US 20120331590A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0331590 A1 Meade et al. (43) Pub. Date: Dec. 27, 2012

(54) USE OF CRY1DAN COMBINATION WITH Publication Classification CRY1BE FORMANAGEMENT OF RESISTANT INSECTS (51) Int. Cl. (75) Inventors: Thomas Meade, Zionsville, IN (US); AOIN37/18 (2006.01) Kenneth Narva, Zionsville, IN (US); Nicholas P. Storer, Kensington, MD AOIH 5/00 (2006.01) (US); Joel J. Sheets, Zionsville, IN AOIC II/00 (2006.01) (US); Aaron T. Woosley, Fishers, IN CI2N5/10 (2006.01) (US); Stephanie L. Burton, Indianapolis, IN (US) AOIGI/00 (2006.01) (73) Assignee: Dow AgroSciences LLC, Indianaoplis, AOIP 7/04 (2006.01) IN (US) AOIH 5/10 (2006.01) (52) U.S. Cl...... 800/302: 514/4.5; 435/419:47/58.1 R (21) Appl. No.: 13/516,665 (22) PCT Fled: Dec. 16, 2010 (57) ABSTRACT (86) PCT NO.: PCT/US10/60829 S371 (c)(1), The Subject invention includes methods and plants for con (2), (4) Date: Aug. 27, 2012 trolling fall army worm insects, said plants comprising a Related U.S. Application Data Cry 1Da insecticidal protein and a Cryl Be insecticidal pro (60) Provisional application No. 61/284.252, filed on Dec. tein, and various combinations of other proteins comprising 16, 2009, provisional application No. 61/284,290, this pair of proteins, to delay or prevent development of filed on Dec. 16, 2009. resistance by the insects. Patent Application Publication Dec. 27, 2012 US 2012/0331590 A1

s

O.O1 O1 1 1O 1OO 1OOO Concentration of Ligand (nM)

Figure l

120 g 1OO 8 O 6 O

O O.O1 0.1 1 10 1OO 1OOO Concentration (nM)

Figure 2 US 2012/0331590 A1 Dec. 27, 2012

USE OF CRY1DA IN COMBINATION WITH identifying insecticidal proteins likely to not exhibit cross CRY1BE FOR MANAGEMENT OF resistance has been suggested (van Mellaert et al. 1999). The RESISTANT INSECTS key predictor of lack of cross resistance inherent in this approach is that the insecticidal proteins do not compete for BACKGROUND OF THE INVENTION receptors in a sensitive insect species. 0001 Humans grow corn for food and energy applica 0007. In the event that two Bt toxins compete for the same tions. Humans also grow many other crops, including Soy receptor, then if that receptor mutates in that insect so that one beans and cotton. Insects eat and damage plants and thereby of the toxins no longer binds to that receptor and thus is no undermine these human efforts. Billions of dollars are spent longer insecticidal against the insect, it might be the case that each year to control insect pests and additional billions are the insect will also be resistant to the second toxin (which lost to the damage they inflict. Synthetic organic chemical competitively bound to the same receptor). That is, the insect insecticides have been the primary tools used to control insect is said to be cross-resistant to both Bt toxins. However, if two pests but biological insecticides, such as the insecticidal pro toxins bind to two different receptors, this could be an indi teins derived from Bacillus thuringiensis (Bt), have played an cation that the insect would not be simultaneously resistant to important role in some areas. The ability to produce insect those two toxins. resistant plants through transformation with Bt insecticidal 0008 For example, Cry 1Fa protein is useful in controlling protein genes has revolutionized modern agriculture and many lepidopteran pests species including the European corn heightened the importance and value of insecticidal proteins borer (ECB; Ostrinia nubilalis (Hübner)) and the FAW, and is and their genes. active against the Sugarcane borer (SCB; Diatraea sacchara 0002. Several Bt proteins have been used to create the lis). The Cry1 Fa protein, as produced in transgenic corn insect-resistant transgenic plants that have been Successfully plants containing event TC1507, is responsible for an indus registered and commercialized to date. These include try-leading insect resistance trait for FAW control. Cry1 Fa is Cry1Ab, Cry1Ac, Cry 1F and Cry3Bb in corn, Cry1Ac and further deployed in the Herculex(R, SmartStaxTM, and Wide Cry2Ab in cotton, and Cry3A in potato. StrikeTM products. 0003. The commercial products expressing these proteins 0009. The ability to conduct (competitive or homologous) express a single protein except in cases where the combined receptor binding studies using Cryl Fa protein is limited insecticidal spectrum of 2 proteins is desired (e.g., Cry1Ab because the most common technique available for labeling and Cry3Bb in corn combined to provide resistance to lepi proteins for detection in receptor binding assays inactivates dopteran pests and rootworm, respectively) or where the inde the insecticidal activity of the Cryl Fa protein. pendent action of the proteins makes them useful as a tool for 0010 Additional Cry toxins are listed at the website of the delaying the development of resistance in Susceptible insect official B.t. nomenclature committee (Crickmore et al.: populations (e.g., Cry1Ac and Cry2Ab in cotton combined to lifesci. Sussex.ac.uk/home/Neil Crickmore/Bt/). There are provide resistance management for tobacco budworm). See currently nearly 60 main groups of “Cry toxins (Cry1 also U.S. Patent Application Publication No. 2009/0313717, Crys9), with additional Cyt toxins and VIP toxins and the which relates to a Cry2 protein plus a Vip3Aa, Cry 1F, or like. Many of each numeric group have capital-letter Sub Cry1A for control of Helicoverpa zea or armigerain. WO groups, and the capital letter Subgroups have lower-cased 2009/132850 relates to Cry1F or Cry1A and Vip3Aa for letter Sub-Subgroups. (Cry1 has A-L, and Cry1A has a-i, for controlling Spodoptera frugiperda. U.S. Patent Application example). Publication No. 2008/0311096 relates in part to Cry1Ab for controlling Cry1 F-resistant ECB. BRIEF SUMMARY OF THE INVENTION 0004 That is, some of the qualities of insect-resistant transgenic plants that have led to rapid and widespread adop 0011. The subject invention relates in part to the surprising tion of this technology also give rise to the concern that pest discovery that Cry 1Da and Cry 1Be do not compete for bind populations will develop resistance to the insecticidal pro ing sites infall armyworm (FAW; Spodoptera frugiperda) gut teins produced by these plants. Several strategies have been cell membrane preparations. As one skilled in the art will Suggested for preserving the utility of Bt-based insect resis recognize with the benefit of this disclosure, plants that pro tance traits which include deploying proteins at a high dose in duce both of these proteins (including insecticidal portions of combination with a refuge, and alternation with, or co-de the full-length proteins) can delay or prevent the development ployment of different toxins (McGaughey et al. (1998), “B.t. of resistance to any of these insecticidal proteins alone. Corn Resistance Management.” Nature Biotechnol. 16:144-146). and soybean are some preferred plants. 0005. The proteins selected for use in an insect resistant 0012. Thus, the subject invention relates in part to the use management (IRM) stack need to exert their insecticidal of a Cry 1Da protein in combination with a Cry 1Be protein. effect independently so that resistance developed to one pro Plants (and acreage planted with Such plants) that produce tein does not confer resistance to the second protein (i.e., both of these proteins are included within the scope of the there is not cross resistance to the proteins). If, for example, a Subject invention. pest population selected for resistance to “Protein A' is sen 0013 The subject invention also relates in part to triple sitive to “Protein B, one would conclude that there is not stacks or "pyramids” of three (or more) toxins, with Cry 1Da cross resistance and that a combination of Protein A and and Cry 1Be being the base pair. In some preferred pyramid Protein B would be effective in delaying resistance to Protein embodiments, the combination of the selected toxins pro A alone. vides three sites of action against FAW. Some preferred “three 0006. In the absence of resistant insect populations, sites of action’ pyramid combinations include the Subject assessments can be made based on other characteristics pre base pair of proteins plus Cry1 Fa, Vip3 Ab, or Cry1E as the Sumed to be related to mechanism of action and cross-resis third protein for targeting FAW. These particular triple stacks tance potential. The utility of receptor-mediated binding in would, according to the Subject invention, advantageously US 2012/0331590 A1 Dec. 27, 2012

and Surprisingly provide three sites of action against FAW. also indicate that the combination of Cry 1Da and Cry1 Be This can help to reduce or eliminate the requirement for proteins can be an effective means to mitigate the develop refuge acreage. ment of resistance in FAW populations to either of these 0014. Additional toxins/genes can also be added accord proteins. Thus, based in part on the data described herein, it is ing to the subject invention. For example, if Cry1 Fa is stacked thought that co-production (stacking) of the Cryl Be and with the subject pair of proteins (both Cry1 Fa and Cry 1Beare Cry 1Da proteins can be used to produce a high dose IRM both active against both FAW and European cornborer Stack for FAW. (ECB)), adding one additional protein to this triple stack 0023. Other proteins can be added to this pair. For wherein the fourth added protein targets ECB, would provide example, the Subject invention also relates in part to triple three sites of action against FAW, and three sites of action stacks or "pyramids” of three (or more) toxins, with Cry 1Da against ECB. This added protein (the fourth protein) could be and Cry 1Be being the base pair. In some preferred pyramid selected from the group consisting of Cry2A, Cry1 I, DIG-3, embodiments, the selected toxins have three separate sites of and Cry1Ab. This would result in a four-protein stack having action against FAW. Some preferred “three sites of action' three sites of action against two insects (ECB and FAW). pyramid combinations include the Subject base pair of pro teins plus Cry1 Fa, Vip3 Ab, or Cry1E as the third protein for DETAILED DESCRIPTION OF THE INVENTION targeting FAW. These particular triple stacks would, accord 0015 The subject invention relates in part to the surprising ing to the Subject invention, advantageously and Surprisingly discovery that Cry 1Da and Cry 1Be do not compete for bind provide three sites of action against FAW. This can help to ing with each other in the gut of fall armyworms (FAW; reduce or eliminate the requirement for refuge acreage. By Spodoptera frugiperda). Thus, a Cryl Da protein can be used 'separate sites of action, it is meant any of the given proteins in combination with a Cryl Be protein in transgenic corn (and do not cause cross-resistance with each other. other plants; e.g., cotton and Soybeans, for example) to delay 0024. Additional toxins/genes can also be added accord or prevent FAW from developing resistance to either of these ing to the subject invention. For example, if Cry1 Fa is stacked proteins alone. The subject pair of proteins can be effective at with the subject pair of proteins (both Cry1 Fa and Cry 1Beare protecting plants (such as maize plants and/or soybean plants) both active against both FAW and European cornborer from damage by Cry-resistant fall armyworm. That is, one (ECB)), adding one additional protein to this triple stack use of the Subject invention is to protect corn and other eco wherein the fourth added protein targets ECB, would provide nomically important plant species from damage and yield three sites of action against FAW, and three sites of action loss caused by fall armyworm populations that could develop against ECB. These added proteins (the fourth protein) could resistance to Cry 1Da or Cry 1Be. be selected from the group consisting of Cry2A, Cry 1I, 0016. The subject invention thus teaches an insect resis DIG-3 (see U.S. Patent Application Ser. No. 61/284.278 (filed tant management (IRM) stack comprising Cryl Da and Dec. 16, 2009) and US 2010 00269223), and Cry1Ab (US Cry1 Be to prevent or mitigate the development of resistance 2008 0311096). This would result in a four-protein stack by FAW to either or both of these proteins. having three sites of action against two insects (ECB and 0017. The present invention provides compositions for FAW). controlling lepidopteran pests comprising cells that produce a 0025 Thus, one deployment option is to use the subject Cry1Da insecticidal protein and a Cryl Be insecticidal pro pair of proteins in combination with a third toxin/gene, and to tein. use this triple stack to mitigate the development of resistance 0018. The invention further comprises a host transformed in FAW to any of these toxins. Accordingly, the subject inven to produce both a Cry 1Da insecticidal protein and a Cry1 Be tion also relates in part to triple stacks or "pyramids” of three insecticidal protein, wherein said host is a microorganism or (or more) toxins. In some preferred pyramid embodiments, a plant cell. The subject polynucleotide(s) are preferably in a the selected toxins have three separate sites of action against genetic construct under control of a non-Bacillus-thuringien FAW sis promoter(s). The Subject polynucleotides can comprise 0026 Included among deployment options of the subject codon usage for enhanced expression in a plant. invention would be to use two, three, or more proteins of the 0019. It is additionally intended that the invention pro Subject proteins in crop-growing regions where FAW can vides a method of controlling lepidopteran pests comprising develop resistant populations. For example, for use of Cry1 Fa contacting said pests or the environment of said pests with an plus Cry1D, see U.S. Patent Application Ser. No. 61/284.252 effective amount of a composition that contains a Cryl Da (filed Dec. 16, 2009), which shows that Cry1D is active core toxin-containing protein and further contains a Cry1 Be against Cry1 F-resistant FAW. This application shows that core toxin-containing protein. Cry 1D does not compete with Cry1F for binding in FAW 0020. An embodiment of the invention comprises a maize membrane preparations. For guidance regarding the use of plant comprising a plant-expressible gene encoding a Cry1 Be Cry1 Fa and Cry 1Be, see U.S. Patent Application Ser. No. insecticidal protein and a plant-expressible gene encoding a 61/284.294 and with Vip3 Ab, see concurrently filed applica Cryl Da insecticidal protein, and seed of Such a plant. tion entitled “COMBINED USE OF Vip3 Ab AND CRY 1 Fa 0021. A further embodiment of the invention comprises a FOR MANAGEMENT OF RESISTANT INSECTS. With maize plant wherein a plant-expressible gene encoding a Cry1 Fa being active against FAW (and European cornborer Cryl Be insecticidal protein and a plant-expressible gene (ECB)). Cry 1Da plus Cry 1Be plus Cry1 Fa would, according encoding a Cryl Da insecticidal protein have been intro to the Subject invention, advantageously and Surprisingly pro gressed into said maize plant, and seed of Such a plant. vide three sites of action against FAW. This can help to reduce 0022. As described in the Examples, competitive receptor or eliminate the requirement for refuge acreage. binding studies using radiolabeled Cry1 Da and Cry 1Be pro (0027 Cry1 Fais deployed in the Herculex(R), SmartStaxTM, teins show that the Cryl Be protein does not compete for and WidesStrikeTM products. The subject pair of genes binding in FAW tissues to which Cry1 Dabinds. These results (Cry1Da and Cry 1Be) could be combined into, for example, US 2012/0331590 A1 Dec. 27, 2012

a Cry1 Fa product such as Herculex(R, SmartStaxTM, and 0034. A person skilled in this art will appreciate that Bt WideStrikeTM. Accordingly, the subject pair of proteins could toxins, even within a certain class such as Cry1 Be, will vary be significant in reducing the selection pressure on these and to some extent in length and the precise location of the tran other commercialized proteins. The Subject pair of proteins sition from core toxin portion to protoxin portion. Typically, could thus be used as in the three gene combinations for corn the Cry 1Be toxins are about 1150 to about 1200 amino acids and other plants (cotton and soybeans, for example). in length. The transition from core toxin portion to protoxin portion will typically occur at between about 50% to about 0028. As discussed above, additional toxins/genes can 60% of the full length toxin. The chimeric toxin of the subject also be added according to the Subject invention. Fortargeting invention will include the full expanse of this N-terminal core ECB, Cry2A, Cry1 I, and/or DIG-3 can be used. See U.S. toxin portion. Thus, the chimeric toxin will comprise at least Patent Application Ser. No. 61/284.278 (filed Dec. 16, 2009). about 50% of the full length of the Cry 1Be protein. This will For the addition of Cry1Ab (for controlling ECB), see U.S. typically be at least about 590 amino acids. With regard to the Patent Application Publication No. 2008/0311096. For the protoxin portion, the full expanse of the Cry1Ab protoxin use of Cry 1E for controlling FAW, see U.S. Patent Applica portion extends from the end of the core toxin portion to the tion Ser. No. 61/284.278 (filed Dec. 16, 2009). C-terminus of the molecule. 0029 Plants (and acreage planted with such plants) that 0035 Genes and Toxins. produce any of the Subject combinations of proteins are 0036. The genes and toxins useful according to the subject included within the scope of the subject invention. Additional invention include not only the full length sequences disclosed toxins/genes can also be added, but the particular stacks dis but also fragments of these sequences, variants, mutants, and cussed above advantageously and Surprisingly provide mul fusion proteins which retain the characteristic pesticidal tiple sites of action against FAW and/or ECB. This can help to activity of the toxins specifically exemplified herein. As used reduce or eliminate the requirement for refuge acreage. A herein, the terms “variants' or “variations of genes refer to field thus planted of over ten acres is thus included within the nucleotide sequences which encode the same toxins or which Subject invention. encode equivalent toxins having pesticidal activity. As used 0030. GENBANK can also be used to obtain the herein, the term “equivalent toxins' refers to toxins having sequences for any of the genes and proteins discussed herein. the same oressentially the same biological activity against the See Appendix A, below. Patents also disclose relevant target pests as the claimed toxins. sequences. For example, U.S. Pat. No. 5,188,960 and U.S. 0037. As used herein, the boundaries represent approxi Pat. No. 5,827.514 describe Cry1 Fa core toxin containing mately 95% (Cry1Da’s and Cry1 Bes), 78% (Cry1D's and proteins suitable for use in carrying out the present invention. Cry1Bs), and 45% (Cry1's) sequence identity, per “Revision U.S. Pat. No. 6,218,188 describes plant-optimized DNA of the Nomenclature for the Bacillus thuringiensis Pesticidal sequences encoding Cry1 Fa core toxin-containing proteins Crystal Proteins. N. Crickmore, D. R. Zeigler, J. Feitelson, that are suitable for use in the present invention. E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean. 0031 Combinations of proteins described herein can be Microbiology and Molecular Biology Reviews (1998) Vol 62: used to control lepidopteran pests. Adult lepidopterans, for 807-813. These cut offs can also be applied to the core toxins example, butterflies and moths, primarily feed on flower nec only. tar and are a significant effector of pollination. Nearly all 0038. It should be apparent to a person skilled in this art lepidopteran larvae, i.e., caterpillars, feed on plants, and that genes encoding active toxins can be identified and many are serious pests. Caterpillars feed on or inside foliage obtained through several means. The specific genes or gene or on the roots or stem of a plant, depriving the plant of portions exemplified herein may be obtained from the isolates nutrients and often destroying the plant's physical Support deposited at a culture depository. These genes, or portions or structure. Additionally, caterpillars feed on fruit, fabrics, and variants thereof, may also be constructed synthetically, for stored grains and flours, ruining these products for sale or example, by use of a gene synthesizer. Variations of genes severely diminishing their value. As used herein, reference to may be readily constructed using standard techniques for lepidopteran pests refers to various life stages of the pest, making point mutations. Also, fragments of these genes can including larval stages. be made using commercially available exonucleases or endo 0032 Some chimeric toxins of the subject invention com nucleases according to standard procedures. For example, prise a full N-terminal core toxin portion of a Bt toxin and, at enzymes such as Bal31 or site-directed mutagenesis can be Some point past the end of the core toxin portion, the protein used to systematically cut off nucleotides from the ends of has a transition to a heterologous protoxin sequence. The these genes. Genes that encode active fragments may also be N-terminal, insecticidally active, toxin portion of a Bt toxin is obtained using a variety of restriction enzymes. Proteases referred to as the “core” toxin. The transition from the core may be used to directly obtain active fragments of these toxin segment to the heterologous protoxin segment can protein toxins. occur at approximately the toxin/protoxinjunction or, in the 0039 Fragments and equivalents which retain the pesti alternative, a portion of the native protoxin (extending past cidal activity of the exemplified toxins would be within the the core toxin portion) can be retained, with the transition to Scope of the Subject invention. Also, because of the redun the heterologous protoxin portion occurring downstream. dancy of the genetic code, a variety of different DNA 0033. As an example, one chimeric toxin of the subject sequences can encode the amino acid sequences disclosed invention, is a full core toxin portion of Cryl Da (amino acids herein. It is well within the skill of a person trained in the art 1 to 601) and/or a heterologous protoxin (amino acids 602 to to create these alternative DNA sequences encoding the same, the C-terminus). In one preferred embodiment, the portion of or essentially the same, toxins. These variant DNA sequences a chimeric toxin comprising the protoxin is derived from a are within the scope of the subject invention. As used herein, Cry1Ab protein toxin. In a preferred embodiment, the portion reference to “essentially the same” sequence refers to of a chimeric toxin comprising the protoxin is derived from a sequences which have amino acid Substitutions, deletions, Cry1Ab protein toxin. additions, or insertions which do not materially affect pesti US 2012/0331590 A1 Dec. 27, 2012 cidal activity. Fragments of genes encoding proteins that 0043. In some instances, non-conservative substitutions retain pesticidal activity are also included in this definition. can also be made. The critical factor is that these substitutions 0040. A further method for identifying the genes encoding must not significantly detract from the biological activity of the toxins and gene portions useful according to the subject the toxin. invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences 0044) Recombinant Hosts. may be detectable by virtue of an appropriate label or may be 0045. The genes encoding the toxins of the subject inven made inherently fluorescent as described in International tion can be introduced into a wide variety of microbial or Application No. WO93/16094. As is well known in the art, if plant hosts. Expression of the toxin gene results, directly or the probe molecule and nucleic acid sample hybridize by indirectly, in the intracellular production and maintenance of forming a strong bond between the two molecules, it can be the pesticide. Conjugal transfer and recombinant transfer can reasonably assumed that the probe and sample have substan be used to create a Bt strain that expresses both toxins of the tial homology. Preferably, hybridization is conducted under subject invention. Other host organisms may also be trans stringent conditions by techniques well-known in the art, as formed with one or both of the toxin genes then used to described, for example, in Keller, G. H. M. M. Manak (1987) accomplish the synergistic effect. With suitable microbial DNA Probes, Stockton Press, New York, N.Y., pp. 169-170. hosts, e.g., Pseudomonas, the microbes can be applied to the Some examples of salt concentrations and temperature com situs of the pest, where they will proliferate and be ingested. binations are as follows (in order of increasing stringency): The result is control of the pest. Alternatively, the microbe 2xSSPE or SSC at room temperature: 1XSSPE or SSC at 42° hosting the toxin gene can be treated under conditions that C.: 0.1XSSPE or SSC at 42° C.: 0.1xSSPE or SSC at 65° C. prolong the activity of the toxin and stabilize the cell. The Detection of the probe provides a means for determining in a treated cell, which retains the toxic activity, then can be known manner whether hybridization has occurred. Such a applied to the environment of the target pest. probe analysis provides a rapid method for identifying toxin encoding genes of the subject invention. The nucleotide seg 0046) Where the Bt toxin gene is introduced via a suitable ments which are used as probes according to the invention can vector into a microbial host, and said host is applied to the be synthesized using a DNA synthesizer and standard proce environment in a living state, it is essential that certain host dures. These nucleotide sequences can also be used as PCR microbes be used. Microorganism hosts are selected which primers to amplify genes of the subject invention. are known to occupy the “phytosphere” (phylloplane, phyl 0041 Variant Toxins. losphere, rhizosphere, and/or rhizoplane) of one or more 0042 Certain toxins of the subject invention have been crops of interest. These microorganisms are selected so as to specifically exemplified herein. Since these toxins are merely be capable of successfully competing in the particular envi exemplary of the toxins of the subject invention, it should be ronment (crop and other insect habitats) with the wild-type readily apparent that the subject invention comprises variant microorganisms, provide for stable maintenance and expres or equivalent toxins (and nucleotide sequences coding for sion of the gene expressing the polypeptide pesticide, and, equivalent toxins) having the same or similar pesticidal activ desirably, provide for improved protection of the pesticide ity of the exemplified toxin. Equivalent toxins will have from environmental degradation and inactivation. amino acid homology with an exemplified toxin. This amino 0047. A large number of microorganisms are known to acid homology will typically be greater than 75%, preferably inhabit the phylloplane (the surface of the plant leaves) and/or be greater than 90%, and most preferably be greater than the rhizosphere (the soil surrounding plant roots) of a wide 95%. The amino acid homology will be highest in critical variety of important crops. These microorganisms include regions of the toxin which account for biological activity or bacteria, algae, and fungi. Of particular interest are microor are involved in the determination of three-dimensional con ganisms, such as bacteria, e.g., genera Pseudomonas, figuration which ultimately is responsible for the biological Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, activity. In this regard, certain amino acid substitutions are Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacte acceptable and can be expected if these substitutions are in num, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, regions which are not critical to activity or are conservative Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., amino acid substitutions which do not affect the three-dimen genera Saccharomyces, Cryptococcus, Kluyveromyces, sional configuration of the molecule. For example, amino Sporobolomyces, Rhodotorula, and Aureobasidium. Of par acids may be placed in the following classes: non-polar, ticular interest are such phytosphere bacterial species as uncharged polar, basic, and acidic. Conservative substitutions Pseudomonas syringae, Pseudomonas fluorescens, Serratia whereby an amino acid of one class is replaced with another marcescens, Acetobacter xylinum, Agrobactenium tumefa amino acid of the same type fall within the scope of the ciens, Rhodopseudomonas spheroides, Xanthomonas subject invention so long as the substitution does not materi campestris, Rhizobium melioti, Alcaligenes entrophus, and ally alter the biological activity of the compound. Below is a Azotobacter vinlandii; and phytosphere yeast species such as listing of examples of amino acids belonging to each class. Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. difluens, C. laurentii, Saccharomy ces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureoba Class of Amino Acid Examples of Amino Acids sidium pollulans. Of particular interest are the pigmented Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp microorganisms. Uncharged Polar Gly, Ser, Thr, Cys, Tyr, ASn, Gln Acidic Asp, Glu 0048. A wide variety of methods is available for introduc Basic Lys, Arg, His ing 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 US 2012/0331590 A1 Dec. 27, 2012

those skilled in the art and are described, for example, in U.S. aging or formation of inclusion bodies; Survival in aqueous Pat. No. 5,135,867, which is incorporated herein by refer environments; lack of mammalian toxicity; attractiveness to CCC. pests for ingestion; ease of killing and fixing without damage 0049 Treatment of Cells. to the toxin; and the like. Other considerations include ease of 0050 Bacillus thuringiensis or recombinant cells express formulation and handling, economics, storage stability, and ing the Bt toxins can be treated to prolong the toxin activity the like. and stabilize the cell. The pesticide microcapsule that is 0055 Growth of Cells. formed comprises the Bt toxin or toxins within a cellular 0056. The cellular host containing the B.t. insecticidal structure that has been stabilized and will protect the toxin gene or genes may be grown in any convenient nutrient when the microcapsule is applied to the environment of the medium, where the DNA construct provides a selective target pest. Suitable host cells may include either prokaryotes advantage, providing for a selective medium so that Substan or eukaryotes, normally being limited to those cells which do tially all or all of the cells retain the B.t. gene. These cells may not produce Substances toxic to higher organisms, such as then be harvested in accordance with conventional ways. mammals. However, organisms which produce Substances Alternatively, the cells can be treated prior to harvesting. toxic to higher organisms could be used, where the toxic 0057 The B.t. cells producing the toxins of the invention substances are unstable or the level of application sufficiently can be cultured using standard art media and fermentation low as to avoid any possibility of toxicity to a mammalian techniques. Upon completion of the fermentation cycle the host. As hosts, of particular interest will be the prokaryotes bacteria can be harvested by first separating the B.t. spores and the lower eukaryotes, such as fungi. and crystals from the fermentation broth by means well 0051. The cell will usually be intact and be substantially in known in the art. The recovered B.t.spores and crystals can be the proliferative form when treated, rather than in a spore formulated into a wettable powder, liquid concentrate, gran form, although in some instances spores may be employed. ules or other formulations by the addition of surfactants, 0052 Treatment of the microbial cell, e.g., a microbe con dispersants, inert carriers, and other components to facilitate taining the B.t. toxin gene or genes, can be by chemical or handling and application for particular target pests. These physical means, or by a combination of chemical and/or formulations and application procedures are all well known in physical means, so long as the technique does not deleteri the art. ously affect the properties of the toxin, nor diminish the 0058. Formulations. cellular capability of protecting the toxin. Examples of 0059 Formulated bait granules containing an attractant chemical reagents are halogenating agents, particularly halo and spores, crystals, and toxins of the B. t. isolates, or recom gens of atomic no. 17-80. More particularly, iodine can be binant microbes comprising the genes obtainable from the used under mild conditions and for sufficient time to achieve B.t. isolates disclosed herein, can be applied to the soil. For the desired results. Other suitable techniques include treat mulated product can also be applied as a seed-coating or root ment with aldehydes, such as glutaraldehyde; anti-infectives, treatment or total plant treatment at later stages of the crop Such as Zephiran chloride and cetylpyridinium chloride; alco cycle. Plant and soil treatments of B.t.cells may be employed hols, such as isopropyl and ethanol; various histologic fixa as wettable powders, granules or dusts, by mixing with vari tives, such as Lugol iodine, Bouin’s fixative, various acids ous inert materials, such as inorganic minerals (phyllosili and Helly's fixative (See: Humason, Gretchen L., Animal cates, carbonates, Sulfates, phosphates, and the like) or TissueTechniques, W. H. Freeman and Company, 1967); or a botanical materials (powdered corncobs, rice hulls, walnut combination of physical (heat) and chemical agents that pre shells, and the like). The formulations may include spreader serve and prolong the activity of the toxin produced in the cell Sticker adjuvants, stabilizing agents, other pesticidal addi when the cell is administered to the host environment. tives, or Surfactants. Liquid formulations may be acqueous Examples of physical means are short wavelength radiation based or non-aqueous and employed as foams, gels, Such as gamma-radiation and X-radiation, freezing, UV irra Suspensions, emulsifiable concentrates, or the like. The ingre diation, lyophilization, and the like. Methods for treatment of dients may include rheological agents, Surfactants, emulsifi microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and ers, dispersants, or polymers. 4,695,462, which are incorporated herein by reference. 0060. As would be appreciated by a person skilled in the 0053. The cells generally will have enhanced structural art, the pesticidal concentration will vary widely depending stability which will enhance resistance to environmental con upon the nature of the particular formulation, particularly ditions. Where the pesticide is in a proform, the method of cell whether it is a concentrate or to be used directly. The pesticide treatment should be selected so as not to inhibit processing of will be present in at least 1% by weight and may be 100% by the proform to the mature form of the pesticide by the target weight. The dry formulations will have from about 1-95% by pest pathogen. For example, formaldehyde will crosslink pro weight of the pesticide while the liquid formulations will teins and could inhibit processing of the proform of a generally be from about 1-60% by weight of the solids in the polypeptide pesticide. The method of treatment should retain liquid phase. The formulations will generally have from about at least a substantial portion of the bio-availability or bioac 10° to about 10" cells/mg. These formulations will be admin tivity of the toxin. istered at about 50 mg (liquid or dry) to 1 kg or more per 0054 Characteristics of particular interest in selecting a hectare. host cell for purposes of production include ease of introduc 0061 The formulations can be applied to the environment ing the B.t. gene or genes into the host, availability of expres of the lepidopteran pest, e.g., foliage or soil, by spraying, sion systems, efficiency of expression, stability of the pesti dusting, sprinkling, or the like. cide in the host, and the presence of auxiliary genetic 0062 Plant Transformation. capabilities. Characteristics of interest for use as a pesticide 0063 A preferred recombinant host for production of the microcapsule include protective qualities for the pesticide, insecticidal proteins of the Subject invention is a transformed Such as thick cell walls, pigmentation, and intracellular pack plant. Genes encoding Bt toxin proteins, as disclosed herein, US 2012/0331590 A1 Dec. 27, 2012 can be inserted into plant cells using a variety of techniques Agrobacterium rhizogenes for the transfer of the DNA into which are well known in the art. For example, a large number the plant cell. Whole plants can then be regenerated from the of cloning vectors comprising a replication system in infected plant material (for example, pieces of leaf, segments Escherichia coli and a marker that permits selection of the of Stalk, roots, but also protoplasts or Suspension-cultivated transformed cells are available for preparation for the inser cells) in a suitable medium, which may contain antibiotics or tion of foreign genes into higher plants. The vectors comprise, biocides for selection. The plants so obtained can then be for example, pBR322. pUC series, M13 mp series, tested for the presence of the inserted DNA. No special pACYC184, interalia. Accordingly, the DNA fragment hav demands are made of the plasmids in the case of injection and ing the sequence encoding the Bt toxin protein can be inserted electroporation. It is possible to use ordinary plasmids. Such into the vector at a suitable restriction site. The resulting as, for example, puC derivatives. plasmid is used for transformation into E. coli. The E. coli 0066. The transformed cells grow inside the plants in the cells are cultivated in a suitable nutrient medium, then har usual manner. They can form germ cells and transmit the Vested and lysed. The plasmid is recovered. Sequence analy transformed trait(s) to progeny plants. Such plants can be sis, restriction analysis, electrophoresis, and other biochemi grown in the normal manner and crossed with plants that have cal-molecular biological methods are generally carried out as the same transformed hereditary factors or other hereditary methods of analysis. After each manipulation, the DNA factors. The resulting hybrid individuals have the correspond sequence used can be cleaved and joined to the next DNA ing phenotypic properties. sequence. Each plasmid sequence can be cloned in the same 0067. In a preferred embodiment of the subject invention, or other plasmids. Depending on the method of inserting plants will be transformed with genes wherein the codon desired genes into the plant, other DNA sequences may be usage has been optimized for plants. See, for example, U.S. necessary. If, for example, the Tior Riplasmid is used for the Pat. No. 5,380,831, which is hereby incorporated by refer transformation of the plant cell, then at least the right border, ence. While some truncated toxins are exemplified herein, it but often the right and the left border of the Ti or Ri plasmid is well-known in the Bt art that 130 kDa-type (full-length) T-DNA, has to be joined as the flanking region of the genes to toxins have an N-terminal half that is the core toxin, and a be inserted. The use of T-DNA for the transformation of plant C-terminal half that is the protoxin “tail.” Thus, appropriate cells has been intensively researched and sufficiently “tails' can be used with truncated/core toxins of the subject described in EP 120516, Lee and Gelvin (2008), Hoekema invention. See e.g. U.S. Pat. No. 6,218,188 and U.S. Pat. No. (1985), Fraley et al., (1986), and An et al., (1985), and is well 6,673.990. In addition, methods for creating synthetic Bt established in the art. genes for use in plants are known in the art (Stewart and 0064. Once the inserted DNA has been integrated in the Burgin, 2007). One non-limiting example of a preferred plant genome, it is relatively stable. The transformation vec transformed plant is a fertile maize plant comprising a plant tor normally contains a selectable marker that confers on the expressible gene encoding a Cry 1Da protein, and further transformed plant cells resistance to a biocide oran antibiotic, comprising a second plant expressible gene encoding a Such as Bialaphos, Kanamycin, G418, Bleomycin, or Hygro Cry 1Be protein. mycin, inter alia. The individually employed marker should 0068 Transfer (or introgression) of the Cry 1Da- and accordingly permit the selection of transformed cells rather Cry 1Be-determined trait(s) into inbred maize lines can be than cells that do not contain the inserted DNA. achieved by recurrent selection breeding, for example by 0065. A large number of techniques are available for backcrossing. In this case, a desired recurrent parent is first inserting DNA into a plant host cell. Those techniques include crossed to a donor inbred (the non-recurrent parent) that transformation with T-DNA using Agrobacterium tumefa carries the appropriate gene(s) for the Cry 1D- and Cry1 Be ciens or Agrobacterium rhizogenes as transformation agent, determined traits. The progeny of this cross is then mated fusion, injection, biolistics (microparticle bombardment), or back to the recurrent parent followed by selection in the electroporation as well as other possible methods. If Agro resultant progeny for the desired trait(s) to be transferred from bacteria are used for the transformation, the DNA to be the non-recurrent parent. After three, preferably four, more inserted has to be cloned into special plasmids, namely either preferably five or more generations of backcrosses with the into an intermediate vector or into a binary vector. The inter recurrent parent with selection for the desired trait(s), the mediate vectors can be integrated into the Tior Riplasmid by progeny will be heterozygous for loci controlling the trait(s) homologous recombination owing to sequences that are being transferred, but will be like the recurrent parent for most homologous to sequences in the T-DNA. The Tior Riplasmid or almost all other genes (see, for example, Poehlman & also comprises the Vir region necessary for the transfer of the Sleper (1995) Breeding Field Crops, 4th Ed., 172-175: Fehr T-DNA. Intermediate vectors cannot replicate themselves in (1987) Principles of Cultivar Development, Vol. 1: Theory Agrobacteria. The intermediate vector can be transferred into and Technique, 360-376). Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both 0069 Insect Resistance Management (IRM) Strategies. in E. coli and in Agrobacteria. They comprise a selection 0070 Roush et al., for example, outlines two-toxin strat marker gene and a linker or polylinker which are framed by egies, also called "pyramiding or 'stacking.” for manage the Right and Left T-DNA border regions. They can be trans ment of insecticidal transgenic crops. (The Royal Society. formed directly into Agrobacteria (Holsters et al., 1978). The Phil. Trans. R. Soc. Lond. B. (1998) 353, 1777-1786). Agrobacterium used as host cell is to comprise a plasmid (0071. On their website, the United States Environmental carrying a vir region. The Vir region is necessary for the Protection Agency (epa.gov/oppbppd1/biopesticides/pips/ transfer of the T-DNA into the plant cell. Additional T-DNA bt corn refuge 2006.htm) publishes the following require may be contained. The bacterium so transformed is used for ments for providing non-transgenic (i.e., non-B.t.) refuges (a the transformation of plant cells. Plant explants can advanta section of non-Bt crops/corn) for use with transgenic crops geously be cultivated with Agrobacterium tumefaciens or producing a single Bt protein active against target pests. US 2012/0331590 A1 Dec. 27, 2012

0072 “The specific structured requirements for corn 0096. Following are examples that illustrate procedures borer-protected Bt (Cry1Ab or Cry 1F) corn products are for practicing the invention. These examples should not be as follows: construed as limiting. All percentages are by weight and all 0073 Structured refuges: 20% non-Lepidopteran Bt Solvent mixture proportions are by Volume unless otherwise corn refuge in Corn Belt; noted. All temperatures are in degrees Celsius. 0074 50% non-Lepidopteran Bt refuge in Cotton Belt 0075 Blocks EXAMPLES (0076 Internal (i.e., within the Bt field) (0077. External (i.e., separate fields within /2 mile (4 Example 1 mile if possible) of the Bt field to maximize random mating) (0078. In-field Strips 'I Labeling of Cry Proteins (0079 Strips must be at least 4 rows wide (preferably 6 rows) to reduce the effects of larval movement (0097. Iodination of Cry toxins. Purified truncated Cry tox 0080. In addition, the National Corn Growers Association, ins were was iodinated using Iodo-Beads or Iodo-gen on their website: (Pierce). Briefly, two Iodo-Beads were washed twice with 0081 (incga.com/insect-resistance-management-fact 500 ul of phosphate buffered saline, PBS (20 mM sodium sheet-bt-corn) phosphate, 0.15 M NaCl, pH 7.5), and placed into a 1.5 ml 0082 also provides similar guidance regarding the refuge centrifuge tube behind lead shielding. To this was added 100 requirements. For example: ul of PBS. In a hood and through the use of proper radioactive I0083) “Requirements of the Corn Borer IRM: handling techniques, 0.5 mCiNa"I (17.4 Ci/mg, Lot 0114, I0084 Plant at least 20% of your corn acres to refuge Amersham) was added to the PBS solution with the Iodo hybrids Bead. The components were allowed to react for 5 minutes at I0085. In cotton producing regions, refuge must be 50% room temperature, then 2-25ug of highly pure truncated Cry I0086 Must be planted within /2 mile of the refuge protein was added to the solution and allowed to react for an hybrids additional 3-5 minutes. The reaction was terminated by I0087. Refuge can be planted as strips within the Bt field; removing the solution from the iodo-beads and applying it to the refuge strips must be at least 4 rows wide a 0.5 ml desalting Zeba spin column (InVitrogen) equilibrated I0088 Refuge may be treated with conventional pesti in PBS. The iodo-bead was washed twice with 10 ul of PBS cides only if economic thresholds are reached for target each and the wash solution also applied to the desalting col insect umn. The radioactive solution was eluted through the desalt I0089 Bt-based sprayable insecticides cannot be used ing column by centrifugation at 1,000xg for 2 min. In the case on the refuge corn of Cry1 Da, the Iodo-gen method was used to conduct the 0090. Appropriate refuge must be planted on every farm radiolabeling procedure. Using this procedure, the cry toxin with Bt corn in 100 mM phosphate buffer (pH 8) was first cleaned of 0091. As stated by Roush et al. (on pages 1780 and 1784 lipopolysaccharides (LPS) by passing it through a small 0.5 right column, for example), stacking or pyramiding of two ml polymyxin column multiple times. To the iodo-gen tube different proteins each effective against the target pests and with little or no cross-resistance can allow for use of a smaller (Pierce Chem. Co.) was added 20 ug of the LPS-free Cry 1Da refuge. Roush Suggests that for a successful stack, a refuge toxin, then 0.5 mCi of Na'I. The reaction mixture was size of less than 10% refuge, can provide comparable resis shaken for 15 min at 25°C. The solution was removed from tance management to about 50% refuge for a single (non the tube, and 50 ul of 0.2M non-radiolabeled NaI added to pyramided) trait. For currently available pyramided Bt corn quench the reaction. The protein was dialyzed vs PBS with 3 products, the U.S. Environmental Protection Agency requires changes of buffer to remove any unbound I. significantly less (generally 5%) structured refuge of non-Bt 0.098 Radio-purity of the iodinated Cry proteins was corn be planted than for single trait products (generally 20%). determined by SDS-PAGE, phosphorimaging and gamma 0092. There are various ways of providing the IRM effects counting. Briefly, 2Lll of the radioactive protein was separated of a refuge, including various geometric planting patterns in by SDS-PAGE. After separation, the gels were dried using a the fields (as mentioned above) and in-bag seed mixtures, as BioRad gel drying apparatus following the manufacturers discussed further by Roush et al. (supra), and U.S. Pat. No. instructions. The driedgels were imaged by wrapping them in 6,551,962. Mylar film (12 um thick), and exposing them under a Molecu 0093. The above percentages, or similar refuge ratios, can lar Dynamics storage phosphor screen (35 cmx43 cm), for 1 be used for the subject double or triple stacks or pyramids. For hour. The plates were developed using a Molecular Dynamics triple stacks with three sites of action against a single target Storm 820 phosphorimager and the imaged analyzed using pest, a goal would be Zero refuge (or less than 5% refuge, for ImageOuantTM software. The radioactive band along with example). This is particularly true for commercial acreage— areas immediately above and below the band were cut from of over 10 acres for example. the gel using a razor blade and counted in a gamma counter. 0094 All patents, patent applications, provisional appli Radioactivity was only detected in the Cry protein band and cations, and publications referred to or cited herein are incor in areas below the band. No radioactivity was detected above porated by reference in their entirety to the extent they are not the band, indicating that all radioactive contaminants con inconsistent with the explicit teachings of this specification. sisted of Smaller protein components than the truncated Cry 0095. Unless specifically indicated or implied, the terms protein. These components most probably represent degrada a', 'an, and “the signify “at least one' as used herein. tion products. US 2012/0331590 A1 Dec. 27, 2012

Example 2 cold binding buffer. The bottom of the centrifuge containing the pellet was cut out and placed into a 13x75-mm glass BBMV Preparation Protocol culture tube. The samples were counted for 5 minutes each in 0099 Preparation and Fractionation of Solubilized the gamma counter. The counts contained in the sample were BBMVS. Subtracted from background counts (reaction with out any 0100 Last instar Spodoptera frugiperda, Ostrinia nubila protein) and was plotted versus BBMV protein concentration. lis, or Heleothis. zea larvae were fasted overnight and then The optimal amount of protein to use was determined to be dissected in the morning after chilling on ice for 15 minutes. 0.15 mg/ml of BBMV protein. The midgut tissue was removed from the body cavity, leaving behind the hindgut attached to the integument. The midgut 0103) To determine the binding kinetics, a saturation curve was placed in 9x volume of ice cold homogenization buffer was generated. Briefly, BBMV's (150 g/ml) were incubated (300 mM mannitol, 5 mM EGTA, 17 mM tris. base, pH 7.5), for 1 hr. at 28°C. with increasing concentrations of 'I Cry supplemented with Protease Inhibitor Cocktail' (Sigma toxin, ranging from 0.01 to 10 nM. Total binding was deter P-2714) diluted as recommended by the supplier. The tissue mined by Sampling 150 of each concentration in triplicate, was homogenized with 15 strokes of a glass tissue homog centrifugation of the sample and counting as described above. enizer. BBMV’s were prepared by the MgCl, precipitation Non-specific binding was determined in the same manner, method of Wolfersberger (1993). Briefly, an equal volume of with the addition of 1,000 nM of the homologous trypsinized a 24 mMMgCl, solution in 300 mM mannitol was mixed with non-radioactive Cry toxin added to the reaction mixture to the midgut homogenate, stirred for 5 minutes and allowed to saturate all non-specific receptor binding sites. Specific bind stand on ice for 15 min. The solution was centrifuged at ing was calculated as the difference between total binding and 2,500xg for 15 min at 4°C. The supernatant was saved and the non-specific binding. pellet suspended into the original volume of 0.5-x diluted 0104 Homologous and heterologous competition binding homogenization buffer and centrifuged again. The two Super assays were conducted using 150 ug/ml BBMV protein and natants were combined, centrifuged at 27,000xg for 30 minat 0.5 nM of the 'I radiolabeled Cry protein. The concentra 4°C. to form the BBMV fraction. The pellet was suspended tion of the competitive non-radiolabeled Cry toxin added to into 10 ml homogenization buffer and Supplemented to pro the reaction mixture ranged from 0.045 to 1,000 nM and were tease inhibitors and centrifuged again at 27,000xg of r30 min added at the same time as the radioactive ligand, to assure true at 4°C. to wash the BBMV's. The resulting pellet was sus binding competition. Incubations were carried out for 1 hr. at pended into BBMV Storage Buffer (10 mM HEPES, 130 mM 28°C. and the amount of 'I Cry proteinbound to its receptor KC1, 10% glycerol, pH 7.4) to a concentration of about 3 toxin measured as described above with non-specific binding mg/ml protein. Protein concentration was determined by subtracted. One hundred percent total binding was deter using the Bradford method (1976) with bovine serum albu mined in the absence of any competitor ligand. Results were min (BSA) as the standard. Alkaline phosphatase determina plotted on a semi-logarithmic plot as percent total specific tion was made prior to freezing the samples using the Sigma assay following manufacturers instructions. The specific binding versus concentration of competitive ligand added. activity of this marker enzyme in the BBMV fraction typi cally increased 7-fold compared to that found in the midgut Example 4 homogenate fraction. The BBMV's were aliquoted into 250 ulal samples, flash frozen in liquid N and stored at -80° C. Summary of Results Final concentration of cocktail components (in M) are AEBSF (500), EDTA (250 mM), Bestatin (32), E-64 (0.35), Leupeptin (0.25), and Aprotinin (0.075). 0105 FIG. 1 shows percent specific binding of 'I Example 3 Cry 1Be (0.5 nM) in BBMV's from FAW versus competition by unlabeled homologous Cry 1Be (O) and heterologous Method to Measure Binding of 'I Cry Proteins to Cryl Da (o). The displacement curve for homologous com BBMV Proteins petition by Cry 1Be results in a sigmoidal shaped curve show ing 50% displacement of the radioligand at about 2 nM of 0101 Binding of 'I Cry Proteins to BBMV's. Cry1 Be. Cry1Da at a concentration of 1,000 nM (2,000-fold 0102) To determine the optimal amount of BBMV protein greater than 'I Cry1 Bebeing displaced) results in less than to use in the binding assays, a Saturation curve was generated. 50% displacement. Error bars represent the range of values 'I radiolabeled Cry protein (0.5 nM) was incubated for 1 hr. obtained from triplicate determinations. at 28°C. with various amounts of BBMV protein, ranging from 0-500 ug/ml in binding buffer (8 mM NaHPO 2 mM 0106 FIG. 2 shows percent specific binding of ''I Cry1Da (0.5 nM) in BBMV's from FAW versus competition KHPO, 150 mM. NaCl, 0.1% bovine serum albumin, pH by unlabeled homologous Cry1 Da (o) and heterologous 7.4). Total volume was 0.5 ml. Bound 'I Cry protein was Cry 1Be (O). The displacement curve for homologous com separated from unbound by sampling 150 ul of the reaction petition by Cry 1Da results in a sigmoidal shaped curve show mixture in triplicate from a 1.5 ml centrifuge tube into a 500 ing 50% displacement of the radioligand at about 1.5 nM of ul centrifuge tube and centrifuging the samples at 14,000xg Cry1Da. Cry1 Bedoes not displace the specific binding of 'I for 6 minutes at room temperature. The Supernatant was gen Cry 1Da at any concentration tested, up to 1,000 nM, or 2,000 tly removed, and the pellet gently washed three times with ice times the concentration of 'I Cry1Da used in the assay.

US 2012/0331590 A1 Dec. 27, 2012 10

-continued

Name AccNo. Authors Year Source Strain Comment 17447 Guan Peng etal 2009 Bt Tn44-1B No NCBI link July 2009 ACM903.19 Lietal 2009 Bt Q-12 AAA22340 Feitelson 1993 Btaizawai PS81I CAAO1880 Anonymous 1995 Bt PS81RR1 AAA22410 Lee & Aronson 1991 Btalesti AA B82749 Kang etal 1997 Bt NTO423 AA D46137 Mustafa 1999 AAQ14326 Tan etal 2OOO AB B76664 Qi etal 2005 Btalesti AAO39719 Wang etal 2002 AA K14339 Nagarathinam etal 2001 Bt kunthala nags3 uncertain sequence CAA29898 Brizzard & Whiteley 1988 Bt thuringiensis HD2 CAA650O3 Soetaert 1996 Bt entomocidus HD110 K63251 Zhang etal 2001 KS1084 Nathan etal 2001 Bt entomocidus HD9 Song etal 2007 Bt Sfw-12 Martins etal 2006 Bt S601 Donovan etal 1994 Bt. EGS847 CAA86568 Bishop etal 1994 Bt morrisoni AA D10292 Kuo etal 2000 Bt wuhanensis HD525 AAM93496 Isakova etal 2002 Bt 834 AAC328SO Payne etal 1998 Bt PS158C2 Baum etal 2003 Xiaodong Sun etal 2009 B No NCBI link July 2009 CAC50778 Arnaut etal 2001 AAQ52380 Baum etal 2003 AAO3972O Wang etal 2002 CAA3O396 Honee etal 1988 Bt entomocidus 60.5 CAA31951 Sanchis etal 1989 Btaizawai 7.29 AAA22343 Feitelson 1993 Btaizawai PS81I CAAO1886 Van Mellaert etal 1990 Bt entomocidus HD110 CAA65457 Strizhow 1996 Btaizawai 7.29 AAF37224 Yu et all 2OOO Bt AF-2 AAGSO438 Aixing etal 2OOO Bt.8 AAMOO264 Chen etal 2001 Bt cOO2 AAL79362 Kao etal 2003 Bt G1O-O1A AAN16462 Lin etal 2003 Bt EOS-2Oa AAX53094 Caietal 2005 Bt C-33 M9788O Kalman etal 993 Bt galleriae HD29 DNA sequence only AAG35409 Song etal 2OOO Bt cOO1 ACDSO894 Huang etal 2008 Bt 087 AAX63901 Thammasittirong etal 2005 BitTA476-1 insufficient sequence CAA38099 Hofte etal 990 Btaizawai HD68 I76415 Payne & Sick 997 DNA sequence only CAA8O234 Lambert 993 Bt. BTSOO349A AAK48937 Lietal 2001 Bt B-Pr-88 ABK35074 Lertwiriyawong etal 2006 Bt. JC291 CAA37933 Visser etal 990 Btkenyae 4F1 CAA39609 Bosse etal 990 Btkenyae AAA22345 Feitelson 991 Btkenyae PS81F AADO4732 Barboza-Corona etal 998 Btkenyae LBIT-147 A15535 Botterman etal 994 DNA sequence only AALSO330 Sun etal 999 BtyBT-032 AAW72936 Huehne etal 2005 Bt JC190 ABX11258 Huang etal 2007 Bt. HZM2 AAA22346 Feitelson 993 Btaizawai PS81A2 AAA22348 Chambers etal 991 Btaizawai EG6346 AAA22347 Feitelson 993 Btaizawai PS81I CAA8O235 Lambert 993 Bt. BTSOO349A BAA25298 Masuda & Asano 998. Bt morrisoni INA67 AAF21767 Song etal 998 Bt morrisoni AAC10641 Payne etal 997 AAO13295 Lietal 2001 Bt B-Pr-88 ACDSO892 Huang etal 2008 Bt O12 ACDSO893 Huang etal 2008 Bt 087 CAA8O233 Lambert 993 Bt. BTSO349A CAA7OSO6 Shevelew etal 997 Bt wuhanensis Cry1Gb1 AAD10291 Kuo & Chak 999 Bt Wuhanensis HD525 Cry1Gb2 AAO 13756 Lietal 2OOO Bt B-Pr-88 Cry1Gc AAQ52381 Baum etal 2003 Cry1 Hal CAA8O236 Lambert 993 Bt. BTSO2O69AA Cry1 Hb1 AAA79694 Koo etal 995 Bt morrisoni BF190 Cry1 H-like AAFO1213 Srifah etal 999 Bt. JC291 insufficient sequence Cry1Ia1 CAA44633 Tailor etal 992 Bt kurstaki Cry1Ia2 AAA22354 Gleave etal 993 Bt kurstaki US 2012/0331590 A1 Dec. 27, 2012 11

-continued

AccNo. Authors Year Source Strain Comment

AAC36999 Shin etal 1995 kurstaki HD1 AABOO958 Kostichka etal 1996 AB88 CAA7O124 Selvapandiyan 1996 61 AAC26910 Zhong etal 1998 kurstaki S101 AAM73516 Porcar etal 2OOO AAK66742 Song etal 2001 AAQ08616 Yao etal 2002 Ly30 AAP86782 Espindola etal 2003 huringiensis CAC85964 Tounsi etal 2003 kurstaki BNS3 AAVS3390 Grossi de Saetal 2005 ABF832O2 Martins etal 2006 ACG63871 Liu & Guo 2008 11 FJ617445 Guan Peng etal 2009 E-1B No NCBI link July 2009 Cry FJ617448 Guan Peng etal 2009 E-1A No NCBI link July 2009 AAA82114 Shin etal 1995 entomocidus BP465 ABW88019 Guan etal 2007 PP61 ACD75515 Liu & Guo 2008 AAC62933 Osman etal 1998 C18 AAE71691 Osman etal 2001 Cry AAD44366 Choi 2OOO d AAG435.26 Song etal 2OOO BTCOO7 AAQ52382 Baum etal 2003 AAC31094 Payne etal 998 insufficient sequence ABG888S9 Lin & Fang ly4a3 insufficient sequence AAA22341 Donovan EGS847 AAA98959 Von Tersch & Gonzalez EGSO92 AAC31092 Payne etal AAQ52372 Baum etal CAC50779 Arnaut etal AABOO376 Koo etal morrisoni BF190 AAS6O191 Jeetal kurstaki K1 AAC31091 Payne etal insufficient sequence AAA22335 Donovan etal kurstaki AAA83516 Widner & Whiteley kurstaki HD1 D86.064 Sasaki etal SOtto DNA sequence only AAC04867 Misra etal kenyae HD549 CAA10671 Yu & Pang SL39 CAA10672 Yu & Pang CAA10670 Yu & Pang CY29 AAO13734 Wei etal Dongbei 66 AAO137SO Zhang etal AAQ04263 Yao etal AAQ52384 Baum etal ABI83671 Tan etal Rpp39 ABLO 1536 Arango etal 46-158-O1 ACFO4939 Hire etal HD-SSO AAA22342 Widner & Whiteley kurstaki HD1 CAA3907S Dankocsiketal kurstaki HD1 AAG36762 Chen etal BTCOO2 AAO13296 Lietal B-Pr-88 AAQ04609 Yao etal AAPS9457 Wang etal AAZ66347 Udayasuriyan etal ABC95996 Huang etal ABC74968 Zhang etal EF157306 Lin etal CAM84575 Saleem etal ABM21764 Lin etal ACG7612O Zhu etal ACG76121 Zhu etal CAA40536 Aronson Shanghai S1 AAG35410 Song etal AAQ52385 Baum etal ABC95997 Huang etal ABC74969 Zhang etal ABC74793 Xia etal Wuhanensis CAL18690 Saleem etal SBSBT1 CAMO932S Saleem etal CMBL-BT1 CAMO9326 Saleem etal CMBL-BT2 ABN 15104 Baietal QCL-1 CAM83895 Saleem etal HD29 CAM83896 Saleem etal CMBL-BT3 AAFO9.583 Choi etal BR30 ABC86927 Huang etal WB10 CAK29.504 Saleem etal 5 2AcT(1) US 2012/0331590 A1 Dec. 27, 2012 12

-continued

Name AccNo. Authors Year Source Strain Comment Cry2Ad4 CAM32331 Saleem et all 2007 Bt CMBL-BT2 Cry2Ad5 CAO78739 Saleem et all 2007 Bt HD29 Cry2Ae1 AAQ52362 Baum et al 2003 Cry2Af1 ABO30519 Beard etal 2007 Bt C81 Cry2Ag ACH916.10 Zhu et all 2008 Bt JF19-2 Cry2Ah EU939453 Zhang etal 2008 B No NCBI link July 2009 Cry2Ah2 ACL80665 Zhang etal 2009 Bt BRC-ZQL3 Cry2Ai F788388 Udayasuriyan etal 2009 B No NCBI link July 2009 Cry3Aa1 AAA22336 Herrnstadt etal 1987 Bt san diego Cry3Aa2 AAA22541 Sekar etal 1987 Bt tenebrionis Cry3Aa3 CAA68482 Hofte etal 1987 Cry3Aa4 AAA22542 McPherson etal 1988 Bt tenebrionis Cry3Aas AAA50255 Donovan etal 1988 Bt morrisoni EG2158 Cry3Aaé AAC43266 Adams etal 1994 Bt tenebrionis Cry3Aa7 CAB41411 Zhang etal 1999 Bt 22 Cry3Aa8 AAS79487 Gao and Cai 2004 BtyM-03 Cry3Aa9 AAWO5659 Bulla and Candas 2004 Bt UTD-OO1 Cry3Aa10 AAU29411 Chen etal 2004 Bt 886 Cry3Aa11 AAW82872 Kurt etal 2005 Bt tenebrionis Mm2 Cry3Aa12 ABY49136 Sezen etal 2008 Bt tenebrionis Cry3Ba1 CAA34983 Sicket all 1990 Bt tolworthi 43F Cry3Ba2 CAAO0645 Peferoen etal 1990 Bt. PGSI2O8 Cry3Bb1 AAA22334 Donovan etal 1992 Bt EG4961 Cry3Bb2 AAA74198 Donovan etal 1995 Bt. EGS144 Cry3Bb3 I15475 Peferoen etal 1995 DNA sequence only Cry3Ca1 CAA42469 Lambert etal 1992 Bt kurstaki Bt109P Cry4Aa1 CAA68485 Ward & Ellar 1987 Bt israelensis Cry4Aa2 BAAO0179 Sen etal 1988 Bt israelensis HD522 Cry4Aa3 CAD3.0148 Berry etal 2002 Bt israelensis Cry4A-like AAY96321 Mahalakshmi etal 2005 Bt LDC-9 insufficient sequence Cry4Ba1 CAA30312 Chungjatpornchai etal 1988 Bt israelensis 4Q2-72 Cry4Ba2 CAA30114 Tungpradubkuletal 1988 Bt israelensis Cry4Ba3 AAA22337 Yamamoto etal 1988 Bt israelensis Cry4Ba4 BAAOO178 Sen et a 1988 Bt israelensis HD522 Cry4Bas CAD3.0095 Berry etal 2002 Bt israelensis Cry4Ba-like ABC47686 Mahalakshmi etal 2005 Bt LDC-9 insufficient sequence Cry4Ca EU646.202 Shu et a 2008 No NCBI link July 2009 Cry4Cb FJ403208 un & Furong 2008 Bt HS18-1 No NCBI link July 2009 Cry4Cb2 FJ597622 un & Furong 2008 BTYWC2-8 No NCBI link July 2009 Cry4Cc FJ4032O7 un & Furong 2008 BtMC28 No NCBI link July 2009 Cry5Aa AAA67694 Narva et all 1994 Bt darmstadiensis PS17 Crys Ab AAA67693 Narva et all 1991 Bt darmstadiensis PS17 Crys Ac I34543 Payne etal 1997 DNA sequence only Crys Ad ABQ82087 Lenane etal 2007 Bt L366 Crys Ba AAA68598 Foncerrada & Narva 1997 Bt PS86Q3 Crys Ba2 ABW88931 Guo et all 2008 YBT 1518 Cry6Aa AAA22357 Narva et all 1993 Bt PSS2A1 Cry6Aa2 AAM46849 Bai et a 2001 YBT 1518 Cry6Aa3 ABHO3377 ia etal 2006 Bt 96418 Cry6Ba AAA22358 Narva et all 1991 Bt PS69D1 Cry7Aa AAA22351 Lambert etal 1992 Bt galleriae PGSI245 Cry7Ab AAA21120 Narva & Fu 1994 Bt dakota HD511 Cry7Ab2 AAA21121 Narva & Fu 1994 Btkumamotoensis 867 Cry7Ab3 ABX24522 Song etal 2008 BtWZ-9 Cry7Ab4 EU380678 Shu etal 2008 B No NCBI link July 2009 Cry7Ab5 ABX79555 Aguirre-Arzola etal 2008 Bt monterrey GM-33 Cry7Ab6 ACI44005 Deng etal 2008 Bt HQ122 Cry7Ab7 FJ94O776 Wang etal 2009 No NCBI link September 2009 Cry7Ab8 GU145299 Feng Jing 2009 No NCBI link November 2009 Cry7Ba ABB70817 Zhang etal 2006 Bthuazhongensis Cry7Ca ABR678.63 Gao et all 2007 Bt BTH-13 Cry7Da ACQ99547 Yi etal 2009 Bt LH-2 Cry8Aa AAA21117 Narva & Fu 1992 Btkumamotoensis Cry8Ab EU044830 Cheng etal 2007 Bt B-JX No NCBI link July 2009 Cry8Ba AAA21118 Narva & Fu 1993 Btkumamotoensis Cry 8Bb CAD57542 Abad etal 2002 Cry8Bc CAD57543 Abad etal 2002 Cry8Ca AAA21119 Sato et al. 1995 Btjaponensis Buibui Cry8Ca2 AAR98783 Shu et all 2004 Bt. HBF-1 Cry8Ca3 EU625349 Du et all 2008 Bt FTL-23 No NCBI link July 2009 Cry8Da BACO7226 Asano etal 2002 Bt galleriae Cry8Da2 BD133574 Asano et all 2002 B DNA sequence only Cry8Da3 BD133575 Asano et all 2002 B DNA sequence only Cry8Db1 BAF93483 Yamaguchi etal 2007 Bt BBT2-5 Cry8Ea AAQ73470 Fuping etal 2003 Bt 185 US 2012/0331590 A1 Dec. 27, 2012 13

-continued

Name AccNo. Authors Year Source Strain Commen EU047597 Liu etal 2007 Bt B-DLL No NCBI link July 2009 AAT48690 Shu etal 2004 Bt 185 also AAW81032 AAT46O73 Shu etal 2004 Bt. HBF-18 ABC42043 Yan etal 2008 Bit 145 FJ198072 Xiaodong etal 2008 Bt FCD114 No NCBI link July 2009 EF465532 Fuping etal 2006 Bt 185 No NCBI link July 2009 EU381044 Yan etal 2008 Bt Su4 No NCBI link July 2009 EU625348 Du etal 2008 Bt FPT2 No NCBI link July 2009 FJ422558 Quezado etal 2008 No NCBI link July 2009 ACN87262 Noguera & Ibarra 2009 Btkenyae F770571 Noguera & Ibarra 2009 Bt canadensis DNA sequence only ABSS3003 Mangena etal 2007 B CAA41122 Shevelew etal 1991 Bt galleriae CAA41425 Gleave etal 1992 Bt DSIRS 17 GQ249293 Suetal 2009 BtSC5(D2) No NCBI link July 2009 GQ249294 Suetal 2009 BitTO3COO1 No NCBI link July 2009 AAQ52376 Baum etal 2003 incomplete sequence CAAS2927 Shevelew etal 1993 Bt galleriae AAV28716 Silva-Werneck etal 2004 Btjaponensis CAA85764 Lambert etal 1996 Bt tolworthi AAQ52375 Baum etal 2003 BAA19948 Asano 1997 Btjaponensis N141 AAB97923 Wasano & Ohba, 1998 Btjaponensis GQ249295 Suetal 2009 BitTO3BOO1 No NCBI link July 2009 GQ249297 Suetal 2009 BitTO3BOO1 No NCBI link July 2009 AAX78439 Flannagan & Abad 2005 Btkurstaki DP1019 BAA34908 Midoh & Oyama 1998 Btaizawai SSK-10 AAO12908 Lietal 2001 Bt B-Hm-16 ABM21765 Lin etal 2006 BtlyA ACE88267 Zhu etal 2008 Btywc5-4 ACFO4743 Zhu etal 2008 BtS ACG63872 Liu & Guo 2008 Bit 11 FJ380927 Sun etal 2008 No NCBI link July 2009 GQ249292 Suetal 2009 GQ249292 No NCBI link July 2009 CACSO78O Arnaut etal 2001 GQ249298 Suetal 2009 BitTO3BOO1 No NCBI link July 2009 AAC63366 Wasano etal 2003 Bt galleriae AAX78440 Flannagan & Abad 2005 Btkurstaki DP1019 GQ249296 Suetal 2009 BitTO3BOO1 No NCBI link August 2009 AAC63366 Wasano etal 998 Bt galleriae insufficient sequence AAA22614 Thorne etal 986 Bt israelensis EOO614 Aran & Toomasu 996 Bt israelensis ONR-60A DNA sequence only CAD3.0098 Berry etal 2002 Bt israelensis DQ167578 Mahalakshmi etal 2006 Bt LDC-9 incomplete sequence AAA22352 Donovan etal 988 Bt israelensis AAA22611 Adams etal 989 Bt israelensis CAD30O81 Berry etal 2002 Bt israelensis DQ166531 Mahalakshmi etal 2007 Bt LDC-9 incomplete sequence CAA60504 Delecluse etal 995 Btjegathesan 367 AAC97162 Orduz etal 998. Bt medellin AAA22355 Narva etal 991 Bt PS33F2 AAA22356 Narva etal 992 Bt PS63B AAA21516 Narva etal 994 Bt Sotto PS801 Cry15Aa AAA22333 Brown & Whiteley 992 Bt thompsoni Cry16Aa CAA638.60 Barloy etal 996 Cb malaysia CH18 Cry 17Aa CAA67841 Barloy etal 998 Cb malaysia CH18 Cry18Aa CAA67SO6 Zhang etal 997 Paenibacillus popilliae Cry 18Ba AAF89667 Pateletal 999 Paenibacillus popilliae Cry18Ca AAF896.68 Pateletal 999 Paenibacillus popilliae Cry19Aa CAA68875 Rosso & Delecluse 996 Btjegathesan 367 Cry19Ba BAA32397 Hwang etal 998 Bt higo Cry20Aa AAB93476 Lee & Gill 997 Bt fukuokaensis Cry20 Ba. ACS936O1 Noguera & Ibarra 2009 Bt higo LBIT-976 Cry20-like GQ14.4333 Yietal 2009 Bty-S DNA sequence only Cry21Aa. I32932 Payne etal 996 DNA sequence only Cry21Aa2 I66477 Feitelson 997 DNA sequence only Cry21 Ba BACO6484 Sato & Asano 2002 Btroskildliensis Cry22Aa I34547 Payne etal 997 DNA sequence only Cry22Aa2 CAD43579 saac etal 2002 B Cry22Aa3 ACD93211 Du etal 2008 Bt FZ-4 Cry22Ab AAKSO456 Baum etal 2OOO Bt EG4140 Cry22Ab2 CAD43577 saac etal 2002 B Cry22Ba CAD43578 saac etal 2002 B Cry23Aa AAF76375 Donovan etal 2OOO B Binary with Cry37Aa1 Cry24Aa AAC61891 Kawalek and Gill 1998 Btjegathesan US 2012/0331590 A1 Dec. 27, 2012 14

-continued

Name AccNo. Authors Year Source Strain Comment Cry24Ba BAD32657 Ohgushi etal 2004 Bt Sotto Cry24Ca CA436OO Beron & Salerno 2005 Bt FCC-41 Cry25Aa AAC61892 Kawalek and Gill 1998 Btjegathesan Cry26Aa AAD25075 Wojciechowska et al 1999 Bt finitimus B-1166 Cry27Aa BAA82796 Saitoh 1999 Bt higo Cry28Aa AAD24189 Wojciechowska et al 1999 Bt finitimus B-1161 Cry28Aa2 AAGOO235 Moore and Debro 2000 Bt finitimus Cry29Aa CAC80985 Delecluse et all 2000 Bt medellin Cry30Aa CAC80986 Delecluse et all 2000 Bt medellin Cry3OBa BADOOOS2 to etal 2003 Bt entomocidus Cry30Ca BAD67157 Ohgushi etal 2004 Bt Sotto Cry30Ca2 ACU24781 Sun and Park 2009 Btjegathesan 367 Cry30Da EF095955 Shu etal 2006 Bty41 No NCBI link July 2009 Cry3ODb1 BAE80088 Kishida et all 2006 Btaizawai BUN1-14 Cry30Ea ACC95445 Fang etal 2007 BtS2160-1 Cry30Ea2 FJ499389 un etal 2008 Btywc2-8 No NCBI link July 2009 Cry3OFa ACI22625 Tan etal 2008 BtMC28 Cry30Ga ACG6002O Zhu et all 2008 Bt HS18-1 Cry31Aa. BAB11757 Saitoh & Mizuki 2OOO Bt 84-HS-1-11 Cry31Aa2 AAL87458 ung and Cote 2OOO BtM15 Cry31Aa3 BAE798.08 Uemorietal 2006 Bt BO195 Cry31Aa4 BAF32571. Yasutake etal 2006 Bt 79-2S Cry31Aas BAF32572 Yasutake etal 2006 Bt 92-10 Cry31Ab BAE79809 Uemorietal 2006 Bt BO195 Cry31Ab2 BAF32570 Yasutake etal 2006 Bt 31-5 Cry31 Ac BAF34368 Yasutake etal 2006 Bt 87-29 Cry32Aa AAG36711 Balasubramanian etal 2001 Btyunnanensis Cry32Ba BAB78601 Takebe etal 2001 B Cry32Ca BAB78602 Takebe etal 2001 B Cry32Da BAB78603 Takebe etal 2001 B Cry33Aa AAL26871 Kim etal 2001 Bt dakota Cry34Aa AAGSO341 Ellis et all 2001 Bt PS801 Binary wi Cry34Aa2 AAK64560 Rupar etal 2001 Bt. EGS899 Binary wi Cry34Aa3 AAT29032 Schnepfetal 2004 Bt PS69Q Binary wi Cry34Aa-4 AAT29030 Schnepfetal 2004 Bt PS185GG Binary wi Cry34Ab AAG41671 Moellenbeck etal 2001 Bt PS149B1 Binary wi Cry34Ac AAGSO118 Ellis et all 2001 Bt PS167H2 Binary wi Cry34Ac2 AAK64562 Rupar et a 2001 Bt EG9444 Binary wi Cry34Ac3 AAT29029 Schnepfetal 2004 Bt KR1369 Binary wi Cry34Ba AAK64565 Rupar et a 2001 Bt EG4851 Binary wi Cry34Ba2 AAT29033 Schnepfetal 2004 Bt PS2O1L3 Binary wi Cry34Ba3 AAT29031 Schnepfetal 2004 Bt PS2O1HEH2 Binary wi Cry35Aa AAGSO342 Ellis et all 2001 Bt PS801 Binary wi Cry35Aa2 AAK64561 Rupar et a 2001 Bt. EGS899 Binary wi Cry35Aa3 AAT29028 Schnepfetal 2004 Bt PS69Q Binary wi Cry35Aa4 AAT29025 Schnepfetal 2004 Bt PS185GG Binary wi Cry35Ab AAG41672 Moellenbeck etal 2001 Bt PS149B1 Binary wi Cry35Ab2 AAK64563 Rupar et a 2001 Bt EG9444 Binary wi Cry35Ab3 AYS36891 AAT29024 2004 Bt KR1369 Binary wi Cry35Ac AAGSO117 Ellis et all 2001 Bt PS167H2 Binary wi Cry35Ba AAK64566 Rupar et a 2001 Bt EG4851 Binary wi Cry35Ba2 AAT29027 Schnepfetal 2004 Bt PS2O1L3 Binary wi Cry35Ba3 AAT29026 Schnepfetal 2004 Bt PS2O1HEH2 Binary wi Cry36Aa AAK64558 Rupar et a 2OO B Cry37Aa AAF76376 Donovan etal 2OOO B Binary wi h Cry23Aa Cry38Aa AAK64559 Rupar et a 2OOO B Cry39Aa BAB72O16 to etal 2001 Btaizawai Cry40Aa BAB72O18 to etal 2001 Btaizawai Cry4OBa BACT7648 to etal 2003 Bun1-14 Cry40Ca EU381045 Shu etal 2008 Bty41 No NCBI link July 2009 Cry4ODa ACF 15199 Zhang etal 2008 BtS2096-2 Cry41Aa. BAD35157 Yamashita et all 2003 Bt A1462 Cry41Ab BAD35163 Yamashita et all 2003 Bt A1462 Cry42Aa BAD35166 Yamashita et all 2003 Bt A1462 Cry43Aa BAD15301 Yokoyama and Tanaka 2003 P. lentimorbus semadara Cry43Aa2 BAD95474 Nozawa 2004 P. popilliae popilliae Cry43 Ba. BAD15303 Yokoyama and Tanaka 2003 P. lentimorbus semadara Cry43-like BAD15305 Yokoyama and Tanaka 2003 P. lentimorbus semadara Cry44Aa BAD08532 Ito etal 2004 Bt entomocidus INA288 Cry45Aa BAD22577 Okumura et all 2004 Bt 89-T-34-22 Cry46Aa BACT9010 Ito etal 2004 Bt dakota Cry46Aa2 BAG68906 Ishikawa etal 2008 Bt A1470 Cry46 Ab BAD35170 Yamagiwa etal 2004 Bt Cry47Aa AAY24695 Kongsuwan etal 2005 Bt CAA890 US 2012/0331590 A1 Dec. 27, 2012 15

-continued

Name AccNo. Authors Year Source Strain Comment

CA18351 ones and Berry 2005 BSIABS9 binary wi CA86545 ones and Berry 2006 BS 47-6B binary wi CA.J86546 ones and Berry 2006 BS NHA1Sb binary wi CA.J86548 ones and Berry 2006 BSLP1G binary wi CA86549 ones and Berry 2006 Bs 2173 binary wi CAHS6541 ones and Berry 2005 BSIABS9 binary wi CA.J86541 ones and Berry 2006 BS 47-6B binary wi CA86543 ones and Berry 2006 BSNEHA1Sb binary wi CA.J86544 ones and Berry 2006 Bs 2173 binary wi CA.J86542 ones and Berry 2006 BSLP1G binary wi BAE86999 Ohgushi etal 2006 SOtto ABI14444 Meng etal 2006 F14-1 EF613489 Song et a 2007 Y41 No NCBI ink July 2009 FJ361760 un etal 2008 BMS9-2 No NCBI ink July 2009 EF633476 Song et a 2007 Y41 No NCBI ink July 2009 FJ361759 un etal 2008 MC28 No NCBI ink July 2009 ACAS21.94 an etal 2009 MC28 ABW88932 Guo et all 2008 BT 1518 AAE33526 Bradfisch et all 2OOO TY41 FJ597621 un & Furong 2008 Ywc2-8 No NCBI ink July 2009 GQ483512 Guan Peng etal 2009 G.7-1 No NCBI ink August 2009 ANC87261 Noguera & Ibarra 2009 kim ANC87260 Noguera & Ibarra 2009 entomocidus ACR43758 Noguera & Ibarra 2009 kim LBIT-980

Vip3Aa1 Vip3Aa AAC37.036 Estruch et all 1996 PNAS 93, AB88 S389-5394 Vip3Aa2 Vip3Ab AAC37037 Estruch et all 1996 PNAS 93, AB424 S389-5394 Vip3Aa3 Vip3Ac Estruch etal U.S. Pat. No. 6,137,033 October 2000 Vip3Aa4 PS36A Sup AAR81079 Feitelson etal 1998 U.S. Pat. No. 6,656,908 Bt PS36A WO9818932 December 2003 (A2, A3) 7 May 1998 Vip3Aas PS81F Sup AAR81080 Feitelson etal 1998 U.S. Pat. No. 6,656,908 WO9818932 December 2003 (A2, A3) 7 May 1998 Jav90 Sup AAR81081 Feitelson et all 1998 U.S. Pat. No. 6,656,908 Bt WO9818932 December 2003 (A2, A3) 7 May 1998 Wip3Aa7 Vip83 AAK95326 Caietal 2001 unpublished BtyBT-833 Wip3Aa8 Vip3A AAK97481 Loguercio etal 2001 unpublished Bt HD12S Wip3Aa9 VipS CAA76665 Selvapandiyan 2001 unpublished Bt A13 etal Wip3Aa10 Vip3V AAN60738 DOSS etal 2002 Protein Expr. Bt Purif. 26, 82-88 Wip3Aa11 Vip3A AAR36859 Liu et all 2003 unpublished Bt C9 Wip3Aa12 Vip3A-WB5 AAM224.56 Wu and Guan 2003 unpublished Bt Wip3Aa13 Vip3A AAL69542 Chen etal 2002 Sheng Wu Bt S184 Gong Cheng Xue Bao 18, 687-692 Wip3Aa14 Vip AAQ12340 Polumetla etal 2003 unpublished Bt tolworthi Wip3Aa15 Vip3A AAPS1131 Wu et all 2004 unpublished BtWBSO Wip3Aa16 Vip3LB AAW 65132 Mesrati et all 2005 FEMS Micro Bt Lett 244, 353-358 Wip3Aa17 Jaw90 Feitelson etal 1999 U.S. Pat. No. 6,603,063 avelin 1990 WO995 7282 August 2003 (A2, A3) 11 Now 1999 US 2012/0331590 A1 Dec. 27, 2012

-continued Wip3Aa18 AAX49395 Cai and Xiao 2005 unpublishe Bt 9816C Vip3Aa19 Vip3ALD DQ241674 Liu et al 2006 unpublishe Bt AL Vip3Aa19 Vip3A-1 DQ539887 Hart etal 2006 unpublishe Vip3Aa20 Vip3A-2 DQ539888 Hart etal 2006 unpublishe Vip3Aa21 Vip ABD84410 Panbangred 2006 unpublishe Bt aizawai Vip3Aa22 Vip3A-LS1 AAY41427 Luetal 2005 unpublishe Bt LS1 Vip3Aa23 Vip3A-LS8 AAY41428 Luetal 2005 unpublishe Bt LS8 Wip3Aa24 BI880913 Song etal 2007 unpublishe BtWZ-7 Wip3Aa25 EF608SO1 Hsieh etal 2007 unpublishe Wip3Aa26 EU294496 Shen and Guo 2007 unpublishe Bt TF9 Wip3Aa27 EU332167 Shen and Guo 2007 unpublishe Bt 16 Wip3Aa28 FJ494817 Xiumei Yu 2008 unpublishe Bt JF23-8 Wip3Aa29 F626674 Xieumei etal 2009 unpublishe Bt JF21-1 Wip3Aa30 FJ626675 Xieumei etal 2009 unpublishe MD2-1 Wip3Aa31 F626676 Xieumei etal 2009 unpublishe F21-1 Wip3Aa32 FJ626677 Xieumei etal 2009 unpublishe MD2-1

Vip3Ab1 Vip3B AAR40284 Feitelson etal 1999 U.S. Pat. No. 6,603,063 Bt KBS9A4-6 WO995.7282 August 2003 (A2, A3) 11 Now 1999 Vip3Ab2 Vip3D AAY88247 Feng and Shen 2006 unpublished Bt

Vib3Ac1 PS49C Narva etal - US application 2004O128716

Vib3A1 PS158C2 Narva etal - US application 2004O128716 Vib3A2 ISP3B CA43276 Van Rie etal 2005 unpublished Bt

Vib3Ae1 ISP3C CAI43277 Van Rie etal 2005 unpublished Bt

Vib3Af1 ISP3A CA43275 Van Rie etal 2005 unpublished Bt Vib3Af2 W3C ADNO8753 Syngenta WO O3,O75655 Vip3Ag1 Vip3B ADN08758 Syngenta WO O2fO78437 Vip3Ag2 FJS56803 Audithoetal 2008 Bt Wip3Ah1. Wip3S DQ832323 Li and Shen 2006 unpublished Bt Wip3Ba1 AAV70653 Rangetal 2004 unpublished V3Bb1 V3Z ADNO8760 Syngenta WO O3,O75655 V3Bb2 EF439819 Akhurst etal 2007

REFERENCE LIST Corn Borer and Fall Armyworm Colonies to Cry1A.105 and Cry 12Ab2. DAI 0830, 2008. Indianapolis, Dow Agro 0109 Heckel, D. G., Gahan, L.J., Baxter, S.W., Zhao, J. Sciences. Derbi Report. Z. Shelton, A. M., Gould, F., and Tabashnik, B. E. (2007). The diversity of Bt resistance genes in species of Lepi 0114. Sheets, J. J. and Storer, N. P. Analysis of Cry1Ac doptera. J Invertebr Pathol 95, 192-197. Binding to Proteins in Brush Border Membrane Vesicles of 0110 Luo, K., Banks, D., and Adang, M.J. (1999). Tox Corn Earworm Larvae (Heleothis zea). Interactions with icity, binding, and permeability analyses of four bacillus Cry1F Proteins and Its Implication for Resistance in the thuringiensis cry1 delta-endotoxins using brush border Field. DAI-0417, 1-26. 2001. Indianapolis, Dow Agro membrane vesicles of Spodoptera exigua and Spodoptera Sciences. frugiperda. Appl. Environ. Microbiol. 65, 457-464. 0115 Tabashnik, B.E., Liu, Y.B., Finson, N., Masson, L., 0111 Palmer, M., Buchkremer, M. Valeva, A, and Bhakdi, and Heckel, D. G. (1997). One gene in diamondback moth S. Cysteine-specific radioiodination of proteins with fluo confers resistance to four Bacillus thuringiensis toxins. rescein maleimide. Analytical Biochemistry 253, 175-179. Proc. Natl. Acad. Sci. U.S.A. 94, 1640-1644. 1997. Ref Type: Journal (Full) 0116 Tabashnik, B. E., Malvar, T., Liu, Y.B., Finson, N., 0112 Sambrook, J. and Russell, D. W. (2001). Molecular Borthakur, D., Shin, B. S., Park, S. H., Masson, L., de Cloning: A Laboratory Manual. Cold Spring Harbor Labo Maagd, R. A., and Bosch, D. (1996). Cross-resistance of ratory). the diamondback moth indicates altered interactions with 0113 Schlenz, M. L., Babcock, J. M., and Storer, N. P. domain II of Bacillus thuringiensis toxins. Appl. Environ. Response of Cry 1F-resistant and Susceptible European Microbiol. 62,2839-2844. US 2012/0331590 A1 Dec. 27, 2012 17

0117 Tabashnik, B. E., Roush, R. T., Earle, E. D., and moth (Lymantria dispar). Arch. Insect Biochem. Physiol Shelton, A. M. (2000). Resistance to Bt toxins. Science 24, 139-147. 287, 42. 0119) Xu, X., Yu, L., and Wu, Y. (2005). Disruption of a 0118 Wolfersberger, M. G. (1993). Preparation and par cadherin gene associated with resistance to Cry1Ac tial characterization of amino acid transporting brush bor {delta-endotoxin of Bacillus thuringiensis in Helicov der membrane vesicles from the larval midgut of the gypsy erpa armigera. Appl Environ Microbiol 71,948-954.

SEQUENCE LISTING

NUMBER OF SEO ID NOS: 2

SEO ID NO 1 LENGTH: 641 TYPE PRT ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Cry1Be

< 4 OOs SEQUENCE: 1.

Met Thir Ser Asn Arg ASn Glu Asn Glu Ile Ile Asn Ala Lell Ser 1. 5 15

Ile Pro Ala Wall Ser Asn His Ser Ala Glin Met Asn Luell Ser Thir Asp 25 3 O

Ala Arg Ile Glu Asp Ser Luell Cys Ile Ala Glu Gly Asn ASn Ile Asp 35 4 O 45

Pro Phe Wall Ser Ala Ser Thir Wall Glin Thir Gly Ile Asn le Ala Gly SO 55 60

Arg Ile Lell Gly Wall Luell Gly Wall Pro Phe Ala Gly Glin le Ala Ser 65 70 8O

Phe Ser Phe Luell Wall Gly Glu Lell Trp Pro Arg Gly Asp Pro 85 90 95

Trp Glu Ile Phe Luell Glu His Wall Glu Glin Luell Ile Arg Glin Glin Wall 105 110

Thir Glu Asn Thir Arg Asp Thir Ala Lell Ala Arg Luell Glin Gly Lell Gly 115 12O 125

ASn Ser Phe Arg Ala Glin Glin Ser Lell Glu Asp Trp Tell Glu Asn 13 O 135 14 O

Arg Asp Asp Ala Arg Thir Arg Ser Wall Lell Tyr Thir Glin Tyr Ile Ala 145 15 O 155 16 O

Luell Glu Lell Asp Phe Luell ASn Ala Met Pro Luell Phe Ala le Arg Asn 1.65 17 O

Glin Glu Wall Pro Luell Luell Met Wall Tyr Ala Glin Ala Ala ASn Lell His 18O 185 190

Luell Lell Lell Luell Arg Asp Ala Ser Lell Phe Gly Ser Glu Phe Gly Lell 195 2 OO

Thir Ser Glin Glu Ile Glin Arg Glu Arg Glin Wall Glu Thir 210 215 22 O

Arg Glu Ser Asp Tyr Ala Arg Trp Tyr Asn Thir Gly Lell Asn 225 23 O 235 24 O

ASn Lell Arg Gly Thir Asn Ala Glu Ser Trp Luell Arg ASn Glin Phe 245 25 O 255

Arg Arg Asp Luell Thir Luell Gly Wall Lell Asp Luell Wall Ala Tell Phe Pro 26 O 265 27 O

Ser Asp Thir Arg Wall Pro Met Asn Thir Ser Ala Glin Lell Thir 28O 285

Arg Glu Ile Thir Asp Pro Ile Gly Arg Thir Asn Ala Pro Ser Gly US 2012/0331590 A1 Dec. 27, 2012 18

- Continued

29 O 295 3 OO Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 3. OS 310 315 32O Ile Glu Ala Ala Val Ile Arg Pro Pro His Lieu. Lieu. Asp Phe Pro Glu 3.25 330 335 Gln Lieu. Thir Ile Phe Ser Val Lieu Ser Arg Trp Ser Asn Thr Glin Tyr 34 O 345 35. O Met Asn Tyr Trp Val Gly. His Arg Lieu. Glu Ser Arg Thir Ile Arg Gly 355 360 365 Ser Leu Ser Thr Ser Thr His Gly Asn Thr Asn Thr Ser Ile Asin Pro 37 O 375 38O Val Thr Lieu. Glin Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Phe 385 390 395 4 OO Ala Gly Ile Asin Ile Lieu. Lieu. Thir Thr Pro Val Asn Gly Val Pro Trp 4 OS 41O 415 Ala Arg Phe Asn Trp Arg Asn Pro Lieu. Asn. Ser Lieu. Arg Gly Ser Lieu 42O 425 43 O Lieu. Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp Ser 435 44 O 445 Glu Thr Glu Lieu Pro Pro Glu Thir Thr Glu Arg Pro Asn Tyr Glu Ser 450 45.5 460 Tyr Ser His Arg Lieu. Ser Asn. Ile Arg Lieu. Ile Ser Gly Asn. Thir Lieu. 465 470 47s 48O Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thir Ile Ser Ser Asp Ser Ile Thr Glin Ile Pro Leu Val Lys Ser Phe SOO 505 51O Asn Lieu. Asn Ser Gly Thr Ser Val Val Ser Gly Pro Gly Phe Thr Gly 515 52O 525 Gly Asp Ile Ile Arg Thr Asn Val Asn Gly Ser Val Lieu. Ser Met Gly 53 O 535 54 O Lieu. Asn. Phe Asn. Asn. Thir Ser Lieu. Glin Arg Tyr Arg Val Arg Val Arg 5.45 550 555 560 Tyr Ala Ala Ser Glin Thr Met Val Lieu. Arg Val Thr Val Gly Gly Ser 565 st O sts Thir Thr Phe Asp Glin Gly Phe Pro Ser Thr Met Ser Ala Asn Glu Ser 58O 585 59 O Lieu. Thir Ser Glin Ser Phe Arg Phe Ala Glu Phe Pro Val Gly Ile Ser 595 6OO 605 Ala Ser Gly Ser Glin Thr Ala Gly Ile Ser Ile Ser Asn. Asn Ala Gly 610 615 62O Arg Glin Thr Phe His Phe Asp Lys Ile Glu Phe Ile Pro Ile Thr Ala 625 630 635 64 O

Thir

<210s, SEQ ID NO 2 &211s LENGTH: 594 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Cry1Da

<4 OOs, SEQUENCE: 2 US 2012/0331590 A1 Dec. 27, 2012 19

- Continued

Met Glu Ile Asn. Asn Glin Asn Glin Wall Pro Tyr Asn Cys Luell Ser 15

Asn Pro Glu Ile Ile Lell Gly Glu Glu Arg Lell Glu Thir Gly Asn 2O 25

Thir Wall Ala Asp Ile Ser Lell Gly Luell Ile ASn Phe Lell Tyr Ser Asn 35 4 O 45

Phe Wall Pro Gly Gly Gly Phe Ile Wall Gly Luell Lell Glu Luell Ile Trp SO 55 6 O

Gly Phe Ile Gly Pro Ser Glin Trp Asp Ile Phe Lell Ala Glin Ile Glu 65 70

Glin Luell Ile Ser Glin Arg Ile Glu Glu Phe Ala Arg Asn Glin Ala Ile 85 90 95

Ser Arg Luell Glu Gly Lell Ser Asn Luell Tyr Wall Wall Arg Ala 1OO 105 11 O

Phe Ser Asp Trp. Glu Asp Pro Thir Asn Pro Ala Lell Arg Glu Glu 115 12 O 125

Met Arg Ile Glin Phe Asn Asp Met Asn Ser Ala Lell Ile Thir Ala Ile 13 O 135 14 O

Pro Luell Phe Arg Val Glin Asn Tyr Glu Wall Ala Lell Lell Ser Wall Tyr 145 150 155 160

Wall Glin Ala Ala Asn Lell His Luell Ser Ile Luell Arg Asp Wall Ser Wall 1.65 17O 17s

Phe Gly Glu Arg Trp Gly Asp Thir Ala Thir Ile Asn Asn Arg Tyr 18O 185 19 O

Ser Asp Luell Thir Ser Lell Ile His Wall Thir Asn His Wall Asp 195

Thir Tyr Asn Gln Gly Lell Arg Arg Luell Glu Gly Arg Phe Luell Ser Asp 21 O 215

Trp Ile Wall Tyr Asn Arg Phe Arg Arg Glin Luell Thir Ile Ser Wall Luell 225 23 O 235 24 O

Asp Ile Wall Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thir Pro Ile 245 250 255

Glin Thir Ala Thr Gin Lell Thir Arg Glu Wall Lell Asp Luell Pro Phe 26 O 265 27 O

Ile Asn Glu Asn Lieu. Ser Pro Ala Ala Ser Pro Thir Phe Ser Ala 285

Ala Glu Ser Ala Ile Ile Arg Ser Pro His Luell Wall Asp Phe Luell Asn 29 O 295 3 OO

Ser Phe Thir Ile Tyr Thir Asp Ser Luell Ala Arg Ala Trp Gly 3. OS 310 315

Gly His Luell Wall Asn Ser Phe Arg Thir Gly Thir Thir Thir Asn Luell Ile 3.25 330 335

Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thir Glu Arg Pro Wall Thir 34 O 345 35. O

Ile Thir Ala Ser Pro Ser Wall Pro Ile Phe Arg Thir Lell Ser Ile 355 360 365

Thir Gly Luell Asp Asn Ser Asn Pro Wall Ala Gly Ile Glu Gly Wall Glu 37 O 375

Phe Glin Asn Thir Ile Ser Arg Ser Ile Arg Ser Gly Pro Ile 385 390 395 4 OO

Asp Ser Phe Ser Glu Lell Pro Pro Glin Asp Ala Ser Wall Ser Pro Ala 4 OS 41O 415 US 2012/0331590 A1 Dec. 27, 2012 20

- Continued

Ile Gly Tyr Ser His Arg Lell His Ala Thir Phe Lieu. Glu Arg Ile 425 43 O

Ser Gly Pro Arg Ile Ala Gly Thir Wall Phe Ser Trp Thir His Arg Ser 435 44 O 445

Ala Ser Pro Thir Asn Glu Wall Ser Pro Ser Arg Ile Thir Glin Ile Pro 450 45.5 460

Trp Wall Ala His Thir Lell Ala Ser Gly Ala Ser Wall Ile Gly 465 470 48O

Pro Gly Phe Thr Gly Gly Asp Ile Luell Thir Arg Asn. Ser Met Gly Glu 485 490 495

Lieu. Gly Thir Luell Arg Wall. Thir Phe Thr Gly Arg Leul Pro Glin Ser SOO 505

Ile Arg Phe Arg Tyr Ala Ser Wall Ala ASn Arg Ser Gly Thir Phe 515 525

Arg Tyr Ser Glin Pro Pro Ser Gly Ile Ser Phe Pro Thir Met 53 O 535 54 O

Asp Ala Gly Glu Pro Lell Thir Ser Arg Ser Phe Ala His Thir Thir Lieu. 5.45 550 555 560

Phe Thr Pro Ile Thr Phe Ser Arg Ala Glin Glu Glu Phe Asp Luell Tyr 565 st O sts

Ile Glin Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Wall. Thir 585 59 O

Ala Thr

1. A transgenic plant comprising DNA encoding a Cryl Da 13. The mixture of seeds of claim 11, wherein said refuge insecticidal protein and DNA encoding a Cry 1Be insecticidal seeds comprise less than 20% of all the seeds in the mixture. protein. 14. The mixture of seeds of claim 11, wherein said refuge 2. The transgenic plant of claim 1, said plant further com seeds comprise less than 10% of all the seeds in the mixture. prising DNA encoding a third core toxin-containing protein, 15. The mixture of seeds of claim 11, wherein said refuge said third protein being selected from the group consisting of seeds comprise less than 5% of all the seeds in the mixture. Cry1 Fa, Vip3Ab, Cry1Ca, and Cry1E. 16. A method of managing development of resistance to a 3. The transgenic plant of claim 2, said plant further com Cry toxin by an insect, said method comprising planting seeds prising DNA encoding Cry 1Fa protein, and DNA encoding a to produce a field of plants of claim 5. fourth protein selected from the group consisting of Cry2A, 17. The field of claim 1, wherein said plants occupy more Cry1 I, DIG-3, and Cry1Ab. than 10 acres. 4. Seed of a plant of claim 1. 18. The plant of claim 1, wherein said plant is selected from 5. A field of plants comprising non-Bt refuge plants and a the group consisting of corn, soybeans, and cotton. plurality of plants of claim 1, wherein said refuge plants 19. The plant of claim 18, wherein said plant is a maize comprise less than 40% of all crop plants in said field. plant. 6. The field of plants of claim 5, wherein said refuge plants comprise less than 30% of all the crop plants in said field. 20. A plant cell of a plant of claim 1, wherein said plant cell 7. The field of plants of claim 5, wherein said refuge plants comprises said DNA encoding said Cry 1Be insecticidal pro comprise less than 20% of all the crop plants in said field. tein and said DNA encoding said Cry1Da insecticidal protein, 8. The field of plants of claim 5, wherein said refuge plants wherein said Cry 1Be insecticidal protein is at least 99% iden comprise less than 10% of all the crop plants in said field. tical with SEQID NO:1, and said Cry 1Da insecticidal protein 9. The field of plants of claim 5, wherein said refuge plants is at least 99% identical with SEQID NO:2. comprise less than 5% of all the crop plants in said field. 21. The plant of claim 1, wherein said Cryl Be insecticidal 10. The field of plants of claim 5, wherein said refuge protein comprises SEQ ID NO:1, and said Cry 1Da insecti plants are in blocks or strips. cidal protein comprises SEQID NO:2. 11. A mixture of seeds comprising refuge seeds from non 22. A method of producing the plant cell of claim 20. Bt refuge plants, and a plurality of seeds of claim 4, wherein 23. A method of controlling a fall armyworm insect by said refuge seeds comprise less than 40% of all the seeds in contacting said insect with a Cryl Be insecticidal protein and the mixture. a Cry 1Da insecticidal protein. 12. The mixture of seeds of claim 11, wherein said refuge seeds comprise less than 30% of all the seeds in the mixture.