WO2011075587A1 - Combined use of cry1da and cry1fa proteins for insect resistance management - Google Patents

Abstract

The subject invention includes methods and plants for controlling lepidopteran insects, said plants comprising Cry1Fa and Cry1Da core toxin containing proteins in combination to delay or prevent development of resistance by the insect(s).

Description

COMBINED USE OF CRY 1 Da AND CRY1Fa PROTEINS

FOR INSECT RESISTANCE MANAGEMENT

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.

[0002] Several Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include CrylAb, CrylAc, CrylF and Cry3Bb in corn, CrylAc 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., CrylAb 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., CrylAc and Cry2Ab in cotton combined to provide resistance management for tobacco budworm).

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

(1998), "5.?. Resistance Management," Nature Biotechnol. 16: 144-146).

[0005] The proteins selected for use in an IRM stack need to exert their insecticidal effect independently so that resistance developed to one protein does not confer resistance to the second protein (i.e., there is not cross resistance to the proteins). If, for example, a pest population selected for resistance to "Protein A" is sensitive to "Protein B", one would conclude that there is not cross resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A alone.

[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, then if that receptor mutates in that insect so that one of the toxins no longer binds to that receptor and thus is no longer insecticidal against the insect, it might be the case that the insect will also be resistant to the second toxin (which competitively bound to the same receptor). That is, the insect is said to be cross-resistant to both Bt toxins. However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to those two toxins.

[0008] Cry 1 Fa is useful in controlling many lepidopteran pests species including the European corn borer (ECB; Ostrinia nubilalis (Hubner)) and the fall armyworm (FAW; Spodoptera frugiperda), and is active against the sugarcane borer (SCB; Diatraea saccharalis). The CrylFa protein, as produced in corn plants containing event TC1507, is responsible for an industry-leading insect resistance trait for FAW control. CrylFa is further deployed in the Herculex^®, SmartStax™, and WideStrike™ products.

[0009] The ability to conduct (competitive or homologous) receptor binding studies using CrylFa protein is limited because the most common technique available for labeling proteins for detection in receptor binding assays inactivates the insecticidal activity of the CrylFa protein.

[0010] 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/). See Appendix A, attached. There are 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. (Cryl has A-L, and CrylA has a-i, for example). Brief Summary of the Invention

[0011] The subject invention relates in part to the surprising discovery that a fall armyworm (Spodoptera frugiperda; FAW) population selected for resistance to the insecticidal activity of the CrylFa protein is not resistant to the insecticidal activity of the CrylDa protein. As one skilled in the art will recognize with the benefit of this disclosure, plants expressing these two insecticidal proteins, or insecticidal portions thereof, will be useful in delaying or preventing the development of resistance to either of these insecticidal proteins alone.

[0012] The subject invention is also supported by the discovery that CrylFa and CrylDa do not compete with each other for binding gut receptors from FAW.

[0013] The subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, with CrylFa and CrylDa toxins being the base pair. One preferred pyramid provides at least two proteins providing non-cross-resistant activity against two pests - the FAW and the ECB (European corn borer; Ostrinia nubilalis): CrylFa plus CrylDa plus one or more anti-ECB toxins such as CrylAb. In some preferred pyramid embodiments, the selected toxins have three separate modes of action against FAW. These preferred "three modes of action" pyramid combinations are CrylFa plus CrylD plus another toxin/gene selected from the group consisting of Vip3Ab, CrylC, CrylBe, and CrylE. Plants (and acreage planted with such plants) that produce these three toxins are included within the scope of the subject invention. Additional toxins/genes can also be added, but these particular triple stacks would, according to the subject invention, advantageously and surprisingly provide three modes of action against FAW. This can help to reduce or eliminate the requirement for refuge acreage. The subject invention also relates generally to the use of three insecticidal proteins (Cry proteins in some preferred embodiments) that do not compete with each other against a single target pest.

[0014] Thus, CrylDa could be used as in the 3 gene combination for corn and other plants (cotton and soybeans, for example). A crylDa gene could be combined into, for example, a CrylFa product such as Herculex^®, Smarts tax™, and Wides Strike™. Accordingly, use of CrylDa could be significant in reducing the selection pressure on other commercialized proteins. BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1: Damage (mean % leaf damage + SEM) to corn leaf segments infested with FAW (blue bars) or rFAW (purple bars). All treatments preceded by the numbers "5163" are leaf segments from plants transformed with a construct containing Cry IDa. Plants in which no Cry IDa expression was detected are grouped on the far left of the graph. Plants in which Cry IDa expression was detected are grouped in the center of the graph. Non-transgenic (i.e., negative) controls are on the far right of the graph and are labeled "B104", "Hill", and "Isoline". A commercial inbred containing CrylFa is the first treatment on right (labeled "Herculex I") and is the same genetic background as the non- transgenic control labeled "Isoline".

[0016] Figure 2: Competition for binding to Spodoptera frugiperda BBMV's by CrylFa core toxin, CrylDa core toxin, and 1251-labeled CrylDa core toxin protein

DETAILED DESCRIPTION OF THE INVENTION

[0017] As reported herein, CrylDa toxin produced in transgenic corn (and other plants; cotton and soybeans, for example) is very effective in controlling fall armyworm (FAW; Spodoptera frugiperda) that have developed resistance to CrylFa activity. Thus, the subject invention relates in part to the surprising discovery that fall armyworm resistant to CrylFa are susceptible (i.e., are not cross-resistant) to CrylDa.

[0018] The subject invention also relates in part to the surprising discovery that CrylDa toxin is effective at protecting plants (such as maize plants) from damage by Cry 1 Fa- resistant fall armyworm. For a discussion of this pest, see e.g. Tabashnik, PNAS (2008), vol. 105 no. 49, 19029-19030.

[0019] The subject invention includes the use of CrylDa toxin to protect corn and other economically important plant species from damage and yield loss caused by fall armyworm feeding or to fall armyworm populations that have developed resistance to CrylFa.

[0020] The subject invention thus teaches an IRM stack to prevent or mitigate the development of resistance by fall armyworm to CrylFa and/or CrylDa. [0021] The present invention provides compositions for controlling lepidopteran pests comprising cells that produce a CrylFa core toxin-containing protein and a CrylDa core toxin-containing protein.

[0022] The invention further comprises a host transformed to produce both a CrylFa core toxin-containing protein and a CrylDa core toxin-containing protein, wherein said host is a microorganism or a plant cell. The subject cry 1 Fa polynucleotide and the subject cry 1 Da polynucleotide are preferably in a genetic construct under control of (operably linked to / comprising) a non-Bacillus-thuringiensis promoter(s). The subject polynucleotides can comprise codon usage for enhanced expression in a plant.

[0023] 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 CrylFa core toxin-containing protein and further contains a CrylDa core toxin-containing protein.

[0024] An embodiment of the invention comprises a maize plant comprising a plant- expressible gene encoding a CrylDa core toxin-containing protein and a plant-expressible gene encoding a CrylFa core toxin-containing protein, and seed of such a plant.

[0025] A further embodiment of the invention comprises a maize plant wherein a plant- expressible gene encoding a CrylDa core toxin-containing protein and a plant-expressible gene encoding a CrylFa core toxin-containing protein have been introgressed into said maize plant, and seed of such a plant.

[0026] Insect receptors. As described in the Examples, competitive receptor binding studies using radiolabeled CrylDa core toxin protein show that the CrylFa core toxin protein does not compete for the high affinity binding site present in FAW insect tissues to which CrylDa binds. These results indicate that the combination of CrylFa and CrylDa proteins is an effective means to mitigate the development of resistance in FAW populations to CrylFa (and likewise, the development of resistance to CrylDa), and would likely increase the level of resistance to this pest in corn plants expressing both proteins.

[0027] Thus, based in part on the data described above and elsewhere herein, it is thought that co-production (stacking) of the CrylDa and CrylFa proteins can be used to produce a high dose IRM stack for FAW. Other proteins can be added to this combination to expand insect-control spectrum. For example in corn, the addition of Cry lAb would create an IRM pyramid for control of European corn borer. [0028] Another deployment option would be to use CrylFa and CrylDa proteins in combination with another, third toxin/gene, and to use this triple stack to mitigate the development of resistance in FAW to any of these toxins. Thus, another deployment option of the subject invention would be to use one, two, or three (or more) of these proteins in crop-growing regions where FAW can develop resistant populations. Accordingly, the subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, with CrylFa and CrylDa toxins being the base pair. One preferred pyramid provides at least two proteins providing non-cross-resistant activity against two pests - the FAW and the ECB (European corn borer; Ostrinia nubilalis) CrylFa plus CrylDa plus one or more ECB toxins such as CrylAb (see US 2008 031 1096), as Cry IF is active against both insects. Other ECB toxins include CrylBe (see USSN 61/284,290; filed December 16, 2009), Cryll (see USSN 61/284,278; filed December 16, 2009), Cry2Aa (see USSN 61/284,278; filed December 16, 2009) and DIG-3 (see US 2010 00269223). In some preferred pyramid embodiments, the selected toxins have three separate modes of action against FAW. These preferred "three modes of action" pyramid combinations are CrylFa plus Cry ID plus another toxin/gene selected from the group consisting of Vip3Ab, CrylC (see USSN

61/284,281; filed December 16, 2009), CrylBe, and CrylE (see USSN 61/284,278; filed December 16, 2009). Plants (and acreage planted with such plants) that produce these three toxins are included within the scope of the subject invention. Additional toxins/genes can also be added, but these particular triple stacks would, according to the subject invention, advantageously and surprisingly provide three modes of action against FAW. This can help to reduce or eliminate the requirement for refuge acreage. A field thus planted of over 10 acres is thus included within the subject invention.

[0029] Thus, CrylDa could be used as in the 3 gene combination for corn that currently in the Development I of the new Trait Development process. CrylFa is in the Herculex^®, SmartStax™, and WidesStrike™ products. Accordingly, use of CrylDa could be significant in reducing the selection pressure on other commercialized proteins.

[0030] Other Vip3 toxins, for example, are listed in the attached Appendix A. Those GENBANK numbers can also be used to obtain the sequences for any of the genes and proteins disclosed or mentioned herein.

[0031] U.S. Patent No. 5, 188,960 and U.S. Patent No. 5,827,514 describe CrylFa 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 CrylFa core toxin- containing proteins that are suitable for use in the present invention.

[0032] Combinations of the toxins described in the subject invention can be used to control lepidopteran pests. Adult lepidopterans, for example, butterflies and moths, primarily feed on flower nectar and are a significant effector of pollination. Nearly all lepidopteran larvae, i.e., caterpillars, feed on plants, and many are serious pests. Caterpillars feed on or inside foliage or on the roots or stem of a plant, depriving the plant of nutrients and often destroying the plant's physical support structure. Additionally, caterpillars feed on fruit, fabrics, and stored grains and flours, ruining these products for sale or severely diminishing their value. As used herein, reference to lepidopteran pests refers to various life stages of the pest, including larval stages.

10033} 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.

[0034] As an example, one chimeric toxin of the subject invention, is a full core toxin portion of CrylFa (amino acids 1 to 601) and a heterologous protoxin (amino acids 602 to the C-terminus). In one preferred embodiment, the portion of a chimeric toxin comprising the protoxin is derived from a CrylAb protein toxin. As a second Example, a second chimeric toxin of the subject invention has the full core toxin portion of Cry 1 Da (amino acids 1 to 619) and a heterologous protoxin (amino acids 620 to the C-terminus). In a preferred embodiment, the portion of a chimeric toxin comprising the protoxin is derived from a CrylAb protein toxin.

[0035] A person skilled in this art will appreciate that Bt toxins, even within a certain class such as Cry IF, will vary to some extent in length and the precise location of the transition from core toxin portion to protoxin portion. Typically, the CrylDa and CrylFa 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 CrylFa Bt toxin protein or at least about 50% of the full length of the Cry IDa Bt toxin protein. This will typically be at least about 590 amino acids. With regard to the protoxin portion, the full expanse of the CrylAb protoxin portion extends from the end of the core toxin portion to the C-terminus of the molecule.

[0036] Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences disclosed but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein. As used herein, the terms "variants" or

"variations" of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term "equivalent toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins.

[0037] As used herein, the boundaries represent approximately 95% (Cry 1 Fa's and IDa's), 78% (CrylF's and CrylD's), and 45% (Cryl 's) sequence identity, per "Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins," N. Crickmore, D.R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D.H. Dean.

Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813. These cut offs can also be applied to the core toxins only (for CrylF and CrylD toxins).

[0038] 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.

[0039] Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments of genes encoding proteins that retain pesticidal activity are also included in this definition.

[0040] A further method for identifying the genes encoding the toxins and gene portions useful according to the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO93/16094. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. Preferably, hybridization is conducted under stringent conditions by techniques well-known in the art, as described, for example, in Keller, G. FL, 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; IX SSPE or SSC at 42° C; 0.1X SSPE or SSC at 42° C; 0. IX 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] A large number of microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,

Methylophilius, Agrobactenum, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytosphere bacterial species as Pseudomonas syringae,

Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobactenium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of particular interest are the pigmented microorganisms.

[0046] 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 US Pat. No. 5135867, which is incorporated herein by reference.

[0047] 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.

[0048] 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.

[0049] Treatment of the microbial cell, e.g., a microbe containing the B.t. toxin gene or genes, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Examples of chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results. Other suitable techniques include treatment with aldehydes, such as glutaraldehyde; anti-infectives, such as zephiran chloride and cetylpyridinium chloride; alcohols, such as isopropyl and ethanol; various histologic fixatives, such as Lugol iodine, Bouin's fixative, various acids and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host environment. Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like. Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.

[0050] 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.

[0051] Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene or genes into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; survival in aqueous environments; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.

[0052] Growth of cells. The cellular host containing the B.t. insecticidal gene or genes may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.

[0053] The B.t. cells producing the toxins of the invention can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art.

[0054] Formulations. Formulated bait granules containing an attractant and spores, crystals, and toxins of the B.t. isolates, or recombinant microbes comprising the genes obtainable from the B.t. isolates disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of B.t. cells may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers. [0055] 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.

[0056] The formulations can be applied to the environment of the lepidopteran pest, e.g., foliage or soil, by spraying, dusting, sprinkling, or the like.

[0057] Plant transformation. A preferred recombinant host for production of the insecticidal proteins of the subject invention is a transformed plant. Genes encoding Bt toxin proteins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in Escherichia coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, inter alia. Accordingly, the DNA fragment having the sequence encoding the Bt toxin protein can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516, Lee and Gelvin (2008), Hoekema (1985), Fraley et al, (1986), and An et al, (1985), and is well established in the art. [0058] 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.

[0059] A large number of techniques is available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics

(microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous

recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA.

Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid

(conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the Right and Left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et ah, 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 ox Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension- cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.

[0060] 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.

1 06 ] 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, US Patent No. 5380831, 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. US Patent No. 6218188 and US Patent No. 6673990. In addition, methods for creating synthetic Bt genes for use in plants are known in the art (Stewart and Burgin, 2007). One non-limiting example of a preferred transformed plant is a fertile maize plant comprising a plant expressible gene encoding a CrylFa protein, and further comprising a second plant expressible gene encoding a Cry 1 Da protein.

[0062] Transfer (or introgression) of the CrylFa- and Cry 1 Da-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 CrylF- and CrylD- determined traits. The progeny of this cross is then mated back to the recurrent parent followed by selection in the resultant progeny for the desired trait(s) to be transferred from the non-recurrent parent. After three, preferably four, more preferably five or more generations of backcrosses with the recurrent parent with selection for the desired trait(s), the progeny will be heterozygous for loci controlling the trait(s) being transferred, but will be like the recurrent parent for most or almost all other genes (see, for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles of Cultivar Development, Vol. 1 : Theory and Technique, 360-376).

[0063] 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).

[0064] 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 (CrylAb or Cry IF) 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"

[0065] In addition, the National Corn Growers Association, on their website:

(ncga.com/insect-resistance-management-fact-sheet-bt-corn)

[0066] 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"

[0067] As stated by Roush et al. (on pages 1780 and 1784 right column, for example), stacking or pyramiding of two different proteins each effective against the target pests and with little or no cross-resistance can allow for use of a smaller refuge. Roush suggests that for a successful stack, a refuge size of less than 10% refuge, can provide comparable resistance management to about 50% refuge for a single (non-pyramided) trait. For currently available pyramided Bt corn products, the U.S. Environmental Protection Agency requires significantly less (generally 5%) structured refuge of non-Bt corn be planted than for single trait products (generally 20%).

[0068] 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. [0069] The above percentages, or similar refuge ratios, can be used for the subject double or triple stacks or pyramids. For triple stacks with three modes of action against a single target pest, a goal would be zero refuge (or less than 5% refuge, for example). This is particularly true for commercial acreage - of over 10 acres for example.

[0070] 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.

[0071] Following are examples that illustrate procedures for practicing the invention.

These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.

[0072] Unless specifically indicated or implied, the terms "a", "an", and "the" signify "at least one" as used herein.

EXAMPLE 1

BIOASSAY DATA

[0073] CrylDa expressed in transgenic corn (pDAS5163) provides protection from feeding by fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith). The same events are more effective in controlling FAW that have developed resistance to CrylFa and are clearly superior to corn plants containing event TCI 507, which is arguably the industry-leading insect resistance trait for FAW control.

[0074] We have also demonstrated that CrylFa (protein from recombinant Pseudomonas fluorescens strain DR1649; plasmid pDAB 1817) and CrylDa (protein from recombinant Pseudomonas fluorescens strain DC782) are both effective at controlling FAW in artificial diet bioassays and that the potency of the combination is greater than is expected from their individual potencies.

[0075] Based on the data described above, co-expressing CrylDa and CrylFa can produce a high dose IRM stack for FAW, other important Spodoptera species, and perhaps other lepidopteran pests. Other proteins can be added to this combination to add spectrum. For example in corn, the addition of CrylAb would create an IRM stack for European corn borer (ECB), Ostrinia nubilalis (Hubner). [0076] As shown in Figure 1, damage (mean % leaf damage + SEM) to corn leaf segments infested with FAW (blue bars) or rFAW (purple bars). All treatments preceded by the numbers "5163" are leaf segments from plants transformed with a construct containing Cry IDa. Plants in which no Cry IDa expression was detected are grouped on the far left of the graph. Plants in which Cry IDa expression was detected are grouped in the center of the graph. Non-trans genie (i.e., negative) controls are on the far right of the graph and are labeled "B104", "Hill", and "Isoline". A commercial inbred containing Cry 1 Fa is the first treatment on right (labeled "Herculex I") and is the same genetic background as the non- transgenic control labeled "Isoline".

[0077] A protoxin chimera consisting of the coding sequence for the trypsin cleaved limit toxin of Cry IDa and the coding sequence for the c-terminal protoxin region of Cry lAb was created and engineered into an expression cassette capable of directing expression in corn (pDAS5163). Corn was transformed using Agrobacterium tumefacians and events containing the Cry lDa/1 Ab chimera were identified. Leaf sections from regenerated plants were bioassayed with wild type fall armyworm (FAW) or larvae from a fall armyworm population that was resistant to CrylFa (rFAW). CrylDa/lAb transformed plants did reduce feeding of FAW but were not as effective as the inbred containing 2 copies of CrylFa (Figure 1). (The CrylDa events tested were hemizygous for the transgene while the converted inbred was homozygous for event TCI 507.) In contrast, the same events containing Cry 1 Da/1 Ab were generally much more effective in reducing the feeding of rFAW than the inbred containing CrylFa (Figure 1).

[0078] The insecticidal activity of CrylFa (protein from recombinant Pseudomonas fluorescens strain DR1649; plasmid pDAB 1817), CrylDa (protein from recombinant P. fluorescens strain DC782), and a 1 : 1 (w:w) combination of the 2 was tested in standard, artificial diet bioassays used to assess potency. Potency estimates were made using LOGIT analysis (JMP^®8.0, SAS Inc. 2008) which produced LC50 estimates and upper and lower limits (95%) for the LC5₀. A test for synergism was conducted using the method described by Tabashnik (1992) by which an expected value for the potency of a combination is calculated using the potencies of each component alone. A combination is considered synergistic when the estimated upper confidence limit of the combination is lower than the calculated expected potency. In the case of fall armyworm (FAW) and a population of fall armyworm that was resistant to CrylFa (rFAW), the upper confidence limits for the LC5₀S of the combination were lower than the estimated potencies (Tables 1 & 2) thereby leading to the conclusion that the combination of Cry lFa and Cry IDa on these 2 populations is synergistic.

[0079] Table 1. Potency estimates, upper and lower limits of the 95% confidence interval (LCL and UCL, respectively), for CrylFa, Cry IDa, and the 1 : 1 (w;w) combination of the 2 on wild type fall armyworm (FAW), Spodoptera frugiperda. The last column contains the expected LC5₀ value based on the potency of each protein alone using the formula described by Tabashnik (1992). Tabashnik BE . Evaluation of synergism among Bacillus

thuringiensis toxins. Applied and Environmental Microbiology 58[10], 3343-3346. 1992. A combination is considered synergistic when the expected value is higher than the upper confidence limit for the combination.

[0080] Table 2. Potency estimates, upper and lower limits of the 95% confidence interval (LCL and UCL, respectively), for CrylFa, Cry IDa, and the 1 : 1 (w;w) combination of the 2 on CrylFa-resistant fall armyworm (rFAW), Spodoptera frugiperda. The last column contains the expected LC5₀ value based on the potency of each protein alone using the formula described by Tabashnik (1992). A combination is considered synergistic when the expected value is higher than the upper confidence limit for the combination.

EXAMPLE 2

SUMMARY OF BINDING DATA

[0081] Competition binding experiments conducted with ¹²⁵I-labeled CrylDa using brush border membrane vesicles (BBMV) isolated from FAW are described below. The results from these experiments demonstrate that CrylDa binds tightly to its receptor and that CrylFa does not compete with CrylDa for binding sites. If resistance to CrylDa could be based on a mutation to the receptor observed in these studies, these data suggest that CrylFa would be a good IRM tool for managing such resistant populations or mitigating the development of such resistance. Results from bioassays with CrylFa-resitant FAW (rFAW) demonstrate that CrylDa is active on this population. Together, these data suggest that CrylFa and CrylDa could be an IRM stack that effectively mitigates the development of resistance to either insecticidal protein.

[0082] Receptor binding assays show that 1251 CrylDa binds tightly to its receptor(s), and can be effectively competed off by unlabeled CrylDa. Neither CrylAb, CrylFa or Cry 1 Be can compete off 1251 CrylDa from its receptor site(s) in FAW BBMV's, indicating that CrylDa has a unique binding site in the midgut of FAW that CrylAb, CrylF and CrylBe do not compete with. Since rFAW are as sensitive to CrylDa as wild type FAW, this indicates that the putative receptor site that is altered in rFAW insects is not the receptor site that CrylDa binds to. Thus, CrylDa is an excellent stacking partner for CrylFa since it interacts at a different target site which is responsible for its biological activity.

[0083] When 1251 CrylDa was added to FAW BBMV's, only non-radiolabeled CrylDa itself was able to displace the bound 1251 CrylDa. The inability of CrylFa, CrylAb, and CrylBe to displace the bound 1251 CrylDa from the BBMV's indicated that in FAW midgut, CrylDa bound to a unique receptor site that CrylFa, CrylAb, and CrylBe do not interact, even though all four of these different Cry toxins are active against FAW larvae.

EXAMPLE 3

Design of chimeric toxins comprising Cryl core toxins and heterologous protoxins

[0084] Chimeric Toxins. Chimeric proteins utilizing the core toxin domain of one Cry toxin fused to the protoxin segment of another Cry toxin have previously been reported, for example, in US Patent No. 5593881 and US Patent No. 5932209.

[0085] CrylDa chimeric protein variants of this invention include chimeric toxins comprising an N-terminal core toxin segment derived from a CrylDa insecticidal toxin fused to a heterologous delta endotoxin protoxin segment at some point past the end of the core toxin segment. The transition from the core toxin to the heterologous protoxin segment can occur at approximately the native core toxin/protoxin junction or, in the alternative, a portion of the native protoxin (extending past the core toxin segment) can be retained, with the transition to the heterologous protoxin occurring downstream. In variant fashion, the core toxin and protoxin segments may comprise exactly the amino acid sequence of the native toxins from which they are derived, or may include amino acid additions, deletions, or substitutions that do not diminish, and may enhance, the biological function of the segments when fused to one another.

[0086] For example, a chimeric toxin of the subject invention comprises a core toxin segment derived from CrylDa and a heterologous protoxin. In a preferred embodiment of the invention, the core toxin segment derived from CrylDa2 (594 amino acids) is fused to a heterologous segment comprising a protoxin segment derived from a CrylAb delta- endotoxin (545 amino acids). The 1139 amino acid sequence of the chimeric protein, herein referred to as CrylDa. It is to be understood that other chimeric fusions comprising CrylDa2 core toxin variants and protoxins derived from CrylAb are within the scope of this invention.

[0087] A second chimeric protein of the invention comprises a core toxin segment derived from CrylFa (603 amino acids) fused to a heterologous segment comprising a protoxin segment derived from a CrylAb delta-endotoxin (545 amino acids). The 1148 amino acid sequence of the chimeric protein, herein called CrylFa.

EXAMPLE 4

Construction of expression plasmids encoding chimeric proteins and expression in

Pseudomonas

[0088] Standard cloning methods [as described in, for example, Sambrook et al, (1989) and Ausubel et al, (1995), and updates thereof] were used in the construction of Pseudomonas fluorescens (Pf) expression construct pDOW2848 engineered to produce a full-length CrylDa chimeric protein. Protein production was performed in Pseudomonas fluorescens strain MB214 (a derivative of strain MB 101; P. fluorescens biovar I), having an insertion of a modified lac operon as disclosed in US Patent No. 5169760. The basic cloning strategy entailed subcloning a DNA fragment encoding the CrylDa protein into plasmid vectors, whereby it is placed under the expression control of the Ptac promoter and the rrnBTlT2 terminator from plasmid pKK223-3 (PL Pharmacia, Milwaukee, WI). One such plasmid was named pDOW2848 and the MB214 isolate harboring this plasmid is named Dpfl50.

[0089] Growth and Expression Analysis in Shake Flasks Production of the CrylDa protein for characterization and insect bioassay was accomplished by shake-flask-grown P.

fluorescens strain Dpfl50. CrylDa protein production driven by the Ptac promoter was conducted as described previously in US Patent No. 5527883. Details of the

microbiological manipulations are available in Squires et ah, (2004), US Patent Application 20060008877, US Patent Application 20080193974, and US Patent Application

20080058262, incorporated herein by reference. Expression was induced by addition of isopropyl- -D-l-thiogalactopyranoside (IPTG) after an initial incubation of 24 hours at 30° with shaking. Cultures were sampled at the time of induction and at various times post- induction. Cell density was measured by optical density at 600 nm (OD₆oo).

[0090] Cell Fractionation and SDS-PAGE Analysis of Shake Flask Samples At each sampling time, the cell density of samples was adjusted to OD₆oo = 20 and 1 mL aliquots were centrifuged at 14000 x g for five minutes. The cell pellets were frozen at -80°.

Soluble and insoluble fractions from frozen shake flask cell pellet samples were generated using EasyLyse™ Bacterial Protein Extraction Solution (EPICENTRE® Biotechnologies, Madison, WI). Each cell pellet was resuspended in 1 mL EasyLyse™ solution and further diluted 1 :4 in lysis buffer and incubated with shaking at room temperature for 30 minutes. The lysate was centrifuged at 14,000 rpm for 20 minutes at 4° and the supernatant was recovered as the soluble fraction. The pellet (insoluble fraction) was then resuspended in an equal volume of phosphate buffered saline (PBS; 11.9 mM Na₂HP0₄, 137 mM NaCl, 2.7 mM KC1, pH7.4).

[0091] Samples were mixed 1 : 1 with 2X Laemmli sample buffer containing β- mercaptoethanol (Sambrook et ah, supra.) and boiled for 5 minutes prior to loading onto Criterion XT Bis-Tris 12% gels (Bio-Rad Inc., Hercules, CA). Electrophoresis was performed in the recommended XT MOPS buffer. Gels were stained with Bio-Safe Coomassie Stain according to the manufacturer's (Bio-Rad) protocol and imaged using the Alpha Innotech Imaging system (San Leandro, CA).

[0092] Inclusion body preparation. Cry 1 Da protein inclusion body (IB) preparations were performed on cells from P. fluorescens fermentations that produced insoluble Bt insecticidal protein, as demonstrated by SDS-PAGE and MALDI-MS (Matrix Assisted Laser Desorption/Ionization Mass Spectrometry). P. fluorescens fermentation pellets were thawed in a 37° water bath. The cells were resuspended to 25% w/v in lysis buffer [50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt (Ethylenediaminetetraacetic acid), 1% Triton X-100, and 5 mM Dithiothreitol (DTT); 5 mL/L of bacterial protease inhibitor cocktail (Catalog # P8465; Sigma-Aldrich, St. Louis, MO) were added just prior to use]. The cells were suspended using a hand-held homogenizer at lowest setting (Tissue Tearor, BioSpec Products, Inc., Bartlesville, OK). Lysozyme (25 mg of Sigma L7651, from chicken egg white) was added to the cell suspension by mixing with a metal spatula, and the suspension was incubated at room temperature for one hour. The suspension was cooled on ice for 15 minutes, then sonicated using a Branson Sonifier 250 (two 1- minute sessions, at 50% duty cycle, 30% output). Cell lysis was checked by microscopy. An additional 25 mg of lysozyme were added if necessary, and the incubation and sonication were repeated. Following confirmation of cell lysis via microscopy, the lysate was centrifuged at 11,500 x g for 25 minutes (4°) to form the IB pellet, and the supernatant was discarded. The IB pellet was resuspended with 100 mL lysis buffer, homogenized with the hand-held mixer and centrifuged as above. The IB pellet was repeatedly washed by resuspension (in 50 mL lysis buffer), homogenization, sonication, and centrifugation until the supernatant became colorless and the IB pellet became firm and off-white in color. For the final wash, the IB pellet was resuspended in sterile-filtered (0.22 μιη) distilled water containing 2 mM EDTA, and centrifuged. The final pellet was resuspended in sterile- filtered distilled water containing 2 mM EDTA, and stored in 1 mL aliquots at -80°.

[0093] SDS-PAGE analysis and quantitation of protein in IB preparations was done by thawing a 1 mL aliquot of IB pellet and diluting 1 :20 with sterile-filtered distilled water. The diluted sample was then boiled with 4X reducing sample buffer [250 mM Tris, pH6.8, 40% glycerol (v/v), 0.4% Bromophenol Blue (w/v), 8% SDS (w/v) and 8% β- mercaptoethanol (v/v)] and loaded onto a Novex® 4-20% Tris-Glycine, 12+2 well gel (Invitrogen) run with IX Tris/Glycine/SDS buffer (BioRad). The gel was run for 60 min at 200 volts then stained with Coomassie Blue (50% G-250/50% R-250 in 45% methanol, 10% acetic acid), and destained with 7% acetic acid, 5% methanol in distilled water.

Quantification of target bands was done by comparing densitometric values for the bands against Bovine Serum Albumin (BSA) standard samples run on the same gel to generate a standard curve.

[0094] Solubilization of Inclusion Bodies. Six mL of CrylDa inclusion body suspension from clone DPfl50 were centrifuged on the highest setting of an Eppendorf model 5415C microfuge (approximately 14,000 x g) to pellet the inclusions. The storage buffer supernatant was removed and replaced with 25 mL of 100 mM sodium carbonate buffer, pHl 1, in a 50 mL conical tube. Inclusions were resuspended using a pipette and vortexed to mix thoroughly. The tube was placed on a gently rocking platform at 4° overnight to extract the target protein. The extract was centrifuged at 30,000 x g for 30 min at 4°, and the resulting supernatant was concentrated 5-fold using an Amicon Ultra- 15 regenerated cellulose centrifugal filter device (30,000 Molecular Weight Cutoff; Millipore). The sample buffer was then changed to 10 mM CAPS [3-(cyclohexamino)l-propanesulfonic acid] pH 10 using disposable PD-10 columns (GE Healthcare, Piscataway, NJ).

[0095] Solubilization and trypsin activation of Inclusion Body protein. In some instances, Cry 1 Da inclusion body suspension from Pf clone DPfl50 was centrifuged on the highest setting of an Eppendorf model 5415C microfuge (approximately 14,000 x g) to pellet the inclusions. The storage buffer supernatant was removed and replaced with 100 mM CAPS, pH 1 1 to provide a protein concentration of approximately 50 mg/mL. The tube was rocked at room temperature for three hours to completely solubilize the protein. Trypsin was added at an amount equal to 5% to 10% (w:w, based on the initial weight of IB powder) and digestion was accomplished by incubation while rocking overnight at 4° or by rocking 90- 120 minutes at room temperature. Insoluble material was removed by centrifugation at 10,000 x g for 15 minutes, and the supernatant was applied to a MonoQ anion exchange column (10 mm by 10 cm). Activated CrylDa protein was eluted (as determined by SDS- PAGE, see below) by a 0% to 100% 1 M NaCl gradient over 25 column volumes. Fractions containing the activated protein were pooled and, when necessary, concentrated to less than 10 mL using an Amicon Ultra- 15 regenerated cellulose centrifugal filter device as above. The material was then passed through a Superdex 200 column (16 mm by 60 cm) in buffer containing 100 mM NaCl. 10% glycerol, 0.5% Tween-20 and 1 mM EDTA. It was determined by SDS-PAGE analysis that the activated (enzymatically truncated) protein elutes at 65 to 70 mL. Fractions containing the activated protein were pooled and concentrated using the centrifugal concentrator as above.

[0096] Gel electrophoresis. The concentrated protein preparations were prepared for electrophoresis by diluting 1 :50 in NuPAGE® LDS sample buffer (Invitrogen) containing 5 mM DTT as a reducing agent and heated at 95° for 4 minutes. The sample was loaded in duplicate lanes of a 4-12% NuPAGE® gel alongside five BSA standards ranging from 0.2 μg to 2 μg/lane (for standard curve generation). Voltage was applied at 200 V using MOPS SDS running buffer (Invitrogen) until the tracking dye reached the bottom of the gel. The gel was stained with 0.2% Coomassie Blue G-250 in 45% methanol, 10% acetic acid, and destained, first briefly with 45% methanol, 10% acetic acid, and then at length with 7% acetic acid, 5% methanol until the background cleared. Following destaining, the gel was scanned with a BioRad Fluor-S Multilmager. The instrument's Quantity One Software v.4.5.2 was used to obtain background-subtracted volumes of the stained protein bands and to generate the BSA standard curve that was used to calculate the concentration of chimeric CrylDa protein in the stock solution.

EXAMPLE 5

Preparation of CrylFa and CrylDa core toxin proteins and isolation of Spodoptera frugiperda brush border membrane vesicles for competitive binding experiments

[0097] The following Examples evaluate the competition binding of Cry 1 core toxin proteins to putative receptors in insect gut tissues. It is shown that 1251-labeled CrylDa core toxin protein binds with high affinity to Brush Border Membrane Vesicles (BBMV's) prepared from Spodoptera frugiperda (fall army worm) and that CrylFa core toxin protein does not compete with this binding.

[0098] Purification of Cry Proteins. A gene encoding a chimeric CrylDa protein was expressed in the Pseudomonas fluorescens expression strain as described in Example 4. In similar fashion, a gene encoding a chimeric protein comprising the CrylFa core toxin (603 amino acids) and CrylAb protoxin (545 amino acids) was expressed in the Pf system. In the CrylFa instance, the expression plasmid was named pDAB 1817, and the P. fluorescens strain that harbors pDAB 1817 was named DPfl29. The proteins were purified by the methods of Example 4, and trypsin digestion to produce activated core toxins from the full- length proteins was then performed, and the products were purified by the methods described in Example 4. Preparations of the trypsin processed (activated core toxin) proteins were >95% pure and had a molecular weight of approximately 65 kDa as determined experimentally by SDS-PAGE. As used herein, the activated core toxin prepared from the CrylDa protein is called the CrylDa core toxin protein, and the activated core toxin prepared from the CrylFa protein is called the CrylFa core toxin protein.

[0099] Preparation and Fractionation of Solubilized BBMV's. Standard methods of protein quantification and SDS-polyacrylamide gel electrophoresis were employed as taught, for example, in Sambrook et al. (1989) and Ausubel et al. (1995), and updates thereof.

[00100] Last instar S. frugiperda larvae were fasted overnight and then dissected after chilling on ice for 15 minutes. The midgut tissue was removed from the body cavity, leaving behind the hindgut attached to the integument. The midgut was placed in a 9X volume of ice cold homogenization buffer (300 mM mannitol, 5 mM EGTA, 17 mM Tris base, pH7.5), supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich P-2714) diluted as recommended by the supplier. The tissue was homogenized with 15 strokes of a glass tissue homogenizer. BBMV's were prepared by the MgC¾ 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°. The supernatant was saved and the pellet suspended into the original volume of 0.5X diluted homogenization buffer and centrifuged again. The two supernatants were combined and centrifuged at 27,000 x g for 30 min at 4° to form the BBMV fraction. The pellet was suspended into BBMV Storage Buffer (10 mM HEPES, 130 mM KC1, 10% glycerol, pH7.4) to a protein concentration of about 3 mg/mL. Protein concentration was determined using Bovine Serum Albumin (BSA) as the standard. Alkaline phosphatase determination (a marker enzyme for the BBMV fraction) was made prior to freezing the samples using the QuantiChrom™ DALP-250 Alkaline Phosphatase Assay Kit (Gentaur Molecular Products, Kampenhout, BE) following the manufacturer's instructions. The specific activity of this enzyme typically increased 7-fold compared to that found in the starting midgut homogenate fraction. The BBMV's were aliquoted into 250 μϊ_^ samples, flash frozen in liquid nitrogen and stored at -80°.

[00101] Electrophoresis. Analysis of proteins by SDS-PAGE was conducted under reducing (i.e. in 5% β-mercaptoethanol, BME) and denaturing (i.e. heated 5 minutes at 90° in the presence of 2% SDS) conditions. Proteins were loaded into wells of a 4% to 20% Tris- Glycine polyacrylamide gel (BioRad; Hercules, CA) and separated at 200 volts for 60 minutes. Protein bands were detected by staining with Coomassie Brilliant Blue R-250 (BioRad) for one hour, and destained with a solution of 5% methanol in 7% acetic acid. The gels were imaged and analyzed using a BioRad Fluro-S Multi Imager™. Relative molecular weights of the protein bands were determined by comparison to the mobilities of known molecular weight proteins observed in a sample of BenchMark™ Protein Ladder (Life Technologies, Rockville, MD) loaded into one well of the gel.

[00102] Iodination of Cry 1 Da core toxin protein. Purified Cry 1 Da core toxin protein was iodinated using Pierce Iodination Beads (Thermo Fisher Scientific, Rockford, IL). Briefly, two Iodination Beads were washed twice with 500 of PBS (20 mM sodium phosphate, 0.15 M NaCl, pH7.5), and placed into a 1.5 mL centrifuge tube with 100 of PBS. 0.5 mCi of 1251-labeled sodium iodide was added, the components were allowed to react for 5 minutes at room temperature, then 1 μg of Cry 1 Da core toxin protein was added to the solution and allowed to react for an additional 3 to 5 minutes. The reaction was terminated by pipetting the solution from the Iodination Beads and applying it to a Zeba™ spin column (Invitrogen) equilibrated in 50 mM CAPS, pHlO.O, 1 mM DTT (dithiothreitol), 1 mM EDTA, and 5% glycerol. The Iodination Beads were washed twice with 10 μΐ, of PBS and the wash solution was also applied to the Zeba™ desalting column. The radioactive solution was eluted through the spin column by centrifuging at 1,000 x g for 2 min. 1251- radiolabeled CrylDa core toxin protein was then dialyzed against 50 mM CAPS, pHlO.O, 1 mM DTT, 1 mM EDTA, and 5% glycerol.

[00103] Imaging. Radio-purity of the iodinated CrylDa core toxin protein was determined by SDS-PAGE and phosphorimaging. Briefly, SDS-PAGE 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 μιη thick) and exposing them under a Molecular Dynamics storage phosphor screen (35 cm x 43 cm) for 1 hour. The plates were developed using a Molecular Dynamics Storm 820 phosphorimager and the image was analyzed using ImageQuant™ software.

EXAMPLE 6

Binding of 1251-labeled Cryl core toxin protein to BBMV's from Spodoptera frugiperda

[00104] A saturation curve was generated to determine the optimal amount of BBMV protein to use in the binding assays with CrylDa and CrylFa core toxin proteins. 0.5 nM of 1251- radiolabeled Cryl core toxin protein was incubated for 1 hr at 28° in binding buffer (8 mM NaHPC-4, 2 mM KH₂P0₄, 150 mM NaCl, 0.1% BSA, pH7.4) with amounts of BBMV protein ranging from 0 μg/mL to 500 μg/mL (total volume of 0.5 mL). 1251-labeled Cryl core toxin protein bound to the BBMV proteins was separated from the unbound fraction by sampling 150 μϊ_^ of the reaction mixture in triplicate into separate 1.5 mL centrifuge tubes and centrifuging the samples at 14,000 x g for 8 minutes at room temperature. The supernatant was gently removed and the pellet was washed three times with ice cold binding buffer. The bottom of the centrifuge tube containing the pellet was cut off, placed into a 13 x 75 mm glass culture tube and the samples were counted for 5 minutes each in the gamma counter. CPM (counts per minute) obtained minus background CPM (reaction with no BBMV protein) was plotted versus BBMV protein concentration. In accordance with results reported by others (Luo et ah, 1999), the optimal concentration of BBMV protein to use in the binding assays was determined to be 150 μg/mL.

EXAMPLE 7

Competitive binding assays to BBMVs from S. frugiperda with core toxin proteins of

CrylDa and CrylFa

[00105] Homologous and heterologous competition binding assays were conducted using 150 μg/mL of S. frugiperda BBMV protein and 0.5 nM of the 1251-radiolabeled CrylDa core toxin protein. Concentrations of the competitive non-radiolabeled CrylFa core toxin protein added to the reaction mixture ranged from 0.045 nM to 1000 nM and were added at the same time as the radioactive CrylDa core toxin protein, to assure true binding competition. Incubations were carried out for 1 hr at 28° and the amount of 1251-labeled CrylDa core toxin protein bound to the BBMV (specific binding) was measured as described above. Non-specific binding was represented by the counts obtained in the presence of 1,000 nM of non-radiolabeled CrylDa core toxin protein. One hundred percent total binding was considered to be the amount of binding in the absence of any competitor CrylFa core toxin protein.

[00106] Receptor binding assays using 1251-labeled CrylDa core toxin protein determined the ability of the CrylFa core toxin protein to displace this radiolabeled ligand from its binding site on BBMV's from 5^*. frugiperda. The results show that the CrylFa core toxin protein did not displace bound 1251-labeled CrylDa core toxin protein from its receptor protein(s) at concentrations as high as 1000 nM (2000 times the concentration of the radioactive binding ligand). As expected, unlabeled CrylDa core toxin protein was able to displace radiolabeled CrylDa core toxin protein from its binding protein(s), exhibiting a sigmoidal dose response curve with 50% displacement occurring at 5 nM.

[00107] It is thus indicated that the CrylDa core toxin protein interacts with a binding site in S. frugiperda BBMV that does not bind the CrylFa core toxin protein. References

[00108] Finney, D.J. 1971. Probit analysis. Cambridge University Press, England.

[00109] Hua, G., L. Masson, J. L. Jurat-Fuentes, G. Schwab, and M. J. Adang. Binding analyses of Bacillus thuringiensis Cry d-endotoxins using brush border membrane vesicles of Ostrinia nubilalis. Applied and Environmental Microbiology 67[2], 872-879. 2001.

[00110] LeOra Software. 1987. POLO-PC. A user's guide to probit and logit analysis.

Berkeley, CA.

[00111] McGaughey, W. FL, F. Gould, and W. Gelernter. Bt resistance management. Nature Biotechnology 16[2], 144-146. 1998

[00112] Marcon, P.R.G.C., L.J. Young, K. Steffey, and B.D. Siegfried. 1999. Baseline susceptibility of the European corn borer, Ostrinia nubilalis (Hubner) (Lepidoptera: Pyralidae) to Bacillus thuringiensis toxins. J. Econ. Entomol. 92 (2): 280-285.

[00113] Robertson, L.J. and H.K. Preisler. 1992. Pesticide bioassays with arthropods. CRC Press, Boca Ranton, FL.

[00114] SAS Institute Inc. 1988. SAS procedures guide, Release 6.03 edition. SAS Institute Inc, Cary, NC.

[00115] Stone, B.F. 1968. A formula for determining degree of dominance in cases of monofactorial inheritance of resistance to chemicals. Bull. WHO 38:325-329.

[00116] Van Mellaert, H., J. Botterman, J. Van Rie, and H. Joos. Transgenic plants for the prevention of development of insects resistant to Bacillus thuringiensis toxins. (Plant Genetic Systems N.V., Belg. 89-401499[400246], 57-19901205. EP. 5-31-1989 Appendix A

List of delta-endotoxins - from Crickmore et al. website (cited in application) Accession Number is to NCBI entry (if available)

2010/060815

10060815

Claims

We claim:

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

DNA encoding a Cry IF insecticidal protein.

2. Seed of a plant of claim 1.

3. A plant of claim 1 wherein DNA encoding a Cry ID insecticidal protein and DNA encoding a CrylF insecticidal protein have been introgressed into said plant.

4. Seed of a plant of claim 3.

5. A field of plants comprising non-Bt refuge plants and a plurality of plants of claim 1, 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-Bt 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 toxin by an insect, said method comprising planting seeds to produce a field of plants of claim 5.

17. The transgenic plant of claim 1, said plant further comprising DNA encoding a CrylAb core toxin-containing protein.

18. A field of plants comprising non-Bt refuge plants and a plurality of transgenic plants of claim 17, wherein said refuge plants comprise less than about 20% of all crop plants in said field.

19. A field of plants comprising a plurality of plants of claim 17, wherein said field comprises less than about 10% refuge plants.

20. A method of managing development of resistance to a Cry toxin by an insect, said method comprising planting seeds to produce a field of plants of claim 19.

21. A composition for controlling lepidopteran pests comprising cells that express

effective amounts of both a Cry IF core toxin-containing protein and a Cry ID core toxin-containing protein.

22. A composition of claim 21 comprising a host transformed to express both a CrylF core toxin-containing protein and a Cry ID core toxin containing protein, wherein said host is a microorganism or a plant cell.

23. A method of controlling lepidopteran pests comprising presenting to said pests or to the environment of said pests an effective amount of a composition of claim 21.

24. A transgenic plant that produces three insecticidal Cry proteins that are insecticidal to the same target insect, said insect having potential to develop resistance to any one of said Cry proteins, and wherein each said Cry protein binds a different gut receptor in said target insect.

25. The plant of claim 24 wherein said insect is fall armyworm.

26. A transgenic plant that produces a CrylFa protein plus a CrylDa protein plus a third protein selected from the group consisting of Vip3A, CrylC, Cry 1 Be, and Cry IE proteins.

27. A method of managing development of resistance to a Cry toxin by an insect, said method comprising planting seeds to produce a field of plants of claim 26.

28. A field of plants comprising non-Bt refuge plants and a plurality of plants of claim 26, wherein said refuge plants comprise less than about 10% of all crop plants in said field.

29. The field of claim 28, wherein said field comprises less than about 5% refuge plants.

30. A method of managing development of resistance to a Cry toxin by an insect, said method comprising planting seeds to produce a field of plants of claim 28 or 29.

31. A mixture of seeds comprising refuge seeds from non-Bt refuge plants, and a

plurality of seeds from a plant of claim 26, wherein said refuge seeds comprise less than 10% of all the seeds in the mixture.

32. A field of any of claims 5, 18, and 28, wherein said plants occupy more than 10 acres.

33. A plant of any of claims 1, 2, 17, 24, and 26, wherein said plant is selected from the group consisting of corn, soybeans, and cotton.

34. A plant of any of claims 1, 2, 17, 24, and 26, wherein said plant is a maize plant.

35. A plant cell of a plant of any of claims 1, 2, 17, 24, 26, 33, and 34, wherein said plant cell comprises said DNA encoding said CrylD insecticidal protein and said DNA encoding said CrylF insecticidal protein, wherein said CrylF insecticidal protein is at least 99% identical with SEQ ID NO: l, and said CrylD insecticidal protein is at least 99% identical with SEQ ID NO:2.

36. A plant of any of claims 1, 2, 17, 24, 26, 33, and 34, wherein said CrylF insecticidal protein comprises SEQ ID NO: l, and said CrylD insecticidal protein comprises SEQ ID NO:2.