Control of spodoptera exigua

Abstract

The present invention provides methods for controlling Spodoptera litura and protecting crops, in particular maize, from economic damage caused by Spodoptera litura. The invention further relates to the use of plants stably transformed with nucleic acid molecules encoding the Cry proteins of the invention, alone or in combination with other insecticidal proteins, for controlling or combating Spodoptera litura.

Description

TECHNICAL FIELD

[0001] The present invention relates to methods for controlling or combating Spodoptera, in particular Spodoptera litura (Lepidoptera, Noctuidae) (also known as common cutworm or oriental leafworm), by using pesticidal proteins and nucleic acid molecules encoding them, in particular by using Bacillus thuringiensis (“Bt”) Cry proteins and cry genes encoding said Cry proteins. BACKGROUND

[0002] Bacillus thuringiensis (Bt) is a gram-positive, spore-forming soil bacterium characterized by its ability to produce crystalline inclusions that are toxic to certain orders and species of plant pests, including insects, but are harmless to plants and other non-target organisms. Thus, compositions comprising Bacillus thuringiensis strains or their insecticidal proteins can be used as environmentally acceptable insecticides to control agricultural insect pests or insect vectors of a wide variety of human or animal diseases.

[0003] Crystal (Cry) proteins from Bacillus thuringiensis have potent insecticidal activity primarily against lepidopteran, dipteran, and coleopteran pest insects. These proteins also show activity against pests in the orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson, J. 1993. The Bacillus Thuringiensis family tree. Advanced Engineered Pesticides. Marcel Dekker, Inc., New York, N.Y.). These proteins were originally classified as Cryl through CryVI, primarily based on their insecticidal activity. The major classes are lepidopteran-specific (I), lepidopteran- and dipteran-specific (II), coleopteran-specific (III), dipteran-specific (IV), and nematode-specific (V) and (VI). The proteins are further classified into subfamilies; more highly related proteins within each family are assigned a subgroup letter, e.g., CrylA, CrylB, CrylC, etc. Proteins that are even more closely related within each subgroup are given names such as CrylCa, CrylCb, etc. The terms "Cry toxin" and "delta-endotoxin" have been used interchangeably with the term "Cry protein." The current nomenclature for Cry proteins and genes is based on amino acid sequence homology, not insect target specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In this more accepted classification, each toxin is assigned a unique name that incorporates a first rank (Arabic numeral), a second rank (capital letter), a third rank (lower case letter), and a fourth rank (another Arabic numeral). In the current classification, the Roman numerals have been exchanged for Arabic numerals in the first rank. For example, "CrylA(a)" under the old nomenclature is now "CrylAa" under the current nomenclature.According to Ibrahim et al. (2010, Bioeng. Bugs, 1 :31-50), Cry toxins can still be classified into six major groups according to their insect host specificity and include: Group 1 - Lepidoptera (e.g., Cryl, Cry9, and Cryl 5); Group 2 - Lepidoptera and Diptera (e.g., Cry2); Group 3 - Coleoptera (Cry3, Cry7, and Cry8); Group 4 - Diptera (Cry4, CrylO, Cryl l, Cryl 6, Cryl 7, Cryl 9, and Cry20); Group 5 - Lepidoptera and Coleoptera (Cryll); and Group 6 - Nematodes (Cry6). The Cryll, Cry2, Cry3, CrylO, and Cryl l toxins (73-82 kDa) are unique in that they appear to be naturally truncated versions of the larger Cryl and Cry4 proteins (130-140 kDa).

[0004] Cry proteins are globular protein molecules that accumulate as protoxins in crystalline form during the sporulation stage of Bt. Upon ingestion by a pest, the crystals are typically solubilized releasing a protoxin that can range in size, for example, 130-140 kDa for many Lepidoptera-active Cry proteins (e.g., Cryl and Cry9), and 60-80 kDa for Coleoptera-active Cry3 proteins and Lepidoptera / Diptera-active Cry2 proteins. Upon solubilization of the crystal by a susceptible insect, the released protoxin is processed in the insect gut by proteases (e.g., trypsin and chymotrypsin) to produce a protease-resistant core Cry protein toxin. This proteolytic processing involves the removal of amino acids from different regions of the various Cry protoxins. For example, a 130-140 kDa Cry protoxin is typically activated by proteolytic removal of a 25-30 amino acid N-terminal peptide and about half of the remaining protein from the C-terminus, resulting in a mature Cry toxin of about 60-70 kDa. The 60-80 kDa protoxins (e.g., Cry2 and Cry3) are also processed, but to a different extent than the larger protoxins. The smaller protoxins typically remove the same or more amino acids from the N-terminus than the larger protoxins, but fewer amino acids from the C-terminus. For example, proteolytic activation of Cry2 family members typically involves removal of about 40-50 N-terminal amino acids. Many of the Cry proteins are quite toxic to a specific target insect, but many have a narrow activity spectrum.

[0005] Cry proteins generally have five conserved sequence domains and three conserved structural domains (see, e.g., de Maagd et al. (2001) Trends Genetics 17:193-199). The first conserved structural domain (referred to as Domain I) typically consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II typically consists of three beta-sheets arranged in a Greek key configuration, and Domain III typically consists of two anti-parallel beta-sheets in a "jelly-roll" conformation (de Maagd et al. 2001, supra). Domains II and III are involved in receptor recognition and binding, and are thus believed to be determinants of toxin specificity.

[0006] Numerous commercially valuable plants, including common agricultural crops, are susceptible to attack by plant pests, including insect and nematode pests, resulting in significant decreases in crop yield and quality. For example, plant pests are a major factor in the loss of important agricultural crops worldwide. In China, approximately 15-20% of the harvestable grain is lost each year to insect pests and disease. Additionally, in the United States alone, approximately $8 billion is lost each year due to infestation by invertebrate pests, including insects. Insect pests are also a burden to vegetable and fruit growers, ornamental flower producers, and home gardeners.

[0007] Insect pests are primarily controlled by intensive application of chemical pesticides, which are active by inhibiting insect growth, preventing insect feeding or reproduction, or causing death. Biological pest control agents, such as Bacillus thuringiensis strains that express pesticidal toxins (e.g., Cry proteins), have also been applied to crop plants with satisfactory results, providing an alternative or supplement to chemical pesticides. Genes encoding some of these Cry proteins have been isolated, and their expression in heterologous hosts (e.g., transgenic plants) has shown to provide another tool for controlling economically important insect pests. Most Cry proteins are active against a very limited spectrum of insect pests. And typically, activity against one insect species is not predictive of activity against a different insect species.

[0008] The fall armyworm (also known as the common cutworm or the Oriental leafworm) is a lepidopteran pest belonging to the family Noctuidae. The fall armyworm is most commonly found in South Asia. However, its natural range also extends from the Oriental and Australasian regions to parts of the Palaearctic region. Countries with the most widely distributed fall armyworm populations include, but are not limited to, China, Indonesia, India, Japan, and Malaysia. The range of the fall armyworm has also extended to non-native areas through international trade. The egg, larval, and / or pupal stages can be present in soil or vegetation being transported across regions. The pupae, in particular, can move long distances due to the relatively long pupation period. Fall armyworm larvae are polyphagous, with 389 host plants, including >30 cultivated crops, such as cotton, corn, soybean, peanut, vegetables, etc. They move in large groups from one host plant to another, causing significant economic losses to many field crops.

[0009] Therefore, good insect control can be achieved, in particular by using chemical pesticides, but in the past decades, the fall armyworm has developed high levels of resistance to insecticides and often has low susceptibility to Cry proteins, e.g., to CrylAc expressed in commercial transgenic cotton plants and to CrylAb expressed in commercial transgenic corn plants (Yinghua et al., 2017. Scientific Reports 7: 41577). Therefore, there is a need for new control methods using pesticidal proteins that can target the fall armyworm, in particular fall armyworm populations that have become resistant to chemical pesticides and those populations with low natural susceptibility to current Cry proteins. To date, there have been no reports of controlling the fall armyworm by using the Cry proteins or engineered Cry proteins of the present invention. SUMMARY

[0010] The present invention provides methods for controlling the fall armyworm and protecting crops, in particular corn, from economic damage caused by the fall armyworm. The present invention further relates to plants, in particular monocot plants, especially corn (Zea mays), stably transformed with a nucleic acid molecule encoding a Cry protein of the present invention, alone or in combination with other insecticidal proteins, for use in controlling or combating the fall armyworm. The present invention still further relates to the use of an insecticidal formulation comprising a Cry protein of the present invention for protecting plants from infestation by the fall armyworm. The present invention also relates to plants, in particular monocot plants, especially corn plants, which can be infested by the fall armyworm and are transformed with an expressible nucleic acid molecule encoding a Cry protein of the present invention to combat or control fall armyworm pest populations.

[0011] According to the present application, a method is provided to combat and / or control insects of the Spodoptera species, in particular Spodoptera litura (the common cutworm or the oriental leafworm), by a step of contacting these insects with a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-10 or an insecticidal fragment thereof.

[0012] Furthermore, according to the present application, the contacting step can be performed with an insecticidal composition comprising: a Cry protein of the present application or an insecticidal fragment thereof, and an acceptable agricultural carrier. Furthermore, the contacting of the insects can be with a plant, especially a monocot plant, particularly a maize plant, stably transformed with an expressible nucleic acid molecule encoding a Cry protein of the present application, such that the transformed plant expresses the Cry protein of the present application or an insecticidal fragment thereof in an amount effective to control the insects.

[0013] Furthermore, plants, especially monocot plants, particularly maize plants, infested with Spodoptera litura are protected from the continued economic damage from this insect by being stably transformed with a gene encoding a Cry protein of the present application.

[0014] Brief description of the sequences in the sequence listing

[0015] SEQ ID NO: 1 is the amino acid sequence of the Cry1 Ja (CryET4) protein.

[0016] SEQ ID NO: 2 is the amino acid sequence of the Cry1 Gb (BT29) protein.

[0017] SEQ ID NO: 3 is the amino acid sequence of the Cry1 Gb-Cry1 If (BT29-BT22) hybrid protein.

[0018] SEQ ID NO: 4 is the amino acid sequence of the Cry1 Gb-Cry1 Fa (BT29-Cry1 Fa) hybrid protein.

[0019] SEQ ID NO: 5 is the amino acid sequence of the Cry9Ca (BT51) protein.

[0020] SEQ ID NO: 6 is the amino acid sequence of the Cry9Ba (BT128) protein.

[0021] SEQ ID NO: 7 is the amino acid sequence of the Cry1 Ab-Cry1 Ca (H04) hybrid protein.

[0022] SEQ ID NO: 8 is the amino acid sequence of the Cry1 Be-Cry1 Ka-Cry1 Be (TIC867-23) hybrid protein.

[0023] SEQ ID NO: 9 is the amino acid sequence of the CrylBe2 (CryET54) protein.

[0024] SEQ ID NO: 10 is the amino acid sequence of the Cry2Aa (BT32) protein. DETAILED DESCRIPTION

[0025] This description is not intended to be a detailed catalog of all the different ways in which the application can be implemented, or all the features that can be added to the instant application. For example, features illustrated with respect to one embodiment can be combined with features illustrated with respect to other embodiments, and the description of features as applied to a specific embodiment is equally applicable to other embodiments. Thus, the following description is intended to be illustrative only and is not intended to be limiting in any way. Numerous variations and additions to the embodiments described can be made and will be apparent to those skilled in the art in light of the instant disclosure. Accordingly, the application as set forth does not intend to be limited to the embodiments described herein, but instead has the full scope defined by the language of the following claims.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0027] As used in this document and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a plant" is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth.

[0028] As used herein, the term "and / or," refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0029] The term "about" is used herein to mean approximately, around, in the region of, or in the vicinity of, when referring to a value. When the term "about" is used in association with a numerical value, it modifies that particular value, by extending the boundary by a margin above or below the stated numerical value. Generally, the term "about" is used herein to modify a numerical value to a margin above or below the stated value, to a variation of plus or minus 20% (preferably 10%) upward or downward. In relation to temperature, the term "about" means ± 1 °C, preferably ± 0.5°C. When the term "about" is used in the context of the present invention (e.g., in combination with a temperature or molecular weight value), the exact value (i.e., without "about") is preferred.

[0030] "Controlling" insects means inhibiting the ability of an insect pest to survive, grow, feed, or reproduce, or limiting insect-related damage or loss in a crop plant, or protecting the yield potential of a crop when grown in the presence of an insect pest, by a toxic effect. "Controlling" insects can or can not mean killing the insect, although it preferably means killing the insect.

[0031] The terms "comprising" or "including," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0032] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) means that the scope of a claim is intended to cover a composition or process which does not materially depart from the basic and novel aspects required by the claim. Thus, the term "consisting essentially of" is not intended to be equated with "comprising" when used in the claims of the present invention.

[0033] As used herein, the term "Cry protein" means an insecticidal protein that can occur in a crystalline form in Bacillus thuringiensis or related bacteria. The term "Cry protein" can refer to a protoxin form or any insecticidal fragment or toxin thereof.

[0034] "Delivering" a composition or toxic protein means that the composition or toxic protein is brought into contact with an insect, which facilitates oral uptake of the composition or toxic protein, resulting in a toxic effect and control of the insect. The composition or toxic protein can be delivered in a number of recognized ways, including but not limited to transgenic plant expression, formulated protein compositions, sprayable protein compositions, bait matrices, or any other recognized protein delivery system in the art.

[0035] "An amount effective to control insects" means a concentration of toxic protein that inhibits the ability of an insect to survive, grow, feed, or reproduce by toxic effect, or limits insect-related damage or loss in a crop plant, or protects the yield potential of a crop when grown in the presence of an insect pest. An amount effective to control insects can or can not mean killing the insect, although it preferably means killing the insect.

[0036] A "gene" is defined herein as a hereditary unit comprising one or more polynucleotides that occupies a specific location on a chromosome or plasmid and contains the genetic instructions for a particular characteristic or trait in an organism.

[0037] As used herein, "pesticidal," "insecticidal," and the like refer to the ability of a Cry protein of the present application to control a pest organism, or the amount of a Cry protein that can control a pest organism as defined herein. Thus, a pesticidal Cry protein can kill or inhibit the ability of a pest organism (e.g., an insect pest) to survive, grow, feed, or reproduce.

[0038] Nucleotides are referred to herein by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Likewise, amino acids are referred to by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0039] The present application is based on the results of toxicity assays performed by feeding Spodoptera eridania (common cutworm) artificial diets comprising purified Cry toxins, and surprisingly showing that certain Cry proteins are toxic to S. eridania (see Example 1). Thus, these active Bt proteins can be used to provide maximum protection against this important pest, and can prevent or reduce the development of insect resistance to Bt insecticidal formulations in the field.

[0040] The "Cry proteins" of the present application can be naturally occurring or engineered, and encompass full-length proteins (protoxins) having the amino acid sequence shown in any of SEQ ID NOS: 1-10 of the Sequence Listing, as well as any insecticidally active fragments thereof.

[0041] The present application also includes polynucleotides that are fragments of the polynucleotides encoding Cry protein protoxins. By "fragment" is meant a portion of the nucleotide sequence encoding a Cry protein. A fragment of the nucleotide sequence can encode a biologically active portion of a Cry protein, a so-called "toxin fragment," or it can be a fragment that can be used as a hybridization probe or PCR primer by using the methods disclosed below. Nucleic acid molecules that are fragments of the nucleotide sequence encoding a Cry protein comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 contiguous nucleotides, or up to the number of nucleotides present in the nucleotide sequences encoding full-length Cry proteins disclosed herein (e.g., 3519 nucleotides for SEQ ID NO: 1), depending on the intended use. By "contiguous" nucleotides is meant nucleotide residues that are immediately adjacent to each other. Some fragments of the nucleotide sequences of the present application will encode toxin fragments that retain the biological activity of the Cry protein, and thus retain insecticidal activity. By "retain insecticidal activity" is meant that the fragment will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 80% of the insecticidal activity of the Cry protein. Methods for measuring insecticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al., (1988) Biochem. J. 252:199-206; Marrone et al., (1985) J. of Economic Entomology 78:290-293; and U.S. Patent No. 5,743,477, all of which are incorporated herein by reference in their entirety.

[0042] Toxin fragments of the Cry proteins of the present application will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, and 450 contiguous amino acids, or up to the total number of amino acids present in the full-length Cry proteins of the present application.

[0043] As used herein, a Cry protein that is "toxic" to an insect pest means that the Cry protein functions as an orally active insect control agent to kill the insect pest, or the Cry protein is capable of disrupting or preventing insect feeding or causing growth inhibition of the insect pest, either of which can or can not result in the death of the insect. When a Cry protein of the present application is delivered to an insect or an insect comes into oral contact with the Cry protein, the result is typically the death of the insect, or the growth of the insect is slowed, or the insect stops feeding such that the insect can no longer access the source of the toxic Cry protein.

[0044] In some embodiments, the present application provides a method of inhibiting the growth of, or killing, a Spodoptera frugiperda pest, the method comprising contacting the Spodoptera frugiperda pest with a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-10, or an insecticidal fragment thereof.

[0045] In some embodiments, the present application provides a method for controlling a population of Spodoptera frugiperda pests, the method comprising contacting the population of pests with an insecticidally effective amount of a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-10, or an insecticidal fragment thereof.

[0046] In further embodiments of the present application, the Spodoptera frugiperda pest or population of pests is further contacted with a second insecticidal protein that is different from the Cry protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-10. In still other embodiments, the second insecticidal protein is selected from the group consisting of a Cry protein, a Vip protein, a protease inhibitor, a lectin, an alpha-amylase, and a peroxidase.

[0047] In other embodiments of the present application, the contacting step, wherein a Cry protein of the present application is contacted with a Spodoptera frugiperda pest, is carried out with a microorganism or a plant that expresses the protein or an insecticidal fragment thereof. In other embodiments, the plant is stably transformed with a nucleic acid molecule that encodes a Cry protein of the present application, or an insecticidal fragment thereof. In still other embodiments, the plant is a monocot or a dicot. In other embodiments, the monocot is a corn plant, or the dicot is a soybean plant.

[0048] In some embodiments, the present application provides a method for protecting a plant from Spodoptera frugiperda pest damage, the method comprising expressing in the plant or cells thereof an insecticidally effective amount of a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-10, or an insecticidal fragment thereof. In other embodiments, the plant is a monocot or a dicot. In still other embodiments, the monocot is a maize plant or the dicot is a soybean plant.

[0049] To be effective against Spodoptera frugiperda, the Cry protein is first orally ingested by the insect. However, the Cry protein can be delivered to the insect in a number of art- recognized ways. Means of orally delivering proteins to insects include, but are not limited to, (1) providing the protein in a transgenic plant, wherein the insect eats (ingests) one or more parts of the transgenic plant, thereby ingesting the polypeptide expressed in the transgenic plant; (2) providing the protein in a formulated protein composition that can be applied to or incorporated into, for example, an insect growth medium; (3) providing the protein in a protein composition that can be applied to a surface, such as spraying onto the surface of a plant part, which is then ingested by the insect when it eats one or more of the sprayed plant parts; (4) a bait matrix; or (5) any other art-recognized protein delivery system. Thus, any method of oral delivery to an insect can be used in the methods of the present application to deliver the toxic Cry proteins of the present application. In some particular embodiments, the Cry proteins of the present application are orally delivered to an insect, wherein the insect ingests one or more parts of a transgenic plant.

[0050] In other embodiments, the Cry proteins of the present application are orally delivered to an insect, wherein the insect ingests one or more parts of a plant that has been sprayed with a composition comprising the Cry proteins of the present application. Delivering the compositions of the present application to the surface of a plant can be performed by using any method known to one of skill in the art for applying a compound, composition, formulation, etc. to the surface of a plant. Some non-limiting examples of delivering to or contacting a plant or part thereof include spraying, dusting, drenching, spreading, misting, atomizing, broadcasting, soaking, soil injection, soil incorporation, wetting through (e.g., root, soil treatment), impregnation, drenching, coating, leaf or stem penetration, side application, or seed treatment, and the like, as well as combinations thereof. These and other procedures for contacting a plant or part thereof with a compound, composition, or formulation are well known to those of skill in the art.

[0051] In some embodiments of the application, the insecticidal Cry proteins of the application are expressed in higher organisms, such as plants. In this case, transgenic plants expressing effective amounts of the insecticidal proteins protect themselves from plant pests, such as insect pests. When a Spodoptera eridania larva begins to feed on such a transgenic plant, it ingests the expressed insecticidal Cry protein. This can prevent the insect from further biting into the plant tissue, or can even harm or kill the insect. The polynucleotide encoding the Cry protein of the application is inserted into an expression cassette, which is then stably integrated into the genome of the plant. In other embodiments, the polynucleotide is included in a non-pathogenic self-replicating virus. Plants transformed according to the application can be monocotyledonous or dicotyledonous plants, and include, but are not limited to, maize (Zea mays), soybean, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, oilseed rape, alfalfa, tobacco, peanut, vegetables (including sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, carrot, eggplant, cucumber, radish, spinach, potato, tomato, asparagus, onion, garlic, melon, pepper, celery, squash, pumpkin, bush squash), fruits (including apple, pear, sea buckthorn, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana), and specialty plants (e.g., Arabidopsis) and woody plants (e.g., conifers and deciduous trees). Preferably, the plants of the application are crop plants, such as maize, soybean, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, sugar beet, sugarcane, tobacco, barley, oilseed rape, and the like. Once the desired polynucleotide has been transformed into a particular plant species, it can be propagated in that species, or moved into other varieties of the same species (including, in particular, commercial varieties) by using traditional breeding techniques.

[0052] Polynucleotides encoding the Cry proteins of the application are expressed in transgenic plants, resulting in the biosynthesis of the encoded Cry proteins (in protoxin or toxin form) in the transgenic plants. In this manner, transgenic plants are produced that have enhanced yield protection in the presence of Spodoptera eridania population pressure. In order to express them in transgenic plants, the nucleotide sequences encoding the Cry proteins can require modification and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants can result from microbial nucleotide sequences having codons that are not favored in plants. It is known in the art that living organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in the application can be changed to conform to plant preferences, while maintaining the amino acids encoded thereby. Furthermore, high expression in plants (e.g., maize plants) is best achieved from coding sequences having at least about 35% GC content, or at least about 45% GC content, or at least about 50% GC content, or at least about 60% GC content. Microbial nucleotide sequences having low GC content can be poorly expressed in plants due to the presence of ATTTA motifs that can destabilize the message and AATAAA motifs that can result in inappropriate polyadenylation. Although certain gene sequences can be expressed adequately in both monocot and dicot plant species, the sequences can be modified to address the specific codon preferences and GC content preferences of monocots or dicots, as these preferences have been shown to be different (Murray et al., Nucl. Acids Res. 17:477-498 (1989)). In addition, the nucleotide sequences are screened for the presence of irregular splice sites that can result in truncation of the message. All changes that need to be made within the nucleotide sequences (e.g., those described above) are made by using techniques of site-directed mutagenesis, PCR, and synthetic gene construction (using methods described, for example, in U.S. Patent Nos. 5,625,136, 5,500,365, and 6,013,523).

[0053] For efficient translation initiation, the sequences adjacent to the initiating methionine can require modification. For example, they can be modified by inclusion of sequences known to be effective in plants. Joshi has proposed consensus sequences suitable for use in plants (NAR 15:6643-6653 (1987)). These consensus sequences are suitable for use with the nucleotide sequences of the application. The sequences are incorporated into constructs comprising the nucleotide sequences up to and including the ATG (while leaving the second amino acid unmodified), or alternatively up to and including the GTC following the ATG (with the possibility of modifying the second amino acid of the transgene).

[0054] Polynucleotide sequences encoding the Cry proteins of the present application can be operably fused to a wide variety of promoters for expression in plants, including constitutive promoters, inducible promoters, temporally-regulated promoters, developmentally-regulated promoters, chemically-regulated promoters, tissue-preferred promoters, and tissue-specific promoters, to make recombinant DNA molecules, i.e., chimeric genes. The choice of promoter will vary depending on the temporal and spatial requirements for expression and also depending on the target species. Thus, expression of the nucleotide sequences of the present application in leaves, in stalks or stems, in ears, in inflorescences (e.g., racemes, panicles, ears, etc.), in roots, or in seedlings is preferred. In many cases, however, protection against more than one type of insect pest is sought, and thus expression in multiple tissues is desirable. While many promoters from dicot plants have been shown to function in monocot plants and vice versa, it is desirable to select a dicot promoter for expression in dicot plants and a monocot promoter for expression in monocot plants. However, there is no limitation on the origin of the selected promoter; it is only necessary that it function to drive expression of the nucleotide sequence in the desired cell.

[0055] Suitable constitutive promoters include, for example, the CaMV 35S promoter (SEQ ID NO: 1546; Odell et al., Nature 313:810-812, 1985); the Arabidopsis At6669 promoter (SEQ ID NO: 1652; see PCT Publication No. WO04081173A2); the maize Ubil (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); the rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81 :581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al., Plant J November, 2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); rice cyclophilin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231 :276-285, 1992); actin 2 (An et al., Plant J. 10(1), 107-121, 1996); the constitutive root tip CT2 promoter (SEQ ID NO: 1535; see also PCT Application No. IL / 2005 / 000627); and Synthetic Super MAS (Ni et al., The Plant Journal 7:661-76, 1995). Other constitutive promoters include those in U.S. Patent Nos. 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. Tissue-specific or tissue-preferred promoters useful for expressing the novel cry protein-encoding sequences of the application in plants, particularly maize, are those that direct expression in roots, pith, leaves, or pollen. Suitable tissue-specific promoters include, but are not limited to, leaf-specific promoters [e.g., by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol.35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993], seed preferred promoters [e.g., from seed-specific genes (Simon et al., Plant Mol. Biol. 5.191, 1985; Scofield et al., J. Biol. Chem. 262:12202, 1987; Baszczynski et al., Plant Mol. Biol. 14:633, 1990), Brazil nut albumin (Pearson' et al., Plant Mol. Biol. 18:235-245, 1992), conglutinin (Ellis et al., Plant Mol. Biol. 10:203-214, 1988), glutelin (rice) (Takaiwa et al., Mol. Gen. Genet. 208:15-22, 1986; Takaiwa et al., FEBS Letts. 221:43-47, 1987), zein (Matzke et al., Plant Mol Biol, 14(3), 323-32, 1990), napA (Stalberg et al., Planta 199:515-519, 1996), wheat SPA (Albani et al, Plant Cell, 9:171-184, 1997), sunflower oleosin (Cummins et al., Plant Mol. Biol. 19:873-876, 1992)], endosperm-specific promoters [e.g., wheat LMW and HMW, gliadin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b, and g hordeins (EMBO J. 3:1409-15, 1984), barley ltrl promoter, barley B1, C, D hordeins (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), barley DOF (Mena et al., The Plant Journal, 116(1):53-62, 1998), Biz2 (EP99106056.7), synthetic promoters (Vicente-Carbajosa et al., Plant J.13:629-640, 1998), rice prolamin NRP33, rice-globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), rice alpha-globulin REB / OHP-1 (Nakase et al., Plant Mol. Biol. 33:513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR gene family (Plant J 12:235-46, 1997), sorghum gamma-sorghum prolamin (Plant Mol. Biol 32:1029-35, 1996)], embryo-specific promoters [e.g., rice OSH1 (Sato et al., Proc. Natl. Acad. Sci. USA, 93:8117-8122), KNOX (Postma-Haarsma et al., Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et al., J. Biochem., 123:386, 1998)], flower-specific promoters [e.g., AtPRP4, chalcone synthase (chsA) (Van der Meer et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217:240-245, 1989), apetala-3], plant reproductive tissues [e.g., OsMADS promoter (U.S. Patent Application 2007 / 0006344)].

[0056] The nucleotide sequence can also be expressed under the control of a chemically regulated promoter. This allows the Cry protein of the invention to be synthesized only when the crop plant is treated with an inducing chemical. Examples of such techniques for chemical induction of gene expression are described in detail in published application EP 0 332 104 and U.S. Patent No. 5,614,395. In one embodiment, the chemically regulated promoter is the tobacco PR-1a promoter.

[0057] Another class of promoters useful in the present application are wound inducible promoters. Numerous promoters have been described that express at wound sites and also at sites of plant pathogen infection. Ideally, such promoters should only be active locally at the site of insect invasion and in this way, the insecticidal protein is only accumulated in the cells where it is needed to be synthesized to kill the invading insect pest. Examples of this class of promoters include those described by Stanford et al., MoI. Gen. Genet. 215:200-208 (1989); Xu et al., Plant Molec. Biol. 22:573-588 (1993); Logemann et al., Plant Cell 1 :151-158 (1989); Rohrmeier & Lehle, Plant Molec. Biol. 22:783-792 (1993); Firek et al., Plant Molec. Biol. 22:129-142 (1993); and Warner et al., Plant J. 3:191-201 (1993).

[0058] Non-limiting examples of promoters useful in the present application that elicit tissue-specific expression patterns include green tissue-specific promoters, root-specific promoters, stem-specific promoters, or flower-specific promoters. Promoters suitable for expression in green tissues include the promoters of many genes involved in photosynthesis, and many of these have been cloned from monocots and dicots. One such promoter is the maize PEPC promoter from the phosphoenolpyruvate carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)). Another promoter for root-specific expression is that described by de Framond (FEBS 290:103-106 (1991) or U.S. Patent No. 5,466,785). Another promoter useful in the present application is the stem-specific promoter described in U.S. Patent No. 5,625,136, which naturally drives expression of the maize trpA gene.

[0059] In addition to selecting an appropriate promoter, a construct for expressing an insecticidal toxin in a plant also requires a suitable transcription terminator operably linked downstream of the heterologous nucleotide sequence. Several such terminators are available and known in the art (e.g., tml from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of the present application.

[0060] Numerous other sequences can be incorporated into the expression cassettes described in the present application. These include sequences that have been shown to enhance expression, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0061] It can be preferred to target the expression of the nucleotide sequences of the present application to different cellular locations in plants. In some cases, localization in the cytosol can be desirable, while in other cases, localization in some subcellular organelle can be preferred. Any mechanism for targeting gene products (e.g., in plants) can be used in the practice of the present application, and such mechanisms are known to exist in plants and the sequences that control their functioning have been characterized in some detail. Sequences that cause targeting of gene products to other cellular compartments have been characterized. Amino terminal sequences can be responsible for targeting a protein of interest to any cellular compartment, such as the vacuole, mitochondria, peroxisomes, protein bodies, endoplasmic reticulum, chloroplasts, amyloplasts, amyloids, protein bodies, apoplast, or the cell wall of a plant (e.g., Unger et al., Plant Molec. Biol. 13:411-418 (1989); Rogers et al., (1985) Proc. Natl. Acad. Sci. USA 82:6512-651; U.S. Patent No. 7,102,057; WO 2005 / 096704, all of which are incorporated herein by reference. Optionally, the signal sequence can be the N-terminal signal sequence from waxy, the N-terminal signal sequence from gamma-zein, a starch binding domain, a C-terminal starch binding domain, a chloroplast targeting sequence (which imports the mature protein into the chloroplast) (Comai et al., (1988) J. Biol. Chem. 263:15104-15109; van den Broeck et al., (1985) Nature 313:358-363; U.S. Patent No. 5,639,949), or a secretion signal sequence from aleurone cells (Koehler & Ho, Plant Cell 2:769-783 (1990)). Additionally, an amino terminal sequence in conjunction with a carboxy terminal sequence together are responsible for vacuolar targeting of gene products (Shinshi et al., (1990) Plant Molec. Biol. 14:357-368). In one embodiment, the signal sequence selected includes a known cleavage site, and the fusion constructed takes into account any amino acids after the cleavage site that are required for cleavage. In some cases, this requirement can be met by adding a small number of amino acids between the cleavage site and the ATG of the transgene or alternatively replacing some amino acids within the transgene sequence. These construction techniques are well known in the art and apply equally to any cellular compartment.

[0062] It will be appreciated that the mechanisms described above for cell targeting can be used not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters, to achieve specific cell targeting goals under the transcriptional regulation of a promoter having a different expression pattern than the expression pattern of the promoter from which the targeting signal is derived.

[0063] Procedures for transforming plants are well known in the art and described throughout the literature. Non-limiting examples of methods for plant transformation include transformation by bacterial-mediated nucleic acid delivery (e.g., via Agrobacterium), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microprojectile bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical), or biological mechanism that results in the introduction of nucleic acid into a plant cell, including any combination thereof. General guidelines known in the art regarding various plant transformation methods include Miki et al. (“Procedures for Introducing Foreign DNA into Plants”, in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0064] For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are suitable, while for direct gene transfer (e.g., particle bombardment, etc.), any vector is suitable and linear DNA comprising only the construct of interest can be used. In the case of direct gene transfer, transformation with a single DNA species or co-transformation (Schocher et al., Biotechnology 4: 1093-1096 (1986)) can be used. For both direct gene transfer and Agrobacterium-mediated transfer, transformation is typically (but not necessarily) performed with a selectable marker, which can be positive selection (phosphomannose isomerase), providing resistance to an antibiotic (kanamycin, hygromycin, or methotrexate) or a herbicide (glyphosate or phosphinothricin). However, the choice of selectable marker is not critical to the present application.

[0065] Agrobacterium-mediated transformation is a widely used method for transforming plants due to its high transformation efficiency and its broad availability to many different species. Agrobacterium-mediated transformation typically involves the transfer of a binary vector carrying the target exogenous DNA to a suitable Agrobacterium strain, which can depend on a complement of the vir gene carried by the host Agrobacterium strain on a co-resident Ti plasmid or on the chromosome (Uknes et al., (1993) Plant Cell 5:159-169). The transfer of recombinant binary vectors to Agrobacterium can be accomplished via a triparental mating procedure, using Escherichia coli carrying the recombinant binary vector and a helper E. coli strain carrying a plasmid capable of moving the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium via nucleic acid transformation. & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[0066] Both dicot and monocot plants can be transformed using Agrobacterium. Methods for Agrobacterium-mediated transformation of rice include well known methods for transformation of rice, such as those described in any of European Patent Application EP 1198985 Al; Aldemita and Hodges (Planta 199:612-617, 1996); Chan et al. (Plant Mol Biol 22(3):491-506, 1993); Hiei et al. (Plant J 6(2):271-282, 1994), the disclosures of which are incorporated herein by reference as if fully set forth. In the case of corn transformation, preferred methods are described in Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1):13-22, 2002), the disclosures of which are incorporated herein by reference as if fully set forth. As examples, the methods are further described in B. Jenes et al., Techniques for Gene Transfer, in Transgenic Plants, Vol. 1, Engineering and Utilization, Eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143; and Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225. The nucleic acid or construct to be expressed is preferably cloned into a vector suitable for transformation of Agrobacterium tumefaciens, such as pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacterium transformed with such a vector can then be used in a known manner for the transformation of plants, for example model plants such as Arabidopsis or crop plants such as tobacco plants, for example by immersion of crushed or chopped leaves in a solution of the Agrobacterium and then cultivating them on a suitable medium.Plant transformation by means of Agrobacterium tumefaciens is described, for example, by Hagen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877, or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants, in Transgenic Plants, Vol. 1, Engineering and Utilization, Eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0067] Plant transformation by recombination Agrobacterium generally involves co-cultivation of Agrobacterium with explants from plants and follows methods well known in the art. Transformed tissues are regenerated on selection media, which carry antibiotic or herbicide resistance markers between the binary plasmid T-DNA borders.

[0068] As discussed previously, another method for transforming plants, plant parts, and plant cells involves propelling inert or biologically active particles into plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050; 5,036,006; and 5,100,792. Generally, this method involves propelling inert or biologically active particles into plant cells under conditions effective to penetrate the outer surface of the cell and provide incorporation into the interior of the cell. When inert particles are used, the vector can be introduced into the cell by coating the particles with a vector comprising the nucleic acid of interest. Alternatively, the cell can be surrounded by the vector, such that the vector is carried into the cell trailing the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria, or bacteriophage, each comprising one or more nucleic acids sought to be introduced) can also be propelled into plant tissues.

[0069] In other embodiments, the polynucleotide encoding a Cry protein of the application can be transformed directly into the plastid genome. The major advantage of plastid transformation is that plastids are generally able to express bacterial genes without significant modification, and that plastids are able to express multiple open reading frames under the control of a single promoter. Plastid transformation techniques are described in detail in U.S. Patent Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT Application No. WO 95 / 16783, and in McBride et al., (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves the introduction of a region of cloned plastid DNA flanking a selectable marker (along with the gene of interest) into suitable target tissue, for example, by using biolistics or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation). The flanking regions, of 1 to 1.5 kb, referred to as targeting sequences, promote homologous recombination with the plastid genome and thus allow replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin or streptomycin can be used as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M. and Maliga, P. (1992) Plant Cell 4, 39-45). The presence of a cloning site between these markers allows the creation of plastid targeting vectors for the introduction of foreign genes (Staub, J. M. and Maliga, P. (1993) EMBO J. 12, 601-606). A significant increase in transformation frequency can be obtained by replacing the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene, which encodes the spectinomycin detoxifying enzyme, aminoglycoside-3'-adenyltransferase (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker has been successfully used for high frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Other selectable markers useful for plastid transformation are known in the art and are included within the scope of the present application. Typically, about 15-20 cell divisions are required after transformation to reach a homoplasmonic state.Plastid expression, in which a gene is inserted into the circular plastid genome by homologous recombination, takes advantage of the large copy number of genes over nuclear expression, allowing expression levels that can easily exceed 10% of total soluble plant protein. In one embodiment, the polynucleotide of the application can be inserted into a plastid-targeted vector and transformed into the plastid genome of the desired plant host. Thus, a homoplasmic plant can be obtained with respect to the plastid genome comprising the nucleotide sequence of the application, which is capable of high expression of the polynucleotide.

[0070] Methods for selecting transformed transgenic plants, plant cells, or plant tissue cultures are routine in the art and can be used in the methods of the application provided herein. For example, the recombinant vectors of the application can further comprise an expression cassette comprising a nucleotide sequence for a selectable marker that can be used to select transformed plants, plant parts, or plant cells. As used herein, "selectable marker" means a nucleotide sequence that, when expressed, imparts a different phenotype to the plant, plant part, or plant cell expressing the marker and thus allows such transformed plants, plant parts, or plant cells to be distinguished from those plants, plant parts, or plant cells that do not have the marker. Such nucleotide sequences can encode a selectable marker or a screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, e.g., by using a selection reagent (e.g., an antibiotic, a herbicide, etc.), or whether the marker is simply a trait that can be identified by observation or testing, e.g., by screening (e.g., an R-locus trait). Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0071] Examples of selectable markers include, but are not limited to, nucleotide sequences encoding neo or nptll, which confer resistance to kanamycin, G418, and the like (Potrykus et al., (1985) Mol. Gen. Genet. 199: 183-188); nucleotide sequences encoding bar, which confer resistance to phosphinothricin; nucleotide sequences encoding an altered 5-enolpyruvate shikimate-3-phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al., (1988) Biotech. 6: 915-922); nucleotide sequences encoding nitrilase (e.g., bxn from Klebsiella ozaenae), which confers resistance to bromoxynil (Stalker et al., (1988) Science 242: 419-423); nucleotide sequences encoding an altered acetolactate synthase (ALS), which confers resistance to imidazolinone, sulfonylurea, or other chemicals that inhibit ALS (European Patent Application No. 154,204); nucleotide sequences encoding an antimethotrexate dihydrofolate reductase (DHFR) (Thillet et al., (1988) J. Biol. Chem. 263: 12500-12508); nucleotide sequences encoding a dalapon dehalogenase, which confers resistance to dalapon; nucleotide sequences encoding a mannose-6-phosphate isomerase (also known as phosphomannose isomerase (PMI)), which confers the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629); nucleotide sequences encoding an altered anthranilate synthase, which confers resistance to 5-methyltryptophan; or nucleotide sequences encoding hph, which confers resistance to hygromycin. Those skilled in the art are able to select a suitable selectable marker for use in the expression cassettes of the present application.

[0072] Additional selectable markers include, but are not limited to, a nucleotide sequence encoding β-glucuronidase, or uidA (GUS), which encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissue (Dellaporta et al., "Molecular cloning of the maize R-nj allele by transposon-tagging with Ac" 263-282, in Chromosome Structure and Function: Impact of New Concepts: 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); a nucleotide sequence encoding β-lactamase, an enzyme for which various chromogenic substrates (e.g., PADAC, chromogenic cephalosporins) are known (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE, which encodes catechol dioxygenase (Zukowsky et al., (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which then condenses to form melanin (Katz et al., (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding β-galactosidase, an enzyme for which chromogenic substrates exist; a nucleotide sequence encoding luciferase (lux), which allows bioluminescent detection (Ow et al., (1986) Science 234:856-859); a nucleotide sequence encoding aequorin, which can be used in calcium-sensitive bioluminescent detection (Prasher et al., (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al., (1995) Plant Cell Reports 14:403-406). Those of skill in the art are capable of selecting a suitable selectable marker for use in the expression cassettes of the present application.

[0073] In addition, as is well known in the art, whole transgenic plants can be regenerated from transformed plant cells, plant tissue cultures, or cultured protoplasts by using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue cultures, or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984) and Vol. II (1986)).

[0074] In addition, the genetic traits described above that are engineered into the transgenic seeds and plants, plant parts, or plant cells of the present application can be transmitted through sexual reproduction or vegetative growth, and thus can be maintained and propagated in subsequent generations of plants. Typically, maintenance and propagation utilizes known agricultural methods that have been developed for meeting specific purposes (e.g., harvesting, sowing, or tilling).

[0075] Accordingly, the polynucleotides can be introduced into a plant, plant part, or plant cell in any number of ways that are well known in the art, as described above. Thus, there is no dependency on a particular method for introducing the polynucleotide(s) into a plant, but rather any method that allows stable integration of the polynucleotide(s) into the plant genome can be used. When more than one polynucleotide is to be introduced, the respective polynucleotides can be assembled as part of a single nucleic acid molecule, or as separate nucleic acid molecules, and can be located on the same or different nucleic acid molecules. Thus, the polynucleotides can be introduced into the cell of interest in a single transformation event, in separate transformation events, or, for example, in the plant, as part of a breeding scheme.

[0076] In some embodiments, the present application provides a method of controlling Spodoptera frugiperda comprising contacting the S. frugiperda with a composition comprising a first insecticidal protein and a second pest control agent different from the first insecticidal protein, wherein the first insecticidal protein is a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-10. In other embodiments, the composition is a formulation for topical application to a plant. In still other embodiments, the composition is a transgenic plant. In further embodiments, the composition is a combination of a formulation for topical application to a transgenic plant. In some embodiments, the formulation comprises the first Cry protein of the present application when the transgenic plant comprises the second pest control agent. In other embodiments, the formulation comprises the second pest control agent when the transgenic plant comprises the first Cry protein of the present application.

[0077] In some embodiments, the second pest control agent can be an agent selected from the group consisting of a chemical pesticide, e.g., an insecticide, a Bacillus thuringiensis (Bt) insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Brevibacillus laterosporus insecticidal protein, a Bacillus sphaericus insecticidal protein, a protease inhibitor (both serine and cysteine types), a lectin, an alpha-amylase, a peroxidase, a cholesterol oxidase, and a double-stranded RNA (dsRNA) molecule.

[0078] In other embodiments, the second pest control agent is a chemical pesticide selected from the group consisting of pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrolides, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas, and juvenile hormone mimics. In other embodiments, the chemical pesticide is selected from the group consisting of abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, bifenazate, buprofezin, carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole, fenothiocarb, fenoxycarb, fenpropathrin, fenpyroximate, fenvalerate, fipronil, fonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophos, hexaflumuron, hydroprene, hydramethylnon, imidacloprid, indoxacarb, jasmolin I, jasmolin II, jasmolin, juvenile hormone I, juvenile hormone II, juvenile hormone III, kelevan, kinoprene, methacrifon, methamidophos, methomyl, methoprene, methoxychlor, metoflumizone, monocrotophos, naled, nithiazin, novaluron, noviflumuron, oxamyl, phorate, phosalone, phosmet, pirimicarb,halofenozide, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde, methamidophos, methidathion, methomyl, methoprene, methoxychlor, monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060), sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin, trichlorfon and triflumuron, aldicarb, oxamyl, fenamiphos, amitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor, etoxazole, fenazaquin,fenbutatin oxide, fenpropathrin, hexythiazox, propargite, pyridaben, and tebufenpyrad. In still other embodiments, the chemical pesticide is selected from the group consisting of: alpha-cypermethrin, beta-cyfluthrin, cyhalothrin, and lambda-cyhalothrin, S- cypermethrin, cypermethrin, esfenvalerate, fenothiocarb, fenoxycarb, furathiocarb, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, avermectin, emamectin, endosulfan, ethiprole, fipronil, flufenoxuron, hexaflumuron, hydroprene, pyriproxyfen, pymetrozine, and amitraz.

[0079] In additional embodiments, the second pest control agent can be one or more of any number of Bacillus thuringiensis insecticidal proteins, including but not limited to Cry proteins, vegetative insecticidal proteins (VIPs), and insecticidal chimeras of any of the foregoing. In other embodiments, the second pest control agent is a Cry protein selected from the group consisting of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Af, Cry1Ag, Cry1Ah, Cry1Ai, Cry1Aj, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Bd, Cry1Be, Cry1Bf, Cry1Bg, Cry1Bh, Cry1Bi, Cry1Ca, Cry1Cb, Cry1Da, Cry1Db, Cry1Dc, Cry1Dd, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Gb, Cry1Gc, Cry1Ha, Cry1Hb, Cry1Hc, Cry1Ia, Cry1Ib, Cry1Ic, Cry1Id, Cry1Ie, Cry1If, Cry1Ig, Cry1Ja, Cry1Jb, Cry1Jc, Cry1Jd, Cry1Ka, Cry1La, Cry1Ma, Cry1Na, Cry1Nb, Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Aj, Cry2Ak, Cry2Al, Cry2Ba, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry4Aa, Cry4Ba, Cry4Ca, Cry4Cb, Cry4Cc, Cry5Aa, Cry5Ab, Cry5Ac, Cry5Ad, Cry5Ba, Cry5Ca, Cry5Da, Cry5Ea, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry7Ac, Cry7Ba, Cry7Bb, Cry7Ca, Cry7Cb, Cry7Da, Cry7Ea, Cry7Fa, Cry7Fb, Cry7Ga, Cry7Gb, Cry7Gc, Cry7Gd, Cry7Ha, Cry7Ia, Cry7Ja, Cry7Ka, Cry7Kb, Cry7La, Cry8Aa, Cry8Ab, Cry8Ac, Cry8Ad, Cry8Ba, Cry8Bb, Cry8Bc, Cry8Ca, Cry8Da, Cry8Db, Cry8Ea, Cry8Fa, Cry8Ga, Cry8Ha, Cry8Ia, Cry8Ib, Cry8Ja, Cry8Ka, Cry8Kb,Cry8La, Cry8Ma, Cry8Na, Cry8Pa, Cry8Qa, Cry8Ra, Cry8Sa, Cry8Ta, Cry9Aa, Cry9Ba, Cry9Bb, Cry9Ca, Cry9Da, Cry9Db, Cry9Dc, Cry9Ea, Cry9Eb, Cry9Ec, Cry9Ed, Cry9Ee, Cry9Fa, Cry9Ga, Cry10Aa, Cry11Aa, Cry11Ba, Cry11Bb, Cry12Aa, Cry13Aa, Cry14Aa, Cry14Ab, Cry15Aa, Cry16Aa, Cry17Aa, Cry18Aa, Cry18Ba, Cry18Ca, Cry19Aa, Cry19Ba, Cry19Ca, Cry20Aa, Cry20Ba, Cry21Aa, Cry21Ba, Cry21Ca, Cry21Da, Cry21Ea, Cry21Fa, Cry21Ga, Cry21Ha, Cry22Aa, Cry22Ab, Cry22Ba, Cry22Bb, Cry23Aa, Cry24Aa, Cry24Ba, Cry24Ca, Cry25Aa, Cry26Aa, Cry27Aa, Cry28Aa, Cry29Aa, Cry29Ba, Cry30Aa, Cry30Ba, Cry30Ca, Cry30Da, Cry30Db, Cry30Ea, Cry30Fa, Cry30Ga, Cry31Aa, Cry31Ab, Cry31Ac, Cry31Ad, Cry32Aa, Cry32Ab, Cry32Ba, Cry32Ca, Cry32Cb, Cry32Da, Cry32Ea, Cry32Eb, Cry32Fa, Cry32Ga, Cry32Ha, Cry32Hb, Cry32Ia, Cry32Ja, Cry32Ka, Cry32La, Cry32Ma, Cry32Mb, Cry32Na, Cry32Oa, Cry32Pa, Cry32Qa, Cry32Ra, Cry32Sa, Cry32Ta, Cry32Ua, Cry33Aa, Cry34Aa, Cry34Ab, Cry34Ac, Cry34Ba, Cry35Aa, Cry35Ab, Cry35Ac, Cry35Ba, Cry36Aa, Cry37Aa, Cry38Aa, Cry39Aa, Cry40Aa, Cry40Ba, Cry40Ca, Cry40Da, Cry41Aa, Cry41Ab, Cry41Ba, Cry42Aa, Cry43Aa, Cry43Ba, Cry43Ca, Cry43Cb, Cry43Cc,Cry44Aa, Cry45Aa, Cry46Aa, Cry46Ab, Cry47Aa, Cry48Aa, Cry48Ab, Cry49Aa, Cry49Ab, Cry50Aa, Cry50Ba, Cry51Aa, Cry52Aa, Cry52Ba, Cry53Aa, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa, Cry56Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba, Cry60Aa, Cry60Ba, Cry61Aa, Cry62Aa, Cry63Aa, Cry64Aa, Cry65Aa, Cry66Aa, Cry67Aa, Cry68Aa, Cry69Aa, Cry69Ab, Cry70Aa, Cry70Ba, Cry70Bb, Cry71Aa, Cry72Aa, and Cry73Aa.

[0080] In further embodiments, the second pest control agent is a Vip3 vegetative insecticidal protein selected from the group consisting of: Vip3Aal, Vip3Aa2, Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vip3Aa8, Vip3Aa9, Vip3AalO, Vip3Aal l, Vip3Aal2, Vip3Aal3, Vip3Aal4, Vip3Aal5, Vip3Aal6, Vip3Aal7, Vip3Aal8, Vip3Aal9, Vip3Aa20, Vip3Aa21, Vip3Aa22, Vip3Aa2, Vip3Aa24, Vip3Aa25, Vip3Aa26, Vip3Aa27, Vip3Aa28, Vip3Aa29, Vip3Aa30, Vip3Aa31, Vip3Aa32, Vip3Aa33, Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38, Vip3Aa39, Vip3Aa40, Vip3Aa41, Vip3Aa42, Vip3Aa43, Vip3Aa44, Vip3Abl, Vip3Ab2, Vip3Acl, Vip3Adl, Vip3Ad2, Vip3Ael, Vip3Afl, Vip3Af2, Vip3Af3, Vip3Agl, Vip3Ag2, Vip3Ag3 HM117633, Vip3Ag4, Vip3Ag5, Vip3Ahl, Vip3Bal, Vip3Ba2, Vip3Bbl, Vip3Bb2, and Vip3Bb3.

[0081] In a still further embodiment, the first Cry protein of the application and the second pest control agent are co-expressed in a transgenic plant. This co-expression of more than one pesticidally effective ingredient in the same transgenic plant can be achieved by genetically engineering the plant to comprise and express all the necessary genes. Alternatively, a first plant "parent 1" can be genetically engineered to express a Cry protein of the application. A second plant "parent 2" can be genetically engineered to express a second pest control agent. By crossing "parent 1" with "parent 2", a progeny plant is obtained that expresses all the genes introduced into "parent 1" and "parent 2".

[0082] In a further embodiment, the application provides a method of producing an anti-pest (e.g., anti-insect) transgenic plant, the method comprising introducing into a plant a polynucleotide, chimeric gene, recombinant vector, expression cassette, or nucleic acid molecule comprising a nucleotide sequence encoding a Cry protein of the application, wherein the nucleotide sequence is expressed in the plant, thereby conferring to the plant resistance to a Spodoptera frugiperda pest and producing an anti-insect transgenic plant. In some embodiments, the introducing is achieved by transforming the plant. In other embodiments, the introducing is achieved by crossing a first plant comprising a chimeric gene, recombinant vector, expression cassette, or nucleic acid molecule of the application with a different, second plant.

[0083] In some embodiments, the application includes a method of providing a farmer with means for controlling a lepidopteran pest, the method comprising supplying or selling to the farmer a plant material, e.g., a seed, comprising a polynucleotide, chimeric gene, expression cassette, or recombinant vector capable of expressing a Cry protein of the application in a plant grown from the seed, as described above.

[0084] Embodiments of the application can be better understood by reference to the following examples. The foregoing and following description of embodiments of the application and various embodiments are not intended to limit the claims but merely to exemplify them. Thus, it will be understood that the claims are not limited to the specific details of these examples. Other embodiments of the application can be practiced without departing from the spirit and scope of the disclosure, which is defined by the appended claims. Example

[0085] Example 1. Activity of Cry proteins against Spodoptera frugiperda

[0086] In artificial diet bioassays, Cry proteins comprising the amino acid sequences of SEQ ID NOs: 1-10 were tested against the Chinese population of the common cutworm, a crop pest in the family Noctuidae (CN-CCW; Spodoptera litura). As shown in Table 1, these Cry proteins have been previously described.

[0087] Table 1. References for Cry protein disclosures

[0088] Cry protein SEQ ID NO: Publication No. CryET4 1 WO199504146 BT29 2 WO2017003811 BT29-BT22 3 WO2018111553 BT29-Cry1Fa 4 WO2018111553 BT51 5 WO2016094159 BT128 6 WO2016094159 H04 7 WO199506730 TIC867-23 8 WO2016061391 CryET54 9 WO200119859 BT32 10 WO2017007679

[0089] Briefly, equal amounts of protein in solution were applied to the surface of artificial insect diets in multi-well plates. After the diet surface dried, CN-CCW larvae were added to each well. The plates were sealed and maintained under ambient laboratory conditions (with respect to temperature, light, and relative humidity). Positive control groups consisted of larvae exposed to a known CN-CCW active Cry protein. Negative control groups consisted of larvae exposed to insect diets treated with buffer solution only and larvae on untreated insect diets; i.e., diets alone. Mortality was assessed after approximately 3-4 days and scored relative to controls.

[0090] Results of the CN-CCW bioassays are shown in column 3 of Table 2, where "-" indicates no activity compared to controls, "+ / -" indicates 0-10% activity compared to controls (this category also includes 0% mortality with strong larval growth inhibition), "+" indicates 10-25% activity compared to controls, "++" indicates 25-75% activity compared to controls, and "+++" indicates 75-100% activity compared to controls. Also shown in Table 2 is an indication of the activity of the Cry proteins against three North American pest insect species in the family Noctuidae, including the black cutworm (BCW; Agrotis ipsilon), the fall armyworm (FAW; Spodoptera frugiperda), and the corn earworm (CEW; Helicoverpa zea). For these three insect species, activity is simply indicated as "+" or "-" without a percentage of mortality indicated (based on published data). Cells labeled "nt" indicate that no published information is available indicating that the Cry protein has been tested against that pest species. These results demonstrate that bioactivity against insects in the same family as Spodoptera litura does not allow accurate prediction of the bioactivity of the Cry proteins against Spodoptera litura.

[0091] Table 2. Results of CN-CCW bioassays with Cry proteins

[0092]

[0093] Example 2. Expression and activity of Cry proteins in maize plants

[0094] Transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19:798-803. Briefly, Agrobacterium strain LBA4404 (pSB1) was transformed with an expression vector comprising two expression cassettes, wherein the first expression cassette comprises a plant-expressible promoter operably linked to a Cry protein-encoding sequence operably linked to a terminator, and the second expression cassette comprises a plant-expressible promoter operably linked to a selectable marker operably linked to a terminator. Expression of the selectable marker allows for the identification of transgenic plants on selection media. Both expression cassettes were cloned into a suitable vector for Agrobacterium-mediated transformation of rice or maize. The transformed Agrobacterium strain was grown on YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), 15 g / L agar, pH 6.8) solid medium at 28°C for 2-4 days. Approximately 0.8 x 10 9 Approximately 0.8 x 10

[0095] Immature embryos from inbred maize lines were excised from 8-12 day old ears into liquid LS-inf + 100 μΜ As. Embryos were rinsed once with fresh infection medium. Then, Agrobacterium solution was added and embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. Then, embryos were transferred to LS As medium with scutellum side up and incubated in the dark for two to three days. Subsequently, approximately 20 to 25 embryos / dish were transferred to LSDc medium supplemented with cefotaxime (250 mg / l) and silver nitrate (1.6 mg / l) and incubated in the dark at approximately 28°C for 10 days.

[0096] Immature embryos that produced embryogenic callus were transferred to LSD1M0.5S medium. The cultures were selected on this medium for about 6 weeks with about 3 weeks of subculture steps. Surviving calli were transferred to Reg1 medium supplemented with mannose. After incubation in light (16 hours light / 8 hours dark regime), green tissue was transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Small plants were transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and allowed to grow in light. After about 2-3 weeks, plants were tested by PCR for the presence of the selectable marker gene and the Bt cry gene. Positive plants from the PCR assay were transferred to a greenhouse for further evaluation.

[0097] Transgenic plants were evaluated for copy number (determined by Taqman analysis), protein expression levels (determined by ELISA), and efficacy against the insect species of interest (in leaf excision bioassays). Specifically, plant tissue (leaves or tassels) from single copy events (V3-V4 stage) were excised and challenged with neonate larvae of H. zea and then incubated at room temperature for 5 days. Results from the transgenic plant tissue bioassays will confirm that the Cry proteins of the present invention are toxic to H. zeta when expressed in transgenic plants. SEQUENCE LISTING <110> SYNGENTA BIOTECH (CHINA) CO., LTD. SYNGENTA CROP PROTECTION LLC <120> Control of Helicoverpa zeta <130> 82021-WO-REG-ORG-P-1 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 1167 <212> PRT <213> Bacillus thuringiensis <400> 1 Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser 1 5 10 15 Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30 Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45 Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60 Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu 65 70 75 80 Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95 Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala 100 105 110 Phe Lys Glu Trp Glu Ser Asp Pro Asp Asn Glu Ala Ala Lys Ser Arg 115 120 125 Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Ile Glu Ala Asn Ile 130 135 140 Pro Ser Phe Arg Ile Ile Gly Phe Glu Val Pro Leu Leu Ser Val Tyr 145 150 155 160 Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile 165 170 175 Phe Gly Glu Arg Trp Gly Leu Thr Thr Lys Asn Val Asn Asp lie Tyr 180 185 190 Asn Arg Gin lie Arg Glu lie His Glu Tyr Ser Asn His Cys Val Asp 195 200 205 Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser lie Ala Gin 210 215 220 Trp Arg lie Tyr Asn Gin Phe Arg Arg Glu Leu Thr Leu Thr Val Leu 225 230 235 240 Asp lie Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro lie 245 250 255 Gln Thr Phe Ser Gin Leu Thr Arg Glu lie Val Thr Ser Pro Val Ser 260 265 270 Glu Phe Tyr Tyr Gly Val lie Asn Ser Gly Asn lie lie Gly Thr Leu 275 280 285 Thr Glu Gin Gin lie Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300 Met lie Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly 305 310 315 320 Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gin Val Ser 325 330 335 Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350 Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365 Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380 Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr 385 390 395 400 Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415 Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430 Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445 Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460 Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro 465 470 475 480 Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495 Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gin Arg Tyr Arg 500 505 510 Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gin Phe Phe Thr Arg 515 520 525 Ile Asn Gly Thr Ser Val Asn Gin Gly Asn Phe Gin Arg Thr Met Asn 530 535 540 Arg Gly Asp Asn Leu Gin Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser 545 550 555 560 Thr Pro Phe Ser Phe Ser Asn Ala Gin Ser Thr Phe Thr Leu Gly Thr 565 570 575 Gln Ala Phe Ser Asn Gin Gin Val Tyr Ile Asp Arg Ile Gin Phe Val 580 585 590 Pro Ala Glu Val Thr Phe Gin Ala Gin Ser Asp Leu Gin Arg Ala Gin 595 600 605 Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gin Leu Gin Leu Gin 610 615 620 Thr Asp Val Thr Asp Tyr Gin Ile Asp Gin Val Ser Asn Leu Val Gin 625 630 635 640 Cys Leu Ser Asp Gin Phe Cys Leu Asp Gin Lys Arg Gin Leu Ser Gin 645 650 655 Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gin 660 665 670 Asp Pro Asn Phe Thr Ser Ile Asn Arg Gin Leu Asp Arg Gly Trp Arg 675 680 685 Gly Ser Thr Asp Ile Thr Ile Gin Gly Gly Asn Asp Val Phe Lys Glu 690 695 700 Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr 705 710 715 720 Leu Tyr Gin Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735 Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gin Asp Leu Glu Val Tyr Leu 740 745 750 Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765 Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780 Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys 785 790 795 800 Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815 Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830 Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845 Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860 Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu 865 870 875 880 Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895 Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910 Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925 Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940 Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp 945 950 955 960 Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975 Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990 Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005 Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020 Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035 Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050 Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065 Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080 Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095 Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110 Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125 Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140 Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155 Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165 <210> 2 <211> 1169 <212> PRT <213> Bacillus thuringiensis <400> 2 Met Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gln 20 25 30 Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe 35 40 45 Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu 65 70 75 80 Leu Ile Asn Gin Arg Ile Thr Glu Phe Ala Arg Gly Gin Ala Ile Gin 85 90 95 Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu 100 105 110 Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val 115 120 125 Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro 130 135 140 Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala 145 150 155 160 Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe 165 170 175 Gly Glu Arg Trp Gly Leu Thr Gin Thr Asn Ile Asn Asp Leu Tyr Ser 180 185 190 Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe 195 200 205 Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr 210 215 220 Arg Phe Gin Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu 225 230 235 240 Phe Pro Asn Tyr Asp lie Arg Thr Tyr Pro lie Pro Thr Lys Ser Gin 245 250 255 Leu Thr Arg Glu lie Tyr Thr Asp Pro lie lie Ser Pro Gly Ala Gin 260 265 270 Ala Gly Tyr Thr Leu Gin Asp Val Leu Arg Glu Pro His Leu Met Asp 275 280 285 Phe Leu Asn Arg Leu lie lie Tyr Thr Gly Glu Tyr Arg Gly lie Arg 290 295 300 His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr 305 310 315 320 Asn lie Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr 325 330 335 Arg Phe lie Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg 340 345 350 Thr Leu Ser Ala Pro lie Phe Arg Asp Glu Pro Gly Ala Asn lie lie 355 360 365 lie Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe lie Gin Pro 370 375 380 Asn Asn Gly Glu Gin Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu 385 390 395 400 Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His 405 410 415 Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro 420 425 430 Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala 435 440 445 Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu 450 455 460 Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Ile Val Lys Gly Pro 465 470 475 480 Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Val Gly Ser Phe 485 490 495 Gly Asp Met Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg 500 505 510 Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Tyr Thr Asn 515 520 525 Ile Asn Gly Thr Thr Ile Asn Ile Gly Asn Phe Ser Ser Thr Met Asp 530 535 540 Ser Gly Asp Asp Leu Gln Tyr Gly Arg Phe Arg Val Ala Gly Phe Thr 545 550 555 560 Thr Pro Phe Thr Phe Ser Asp Ala Met Ser Thr Phe Thr Ile Gly Ala 565 570 575 Phe Ser Phe Ser Ser Asn Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe 580 585 590 Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala 595 600 605 Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu 610 615 620 Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val 625 630 635 640 Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser 645 650 655 Glu Lys Val Lys His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu 660 665 670 Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp 675 680 685 Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys 690 695 700 Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr 705 710 715 720 Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg 725 730 735 Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr 740 745 750 Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr 755 760 765 Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly 770 775 780 Glu Pro Asn Arg Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp 785 790 795 800 Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe 805 810 815 Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly 820 825 830 Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu 835 840 845 Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu 850 855 860 Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys 865 870 875 880 Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val 885 890 895 Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr 900 905 910 Asn Ile Ala Ile Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg 915 920 925 Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala 930 935 940 Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr 945 950 955 960 Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser 965 970 975 Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His 980 985 990 Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu 995 1000 1005 Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala 1010 1015 1020 Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 1025 1030 1035 Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu 1040 1045 1050 Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala 1055 1060 1065 Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly 1070 1075 1080 Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr 1085 1090 1095 Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp 1100 1105 1110 Asn Thr Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu 1115 1120 1125 Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr 1130 1135 1140 Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile 1145 1150 1155 Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165 <210> 3 <211> 1169 <212> PRT <213> Artificial Sequence <220> <223> BT29-BT22 <400> 3 Met Glu lie Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 Asn Pro Glu Ser Glu lie Leu Asn Val Ala lie Phe Ser Ser Glu Gin 20 25 30 Val Ala Glu lie His Leu Lys lie Thr Arg Leu lie Leu Glu Asn Phe 35 40 45 Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu lie Trp Gly 50 55 60 lie Phe Asn Glu Asp Gin Trp Ser Ala Phe Leu Arg Gin Val Glu Glu 65 70 75 80 Leu lie Asn Gin Arg lie Thr Glu Phe Ala Arg Gly Gin Ala lie Gin 85 90 95 Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr lie Leu Ala Leu 100 105 110 Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val 115 120 125 Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro 130 135 140 Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala 145 150 155 160 Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe 165 170 175 Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser 180 185 190 Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe 195 200 205 Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr 210 215 220 Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu 225 230 235 240 Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln 245 250 255 Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln 260 265 270 Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp 275 280 285 Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg 290 295 300 His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr 305 310 315 320 Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr 325 330 335 Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg 340 345 350 Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile 355 360 365 Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro 370 375 380 Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu 385 390 395 400 Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His 405 410 415 Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro 420 425 430 Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala 435 440 445 Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu 450 455 460 Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro 465 470 475 480 Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe 485 490 495 Ala Phe Ser Asn Val Asn Leu Asp Trp Asn Leu Ser Gln Arg Tyr Arg 500 505 510 Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Met Tyr Val Thr 515 520 525 Ile Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asn 530 535 540 Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asp 545 550 555 560 Thr Ala Phe Thr Phe Pro Thr Lys Ala Ser Ser Leu Thr Val Gly Ala 565 570 575 Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu 580 585 590 Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Lys Ala 595 600 605 Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu 610 615 620 Lys Thr Asp Val Thr Asp Tyr His Ile Asp Lys Val Ser Asn Leu Val 625 630 635 640 Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser 645 650 655 Glu Lys Val Lys His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu 660 665 670 Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp 675 680 685 Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys 690 695 700 Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr 705 710 715 720 Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg 725 730 735 Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr 740 745 750 Leu lie Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr 755 760 765 Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro lie Gly Lys Cys Gly 770 775 780 Glu Pro Asn Arg Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp 785 790 795 800 Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe 805 810 815 Ser Leu Asp lie Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly 820 825 830 Val Trp Val lie Phe Lys lie Lys Thr Gin Asp Gly His Ala Arg Leu 835 840 845 Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu 850 855 860 Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys 865 870 875 880 Leu Glu Leu Glu Thr Asn lie Val Tyr Lys Glu Ala Lys Lys Ser Val 885 890 895 Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Arg Leu Gin Ala Asp Thr 900 905 910 Asn Ile Ala Ile Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg 915 920 925 Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala 930 935 940 Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr 945 950 955 960 Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser 965 970 975 Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His 980 985 990 Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu 995 1000 1005 Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala 1010 1015 1020 Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 1025 1030 1035 Glu Asp Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu 1040 1045 1050 Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala 1055 1060 1065 Thr Gin Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly 1070 1075 1080 Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr 1085 1090 1095 Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp 1100 1105 1110 Asn Thr Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu 1115 1120 1125 Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr 1130 1135 1140 Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile 1145 1150 1155 Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165 <210> 4 <211> 1169 <212> PRT <213> Artificial Sequence <220> <223> BT29-CrylFa <400> 4 Met Glu Ile Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 Asn Pro Glu Ser Glu Ile Leu Asn Val Ala Ile Phe Ser Ser Glu Gin 20 25 30 Val Ala Glu Ile His Leu Lys Ile Thr Arg Leu Ile Leu Glu Asn Phe 35 40 45 Leu Pro Gly Gly Ser Phe Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 Ile Phe Asn Glu Asp Gln Trp Ser Ala Phe Leu Arg Gln Val Glu Glu 65 70 75 80 Leu Ile Asn Gln Arg Ile Thr Glu Phe Ala Arg Gly Gln Ala Ile Gln 85 90 95 Arg Leu Val Gly Phe Gly Arg Ser Tyr Asp Glu Tyr Ile Leu Ala Leu 100 105 110 Lys Glu Trp Glu Asn Asp Pro Asp Asn Pro Ala Ser Lys Glu Arg Val 115 120 125 Arg Thr Arg Phe Arg Thr Thr Asp Asp Ala Leu Leu Thr Gly Val Pro 130 135 140 Leu Met Ala Ile Pro Gly Phe Glu Leu Ala Thr Leu Ser Val Tyr Ala 145 150 155 160 Gln Ser Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ala Val Phe Phe 165 170 175 Gly Glu Arg Trp Gly Leu Thr Gln Thr Asn Ile Asn Asp Leu Tyr Ser 180 185 190 Arg Leu Lys Asn Ser Ile Arg Asp Tyr Thr Asn His Cys Val Arg Phe 195 200 205 Tyr Asn Ile Gly Leu Gly Asn Leu Asn Val Ile Arg Pro Glu Tyr Tyr 210 215 220 Arg Phe Gln Arg Glu Leu Thr Ile Ser Val Leu Asp Leu Val Ala Leu 225 230 235 240 Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Pro Thr Lys Ser Gln 245 250 255 Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Ile Ser Pro Gly Ala Gln 260 265 270 Ala Gly Tyr Thr Leu Gln Asp Val Leu Arg Glu Pro His Leu Met Asp 275 280 285 Phe Leu Asn Arg Leu Ile Ile Tyr Thr Gly Glu Tyr Arg Gly Ile Arg 290 295 300 His Trp Ala Gly His Glu Val Glu Ser Ser Arg Thr Gly Met Met Thr 305 310 315 320 Asn Ile Arg Phe Pro Leu Tyr Gly Thr Ala Ala Thr Ala Glu Pro Thr 325 330 335 Arg Phe Ile Thr Pro Ser Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg 340 345 350 Thr Leu Ser Ala Pro Ile Phe Arg Asp Glu Pro Gly Ala Asn Ile Ile 355 360 365 Ile Arg Tyr Arg Thr Ser Leu Val Glu Gly Val Gly Phe Ile Gln Pro 370 375 380 Asn Asn Gly Glu Gln Leu Tyr Arg Val Arg Gly Thr Leu Asp Ser Leu 385 390 395 400 Asp Gln Leu Pro Leu Glu Gly Glu Ser Ser Leu Thr Glu Tyr Ser His 405 410 415 Arg Leu Cys His Val Arg Phe Ala Gln Ser Leu Arg Asn Ala Glu Pro 420 425 430 Leu Asp Tyr Ala Arg Val Pro Met Phe Ser Trp Thr His Arg Ser Ala 435 440 445 Thr Pro Thr Asn Thr Ile Asp Pro Asp Val Ile Thr Gln Ile Pro Leu 450 455 460 Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro 465 470 475 480 Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe 485 490 495 Ala Tyr Thr lie Val Asn lie Asn Gly Gin Leu Pro Gin Arg Tyr Arg 500 505 510 Ala Arg lie Arg Tyr Ala Ser Thr Thr Asn Leu Arg lie Tyr Val Thr 515 520 525 Val Ala Gly Glu Arg lie Phe Ala Gly Gin Phe Asn Lys Thr Met Asp 530 535 540 Thr Gly Asp Pro Leu Thr Phe Gin Ser Phe Ser Tyr Ala Thr lie Asn 545 550 555 560 Thr Ala Phe Thr Phe Pro Met Ser Gin Ser Ser Phe Thr Val Gly Ala 565 570 575 Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr lie Asp Arg Phe Gin Leu 580 585 590 lie Pro Val Thr Ala Thr Phe Gin Ala Glu Tyr Asp Leu Gin Lys Ala 595 600 605 Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gin lie Gly Leu 610 615 620 Lys Thr Asp Val Thr Asp Tyr His lie Asp Lys Val Ser Asn Leu Val 625 630 635 640 Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Gin Leu Ser 645 650 655 Glu Lys Val Lys His Ala Lys Arg Leu Cys Asp Glu Arg Asn Leu Leu 660 665 670 Gln Asp Pro Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp 675 680 685 Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys 690 695 700 Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr 705 710 715 720 Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg 725 730 735 Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr 740 745 750 Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr 755 760 765 Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly 770 775 780 Glu Pro Asn Arg Cys Ala Thr His Leu Glu Trp Asn Pro Asp Leu Asp 785 790 795 800 Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe 805 810 815 Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly 820 825 830 Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu 835 840 845 Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu 850 855 860 Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys 865 870 875 880 Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Lys Ser Val 885 890 895 Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr 900 905 910 Asn Ile Ala Ile Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg 915 920 925 Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala 930 935 940 Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Tyr Ser Leu Tyr 945 950 955 960 Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser 965 970 975 Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gin Asn Asn His 980 985 990 Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gin Gin 995 1000 1005 Val Arg Val Cys Pro Gly Arg Gly Tyr He Leu Arg Val Thr Ala 1010 1015 1020 Tyr Lys Gin Gly Tyr Gly Gin Gin Cys Val Thr He His Gin He 1025 1030 1035 Glu Asp Asn Thr Asp Gin Leu Gin Phe Ser Gin Cys Val Glu Gin 1040 1045 1050 Glu He Tyr Pro Gin Gin Thr Val Thr Cys Gin Asp Tyr Thr Ala 1055 1060 1065 Thr Gin Gin Gin Tyr Gin Gin Thr Tyr Thr Ser Gin Gin Gin Gly 1070 1075 1080 Tyr Asp Gly Ala Tyr Gin Gin Gin Ser Ser Val Pro Gin Gin Tyr 1085 1090 1095 Ala Ser Ala Tyr Gin Gin Gin Gin Gin Tyr Thr Asp Gly Arg Arg Gin 1100 1105 1110 Asn Thr Cys Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin 1115 1120 1125 Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr 1130 1135 1140 Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile 1145 1150 1155 Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165 <210> 5 <211> 660 <212> PRT <213> Bacillus thuringiensis <400> 5 Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His 1 5 10 15 Cys Gly Cys Pro Ser Asp Asp Asp Val Lys Tyr Pro Leu Ala Ser Asp 20 25 30 Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Met 35 40 45 Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Ile 50 55 60 Ser Gly Arg Asp Ala Val Gln Thr Ala Leu Thr Val Val Gly Arg Ile 65 70 75 80 Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gln Ile Val Ser Phe Tyr 85 90 95 Gln Phe Leu Leu Asn Thr Leu Trp Pro Val Asn Asp Thr Ala Ile Trp 100 105 110 Glu Ala Phe Met Arg Gln Val Glu Glu Leu Val Asn Gln Gln Ile Thr 115 120 125 Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp 130 135 140 Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp Leu Ala Asp Arg 145 150 155 160 Asn Asp Thr Arg Asn Leu Ser Val Val Arg Ala Gln Phe Ile Ala Leu 165 170 175 Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala Val Asn Gly Gln 180 185 190 Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Val Asn Leu His Leu 195 200 205 Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Glu Gly Trp Gly Phe Thr 210 215 220 Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg Gln Leu Glu Leu Thr Ala 225 230 235 240 Arg Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr Asn Thr Gly Leu Asp Arg 245 250 255 Leu Arg Gly Thr Asn Thr Glu Ser Trp Leu Arg Tyr His Gin Phe Arg 260 265 270 Arg Glu Met Thr Leu Val Val Leu Asp Val Val Ala Leu Phe Pro Tyr 275 280 285 Tyr Asp Val Arg Leu Tyr Pro Thr Gly Ser Asn Pro Gin Leu Thr Arg 290 295 300 Glu Val Tyr Thr Asp Pro Ile Val Phe Asn Pro Pro Ala Asn Val Gly 305 310 315 320 Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330 335 Glu Asn Ala Phe Ile Arg Pro Pro His Leu Phe Asp Arg Leu Asn Ser 340 345 350 Leu Thr Ile Ser Ser Asn Arg Phe Pro Val Ser Ser Asn Phe Met Asp 355 360 365 Tyr Trp Ser Gly His Thr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Ala 370 375 380 Val Gin Glu Asp Ser Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile 385 390 395 400 Asn Pro Gly Val Asp Gly Thr Asn Arg Ile Glu Ser Thr Ala Val Asp 405 410 415 Phe Arg Ser Ala Leu lie Gly lie Tyr Gly Val Asn Arg Ala Ser Phe 420 425 430 Val Pro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro Ala Asn Gly Gly 435 440 445 Cys Arg Asp Leu Tyr Asp Thr Asn Asp Glu Leu Pro Pro Asp Glu Ser 450 455 460 Thr Gly Ser Ser Thr His Arg Leu Ser His Val Thr Phe Phe Ser Phe 465 470 475 480 Gln Thr Asn Gln Ala Gly Ser lie Ala Asn Ala Gly Ser Val Pro Thr 485 490 495 Tyr Val Trp Thr Arg Arg Asp Val Asp Leu Asn Asn Thr lie Thr Pro 500 505 510 Asn Arg lie Thr Gin Leu Pro Leu Val Lys Ala Ser Ala Pro Val Ser 515 520 525 Gly Thr Thr Val Leu Lys Gly Pro Gly Phe Thr Gly Gly Gly lie Leu 530 535 540 Arg Arg Thr Thr Asn Gly Thr Phe Gly Thr Leu Arg Val Thr Val Asn 545 550 555 560 Ser Pro Leu Thr Gin Gin Tyr Arg Leu Arg Val Arg Phe Ala Ser Thr 565 570 575 Gly Asn Phe Ser Ile Arg Leu Leu Arg Gly Gly Val Ser Ile Gly Asp 580 585 590 Val Arg Leu Gly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu 595 600 605 Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro 610 615 620 Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gly 625 630 635 640 Val Ser Thr Gly Gly Glu Tyr Tyr Ile Asp Arg Ile Glu Ile Val Pro 645 650 655 Val Asn Pro Ala 660 <210> 6 <211> 1147 <212> PRT <213> Bacillus thuringiensis <400> 6 Met Asp Leu Asp Gly Asn Lys Thr Glu Thr Glu Thr Glu Ile Val Asn 1 5 10 15 Gly Ser Glu Ser Ser Ile Asp Pro Ser Ser Val Ser Tyr Ala Gly Asn 20 25 30 Asn Ser Tyr Ser Ser Ala Leu Asn Leu Asn Ser Cys Gln Asn Arg Gly 35 40 45 Ile Ala Gin Trp Val Asn Thr Leu Gly Gly Ala Ile Gly Gin Ala Val 50 55 60 Ser Ile Gly Thr Ser Ile Ile Ser Leu Leu Ala Ala Pro Thr Leu Thr 65 70 75 80 Gly Ser Ile Ser Leu Ala Phe Asn Leu Ile Arg Arg Met Gly Thr Gly 85 90 95 Ser Asn Gly Ser Ser Ile Ser Asp Leu Ser Ile Cys Asp Leu Leu Ser 100 105 110 Ile Ile Asn Leu Arg Val Ser Gin Ala Val Leu Asn Asp Gly Ile Ala 115 120 125 Asp Phe Asn Gly Ser Val Ala Val Tyr Asp Leu Tyr Leu His Ala Leu 130 135 140 Arg Ser Trp Asn Asn Asn Pro Asn Ala Ala Thr Ala Glu Glu Leu Arg 145 150 155 160 Thr Arg Phe Arg Ile Ala Asp Ser Glu Phe Glu Arg Ile Leu Thr Arg 165 170 175 Gly Ser Leu Thr His Gly Gly Ser Leu Ala Arg Gin Asp Ala Gin Val 180 185 190 Leu Leu Leu Pro Ser Phe Val Asn Ala Ala Tyr Leu His Leu Leu Ile 195 200 205 Leu Arg Asp Ala Ser Arg Tyr Gly Ala Ser Trp Gly Leu Phe Asn Thr 210 215 220 Thr Pro His Ile Asn Tyr Pro Val Arg Leu Gln Gln Leu Ile Gly Ser 225 230 235 240 Tyr Thr His Tyr Cys Thr His Trp Tyr Asn Gln Gly Leu Asn Glu Ile 245 250 255 Arg Gln Arg Gly Asn Thr Ala Val Asn Trp Leu Glu Phe His Arg Tyr 260 265 270 Arg Arg Asp Met Thr Leu Met Val Leu Asp Val Val Ser Leu Phe Ser 275 280 285 Ala Leu Asp Thr Ile Arg Tyr Pro Asn Ala Thr Val Val Gln Leu Ser 290 295 300 Arg Thr Val Tyr Thr Asp Pro Ile Gly Phe Val Asn Arg Gly Ser Gly 305 310 315 320 Asn Arg Leu Ser Trp Phe Asp Trp Arg Asn Gln Ala Asn Phe Ser Thr 325 330 335 Leu Glu Ser Glu Met Pro Thr Pro Ser Ser Pro Leu Ser Leu Asn His 340 345 350 Met Ser Ile Phe Thr Gly Pro Leu Thr Leu Pro Val Ser Pro Asn Thr 355 360 365 His Arg Ala Arg Val Trp Tyr Gly Asn Gln Asn Met Phe Thr Thr Gly 370 375 380 Ser Gln Asn Ser Gly Gln Thr Thr Asn Ser Ile Gln Asn Ile Ser Gly 385 390 395 400 Leu Glu Ile Phe Arg Ile Asp Ser Gln Ala Cys Asn Leu Asn Asn Asn 405 410 415 Ser Tyr Gly Val Asn Arg Ala Glu Phe Phe His Gly Ala Ser Gln Gly 420 425 430 Ser Gln Arg Ser Val Tyr Gln Gly Tyr Ile Arg Gln Ser Gly Leu Asp 435 440 445 Asn Pro Val Val Met Asn Leu Gln Ser Phe Leu Pro Gly Glu Asn Ser 450 455 460 Ala Thr Pro Thr Ala Gln Asp Tyr Thr His Ile Leu Ser Asn Pro Val 465 470 475 480 Asn Ile Arg Gly Gly Leu Arg Gln Ile Val Ala Asp Arg Arg Ser Ser 485 490 495 Val Val Val Tyr Gly Trp Thr His Lys Ser Leu Ser Arg Arg Ser Leu 500 505 510 Val Ala Pro Asp Gln Ile Thr Gln Val Pro Ala Val Lys Ala Ser Pro 515 520 525 Ser Ser His Cys Thr Ile Ile Ala Gly Pro Gly Phe Thr Gly Gly Asp 530 535 540 Leu Val Ser Leu Gln Pro Asn Gly Gln Leu Val Ile Pro Phe Gln Val 545 550 555 560 Ser Ala Pro Glu Thr Asn Tyr His Ile Arg Ile Cys Tyr Val Ser Thr 565 570 575 Ser Asp Cys Ser Ile Asn Thr Ile Cys Asn Asp Glu Thr His Leu Ser 580 585 590 Thr Leu Pro Ser Thr Thr Ser Ser Leu Glu Asn Leu Gln Cys Asn His 595 600 605 Leu His Tyr Phe Asn Val Gly Thr Phe Lys Pro Thr Ile Asp Ser Lys 610 615 620 Leu Thr Leu Val Asn Thr Ser Pro Asn Ala Asn Ile Ile Ile Asp Lys 625 630 635 640 Ile Glu Phe Ile Pro Val Asp Thr Ala Gln Gln Gln Asn Glu Asp Leu 645 650 655 Glu Ala Ala Lys Lys Ala Val Ala Ser Leu Phe Thr Arg Thr Arg Asp 660 665 670 Gly Leu Gln Val Asn Val Lys Asp Tyr Gln Val Asp Gln Ala Ala Asn 675 680 685 Leu Val Ser Cys Leu Ser Asp Glu Gin Tyr Gly Tyr Asp Lys Lys Met 690 695 700 Leu Leu Glu Ala Val Arg Ala Ala Lys Arg Leu Ser Arg Glu Arg Asn 705 710 715 720 Leu Leu Gin Asp Pro Asp Phe Asn Thr He Asn Ser Thr Glu Glu Asn 725 730 735 Gly Trp Lys Ala Ser Asn Gly Val Thr He Ser Glu Gly Gly Pro Phe 740 745 750 Tyr Lys Gly Arg Ala He Gin Leu Ala Ser Ala Arg Glu Asn Tyr Pro 755 760 765 Thr Tyr He Tyr Gin Lys Val Asp Ala Ser Glu Leu Lys Pro Tyr Thr 770 775 780 Arg Tyr Arg Leu Asp Gly Phe Val Lys Ser Ser Gin Asp Leu Glu He 785 790 795 800 Asp Leu He His His His Lys Val His Leu Val Lys Asn Val Pro Asp 805 810 815 Asn Leu Val Ser Asp Thr Tyr Pro Asp Asp Ser Cys Ser Gly He Asn 820 825 830 Arg Cys Gin Glu Gin Gin Met Val Asn Ala Gin Leu Glu Thr Glu His 835 840 845 His His Pro Met Asp Cys Cys Glu Ala Ala Gin Thr His Glu Phe Ser 850 855 860 Ser Tyr He Asp Thr Gly Asp Leu Asn Ser Ser Val Asp Gin Gly He 865 870 875 880 Trp Ala He Phe Lys Val Arg Thr Thr Asp Gly Tyr Ala Thr Leu Gly 885 890 895 Asn Leu Glu Leu Val Glu Val Gly Pro Leu Ser Gly Glu Ser Leu Glu 900 905 910 Arg Glu Gin Arg Asp Asn Thr Lys Trp Ser Ala Glu Leu Gly Arg Lys 915 920 925 Arg Ala Glu Thr Asp Arg Val Tyr Gin Asp Ala Lys Gin Ser He Asn 930 935 940 His Leu Phe Val Asp Tyr Gin Asp Gin Gin Leu Asn Pro Glu He Gly 945 950 955 960 Met Ala Asp He Met Asp Ala Gin Asn Leu Val Ala Ser He Ser Asp 965 970 975 Val Tyr Ser Asp Ala Val Leu Gin He Pro Gly He Asn Tyr Glu He 980 985 990 Tyr Thr Glu Leu Ser Asn Arg Leu Gin Gin Ala Ser Tyr Leu Tyr Thr 995 1000 1005 Ser Arg Asn Ala Val Gin Asn Gly Asp Phe Asn Asn Gly Leu Asp 1010 1015 1020 Ser Trp Asn Ala Thr Ala Gly Ala Ser Val Gin Gin Asp Gly Asn 1025 1030 1035 Thr His Phe Leu Val Leu Ser His Trp Asp Ala Gin Val Ser Gin 1040 1045 1050 Gln Phe Arg Val Gin Pro Asn Cys Lys Tyr Val Leu Arg Val Thr 1055 1060 1065 Ala Glu Lys Val Gly Gly Gly Asp Gly Tyr Val Thr Ile Arg Asp 1070 1075 1080 Asp Ala His His Thr Glu Thr Leu Thr Phe Asn Ala Cys Asp Tyr 1085 1090 1095 Asp Ile Asn Gly Thr Tyr Val Thr Asp Asn Thr Tyr Ile Thr Lys 1100 1105 1110 Glu Val Val Phe His Pro Glu Thr Gin His Met Trp Val Glu Val 1115 1120 1125 Asn Glu Thr Glu Gly Ala Phe His Leu Asp Ser Ile Glu Phe Val 1130 1135 1140 Glu Thr Glu Lys 1145 <210> 7 <211> 1193 <212> PRT <213> Artificial Sequence <220> <223> H04 <400> 7 Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu Met Arg lie Gin Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 lie Pro Leu Phe Ala Val Gin Asn Tyr Gin Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Val Gin Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175 Val Phe Gly Gin Arg Trp Gly Phe Asp Ala Ala Thr lie Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu lie Gly Asn Tyr Thr Asp His Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp lie Arg Tyr Asn Gin Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp lie Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro 245 250 255 lie Arg Thr Val Ser Gin Leu Thr Arg Glu lie Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gin Gly lie Glu 275 280 285 Gly Ser lie Arg Ser Pro His Leu Met Asp lie Leu Asn Ser lie Thr 290 295 300 Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg lie Val Ala 340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 Arg Pro Phe Asn lie Gly lie Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu lie Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser lie lie 435 440 445 Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Thr Leu Thr Asn 450 455 460 Thr Ile Asp Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe 465 470 475 480 Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln 500 505 510 Val Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg 515 520 525 Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala 530 535 540 Ser Thr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys 545 550 555 560 Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr 565 570 575 Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly 580 585 590 Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu 595 600 605 Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615 620 Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe 625 630 635 640 Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His 645 650 655 Ile Asp Gln Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys 660 665 670 Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg 675 680 685 Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile 690 695 700 Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile 705 710 715 720 Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Gln Gly 725 730 735 Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Pro Ile Asp Glu 740 745 750 Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr He Gin Gin 755 760 765 Asp Ser Gin Asp Leu Glu He Tyr Leu He Arg Tyr Asn Ala Lys His 770 775 780 Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala 785 790 795 800 Pro Ser Pro He Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His 805 810 815 Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys 820 825 830 Cys Ala His His Ser His His Phe Ser Leu Asp He Asp Val Gly Cys 835 840 845 Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val He Phe Lys He Lys 850 855 860 Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu 865 870 875 880 Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys 885 890 895 Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn He Val 900 905 910 Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln 915 920 925 Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala 930 935 940 Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser 945 950 955 960 Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg 965 970 975 Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn 980 985 990 Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val 995 1000 1005 Asp Val Glu Glu Gln Asn Asn His Arg Ser Val Leu Val Val Pro 1010 1015 1020 Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly 1025 1030 1035 Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly 1040 1045 1050 Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu 1055 1060 1065 Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn 1070 1075 1080 Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Glu Glu Tyr Glu 1085 1090 1095 Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr Glu 1100 1105 1110 Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu Glu 1115 1120 1125 Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn 1130 1135 1140 Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr 1145 1150 1155 Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu 1160 1165 1170 Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu 1175 1180 1185 Leu Leu Met Glu Glu 1190 <210> 8 <211> 1231 <212> PRT <213> Artificial Sequence <220> <223> TIC867-23 <400> 8 Met Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asn Leu Ser Thr Asp 20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp 35 40 45 Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Ile Ala Ser 65 70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr Arg Asp Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Asn Ser Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asp Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn 165 170 175 Gln Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln Val Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg Val Tyr Pro Met Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Val Ile Arg Pro Pro His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Phe Ser Val Leu Ser Arg Trp Ser Asn Thr Gln Tyr 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu Glu Ser Arg Thr Ile Arg Gly 355 360 365 Ser Leu Ser Thr Ser Thr His Gly Asn Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Phe 385 390 395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile Ser Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ser Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His 500 505 510 Thr Leu Gln Ser Gly Thr Thr Val Val Lys Gly Pro Gly Phe Thr Gly 515 520 525 Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Phe Ser Asn 530 535 540 Val Asn Leu Asp Phe Asn Leu Ser Gln Arg Tyr Arg Ala Arg Ile Arg 545 550 555 560 Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu 565 570 575 Arg Ile Phe Ala Gly Gln Phe Asp Lys Thr Met Asp Ala Gly Ala Pro 580 585 590 Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr 595 600 605 Phe Pro Glu Arg Ser Ser Ser Leu Thr Val Gly Ala Asp Thr Phe Ser 610 615 620 Ser Gly Asn Glu Val Tyr Val Asp Arg Phe Glu Leu Ile Pro Val Thr 625 630 635 640 Ala Thr Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln 645 650 655 Glu Ala Val Asn Ala Leu Phe Thr Asn Thr Asn Pro Arg Arg Leu Lys 660 665 670 Thr Gly Val Thr Asp Tyr His Ile Asp Glu Val Ser Asn Leu Val Ala 675 680 685 Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Leu Glu 690 695 700 Lys Val Lys Tyr Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln 705 710 715 720 Asp Pro Asn Phe Thr Ser Ile Asn Lys Gln Pro Asp Phe Ile Ser Thr 725 730 735 Asn Glu Gln Ser Asn Phe Thr Ser Ile His Glu Gln Ser Glu His Gly 740 745 750 Trp Trp Gly Ser Glu Asn Ile Thr Ile Gln Glu Gly Asn Asp Val Phe 755 760 765 Lys Glu Asn Tyr Val Ile Leu Pro Gly Thr Phe Asn Glu Cys Tyr Pro 770 775 780 Thr Tyr Leu Tyr Gin Lys lie Gly Glu Ala Glu Leu Lys Ala Tyr Thr 785 790 795 800 Arg Tyr Gin Leu Ser Gly Tyr lie Glu Asp Ser Gin Asp Leu Glu lie 805 810 815 Tyr Leu lie Arg Tyr Asn Ala Lys His Glu Thr Leu Asp Val Pro Gly 820 825 830 Thr Glu Ser Val Trp Pro Leu Ser Val Glu Ser Pro lie Gly Arg Cys 835 840 845 Gly Glu Pro Asn Arg Cys Ala Pro His Phe Glu Trp Asn Pro Asp Leu 850 855 860 Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His 865 870 875 880 Phe Ser Leu Asp lie Asp Val Gly Cys lie Asp Leu His Glu Asn Leu 885 890 895 Gly Val Trp Val Val Phe Lys lie Lys Thr Gin Glu Gly His Ala Arg 900 905 910 Leu Gly Asn Leu Glu Phe lie Glu Glu Lys Pro Leu Leu Gly Glu Ala 915 920 925 Leu Ser Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu 930 935 940 Lys Leu Gin Leu Glu Thr Lys Arg Val Tyr Thr Glu Ala Lys Glu Ala 945 950 955 960 Val Asp Ala Leu Phe Val Asp Ser Gin Tyr Asp Arg Leu Gin Ala Asp 965 970 975 Thr Asn Ile Gly Met Ile His Ala Ala Asp Lys Leu Val His Arg Ile 980 985 990 Arg Glu Ala Tyr Leu Ser Glu Leu Ser Val Ile Pro Gly Val Asn Ala 995 1000 1005 Glu Ile Phe Glu Glu Leu Glu Gly Arg Ile Ile Thr Ala Ile Ser 1010 1015 1020 Leu Tyr Asp Ala Arg Asn Val Val Lys Asn Gly Asp Phe Asn Asn 1025 1030 1035 Gly Leu Ala Cys Trp Asn Val Lys Gly His Val Asp Val Gin Gin 1040 1045 1050 Ser His His Arg Ser Val Leu Val Ile Pro Glu Trp Glu Ala Glu 1055 1060 1065 Val Ser Gin Ala Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu 1070 1075 1080 Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr 1085 1090 1095 Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Lys Asn 1100 1105 1110 Cys Glu Glu Glu Glu Val Tyr Pro Thr Asp Thr Gly Thr Cys Asn 1115 1120 1125 Asp Tyr Thr Ala His Gln Gly Thr Ala Ala Cys Asn Ser Arg Asn 1130 1135 1140 Ala Gly Tyr Glu Asp Ala Tyr Glu Val Asp Thr Thr Ala Ser Val 1145 1150 1155 Asn Tyr Lys Pro Thr Tyr Glu Glu Glu Thr Tyr Thr Asp Val Arg 1160 1165 1170 Arg Asp Asn His Cys Glu Tyr Asp Arg Gly Tyr Val Asn Tyr Pro 1175 1180 1185 Pro Val Pro Ala Gly Tyr Met Thr Lys Glu Leu Glu Tyr Phe Pro 1190 1195 1200 Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Lys 1205 1210 1215 Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1220 1225 1230 <210> 9 <211> 1227 <212> PRT <213> Bacillus thuringiensis <400> 9 Met Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asn Leu Ser Thr Asp 20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp 35 40 45 Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Ile Ala Ser 65 70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu His Val Glu His Leu Ile Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr Arg Asp Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Asn Ser Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asp Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn 165 170 175 Gln Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln Val Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg Val Tyr Pro Met Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Val Ile Arg Pro Pro His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Phe Ser Val Leu Ser Arg Trp Ser Asn Thr Gln Tyr 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu Glu Ser Arg Thr Ile Arg Gly 355 360 365 Ser Leu Ser Thr Trp Thr His Gly Asn Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Phe 385 390 395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile Ser Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ser Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ser Phe 500 505 510 Asn Leu Asn Ser Gly Thr Ser Val Val Ser Gly Pro Gly Phe Thr Gly 515 520 525 Gly Asp Ile Ile Arg Thr Asn Val Asn Gly Ser Val Leu Ser Met Gly 530 535 540 Leu Asn Phe Asn Asn Thr Ser Leu Gln Arg Tyr Arg Val Arg Val Arg 545 550 555 560 Tyr Ala Ala Ser Gln Thr Met Val Leu Arg Val Thr Val Gly Gly Ser 565 570 575 Thr Thr Phe Asp Gln Gly Phe Pro Ser Thr Met Ser Ala Asn Glu Ser 580 585 590 Leu Thr Ser Gln Ser Phe Arg Phe Ala Glu Phe Pro Val Gly Ile Ser 595 600 605 Ala Ser Gly Ser Gln Thr Ala Gly Ile Ser Ile Ser Asn Asn Ala Gly 610 615 620 Arg Gln Thr Phe His Phe Asp Lys Ile Glu Phe Ile Pro Ile Thr Ala 625 630 635 640 Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gin Glu Ala Val Asn 645 650 655 Ala Leu Phe Thr Asn Thr Asn Pro Arg Arg Leu Lys Thr Gly Val Thr 660 665 670 Asp Tyr His Ile Asp Glu Val Ser Asn Leu Val Ala Cys Leu Ser Asp 675 680 685 Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Leu Glu Lys Val Lys Tyr 690 695 700 Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe 705 710 715 720 Thr Ser Ile Asn Lys Gin Pro Asp Phe Asn Ser Asn Asn Glu Gin Ser 725 730 735 Asn Phe Thr Ser Ile His Glu Gin Ser Glu His Gin Trp Trp Gin Gin 740 745 750 Glu Asn Ile Thr Ile Gin Glu Gin Asn Asp Val Phe Lys Glu Asn Tyr 755 760 765 Val Thr Leu Pro Gly Thr Phe Asn Glu Cys Tyr Pro Thr Tyr Leu Tyr 770 775 780 Gln Lys Ile Gly Glu Ala Glu Leu Lys Ala Tyr Thr Arg Tyr Gin Leu 785 790 795 800 Ser Gly Tyr Ile Glu Asp Ser Gin Asp Leu Glu Ile Tyr Leu Ile Arg 805 810 815 Tyr Asn Ala Lys His Glu Thr Leu Asp Val Pro Gly Thr Glu Ser Val 820 825 830 Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu Pro Asn 835 840 845 Arg Cys Ala Pro His Phe Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys 850 855 860 Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp 865 870 875 880 Ile Asp Val Gly Cys Ile Asp Leu His Glu Asn Leu Gly Val Trp Val 885 890 895 Val Phe Lys Ile Lys Thr Gin Glu Gly His Ala Arg Leu Gly Asn Leu 900 905 910 Glu Phe Ile Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ser Arg Val 915 920 925 Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Gin Leu 930 935 940 Glu Thr Lys Arg Val Tyr Thr Glu Ala Lys Glu Ala Val Asp Ala Leu 945 950 955 960 Phe Val Asp Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Gly 965 970 975 Met Ile His Ala Ala Asp Lys Leu Val His Arg Ile Arg Glu Ala Tyr 980 985 990 Leu Ser Glu Leu Ser Val Ile Pro Gly Val Asn Ala Glu Ile Phe Glu 995 1000 1005 Glu Leu Glu Gly Arg Ile Ile Thr Ala Ile Ser Leu Tyr Asp Ala 1010 1015 1020 Arg Asn Val Val Lys Asn Gly Asp Phe Asn Asn Gly Leu Ala Cys 1025 1030 1035 Trp Asn Val Lys Gly His Val Asp Val Gln Gln Ser His His Arg 1040 1045 1050 Ser Val Leu Val Ile Pro Glu Trp Glu Ala Glu Val Ser Gln Ala 1055 1060 1065 Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala 1070 1075 1080 Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 1085 1090 1095 Glu Asn Asn Thr Asp Glu Leu Lys Phe Lys Asn Cys Glu Glu Glu 1100 1105 1110 Glu Val Tyr Pro Thr Asp Thr Gly Thr Cys Asn Asp Tyr Thr Ala 1115 1120 1125 His Gln Gly Thr Ala Val Cys Asn Ser Arg Asn Ala Gly Tyr Glu 1130 1135 1140 Asp Ala Tyr Glu Val Asp Thr Thr Ala Ser Val Asn Tyr Lys Pro 1145 1150 1155 Thr Tyr Glu Glu Glu Thr Tyr Thr Asp Val Arg Arg Asp Asn His 1160 1165 1170 Cys Glu Tyr Asp Arg Gly Tyr Val Asn Tyr Pro Pro Val Pro Ala 1175 1180 1185 Gly Tyr Met Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys 1190 1195 1200 Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Lys Phe Ile Val Asp 1205 1210 1215 Ser Val Glu Leu Leu Leu Met Glu Glu 1220 1225 <210> 10 <211> 633 <212> PRT <213> Bacillus thuringiensis <400> 10 Met Asn Ser Val Leu Asn Ser Gly Arg Thr Thr lie Cys Asp Ala Tyr 1 5 10 15 Asn Val Ala Ala His Asp Pro Phe Ser Phe Gin His Lys Ser Leu Asp 20 25 30 Thr Val Gin Lys Glu Trp Thr Glu Trp Lys Lys Asn Asn His Ser Leu 35 40 45 Tyr Leu Asp Pro lie Val Gly Thr Val Ala Ser Phe Leu Leu Lys Lys 50 55 60 Val Gly Ser Leu Val Gly Lys Arg lie Leu Ser Glu Leu Arg Asn Leu 65 70 75 80 lie Phe Pro Ser Gly Ser Thr Asn Leu Met Gin Asp lie Leu Arg Glu 85 90 95 Thr Glu Gin Phe Leu Asn Gin Arg Leu Asn Thr Asp Thr Leu Ala Arg 100 105 110 Val Asn Ala Glu Leu lie Gly Leu Gin Ala Asn lie Arg Glu Phe Asn 115 120 125 Gin Gin Val Asp Asn Phe Leu Asn Pro Thr Gin Asn Pro Val Pro Leu 130 135 140 Ser lie Thr Ser Ser Val Asn Thr Met Gin Gin Leu Phe Leu Asn Arg 145 150 155 160 Leu Pro Gin Phe Gin lie Gin Gly Tyr Gin Leu Leu Leu Leu Pro Leu 165 170 175 Phe Ala Gin Ala Ala Asn Leu His Leu Ser Phe lie Arg Asp Val lie 180 185 190 Leu Asn Ala Asp Glu Trp Gly lie Ser Ala Ala Thr Leu Arg Thr Tyr 195 200 205 Arg Asp Tyr Leu Arg Asn Tyr Thr Arg Asp Tyr Ser Asn Tyr Cys lie 210 215 220 Asn Thr Tyr Gin Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu His Asp 225 230 235 240 Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val 245 250 255 Ser lie Trp Ser Leu Phe Lys Tyr Gin Ser Leu Met Val Ser Ser Gly 260 265 270 Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gin Gin Thr Gin Ser Phe 275 280 285 Thr Ser Gin Asp Trp Pro Phe Leu Tyr Ser Leu Phe Gin Val Asn Ser 290 295 300 Asn Tyr lie Leu Ser Gly lie Ser Gly Thr Arg Leu Ser lie Thr Phe 305 310 315 320 Pro Asn Ile Gly Gly Leu Pro Gly Ser Thr Thr Thr His Ser Leu Asn 325 330 335 Ser Ala Arg Val Asn Tyr Ser Gly Gly Val Ser Ser Gly Leu Ile Gly 340 345 350 Ala Thr Asn Leu Asn His Asn Phe Asn Cys Ser Thr Val Leu Pro Pro 355 360 365 Leu Ser Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Thr Asp Arg 370 375 380 Glu Gly Val Ala Thr Ser Thr Asn Trp Gln Thr Glu Ser Phe Gln Thr 385 390 395 400 Thr Leu Ser Leu Arg Cys Gly Ala Phe Ser Ala Arg Gly Asn Ser Asn 405 410 415 Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu Val 420 425 430 Ile Arg Asn Glu Asp Leu Thr Arg Pro Leu His Tyr Asn Gln Ile Arg 435 440 445 Asn Ile Glu Ser Pro Ser Gly Thr Pro Gly Gly Ala Arg Ala Tyr Leu 450 455 460 Val Ser Val His Asn Arg Lys Asn Asn Ile Tyr Ala Ala Asn Glu Asn 465 470 475 480 Gly Thr Met Ile His Leu Ala Pro Glu Asp Tyr Thr Gly Phe Thr Ile 485 490 495 Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile 500 505 510 Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln Asn 515 520 525 Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn 530 535 540 Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val Thr 545 550 555 560 Ile Asn Gly Arg Val Tyr Thr Ala Thr Asn Val Asn Thr Thr Thr Asn 565 570 575 Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn Ile 580 585 590 Gly Asn Ile Val Ala Ser Ser Asn Ser Asp Val Pro Leu Asp Ile Asn 595 600 605 Val Thr Leu Asn Ser Gly Thr Gln Phe Asp Leu Met Asn Ile Met Leu 610 615 620 Val Pro Thr Asn Ile Ser Pro Leu Tyr 625 630

Claims

1. A method of inhibiting the growth of, or killing, a Spodoptera frugiperda (Sf) pest, the method comprising contacting the Sf pest with a Cry protein comprising the amino acid sequence set forth in SEQ ID NO:

3. Spodoptera litura ) 2. The method of claim 1, wherein the contacting step is performed with a microorganism or a plant expressing the protein.

3. The method of claim 2, wherein the plant is stably transformed with a DNA sequence encoding the protein.

4. The method of claim 3, wherein the plant is a monocot or a dicot.

5. The method of claim 4, wherein the monocot is a maize plant or the dicot is a soybean plant.

6. A method for controlling a Spodoptera litura pest population, the method comprising contacting the pest population with an insecticidally effective amount of a Cry protein comprising the amino acid sequence set forth in SEQ ID NO:

3.

7. The method of claim 1 or 6, wherein the Spodoptera litura pest or pest population is further contacted with a second insecticidal protein different from the Cry protein comprising the amino acid sequence set forth in SEQ ID NO:

3.

8. The method of claim 7, wherein the second insecticidal protein is selected from the group consisting of a Cry protein, a Vip protein, a protease inhibitor, a lectin, an alpha- amylase, and a peroxidase.

9. A method for protecting a plant from Spodoptera litura pests, the method comprising expressing in the plant or a cell thereof an insecticidally effective amount of a Cry protein comprising the amino acid sequence set forth in SEQ ID NO: 3.

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