CN113179823A - Control of black cutworms - Google Patents

Abstract

The present invention provides methods for controlling black cutworms (Agrotis ipsilon) and protecting crops, particularly corn, from economic damage caused by black cutworms. 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 black cutworm.

Description

Control of black cutworms

Technical Field

The present invention relates to methods for controlling or combating Trichoplusia (Agrotis), in particular Trichoplusia (Agrotis ipsilon) (Lepidoptera), Trichoplusia (Noctuidae) (also known as black cutworm (black cutworm)), 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

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming soil bacterium characterized by its ability to produce crystalline inclusions that are specifically toxic to certain orders and species of plant pests, including insects, but not harmful to plants and other non-target organisms. Thus, compositions comprising bacillus thuringiensis strains or insecticidal proteins thereof can be used as environmentally acceptable insecticides to control agricultural insect pests or insect vectors of a wide variety of human or animal diseases.

Crystal (Cry) proteins from bacillus thuringiensis have potent insecticidal activity against predominantly lepidopteran, dipteran, and coleopteran pest insects. These proteins also show activity against pests in the orders Hymenoptera (Hymenoptera), Homoptera (Homoptera), pediculoptera (phiraptera), Mallophaga (Mallophaga) and acarina (Acari) and other invertebrates such as phylum lineatum (Nemathelminthes), phylum Platyhelminthes and phylum sarcophaga (sarcophaga) (feitesson, j.1993.the Bacillus thuningiensis family tree, advanced Engineered pesticides. marcel Dekker, inc., New York, n.y.). These proteins were originally classified as CryI to CryVI, based primarily on their insecticidal activity. The main 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; within each family, more highly related proteins are assigned grouping letters, such as CryIA, CryIB, CryIC, and the like. Within each packet, the more closely related proteins are given names such as cryic (a), cryic (b), and the like. The terms "Cry toxin" and "delta-endotoxin" have been used interchangeably with the term "Cry protein". 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 (lowercase letter), and a fourth rank (another arabic numeral). In the current taxonomy, the roman numerals have been transposed to arabic numerals in the first level. For example, "CryIA (a)" under the old nomenclature is now "Cry 1 Aa" under the current nomenclature. According to Ibrahim et al (2010, bioenng. bugs,1:31-50), Cry toxins can still be classified into six major classes according to their insect host specificity, and include: group 1-lepidoptera (e.g., Cry1, Cry9, and Cry 15); group 2-lepidoptera and diptera (e.g., Cry 2); group 3 — coleoptera (Cry3, Cry7, and Cry 8); group 4-twin wings (Cry4, Cry10, Cry11, Cry16, Cry17, Cry19, and Cry 20); group 5-lepidopteran and coleopteran species (Cry 1I); and group 6-nematodes (Cry 6). The Cry1I, Cry2, Cry3, Cry10 and Cry11 toxins (73-82kDa) are unique in that they appear to be natural truncations of the larger Cry1 and Cry4 proteins (130-.

Cry proteins are globular protein molecules that accumulate as protoxins in crystal form during the sporulation phase of Bt. Upon ingestion by pests, the crystals typically dissolve to release protoxins which may range in size, for example, from 130-140kDa for many lepidopteran active Cry proteins (e.g., Cry1 and Cry9) and from 60-80kDa for coleopteran active Cry3 proteins and lepidopteran/dipteran active Cry2 proteins. After the crystals are solubilized by susceptible insects, the released protoxin is processed in the insect gut by proteases (e.g., trypsin and chymotrypsin) to produce protease-resistant core Cry protein toxins. This proteolytic processing involves the removal of amino acids from different regions of various Cry protoxins. For example, the 130-plus 140kDa Cry protoxin is typically activated by: proteolytically removes the 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 approximately 60-70 kDa. Protoxins of 60-80kDa (e.g., Cry2 and Cry3) are also processed, but to a different extent than the larger protoxins. Smaller protoxins typically remove the same or more amino acids from the N-terminus, but less amino acids from the C-terminus than larger protoxins. For example, proteolytic activation of members of the Cry2 family typically involves removal of approximately 40-50N-terminal amino acids. Many of the Cry proteins are quite toxic to specific target insects, but many have a narrow spectrum of activity.

Cry proteins typically have five conserved sequence domains and three conserved domains (see, e.g., de Maagd et al, (2001) Trends Genetics 17: 193-199). The first conserved domain (referred to as domain I) typically consists of seven alpha helices and involves membrane insertion and pore formation. Domain II typically consists of three β -sheets arranged in a greek key configuration, and domain III typically consists of two antiparallel β -sheets constructed in a "pancake-type" configuration (de Maagd et al, 2001, supra). Domains II and III are involved in receptor recognition and binding and are therefore considered determinants of toxin specificity.

Many commercially valuable plants, including common crops, are vulnerable to attack by plant pests, including insect and nematode pests, resulting in a significant reduction in crop yield and quality. For example, plant pests are a major cause of loss of important crops of the world. Approximately 15-20% of the harvestable grain is lost to insect pests and diseases each year in china. In addition, in the united states alone, there are approximately 80 billion dollars lost annually due to infestation by invertebrate pests (including insects). Insect pests are also a burden for vegetable and fruit growers, ornamental flower producers, and home gardeners.

Insect pests are controlled primarily 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 expressing pesticidal toxins (e.g., Cry proteins), have also been applied to crop plants with satisfactory results, providing alternatives or supplements to chemical pesticides. Genes encoding some of these Cry proteins have been isolated, and their expression in heterologous hosts (e.g., transgenic plants) has been 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 cannot be predicted against a different insect species.

Black cutworms, Agrotis ipsilon (Hufnagel) (lepidoptera; noctuidae), are key pests of cotton, maize, potatoes, kidney beans and other crops. Its long-range migratory behavior has allowed the species to spread throughout africa, europe, asia and america. Agrotis parvus is an important global maize pest present in every continent where maize is cultivated. Moths typically lay eggs in weeds, and when the weed host is destroyed, the larvae will switch from feeding on weeds to feeding on corn. Black cutworms can cause significant damage to unprotected corn fields by cutting off seedlings or digging channels into the base of older plants and destroying the growing point. Agrotis microti is known to be rather recalcitrant to Bt Cry proteins. However, a few commercial Bt maize events expressing Cry proteins were slightly effective against black cutworm; however, none provide complete control. For example, in the pure Cry1F maize line, the frequency of 5-10% of plants cut due to black cutworm is not surprising.

Good insect control can thus be achieved, in particular, by using chemical pesticides, but like other pests, black cutworms have the ability to develop high levels of resistance to insecticides and to certain Cry proteins. Thus, there is a need for new control methods that use pesticidal proteins that can target agrotis parthenocissus (particularly agrotis parthenocissus populations that have become resistant to chemical pesticides and particularly those that are resistant or recalcitrant to the current Cry proteins). To date, there has been no report of controlling black cutworm by using the Cry protein of the invention or an engineered Cry protein.

Summary of The Invention

The present invention provides methods for controlling black cutworms and protecting crops, particularly corn, from economic damage caused by black cutworms. The invention further relates to the use of plants, in particular monocotyledonous plants, in particular maize (Zea mays), stably transformed with a nucleic acid molecule encoding a Cry protein of the invention (alone or in combination with other insecticidal proteins) for controlling or combating agrotis microti. The invention further relates to the use of insecticidal preparations comprising a Cry protein according to the invention for protecting plants against attack by black cutworm. The invention also relates to plants, especially monocot plants, in particular maize plants, which can be infested with black cutworm and transformed with an expressible nucleic acid molecule encoding a Cry protein of the invention to combat or control black cutworm pest populations.

According to the present invention, there is provided a method for combating and/or controlling insects of the species Trichoplusia, in particular cutworm (black cutworm), by contacting these insects with a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 6 or an insecticidal fragment thereof.

Furthermore, according to the invention, said contacting step may be carried out with an insecticidal composition comprising: a Cry protein of the invention, or an insecticidal fragment thereof, and an acceptable agricultural carrier. Furthermore, the insect contact can be with a plant, particularly a monocot plant, particularly a maize plant, stably transformed with an expressible nucleic acid molecule encoding a Cry protein of the invention, such that the transformed plant expresses the Cry protein of the invention, or an insecticidal fragment thereof, in an amount effective to control insects.

Furthermore, plants, especially monocotyledonous plants, in particular maize plants, which are infested with agrotis microti, are protected from continuing economic damage from this insect by stable transformation with the genes encoding the Cry proteins of the invention.

Brief description of the sequences in the sequence listing

SEQ ID NO. 1 is the amino acid sequence of the Cry1Ig (BT25) protein.

SEQ ID NO. 2 is the amino acid sequence of Cry1Ja (CryET4) protein.

SEQ ID NO. 3 is the amino acid sequence of Cry1Bb-Cry1Ca-Cry1Ac (TIC860) hybrid Cry protein.

The SEQ ID NO. 4 is the amino acid sequence of Cry1Be-Cry1Ka-Cry1Ab (TIC867) hybrid Cry protein.

SEQ ID NO. 5 is the amino acid sequence of Cry1Be-Cry1Ca-Cry1Ab (TIC868) hybrid protein.

SEQ ID NO. 6 is the amino acid sequence of Cry1Be2(CryET54) protein.

Detailed Description

This description is not intended to be an exhaustive list of all the different ways in which the invention may be practiced or to add all the features of the invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the present invention contemplates that, in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. In addition, numerous variations and additions to the various embodiments set forth herein will be apparent to those skilled in the art in view of this disclosure without departing from the present invention. Accordingly, the following description is intended to illustrate certain specific embodiments of the invention and is not intended to be exhaustive or to limit all permutations, combinations and variations thereof.

Unless defined otherwise, 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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, 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.

As used herein, the word "and/or" means 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").

The term "about" is used herein to mean approximately, left-right, or near. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Generally, the term "about" is used herein to modify a numerical value above or below the stated value, with a 20% (preferably 10%) change up or down (higher or lower). With respect to temperature, the term "about" means ± 1 ℃, preferably ± 0.5 ℃. 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.

By "controlling" an insect is meant inhibiting the ability of an insect pest to survive, grow, feed or reproduce by toxic effects, or limiting insect-related damage or loss in crop plants, or protecting the yield potential of a crop when grown in the presence of an insect pest. "controlling" an insect may or may not mean killing the insect, although it preferably means killing the insect.

The terms "comprises" or "comprising," 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.

As used herein, the transitional phrase "consisting essentially of" (and grammatical variants) means that the scope of the claims is to be interpreted as encompassing the explicitly recited materials or steps recited in the claims, as well as those materials or steps that do not materially alter the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of …" is not intended to be construed as equivalent to "comprising" when used in the claims of this invention.

As used herein, the term "Cry protein" means an insecticidal protein that can appear in crystal form in bacillus thuringiensis or related bacteria. The term "Cry protein" can refer to the protoxin form or any insecticidal fragment or toxin thereof.

By "delivering" a composition or toxic protein is meant that the composition or toxic protein comes into contact with the insect, which facilitates oral ingestion of the composition or toxic protein, resulting in toxic effects and control of the insect. The composition or toxic protein may 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 art recognized protein delivery system.

By "effective insect controlling amount" is meant a concentration of a toxic protein that inhibits the ability of an insect to survive, grow, feed or reproduce by a toxic effect, or limits insect-related damage or loss in crop plants, or protects the yield potential of a crop when grown in the presence of an insect pest. An "effective insect controlling amount" may or may not mean killing the insect, although it preferably means killing the insect.

A "gene" is defined herein as a genetic unit comprising one or more polynucleotides, which occupy a specific position on a chromosome or plasmid, and which comprise genetic instructions for a specific feature or trait in an organism.

As used herein, "pesticidal", "insecticidal", and the like, refer to the ability of a Cry protein of the invention to control a pest organism, or can control the amount of a Cry protein of a pest organism as defined herein. Accordingly, the pesticidal Cry proteins can kill or inhibit the ability of a pest organism (e.g., an insect pest) to survive, grow, feed, or multiply.

Nucleotides are designated herein by the following standard abbreviations: adenine (a), cytosine (C), thymine (T) and guanine (G). Likewise, amino acids are designated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 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).

The present invention is based on the results of a toxicity assay performed by feeding cutworm black (black cutworm) with an artificial diet comprising purified Cry toxins derived from bacillus thuringiensis, and surprisingly shows that certain Cry proteins are toxic to cutworm (see example 1). Thus, these active Bt proteins can be used to provide maximum protection against the important pest and can prevent or reduce the development of insect resistance to Bt insecticidal formulations in the field.

The "Cry proteins" of the invention may be naturally occurring or engineered and encompass full length proteins (protoxins) having the amino acid sequence shown in any one of SEQ ID NOs: 1-6 of the sequence Listing, as well as any insecticidally active fragments thereof.

The invention also includes polynucleotides that are fragments of polynucleotides encoding Cry protein protoxins. By "fragment" is meant a portion of the nucleotide sequence encoding the Cry protein. A fragment of a nucleotide sequence may encode a biologically active portion of a Cry protein, a so-called "toxin fragment," or it may 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 a 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 consecutive nucleotides, or up to the number of nucleotides present in the nucleotide sequence encoding the full-length Cry protein disclosed herein (e.g., 3519 nucleotides for SEQ ID NO: 1), depending on the intended use. "contiguous" nucleotides means nucleotide residues that are immediately adjacent to each other. Some fragments of the nucleotide sequences of the present invention will encode toxin fragments that retain the biological activity of the Cry proteins, and thus, the insecticidal activity. By "retains insecticidal activity" is meant that the fragment will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, and 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, e.g., Czapla and Lang (1990) J.Econ.Entomol.83: 2480-2485; andrews et al, (1988) biochem. J.252: 199-206; marron et al, (1985) J.of Economic Entomogy 78: 290-293; and U.S. patent No. 5,743,477, all of which are incorporated herein by reference in their entirety.

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

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 that the Cry protein is capable of disrupting or arresting insect feeding or causing growth inhibition to the insect pest, both of which may or may not cause death of the insect. When a Cry protein of the invention is delivered to an insect or an insect comes into oral contact with said Cry protein, the result is typically death of said insect, or slow growth of said insect, or cessation of feeding by said insect makes available to said insect a source of said toxic Cry protein.

In some embodiments, the present invention provides methods of inhibiting the growth of or killing a black cutworm pest, comprising contacting the black cutworm pest with a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs 1-6 or an insecticidal fragment thereof.

In some embodiments, the present invention provides methods for controlling a black cutworm pest population comprising contacting the pest population with an insecticidally effective amount of a Cry protein or insecticidal fragment thereof comprising the amino acid sequence of any one of SEQ ID NOs 1-6.

In a further embodiment of the invention, the black cutworm pest or pest population 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 1-6. In still other embodiments, the second insecticidal protein is selected from the group consisting of: cry proteins, Vip proteins, protease inhibitors, lectins, alpha-amylases, and peroxidases.

In other embodiments of the invention, the contacting step, wherein the Cry protein of the invention is contacted with a black cutworm pest, is performed with a microorganism or plant that expresses the protein or insecticidal fragment thereof. In other embodiments, the plants are stably transformed with a nucleic acid molecule encoding a Cry protein of the invention, or an insecticidal fragment thereof. In yet other embodiments, the plant is a monocot or a dicot. In other embodiments, the monocot plant is a maize plant, or the dicot plant is a soybean plant.

In some embodiments, the present invention provides methods for protecting a plant from a black cutworm pest, comprising expressing in the plant or cell thereof an insecticidally effective amount of a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs 1-6 or an insecticidal fragment thereof. In other embodiments, the plant is a monocot or a dicot. In still other embodiments, the monocot plant is a maize plant, or the dicot plant is a soybean plant.

To be effective against black cutworm, the Cry proteins are first taken orally by the insects. However, the Cry proteins can be delivered to the insects in a number of recognized ways. Means for oral delivery of a protein to an insect 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 a 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, e.g., sprayed onto the surface of plant parts, and then ingested by the insect as the insect 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 insects can be used in the methods of the invention to deliver a toxic Cry protein of the invention. In some particular embodiments, a Cry protein of the invention is delivered orally to an insect, wherein the insect ingests one or more parts of a transgenic plant.

In other embodiments, a Cry protein of the invention is delivered orally to an insect, wherein said insect ingests one or more portions of a plant sprayed with a composition comprising a Cry protein of the invention. Delivery of the compositions of the present invention to the surface of the plant may be carried out by using any method known to those skilled in the art for applying compounds, compositions, formulations, etc. to the surface of the plant. Some non-limiting examples of delivery to or contact with a plant or portion thereof include spraying, dusting, spraying, spreading, misting, atomizing, broadcasting, soaking, soil injection, soil incorporation, drenching (e.g., root, soil treatment), dipping, pouring, cladding, leaf or stem infiltration, side application, or seed treatment, and the like, as well as combinations thereof. These and other procedures for contacting plants or parts thereof with compounds, compositions or formulations are well known to those skilled in the art.

In some embodiments of the invention, the insecticidal Cry proteins of the invention are expressed in higher organisms (e.g., plants). In this case, transgenic plants expressing an effective amount of an insecticidal protein protect themselves from plant pests, such as insect pests. When the black cutworm larvae begin to feed on such transgenic plants, they take up the expressed insecticidal Cry protein. This may prevent the insect from further biting into the plant tissue, or may even injure or kill the insect. The polynucleotide encoding the Cry protein of the invention 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 present invention may be monocotyledonous or dicotyledonous plants and include, but are not limited to, maize (maize), soybean, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugarbeet, cotton, sugarcane, oilseed rape, alfalfa, tobacco, peanuts, vegetables (including sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, kohlrabi, turnip, carrot, eggplant, cucumber, radish, spinach, potato, tomato, asparagus, onion, garlic, melon, capsicum, celery, squash, pumpkin), fruits (including apple, pear, quince, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana) and specialty plants (e.g., Arabidopsis (Arabidopsis)) and woody plants (e.g., coniferous trees and deciduous trees). Preferably, the plants of the invention are crop plants, such as maize, soybean, sorghum, wheat, sunflower, tomato, crucifers, capsicum, 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 to other varieties of the same species (including particularly commercial varieties) by using conventional breeding techniques.

The polynucleotides encoding the Cry proteins of the invention are expressed in transgenic plants, resulting in the biosynthesis of the Cry proteins encoded (in protoxin or toxin form) in said transgenic plants. In this way, transgenic plants with enhanced yield protection in the presence of agrotis pilosus population pressure were generated. In order to express them in transgenic plants, the nucleotide sequences encoding the Cry proteins may need to be modified and optimized. Although in many cases genes from microorganisms can be expressed at high levels in plants without modification, low expression in transgenic plants may be due to microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that living organisms have a specific preference for codon usage, and that the codons of the nucleotide sequences described in the present invention can be varied to fit the plant preference while retaining the amino acids encoded thereby. Furthermore, high expression in plants (e.g., maize plants) is optimally 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 with low GC content may be poorly expressed in plants due to the presence of ATTTA motifs which may destabilize the information and AATAAA motifs which may cause inappropriate polyadenylation. Although certain gene sequences can be expressed adequately in both monocot and dicot species, the sequences can be modified to address 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, nucleotide sequences are screened for the presence of an irregular splice site that may result in truncation of the message. All changes that need to be made within the nucleotide sequence (e.g., those described above) are made by techniques using well known site-directed mutagenesis, PCR, and synthetic gene construction (using methods such as those described in U.S. patent nos. 5,625,136, 5,500,365, and 6,013,523).

For efficient translation initiation, the sequence adjacent to the initiating methionine may need to be modified. For example, they may be modified by including sequences known to be effective in plants. Suitable consensus sequences for plants have been proposed by Joshi (NAR 15:6643-6653 (1987)). These consensus sequences are suitable for use with the nucleotide sequences of the present invention. The sequence is incorporated into a construct comprising the nucleotide sequence up to and including the ATG (with the second amino acid being unmodified), or alternatively up to and including the GTC following the ATG (with the possibility of modifying the second amino acid of the transgene).

The polynucleotide sequences encoding the Cry proteins of the invention 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 produce 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 invention in leaves, in stalks or stems, in ears, in inflorescences (e.g., panicles, cobs, etc.), in roots, or in seedlings is preferred. However, in many cases, it is desirable to seek protection against more than one type of insect pest, and thus expression in a variety of tissues. While many promoters from dicots have been shown to be functional in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots and monocot promoters for expression in monocots. However, there is no limitation on the origin of the promoter selected; they are sufficient to be operable in driving expression of the nucleotide sequence in the desired cell.

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. WO04081173A 2); maize Ubi 1(Christensen et al, Plant mol. biol.18:675-689, 1992); rice actin (McElroy et al, Plant Cell 2:163-171, 1990); pEMU (Last et al, the or. 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-; ubiquitin (Christensen et al, Plant mol. biol.18:675-689, 1992); rice cyclophilins (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. Pat. 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-preferential promoters useful for expressing the novel cry protein coding sequences of the invention 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., as described 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), Brazilian nut albumin (Pearson' et al, Plant Mol. Biol.18:235-245,1992), legumin (Ellis et al, Plant Mol. biol.10:203-214,1988), gluten (rice) (Takaiwa et al, Mol. Genet.208:15-22,1986; Takaiwa et al, FEke, BS letters 221:43-47,1987, Gen. (Gen.) Alkalwa et al, wheat gluten protein 8719: 11-11, wheat gluten protein 121; wheat gluten, wheat protein 121; wheat protein 121, wheat protein 102: 23, wheat protein 121; wheat protein; Skinra et al, wheat protein; Skinra, etc. (19, Skinra, Skin, wheat a, B and g gliadins (EMB03:1409-, 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 oleosins (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.t.217: 240-245,1989), apetala-3], Plant tissues [ e.g., OsMADS promoter (U.S. 2007/0006344.S. ].

The nucleotide sequence may also be expressed under the control of a chemically regulated promoter. This allows the Cry proteins of the invention to be synthesized only when the crop plants are treated with an induction chemical. Examples of such techniques for chemical induction of gene expression are described in detail in published application EP 0332104 and U.S. patent No. 5,614,395. In one embodiment, the chemically regulated promoter is a tobacco PR-1a promoter.

Another class of promoters useful in the present invention are wound-inducible promoters. Numerous promoters have been described that express at the site of a wound and also at the site of a plant pathogen infection. Ideally, such promoters should be active only locally at the insect infestation site, and in this way, the insecticidal proteins accumulate only in cells that need to synthesize the insecticidal proteins to kill the invading insect pest. Examples of promoters of this class include those described by: stanford et al, mol.Gen.Genet.215:200-208 (1989); xu et al, Plant mol.biol.22: 573-588 (1993); logemann et al, Plant Cell 1:151-158 (1989); rohrmeier & Lehle, Plant Molec.biol.22: 783. Bucking792 (1993); firek et al, Plant mol.biol.22: 129-142 (1993); and Warner et al, Plant J.3:191-201 (1993).

Non-limiting examples of promoters useful in the present invention that cause 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 promoters of many genes involved in photosynthesis regulation, and many of them have been cloned from monocotyledons and dicotyledons. One such promoter is the maize PEPC promoter from the phosphoenolcarboxylase gene (Hudspeth & Grula, Plant molecular. 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. Pat. No. 5,466,785). Another promoter useful in the present invention is the stem-specific promoter described in U.S. patent No. 5,625,136, which naturally drives expression of the maize trpA gene.

In addition to selecting an appropriate promoter, constructs for expressing insecticidal toxins in plants also require an appropriate transcription terminator to be 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 which is known to function in plants may be used in the context of the present invention.

Numerous other sequences can be incorporated into the expression cassettes described in the present invention. 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).

It may be preferred to target expression of the nucleotide sequences of the present invention to different cellular locations in a plant. In some cases, localization in the cytosol may be desirable, while in other cases, localization in some subcellular organelles may be preferred. Any mechanism for targeting gene products (e.g., in plants) can be used in the practice of the present invention, and such mechanisms are known to exist in plants and the sequences that control the functioning of these mechanisms have been characterized in greater detail. Sequences have been characterized that cause targeting of gene products to other cellular compartments. The amino-terminal sequence may be responsible for targeting the protein of interest to any cellular compartment, e.g., vacuole, mitochondria, peroxisomes, proplastids, endoplasmic reticulum, chloroplast, starch granules, amyloplasts, apoplast, or cell wall of a Plant (e.g., Unger et al, Plant mol. biol.13:411-418 (1989); Rogers et al, (1985) Proc. Natl. Acad. Sci. USA 82: 6512-651; U.S. Pat. No. 7,102,057; WO 2005/096704, all of which are incorporated herein by reference; optionally, the signal sequence may be an N-terminal signal sequence from waxy, an 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 chloroplasts (Comai et al, (1988) J.biol. 151m. 04: Cheden. 09; van Broeck et al, (1985) nature 313: 358-363; U.S. Pat. No. 5,639,949) or secretory signal sequences from aleurone cells (Koehler & Ho, Plant Cell 2:769-783 (1990)). In addition, the amino-terminal sequence together with the carboxy-terminal sequence is responsible for vacuolar targeting of the gene product (Shinshi et al, (1990) Plant mol. biol.14: 357-368). In one embodiment, the selected signal sequence 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 transgenic ATG or alternatively replacing some amino acids within the transgenic sequence. These construction techniques are well known in the art and apply equally to any cellular compartment.

It will be appreciated that the mechanisms described above for cell targeting may be used not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters, to affect specific cell targeting targets under the transcriptional regulation of promoters having an expression pattern different from that of the promoter from which the targeting signal is derived.

Procedures for transforming plants are well known in the art and are described throughout the literature. Non-limiting examples of methods for plant transformation include transformation by: bacteria-mediated nucleic acid delivery (e.g., via Agrobacterium), virus-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 for various Plant transformation Methods known in the art include Miki et al ("Procedures for Introducing DNA for insect 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 Rakowczy-Trojanowska (cell. mol. biol. Lett.7: 849-.

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

Agrobacterium-mediated transformation is a commonly used method for transforming plants because of its high transformation efficiency and because of its wide availability with many different species. Agrobacterium-mediated transformation typically involves transfer of a binary vector carrying the exogenous DNA of interest to a suitable Agrobacterium strain, which may rely on a complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or on a chromosome (Uknes et al, (1993) Plant Cell 5: 159-169). Transfer of the recombinant binary vector to agrobacterium may be accomplished by a triparental mating procedure using Escherichia coli (Escherichia coli) carrying the recombinant binary vector, a helper Escherichia coli strain carrying a plasmid capable of mobilizing the recombinant binary vector to the target agrobacterium strain. Alternatively, the recombinant binary vector may be transferred to Agrobacterium by nucleic acid transformation (A)

&Willmitzer(1988)Nucleic Acids Res.16:9877)。

Both dicotyledonous and monocotyledonous plants can be transformed by using Agrobacterium. Methods for agrobacterium-mediated transformation of rice include well-known methods for rice transformation, such as those described in any of the following: european patent application EP 1198985a 1; aldemita and Hodges (Planta 199: 612. sup. -. 617. sup.,1996); chan et al (Plant Mol Biol 22(3):491-506, 1993); hiei et al (Plant J6 (2):271-282,1994), the disclosure of which is incorporated by reference as if fully set forth. In the case of maize 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 disclosure of which is incorporated herein by reference as if fully set forth. As an example, the method is further described in the following documents: 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 mol. 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, for example pBin19(Bevan et al, Nucl. acids Res.12(1984) 8711). The agrobacterium transformed with such a vector can then be used in a known manner for the transformation of plants, for example plants used as models (e.g. arabidopsis thaliana) or crop plants (e.g. tobacco plants), for example by immersing the comminuted leaves or the minced leaves in an agrobacterium solution and then cultivating them in a suitable medium. Plant transformation with the aid 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 Ultilization, compiled in S.D.Kung and R.Wu, Academic Press,1993, pages 15-38.

Plant transformation by recombinant agrobacterium typically involves co-cultivation of agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissues, which carry antibiotic or herbicide resistance markers between the binary plasmid T-DNA borders, are regenerated on selection medium.

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

In other embodiments, the polynucleotides encoding the Cry proteins of the invention can be transformed directly into the plastid genome. The main advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without major modification, and plastids are capable of expressing multiple open reading frames under the control of a single promoter. Plastid transformation techniques are described extensively in U.S. Pat. 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-. The basic technique for chloroplast transformation involves introducing a region of cloned plastid DNA flanking a selectable marker (along with the gene of interest) into a suitable target tissue, for example, by using biolistic or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation). The 1 to 1.5kb flanking regions, called targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastid group (plastome). Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin or streptomycin could be used as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Malaga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-. The presence of cloning sites 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 gene with a dominant selectable marker (bacterial aadA gene, which encodes the spectinomycin detoxification enzyme, aminoglycoside-3' -adenylyl transferase) (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90,913 + 917). Previously, this marker has been successfully used for the high frequency transformation of the plastidic genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. acids Res.19: 4083-. Other selectable markers useful for plastid transformation are known in the art and are included within the scope of the invention. Typically, about 15-20 cell division cycles are required after transformation to achieve a homogeneous state. Plastid expression, in which a gene is inserted by homologous recombination into all the thousands of copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number of genes over nuclear expression, allowing expression levels that can easily exceed 10% of the total soluble plant protein. In one embodiment, the polynucleotides of the invention may be inserted into a plastid targeting vector and transformed into the plastid genome of the desired plant host. Thus, plants of the same type as the plastid genome comprising the nucleotide sequence of the invention are obtained, which are capable of high expression of the polynucleotide.

Methods of selecting transformed transgenic plants, plant cells, or plant tissue cultures are conventional in the art and may be used in the methods of the invention provided herein. For example, the recombinant vectors of the invention may also include 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, a "selectable marker" means a nucleotide sequence that, when expressed, confers a different phenotype on a 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 may encode a selectable or screenable marker, depending on whether the marker confers a trait that can be selected by chemical means, e.g., through the use of a selection agent (e.g., an antibiotic, 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 may be used among the expression cassettes described herein.

Examples of selectable markers include, but are not limited to: a nucleotide sequence encoding neo or nptII, which confers resistance to kanamycin, G418, etc. (Potrykus et al, (1985) mol.Gen.Genet.199: 183-188); a nucleotide sequence encoding bar which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase which confers resistance to glyphosate (Hinche et al, (1988) Biotech.6: 915-922); a nucleotide sequence encoding a nitrilase, such as bxn from Klebsiella pneumoniae (Klebsiella ozaenae), which confers resistance to bromoxynil (Stalker et al, (1988) Science 242: 419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinones, sulfonylureas, or other ALS-inhibiting chemicals (european patent application No. 154204); a nucleotide sequence encoding dihydrofolate reductase (DHFR) against methotrexate (Thillet et al (1988) J.biol.chem.263: 12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding mannose-6-phosphate isomerase (also known as phosphomannose isomerase (PMI)) which confers the ability to metabolize mannose (U.S. Pat. nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyltryptophan; or a nucleotide sequence encoding hph, which confers resistance to hygromycin. One skilled in the art will be able to select suitable selectable markers for use in the expression cassettes of the invention.

Additional selectable markers include, but are not limited to: a nucleotide sequence encoding a β -glucuronidase, or uida (gus), which encodes an enzyme for which various chromogenic substrates are known; nucleotide sequences of the R-locus which encode products which modulate the production of anthocyanidin pigment (red) in plant tissue (Dellaporta et al, "Molecular cloning of the mail R-nj insulator by transloson-tagging with Ac" 263-282, in Chromosome Structure and Function: Impact of New Concepts, 18th Stacker Genetics Symposium (edited by Gustafson & applications, Plenum Press 1988)); nucleotide sequences encoding beta-lactamase, an enzyme for which various chromogenic substrates (e.g., PADAC, chromogenic cephalosporins) are known (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75: 3737-; a nucleotide sequence encoding xylE, which encodes a 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 aggregates to form melanin) (Katz et al, (1983) J.Gen.Microbiol.129: 2703-2714); a nucleotide sequence encoding beta-galactosidase, an enzyme for which a chromogenic substrate is present; a nucleotide sequence encoding a luciferase enzyme (lux) which allows for bioluminescent detection (Ow et al, (1986) Science 234: 856-859); nucleotide sequences encoding aequorin, which can be used in a calcium-sensitive bioluminescence assay (Prasher et al, (1985) biochem. Biophys. Res. Comm.126: 1259-1268); or a nucleotide sequence encoding a green fluorescent protein (Niedz et al, (1995) Plant Cell Reports 14: 403-. One skilled in the art will be able to select suitable selectable markers for use in the expression cassettes of the invention.

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 starting 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 (eds.) (Cell Culture and social Cell Genetics of Plants, Acad.Press, Orlando, Vol.I (1984) and Vol.II (1986)).

In addition, the genetic traits described above which are engineered into the transgenic seeds and plants, plant parts or plant cells of the invention can be transmitted by sexual reproduction or vegetative growth and can therefore be maintained and propagated in progeny plants. Generally, maintenance and propagation utilize known agricultural methods developed to meet specific objectives (e.g., harvesting, sowing, or farming).

Thus, the polynucleotide may be introduced into the plant, plant part, or plant cell in any number of ways well known in the art, as described above. Thus, independent of the particular method used to introduce the polynucleotide or polynucleotides into the plant, any method that allows for stable integration of the polynucleotide or polynucleotides into the plant genome may be used. When more than one polynucleotide is to be introduced, the respective polynucleotides may be assembled as part of a single nucleic acid molecule, or as separate nucleic acid molecules, and may be located on the same or different nucleic acid molecules. Thus, the polynucleotides may be introduced into the cell of interest in a single transformation event, in separate transformation events, or as part of a breeding scheme, e.g., in a plant.

In some embodiments, the present invention provides methods of controlling black cutworm, comprising contacting the black cutworm 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 of SEQ ID NOs 1-6. In other embodiments, the composition is a formulation for topical application to a plant. In yet other embodiments, the composition is a transgenic plant. In a further embodiment, the composition is a combination of formulations that are topically applied to the transgenic plant. In some embodiments, when the transgenic plant comprises the second pest control agent, the formulation comprises the first Cry protein of the invention. In other embodiments, when the transgenic plant comprises the first Cry protein of the invention, the formulation comprises the second pest control agent.

In some embodiments, the second pest control agent may be an agent selected from the group consisting of: chemical pesticides such as insecticides, Bacillus thuringiensis (Bt) insecticidal proteins, Xenorhabdus (Xenorhabdus) insecticidal proteins, Photorhabdus (phorhabdus) insecticidal proteins, Bacillus laterosporus (Brevibacillus laterosporus) insecticidal proteins, Bacillus sphaericus (Bacillus sphaericus) insecticidal proteins, protease inhibitors (both serine and cysteine types), lectins, alpha-amylases, peroxidases, cholesterol oxidases, and double stranded rna (dsrna) molecules.

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 (abamectin), acephate (acephate), acetamiprid (acetamiprid), sulfadimidine (amidoflumete) (S-1955), avermectin (avermectins), azadirachtin (azadirachtin), bayphos (azinphos-methyl), bifenthrin (bifenthrin), bifenazate (bifenazate), buprofezin (buprofefazine), carbofuran (carbofuran), chlorfenapyr (chlorfenapyr), chlorfluazuron (chlorfluazuron), chlorpyrifos (chloropyrifos), chlorpyrifos (chlorpyrifos), chlorpyrifos-methyl, chromafenozide (chlorofenthiuron), clothianidin (cypermethrin), cyhalothrin (cyhalothrin), cyfluthrin (cyhalothrin), cyhalothrin (cyhalothrin), cyhalothrin (cyhalothrin), cyhalothrin (cyhalothrin), cyhalothrin (cyhalothrin), cyhalothrin (cyhalothrin ), cyhalothrin (cyhalothrin), cyhalothrin (cyhalothrin ), cyhalothrin), cyhalothrin (cyhalothrin, cyhalothri, Endosulfan (endosulfan), fenvalerate (esfenvalerate), ethiprole (ethiprole), fenoxycarb (fenothiocarb), fenoxycarb (fenoxycarb), fenpropathrin (fenpropathrin), fenpyroximate (fenpyroximate), fenvalerate (fenvalerate), fipronil (fipronil), flonicamid (flonicamid), flufenvalerate (fluythrinate), tau-fluvalinate (tau-fluvalinate), pyrimethanil (UR-50701), flufenoxuron (flufenoxuron), phos (thiophosphorus), chlorfenapyr (halofenozide), flufenoxuron (hexaflumuron), imidacloprid (imidacloprid), indoxacarb (indoxacarb), isofenphos (isophos), thion (fenpyrazofos), thion (fenthion), thion (methoxyfenozide) (XD), thion (methoxyfenozide (XD) (methoxyfenozide), thion (D (XD) (methoxyfenozide), methoxyfenozide (D (methoxyfenozide), methoxyfenozide (XD) (methoxyfenozide), methoxyfenozide (metofen) (MTP (metofen) (XD (MTP (MTX), methoxyfenozide (metofen (MTP) (MTP), MTP (MTP), MTP (MTP), MTX-D), MTP (MTX-D), MTX-N (MTP (MTX-N (MTX-D), MTX-N (MTP (MTX-N), MTP (MTP), MTP (MTP), MTP (MTP), MTP (MTP), MTP (MTP), MTP (MTP), MTP (MTP), MTP (MTP), MTP (methyl), MTP (MTP), MTP (MTP), MTP (MTP), MTP (MTP), N), MTP (methyl), MTP (methyl), N), MTP (MTP), MTP (methyl) and MTP), MTP (methyl), MTP (MTP), MTP (, Oxamyl, parathion-methyl, permethrin, phorate, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyriproxyfen, flutenone, teflubenzuron, tefluthrin, terbuthion, tebufenofos, thiofenozide, tebufenozide, tefluthrin, tebufenozide, tefluthrin, tebufenpyrazone, tebufenpyrad, tefluthrin, tebufenpyrazofos, tefluthrin, tebufenthion, tefluthrin, thifens, thifenuron, thiacloprid, thiaclopramide, bensulbencarb, bencarb, thiuron, bencarb, thiuron, bencarb, thiuron, bencarb, thiuron, cyhexatin (cyhexatin), dicofol (dicofol), ubiquitin (dienochlor), etoxazole (etoxazole), fenazaquin (fenazaquin), fenbutatin oxide (fenbutatin oxide), fenpropathrin, fenpyroximate, hexythiazox (hexythiazox), propargite (propargite), pyridaben (pyridaben), and tebufenpyrad (tebufenpyrad). In still other embodiments, the chemical pesticide is selected from the group consisting of: cypermethrin, cyhalothrin, cyfluthrin and beta-cyfluthrin, S-fenvalerate, tetrabromthrin, fenoxycarb, methomyl, oxamyl, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, emamectin benzoate, endosulfan, ethiprole, fipronil, flufenoxuron, triflumuron, bendiocarb, pyriproxyfen, pymetrozine and amitraz.

In additional embodiments, the second pest control agent may 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 insecticidal proteins. In other embodiments, the second pest control agent is a Cry protein selected from the group consisting of: cry1, Cry2, Cry7, Cry8, Cry7, Cry8, Cry1, Cry2, Cry1, Cry2, Cry1, Cry2, Cry4, Cry5, Cry7, Cry2, Cry7, Cry8, Cry7, Cry8, Cry7, Cry8, Cry7, Cry8, Cry7, Cry8, Cry7, cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry21, Cry32, Cry21, Cry32, Cry21, Cry32, Cry21, Cry32, Cry21, Cry32, 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 Cry73 Aa.

In further embodiments, the second pest control agent is a Vip3 vegetative insecticidal protein selected from the group consisting of: vip3Aa, Vip3Ab, Vip3Aa, Vip3Ab, Vip3Aa, Vip3 Ab.

In a still further embodiment, said first Cry protein of the invention and said second pest control agent are co-expressed in a transgenic plant. This co-expression of more than one pesticidally active ingredient in the same transgenic plant can be achieved by genetically engineering the plant to contain and express all the necessary genes. Alternatively, a plant "parental 1" can be genetically engineered to express a Cry protein of the invention. A second plant "parent 2" may be genetically engineered to express a second pest control agent. By crossing "parent 1" with "parent 2", progeny plants expressing all the genes introduced into "parent 1" and "parent 2" are obtained.

In a further embodiment, the invention provides a method of producing a pest-resistant (e.g., insect-resistant) 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 invention, wherein said nucleotide sequence is expressed in said plant, thereby conferring resistance to said plant to a black cutworm pest, and producing a pest-resistant transgenic plant. In some embodiments, said introducing is effected by transforming said plant. In other embodiments, the introduction is achieved by crossing a first plant comprising a chimeric gene, recombinant vector, expression cassette or nucleic acid molecule of the invention with a second, different plant.

In some embodiments, the invention includes a method of providing a farmer with a means for controlling lepidopteran pests, the method comprising supplying or selling to the farmer plant material, such as seeds, comprising a polynucleotide, chimeric gene, expression cassette or recombinant vector capable of expressing a Cry protein of the invention in plants grown from said seeds, as described above.

Embodiments of the invention may be better understood by reference to the following examples. The foregoing and following description of embodiments of the invention, as well as the various embodiments, are not intended to limit the claims, but are merely illustrative thereof. It is therefore to be understood that the claims are not to be limited to the specific details of these embodiments. Those skilled in the art will appreciate that other embodiments of the invention can be practiced without departing from the spirit and scope of the present disclosure, which is defined by the appended claims.

Examples

Example 1 Activity of Cry proteins against black cutworm

In an artificial diet bioassay, a Cry protein comprising the amino acid sequence of SEQ ID NOS: 1-6 and a Cry protein designated BT128 in the literature (Cry9Ba) was tested against a Chinese population of black cutworms (CN-BCW; agrotis microti) which is a crop pest in the family Spodoptera. As shown in table 1, these Cry proteins have been previously described.

TABLE 1 references to Cry protein disclosures

Cry proteins	SEQ ID NO:	Publication number
			BT25	1	WO2017003811
CryET4	2	WO199504146
			TIC860	3	WO2016061391
TIC867	4	WO2016061391
			TIC868	5	WO2016061391
CryET54	6	WO200119859
			BT128(Cry9Ba)	NA	WO2016094159

Essentially, equal amounts of protein in solution were applied to the surface of the artificial insect diet in the multi-well plate. After the dietary surface was dried, CN-BCW larvae were added to each well. The plates were sealed and maintained at ambient laboratory conditions (with respect to temperature, light and relative humidity). The positive control group consisted of larvae exposed to a Cry protein known to be CN-BCW active. The negative control group consisted of larvae exposed to the insect diet treated with buffer solution only and larvae on the untreated insect diet; i.e. an individual meal. Mortality was assessed after approximately 3-4 days and scored relative to controls.

The results of the CN-BCW bioassay are shown in column 3 of table 2, where "-" indicates no activity compared to the control, "+/-" indicates 0-10% activity compared to the control (this category also includes 0% mortality with strong larval growth inhibition), "+" indicates 10-25% activity compared to the control, "+ +" indicates 25-75% activity compared to the control, and "+ + + + +" indicates 75-100% activity compared to the control. Also shown in Table 2 is an indication of the activity of the Cry proteins against a North American strain of black cutworm (NA-BCW; black cutworm). For this insect species, the activity is simply indicated as "+" or "-", with no indication of the percent mortality (based on published data). The cell labeled "nt" indicates that there is no published information indicating that the Cry protein has been tested against NA-BCW. These results clearly demonstrate that the lack of biological activity or biological activity of north american populations directed to agrotis ypsilon does not accurately predict the lack of biological activity or biological activity of the same Cry protein against chinese populations of the same species (agrotis ypsilon).

TABLE 2 results of BCW bioassay with Cry proteins

Example 2 expression and Activity of Cry proteins in maize plants

Transformation of immature maize embryos is performed essentially as described in Negrotto et al, 2000, Plant Cell Reports 19: 798-. 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 coding 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 the identification of transgenic plants on selective media. Both expression cassettes were cloned into appropriate vectors for agrobacterium-mediated transformation of rice or maize. The transformed Agrobacterium strain was grown for 2-4 days at 28 ℃ on YEP (yeast extract (5g/L), peptone (10g/L), NaCl (5g/L), 15g/L agar, pH 6.8) solid medium. Will be about 0.8 x 10⁹The individual Agrobacterium cells were suspended in LS-inf medium supplemented with 100. mu.M As. The bacteria were pre-induced in this medium for approximately 30-60 minutes.

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

Immature embryos producing embryogenic callus were transferred to lsd1m0.5s medium. The cultures were selected on this medium for approximately 6 weeks with approximately 3 weeks of subculture steps. Surviving calli were transferred to Reg1 medium supplemented with mannose. After culturing in light (16 hour light/8 hour dark regime), green tissues were transferred to Reg2 medium without growth regulators and incubated for approximately 1-2 weeks. The plantlets were transferred to a Magenta GA-7 box (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 the greenhouse for further evaluation.

Transgenic plants were evaluated for copy number (as determined by Taqman analysis), protein expression level (as determined by ELISA), and efficacy against the insect species of interest (in leaf excision bioassay). Specifically, plant tissue (leaves or silks) were excised from single copy events (stages V3-V4) and infested with neonatal larvae of black cutworm, followed by incubation at room temperature for 5 days. The results of transgenic plant tissue bioassays will confirm that the Cry proteins of the invention are toxic to black cutworm when expressed in transgenic plants.

Sequence listing

<110> Syngenta Biotechnology CHina, CO

CAO, Guangyu

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Val Gln Ser Leu Pro Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu

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Pro Glu Ala Thr Gly Gln Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu

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Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu

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Phe Lys Glu Trp Glu Ser Asp Pro Asp Asn Glu Ala Ala Lys Ser Arg

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Pro Ser Phe Arg Ile Ile Gly Phe Glu Val Pro Leu Leu Ser Val Tyr

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Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr

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Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro

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Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser

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Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu

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Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr

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Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly

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Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu

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Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val

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Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr

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Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala

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Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr

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Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp

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Ser Val Glu Leu Leu Leu Met Glu Glu

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Ala Arg Ile Glu Asp Ser Leu Cys Val Ala Glu Val Asn Asn Ile Asp

35 40 45

Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly

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Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser

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Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro

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Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile Arg Gln Gln Val

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Thr Glu Asn Thr Arg Asn Thr Ala Ile Ala Arg Leu Glu Gly Leu Gly

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Arg Gly Tyr Arg Ser Tyr Gln Gln Ala Leu Glu Thr Trp Leu Asp Asn

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Arg Asn Asp Ala Arg Ser Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala

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Glu Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His

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Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly Met

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Ala Ser Ser Asp Val Asn Gln Tyr Tyr Gln Glu Gln Ile Arg Tyr Thr

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Glu Glu Tyr Ser Asn His Cys Val Gln 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

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Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro

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Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr

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Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly

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Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala

305 310 315 320

Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp Phe Pro Glu

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Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp Ser Ser Thr Gln His

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Met Asn Tyr Trp Val Gly His Arg Leu Asn Phe Arg Pro Ile Gly Gly

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Thr Leu Asn Thr Ser Thr Gln Gly Leu Thr Asn Asn Thr Ser Ile Asn

370 375 380

Pro Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser

385 390 395 400

Asn Ala Gly Thr Asn Ile Leu Phe Thr Thr Pro Val Asn Gly Val Pro

405 410 415

Trp Ala Arg Phe Asn Phe Ile Asn Pro Gln Asn Ile Tyr Glu Arg Gly

420 425 430

Ala Thr Thr Tyr Ser Gln Pro Tyr Gln Gly Val Gly Ile Gln Leu Phe

435 440 445

Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr

450 455 460

Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Leu Ile Ile Gly Asn

465 470 475 480

Thr Leu Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg

485 490 495

Thr Asn Thr Ile Gly Pro Asn Arg Ile Asn Gln Ile Pro Leu Val Lys

500 505 510

Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe

515 520 525

Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser

530 535 540

Leu Gln Val Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg

545 550 555 560

Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly

565 570 575

Ala Ala Ser Thr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu

580 585 590

Gln Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg

595 600 605

Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile

610 615 620

Ile Gly Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser

625 630 635 640

Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr

645 650 655

Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala

660 665 670

Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val Thr Asp

675 680 685

Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser Asp Glu

690 695 700

Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala

705 710 715 720

Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn Phe Lys

725 730 735

Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr Gly Ile

740 745 750

Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu

755 760 765

Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile

770 775 780

Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr

785 790 795 800

Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala

805 810 815

Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu

820 825 830

Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala

835 840 845

Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly

850 855 860

Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val

865 870 875 880

Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe Lys

885 890 895

Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu

900 905 910

Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala

915 920 925

Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn

930 935 940

Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn

945 950 955 960

Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His

965 970 975

Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro Glu

980 985 990

Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu Glu

995 1000 1005

Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val

1010 1015 1020

Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val

1025 1030 1035

Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val

1040 1045 1050

Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg

1055 1060 1065

Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys

1070 1075 1080

Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn

1085 1090 1095

Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile

1100 1105 1110

Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln

1115 1120 1125

Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn

1130 1135 1140

Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu

1145 1150 1155

Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn

1160 1165 1170

Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr

1175 1180 1185

Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu

1190 1195 1200

Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu

1205 1210 1215

Leu Leu Met Glu Glu

1220

<210> 4

<211> 1187

<212> PRT

<213> Artificial sequence

<220>

<223> TIC867

<400> 4

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 Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val

645 650 655

Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val

660 665 670

Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser

675 680 685

Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys

690 695 700

His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn

705 710 715 720

Phe Arg Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr

725 730 735

Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val

740 745 750

Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln

755 760 765

Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg

770 775 780

Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr

785 790 795 800

Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp

805 810 815

Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cys Ala His His Ser His

820 825 830

His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp

835 840 845

Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala

850 855 860

Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu

865 870 875 880

Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg

885 890 895

Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu

900 905 910

Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala

915 920 925

Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser

930 935 940

Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn

945 950 955 960

Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser

965 970 975

Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly

980 985 990

Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn

995 1000 1005

Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val

1010 1015 1020

Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg

1025 1030 1035

Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile

1040 1045 1050

His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys

1055 1060 1065

Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp

1070 1075 1080

Tyr Thr Ala Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg

1085 1090 1095

Asn Arg Gly Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro

1100 1105 1110

Ala Asp Tyr Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly

1115 1120 1125

Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr

1130 1135 1140

Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe

1145 1150 1155

Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly

1160 1165 1170

Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1175 1180 1185

<210> 5

<211> 1199

<212> PRT

<213> Artificial sequence

<220>

<223> TIC868

<400> 5

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 Asn Gln Ile Pro Leu Val Lys Gly Phe

500 505 510

Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly

515 520 525

Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln

530 535 540

Val Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg

545 550 555 560

Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala

565 570 575

Ser Thr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys

580 585 590

Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr

595 600 605

Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly

610 615 620

Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu

625 630 635 640

Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu

645 650 655

Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Glu Leu Phe

660 665 670

Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His

675 680 685

Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys

690 695 700

Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg

705 710 715 720

Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile

725 730 735

Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile

740 745 750

Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly

755 760 765

Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu

770 775 780

Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu

785 790 795 800

Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His

805 810 815

Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala

820 825 830

Pro Ser Pro Ile Gly Lys Cys Ala His His Ser His His Phe Ser Leu

835 840 845

Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp

850 855 860

Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn

865 870 875 880

Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg

885 890 895

Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu

900 905 910

Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala

915 920 925

Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile

930 935 940

Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala

945 950 955 960

Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe

965 970 975

Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala

980 985 990

Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp

995 1000 1005

Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg

1010 1015 1020

Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu

1025 1030 1035

Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala

1040 1045 1050

Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile

1055 1060 1065

Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu

1070 1075 1080

Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala

1085 1090 1095

Thr Gln Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly

1100 1105 1110

Tyr Asp Gly Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr

1115 1120 1125

Ala Ser Ala Tyr Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp

1130 1135 1140

Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu

1145 1150 1155

Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr

1160 1165 1170

Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile

1175 1180 1185

Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu

1190 1195

<210> 6

<211> 1227

<212> PRT

<213> Bacillus thuringiensis

<400> 6

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 Gln 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 Gln Asp Pro Asn Phe

705 710 715 720

Thr Ser Ile Asn Lys Gln Pro Asp Phe Asn Ser Asn Asn Glu Gln Ser

725 730 735

Asn Phe Thr Ser Ile His Glu Gln Ser Glu His Gly Trp Trp Gly Ser

740 745 750

Glu Asn Ile Thr Ile Gln Glu Gly 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 Gln Leu

785 790 795 800

Ser Gly Tyr Ile Glu Asp Ser Gln 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 Gln 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 Gln 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

Claims (10)

1. A method of inhibiting the growth of or killing a black cutworm (Agrotis ipsilon) pest, said method comprising contacting said black cutworm with a Cry protein comprising an amino acid sequence of any one of SEQ ID NOs 1-6 or an insecticidal fragment thereof.

2. A method for controlling a black cutworm pest population, said method comprising contacting said pest population with an insecticidally effective amount of a Cry protein comprising an amino acid sequence of any one of SEQ ID NOs 1-6, or an insecticidal fragment thereof.

3.The method of claim 1 or 2, wherein the black cutworm pest or pest population is further contacted with a second insecticidal protein that is different from the Cry protein comprising the amino acid sequence of any of SEQ ID NOs 1-6.

4. The method of claim 3, wherein the second insecticidal protein is selected from the group consisting of: cry proteins, Vip proteins, protease inhibitors, lectins, alpha-amylases, and peroxidases.

5. The method of any one of claims 1 to 4, wherein the contacting step is performed with a microorganism or plant expressing the protein or insecticidal fragment thereof.

6. The method of claim 5, wherein said plant is stably transformed with a DNA sequence encoding said protein or insecticidal fragment thereof.

7. The method of claim 6, wherein the plant is a monocot or a dicot.

8. The method of claim 7, wherein the monocot is a maize plant or the dicot is a soybean plant.

9. A method for protecting a plant from a black cutworm pest, the method comprising expressing in the plant or cell thereof an insecticidally effective amount of a Cry protein comprising the amino acid sequence of any one of SEQ ID NOs 1-6 or an insecticidal fragment thereof.

10. A method for controlling a black cutworm pest population, said method comprising contacting said pest population with an insecticidally effective amount of a Cry protein comprising an amino acid sequence of any one of SEQ ID NOs 1-6, or an insecticidal fragment thereof.