQ ID NO:2.

22. A method of producing the plant cell of claim 20.

23. A method of controlling a European cornborer insect by contacting said insect with a CrylBe insecticidal protein and a CrylAb insecticidal protein.