WO2022155872A1

WO2022155872A1 - Control of noctuid, crambid, and pyralid pests

Info

Publication number: WO2022155872A1
Application number: PCT/CN2021/073190
Authority: WO
Inventors: Guangyu Cao; Chunping Luo; Qianqian Duan; Ying LONG
Original assignee: Syngenta Biotechnology China Co., Ltd.; Syngenta Crop Protection Ag
Priority date: 2021-01-22
Filing date: 2021-01-22
Publication date: 2022-07-28
Also published as: CN116829163A

Abstract

Provided are methods of controlling Lepidoptera such as Noctuidae, Crambidae, and Pyralidae and to protect crops, particularly corn, against economic damage caused by Lepidoptera such as Noctuidae, Crambidae, and Pyralidae. Further provided is the usage of plants stably transformed with a nucleic acid molecule that encodes a Cry protein, either alone or in combination with other insecticidal proteins to control or combat Lepidoptera such as Noctuidae, Crambidae, and Pyralidae.

Description

CONTROL OF NOCTUID, CRAMBID, AND PYRALID PESTS

FIELD OF THE INVENTION

This invention relates to compositions and methods to control or combat Noctuidae, Crambidae and Pyralidae pests using pesticidal proteins and the nucleic acid molecules that encode them.

BACKGROUND OF THE INVENTION

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 are harmless to plants and other non-target organisms. For this reason, compositions comprising Bacillus thuringiensis strains, or their insecticidal proteins can be used as environmentally-acceptable insecticides to control agricultural insect pests or insect vectors of a 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 have shown activity against pests in the Orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari pest orders, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson, J. 1993. The Bacillus Thuringiensis family tree. In 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 major classes were Lepidoptera-specific (I) , Lepidoptera-and Diptera-specific (II) , Coleoptera-specific (III) , Diptera-specific (IV) , and nematode-specific (V) and (VI) . The proteins were further classified into subfamilies; more highly related proteins within each family were assigned divisional letters such as CryIA, CryIB, CryIC, etc. Even more closely related proteins within each division were given names such as CryIC (a) , CryIC (b) , etc. 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 upon amino acid sequence homology rather than 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 incorporating a primary rank (an Arabic number) , a secondary rank (an uppercase letter) , a tertiary rank (a lowercase letter) , and a quaternary rank (another Arabic number) . In the current classification, Roman numerals have been exchanged for Arabic numerals in the primary rank. For example, “CryIA (a) ” under the older nomenclature is now “Cry1Aa” under the current nomenclature. According to Ibrahim et al. (2010, Bioeng. Bugs, 1: 31-50) , the Cry toxins can still be separated into six major classes according to their insect host specificities and include: Group 1-lepidopteran e.g., Cry1, Cry9 and Cry15) ; group 2-lepidopteran and dipteran (e.g., Cry2) ; group 3-coleopteran (Cry3, Cry7 and Cry8) ; group 4-dipteran (Cry4, Cry10, Cry11, Cry16, Cry17, Cry19 and Cry20) ; group 5-lepidopteran and coleopteran (Cry1I) ; and group 6-nematodes (Cry6) . The Cry1I, Cry2, Cry3, Cry10 and Cry11 toxins (73–82 kDa) are unique because they appear to be natural truncations of the larger Cry1 and Cry4 proteins (130–140 kDa) .

Cry proteins are globular protein molecules which accumulate as protoxins in crystalline form during the sporulation stage of Bt. After ingestion by a pest, the crystals are typically solubilized to release protoxins, which can range in size, for example, from 130-140 kDa for many of the lepidopteran-active Cry proteins, such as Cry1 and Cry9, and 60-80 kDa for the coleopteran-active Cry3 proteins and the lepidopteran/dipteran-active Cry2 proteins. After the crystals are solubilized by a susceptible insect the released protoxins are processed by proteases in the insect gut, for example trypsin and chymotrypsin, to produce a protease-resistant core Cry protein toxin. This proteolytic processing involves the removal of amino acids from different regions of the various Cry protoxins. For example, Cry protoxins that are 130-140 kDa are typically activated through the proteolytic removal of an N-terminal peptide of 25-30 amino acids and approximately half of the remaining protein from the C-terminus resulting in an approximately 60-70 kDa mature Cry toxin. The protoxins that are 60-80 kDa, e.g. Cry2 and Cry3, are also processed but not to the same extent as the larger protoxins. The smaller protoxins typically have equal or more amino acids removed from the N-terminus than the larger protoxins but less amino acids removed from the C-terminus. For example, proteolytic activation of Cry2 family members typically involves the removal of approximately 40-50 N-terminal amino acids. Many of the Cry proteins are quite toxic to specific target insects, but many have narrow spectrums of activity.

Cry proteins generally have five conserved sequence domains, and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17: 193-199) . The first conserved structural domain, called Domain I, typically consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II typically consists of three beta-sheets arranged in a Greek key configuration, and domain III typically consists of two antiparallel beta-sheets in ‘jelly-roll’ formation (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.

Numerous commercially valuable plants, including common agricultural crops, are susceptible to attack by plant pests including insect and nematode pests, causing substantial reductions in crop yield and quality. For example, plant pests are a major factor in the loss of the world's important agricultural crops. About 15-20 percent of harvestable grain in China is lost every year to insect pests and diseases. In addition, about $8 billion are lost every year in the United States alone due to infestations of invertebrate pests including insects. Insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and to home gardeners.

Insect pests are mainly controlled by intensive applications of chemical pesticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Biological pest control agents, such as Bacillus thuringiensis strains expressing pesticidal toxins such as Cry proteins, have also been applied to crop plants with satisfactory results, offering an alternative or compliment to chemical pesticides. The genes coding for some of these Cry proteins have been isolated and their expression in heterologous hosts such as transgenic plants have been shown to provide another tool for the control of economically important insect pests. Most Cry proteins are active against a very limited spectra of insect pests. And typically, activity against one insect species does not predict activity against a different insect species.

Lepidopteran pests, including the Noctuid, Crambid, and Pyralid pests of the disclosure, continue to be an issue in China and other countries where such pests are present. Additionally, the threat of development of resistance to existing insecticidal proteins means that the introduction of new insecticidal proteins is important. Thus, there remains a need to identify insecticidal proteins that are capable of controlling Lepidopteran pests such as the Noctuid, Crambid, and Pyralid pests of the disclosure.

SUMMARY OF THE INVENTION

The present disclosure provides compositions and methods of controlling Lepidopteran pests such as Noctuid, Crambid, and Pyralid pests and to protect crops, particularly corn and rice, against economic damage caused by such pests. This disclosure further relates to the use of plants, especially monocotyledonous plants, particularly corn (maize, Zea mays) and rice (Oryza sativa) , stably transformed with a nucleic acid molecule that encodes a Cry protein of the disclosure, either alone or in combination with other insecticidal proteins to control or combat Lepidoptera such as Noctuidae, Crambidae, and Pyralidae. This disclosure still further relates to the use of insecticidal formulations containing the Cry proteins of the disclosure to protect plants from Lepidoptera such as Noctuidae, Crambidae, and Pyralidae. This disclosure also relates to a plant, especially a monocot plant, particularly a corn or rice plant, infestable by Lepidoptera such as Noctuidae, Crambidae, and Pyralidae and transformed with an expressible nucleic acid molecule that encodes a Cry protein of the disclosure to combat or control Lepidoptera such as Noctuidae, Crambidae, and Pyralidae pest populations.

In accordance with this disclosure, a method is provided to combat and/or control Lepidoptera such as Noctuidae, Crambidae, and Pyralidae insects of the species Helicoverpa armigera (Cotton bollworm/Old World bollworm) , Mythimna separata (Oriental armyworm) , Athetis lepigone (Two-spotted armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , Agrotis ipsilon (Black cutworm) , Conogethes punctiferalis (Yellow peach moth) , Chilo suppressalis (Striped stem borer) , Sesamia inferens (Pink stem borer) , or Ostrinia furnacalis (Asian corn borer) by the step of contacting these insects with a Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3, or an insecticidal fragment thereof. In some embodiments, the Agrotis ipsilon (Black cutworm) is from a population of Agrotis ipsilon (Black cutworm) from China.

In some embodiments, the contacting step can be carried out with an insecticidal composition comprising: the Cry protein of the disclosure, or insecticidal fragment thereof, and an acceptable agricultural carrier. In some embodiments, the contacting of the insects can be with a plant, especially a monocotyledonous plant, particularly a corn or rice plant, stably transformed with an expressible nucleic acid molecule that encodes a Cry protein of the disclosure, so that the transformed plant expresses the Cry protein of the disclosure, or an insecticidal fragment thereof, in an effective insect-controlling amount.

Moreover, a plant, especially a monocotyledonous plant, particularly a corn or rice plant, infested by Lepidoptera such as Noctuidae, Crambidae, and Pyralidae insects, is protected from sustaining economic damage from this insect by having been stably transformed with a gene that encodes a Cry protein of the disclosure.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is an amino acid sequence of a BT29 protein.

SEQ ID NO: 2 is an amino acid sequence of a BT29-BT22 chimeric protein.

SEQ ID NO: 3 is an amino acid sequence of a BT29-Cry1Fa chimeric protein.

DETAILED DESCRIPTION OF THE INVENTION

This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant 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 invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 reference 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” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative, “or. ”

The term “about” is used herein to mean approximately, roughly, around, or in the region of. 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. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (higher or lower) . With regard to a temperature the term “about” means ± 1 ℃, preferably ± 0.5℃. Where the term “about” is used in the context of this invention (e.g., in combinations with temperature or molecular weight values) the exact value (i.e., without “about” ) is preferred.

To “control” insects means to inhibit, through a toxic effect, the ability of insect pests to survive, grow, feed, or reproduce, or to limit insect-related damage or loss in crop plants or to protect the yield potential of a crop when grown in the presence of insect pests. To “control” insects may or may not mean killing the insects, although it preferably means killing the insects.

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 a claim is to be interpreted to encompass the specified materials or steps recited in the claim” and those that do not materially alter the basic and novel characteristic (s) ” of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising. ”

As used herein, the term “Cry protein” means an insecticidal protein that may occur in crystalline form in Bacillus thuringiensis or related bacteria. The term “Cry protein” can refer to the protoxin form or any insecticidal fragment or toxin thereof.

To “deliver” a composition or toxic protein means that the composition or toxic protein comes in contact with an insect, which facilitates the oral ingestion of the composition or toxic protein, resulting in a toxic effect and control of the insect. The composition or toxic protein can be delivered in many recognized ways, including but not limited to, transgenic plant expression, formulated protein composition (s) , sprayable protein composition (s) , a bait matrix, or any other art-recognized protein delivery system.

“Effective insect-controlling amount” means that concentration of a toxic protein that inhibits, through a toxic effect, the ability of insects to survive, grow, feed or reproduce, or limits insect-related damage or loss in crop plants or protects the yield potential of a crop when grown in the presence of insect pests. “Effective insect-controlling amount” may or may not mean killing the insects, although it preferably means killing the insects.

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

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

Nucleotides are indicated herein by the following standard abbreviations: adenine (A) , cytosine (C) , thymine (T) , and guanine (G) . Amino acids are likewise indicated 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; 1) , 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) .

This invention is based on the result of toxicity assays which were conducted by feeding certain Noctuid, Crambid, and Pyralid insects an artificial diet containing a purified Cry toxin and which surprisingly showed that certain Cry proteins were toxic to one or more of the tested insects (see Example 1) . Therefore, these active Cry proteins can be used to provide maximum protection against such pests and can prevent or reduce the development of insect resistance to Cry insecticidal formulations in the field.

The “Cry proteins” of this disclosure can be naturally occurring or engineered and encompass the full-length protein (protoxin) having the amino acid sequence shown in any of SEQ ID NOs: 1-3 of the Sequence Listing, as well as any insecticidally active fragment thereof.

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

A toxin fragment of a Cry protein of the disclosure will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, and 450 contiguous amino acids, or up to the total number of amino acids present in a full-length Cry protein of the disclosure.

As used herein, a Cry protein that is “toxic” to an insect pest is meant that the Cry protein functions as an orally active insect control agent to kill the insect pest, or the Cry protein is able to disrupt or deter insect feeding, or causes growth inhibition to the insect pest, both of which may or may not cause death of the insect. When a Cry protein of the disclosure is delivered to an insect or an insect comes into oral contact with the Cry protein, the result is typically death of the insect, or the insect’s growth is slowed, or the insect stops feeding upon the source that makes the toxic Cry protein available to the insect.

In some embodiments, the disclosure provides a method of inhibiting the growth or killing a pest, comprising contacting the pest with a Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of Helicoverpa armigera (Cotton bollworm/Old World bollworm) , Mythimna separata (Oriental armyworm) , Athetis lepigone (Two-spotted armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , Agrotis ipsilon (Black cutworm) , Conogethes punctiferalis (Yellow peach moth) , Chilo suppressalis (Striped stem borer) , Sesamia inferens (Pink stem borer) , and Ostrinia furnacalis (Asian corn borer) . In some embodiments, the Agrotis ipsilon (Black cutworm) is from a population of Agrotis ipsilon (Black cutworm) from China.

In some embodiments, the disclosure provides a method for controlling a pest population, comprising contacting the pest population with an insecticidally-effective amount of a Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of Helicoverpa armigera (Cotton bollworm/Old World bollworm) , Mythimna separata (Oriental armyworm) , Athetis lepigone (Two-spotted armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , Agrotis ipsilon (Black cutworm) , Conogethes punctiferalis (Yellow peach moth) , Chilo suppressalis (Striped stem borer) , Sesamia inferens (Pink stem borer) , and Ostrinia furnacalis (Asian corn borer) . In some embodiments, the Agrotis ipsilon (Black cutworm) is from a population of Agrotis ipsilon (Black cutworm) from China.

In further embodiments of the disclosure, the pest or pest population is further contacted with a second insecticidal protein different than the Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3. In still other embodiments, the second insecticidal protein is selected from the group consisting of a Bacillus thuringiensis (Bt) insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Brevibacillus laterosporus insecticidal protein, a Bacillus sphaericus insecticidal protein, a perforin, a protease inhibitor (both serine and cysteine types) , a lectin, an alpha-amylase, a peroxidase, a cholesterol oxidase and a double stranded RNA (dsRNA) molecule.

In other embodiments of the disclosure, the contacting step, whereby a Cry protein of the disclosure comes into contact with a pest, is carried out with a microorganism or a plant expressing said protein, or insecticidal fragment thereof. In other embodiments, the plant is stably transformed with a nucleic acid molecule that encodes the Cry protein of the disclosure, or an insecticidal fragment thereof. In still other embodiments, the plant is a monocotyledonous or dicotyledonous plant. In other embodiments, the monocotyledonous plant is a corn or rice plant, or the dicotyledonous plant is a soybean plant.

In some embodiments, the disclosure provides a method for protecting a plant from a pest, comprising expressing in the plant or cell thereof, an insecticidally-effective amount of a Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of Helicoverpa armigera (Cotton bollworm/Old World bollworm) , Mythimna separata (Oriental armyworm) , Athetis lepigone (Two-spotted armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , Agrotis ipsilon (Black cutworm) , Conogethes punctiferalis (Yellow peach moth) , Chilo suppressalis (Striped stem borer) , Sesamia inferens (Pink stem borer) , and Ostrinia furnacalis (Asian corn borer) . In some embodiments, the Agrotis ipsilon (Black cutworm) is from a population of Agrotis ipsilon (Black cutworm) from China.

To be effective against the insect pests of the disclosure, the Cry protein is first orally ingested by the insect. However, the Cry protein can be delivered to the insect in many recognized ways. The ways to deliver a protein orally to an insect include, but are not limited to, providing the protein (1) in a transgenic plant, wherein the insect eats (ingests) one or more parts of the transgenic plant, thereby ingesting the polypeptide that is expressed in the transgenic plant; (2) in a formulated protein composition (s) that can be applied to or incorporated into, for example, insect growth media; (3) in a protein composition (s) that can be applied to the surface, for example, sprayed, onto the surface of a plant part, which is 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 an insect can be used in a method of the disclosure to deliver the toxic Cry proteins of the disclosure. In some particular embodiments, the Cry protein of the disclosure is delivered orally to an insect, wherein the insect ingests one or more parts of a transgenic plant.

In other embodiments, the Cry protein of the disclosure is delivered orally to an insect, wherein the insect ingests one or more parts of a plant sprayed with a composition comprising the Cry proteins of the disclosure. Delivering the compositions of the disclosure to a plant surface can be done using any method known to those of skill in the art for applying compounds, compositions, formulations and the like to plant surfaces. Some non-limiting examples of delivering to or contacting a plant or part thereof include spraying, dusting, sprinkling, scattering, misting, atomizing, broadcasting, soaking, soil injection, soil incorporation, drenching (e.g., root, soil treatment) , dipping, pouring, coating, leaf or stem infiltration, side dressing or seed treatment, and the like, and combinations thereof. These and other procedures for contacting a plant or part thereof with compound (s) , composition (s) or formulation (s) are well-known to those of skill in the art.

In some embodiments of the disclosure, an insecticidal Cry protein of the disclosure is expressed in a higher organism, for example, a plant. In this case, transgenic plants expressing effective amounts of the insecticidal protein protect themselves from plant pests such as insect pests. When an insect pest larva starts feeding on such a transgenic plant, it ingests the expressed insecticidal Cry protein. This can deter the insect from further biting into the plant tissue or may even harm or kill the insect. A polynucleotide that encodes a Cry protein of the disclosure is inserted into an expression cassette, which is then stably integrated in the genome of the plant. In other embodiments, the polynucleotide is included in a non-pathogenic self-replicating virus. Plants transformed in accordance with the disclosure may be monocots or dicots and include, but are not limited to, corn (maize) , soybean, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, oilseed rape, alfalfa, tobacco, peanuts, vegetables, including, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, carrot, eggplant, cucumber, radish, spinach, potato, tomato, asparagus, onion, garlic, melons, pepper, celery, squash, pumpkin, zucchini, fruits, including, apple, pear, quince, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, and specialty plants, such as Arabidopsis, and woody plants such as coniferous and deciduous trees. Preferably, plants of the of the disclosure are crop plants such as maize, soybean, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, sugar beet, sugarcane, tobacco, barley, oilseed rape, and the like. Once a desired polynucleotide has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.

A polynucleotide encoding a Cry protein of the disclosure is expressed in transgenic plants, thus causing the biosynthesis of the encoded Cry protein, either in protoxin or toxin form, in the transgenic plants. In this way, transgenic plants with enhanced yield protection in the presence of a population of insect pest pressure are generated. For their expression in transgenic plants, the nucleotide sequences that encode the Cry protein may require modification and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that living organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in this disclosure can be changed to conform with plant preferences, while maintaining the amino acids encoded thereby. Furthermore, high expression in plants, for example corn plants, is best achieved from coding sequences that have at least about 35%GC content, or at least about 45%, or at least about 50%, or at least about 60%. Microbial nucleotide sequences that have low GC contents may express poorly in plants due to the existence of ATTTA motifs that may destabilize messages, and AATAAA motifs that may cause inappropriate polyadenylation. Although certain gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17: 477-498 (1989) ) . In addition, the nucleotide sequences are screened for the existence of illegitimate splice sites that may cause message truncation. All changes required to be made within the nucleotide sequences such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described for example in US Patent Nos. 5,625,136; 5,500,365 and 6,013,523.

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

The polynucleotide sequence that encodes a Cry protein of the disclosure, can be operably fused to a variety of promoters for expression in plants including constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters to prepare 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 this disclosure in leaves, in stalks or stems, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc. ) , in roots, or seedlings is preferred. In many cases, however, protection against more than one type of insect pest is sought, and thus expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired cell.

Suitable constitutive promoters include, for example, CaMV 35S promoter (; Odell et al., Nature 313: 810-812, 1985) ; Arabidopsis At6669 promoter (see PCT Publication No. W004081173A2) ; 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., Theor. Appl. Genet. 81: 581-588, 1991) ; CaMV 19S (Nilsson et al., Physiol. Plant 100: 456-462, 1997) ; GOS2 (de Pater et al., Plant J November; 2 (6) : 837-44, 1992) ; ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675-689, 1992) ; Rice cyclophilin (Bucholz et al., Plant Mol Biol. 25 (5) : 837-43, 1994) ; Maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231: 276-285, 1992) ; Actin 2 (An et al., Plant J. 10 (1) ; 107-121, 1996) , constitutive root tip CT2 promoter (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 the expression of the Cry protein coding sequences of the disclosure in plants, particularly maize, are those that direct expression in root, pith, leaf or pollen. Suitable tissue-specific promoters include, but not limited to, leaf-specific promoters [such as described, for example, by Yamamoto et al., Plant J. 12: 255-265, 1997; Kwon et al., Plant Physiol. 105: 357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35: 773-778, 1994; Gotor et al., Plant J. 3: 509-18, 1993; Orozco et al., Plant Mol. Biol. 23: 1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90: 9586-9590, 1993] , seed-preferred promoters [e.g., from seed specific genes (Simon, et al., Plant Mol. Biol. 5.191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990) , Brazil Nut albumin (Pearson' et al., Plant Mol. Biol. 18: 235-245, 1992) , legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988) , Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987) , Zein (Matzke et al., Plant Mol Biol, 143) . 323-32 1990) , napA (Stalberg, et al., Planta 199: 515-519, 1996) , Wheat SPA (Albanietal, Plant Cell, 9: 171-184, 1997) , sunflower oleosin (Cummins, etal., Plant Mol. Biol. 19: 873-876, 1992) ] , endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216: 81-90, 1989; NAR 17: 461-2) , wheat a, b and g gliadins (EMB03: 1409-15, 1984) , Barley ltrl promoter, barley B1, C, D hordein (Theor Appl Gen 98: 1253-62, 1999; Plant J 4: 343-55, 1993; Mol Gen Genet 250: 750-60, 1996) , Barley DOF (Mena et al., The Plant Journal, 116 (1) : 53-62, 1998) , Biz2 (EP99106056.7) , Synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998) , rice prolamin NRP33, rice -globulin Glb-1 (Wu et al., Plant Cell Physiology 39 (8) 885-889, 1998) , rice alpha-globulin REB/OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997) , rice ADP-glucose PP (Trans Res 6: 157-68, 1997) , maize ESR gene family (Plant J 12: 235-46, 1997) , sorgum gamma-kafirin (Plant Mol. Biol 32: 1029-35, 1996) ] , embryo specific promoters [e.g., rice OSH1 (Sato et al., Proc. Nati. Acad. Sci. USA, 93: 8117-8122) , KNOX (Postma-Haarsma of al, Plant Mol. Biol. 39: 257-71, 1999) , rice oleosin (Wu et at, J. Biochem., 123: 386, 1998) ] , flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990) , LAT52 (Twell et al., Mol. Gen Genet. 217: 240-245; 1989) , apetala-3, plant reproductive tissues [e.g., OsMADS promoters (U.S. Patent Application 2007/0006344) ] .

The nucleotide sequences can also be expressed under the regulation of promoters that are chemically regulated. This enables the Cry proteins of the disclosure to be synthesized only when the crop plants are treated with the inducing chemicals. Examples of such technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 and US Patent No. 5,614,395. In one embodiment, the chemically regulated promoter is the tobacco PR-1a promoter.

Another category of promoters useful in the disclosure is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. Ideally, such a promoter should only be active locally at the sites of insect invasion, and in this way the insecticidal proteins only accumulate in cells that need to synthesize the insecticidal proteins to kill the invading insect pest. Examples of promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989) , Xu et al. Plant Molec. Biol. 22: 573-588 (1993) , Logemann et al. Plant Cell 1: 151-158 (1989) , Rohrmeier &Lehle, Plant Molec. Biol. 22: 783-792 (1993) , Firek et al. Plant Molec. Biol. 22: 129-142 (1993) , and Warner et al. Plant J. 3: 191-201 (1993) .

Non-limiting examples of promoters that cause tissue specific expression patterns that are useful in the disclosure include green tissue specific, root specific, stem specific, or flower specific. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. One such promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth &Grula, Plant Molec. Biol. 12: 579-589 (1989) ) . Another promoter for root specific expression is that described by de Framond (FEBS 290: 103-106 (1991) or US Patent No. 5,466,785) . Another promoter useful in the disclosure is the stem specific promoter described in U.S. Pat. No. 5,625,136, which naturally drives expression of a maize trpA gene.

In addition to the selection of a suitable promoter, constructs for expression of an insecticidal toxin in plants require an appropriate transcription terminator to be operably linked downstream of the Cry protein coding sequences of the disclosure. Several such terminators are available and known in the art (e.g. tml from CaMV, E9 from rbcS) . Any available terminator known to function in plants can be used in the context of this disclosure.

Numerous other sequences can be incorporated into expression cassettes described in this disclosure. 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 preferable to target expression of the nucleotide sequences of the present disclosure to different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be preferred. Any mechanism for targeting gene products, e.g., in plants, can be used to practice this invention, and such mechanisms are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. Sequences have been characterized which cause the targeting of gene products to other cell compartments Amino terminal sequences can be responsible for targeting a protein of interest to any cell compartment, such as, a vacuole, mitochondrion, peroxisome, protein bodies, endoplasmic reticulum, chloroplast, starch granule, amyloplast, apoplast or cell wall of a plant (e.g. Unger et. al. Plant Molec. Biol. 13: 411-418 (1989) ; Rogers et. al. (1985) Proc. Natl. Acad. Sci. USA 82: 6512-651; U.S. Pat. No. 7,102,057; WO 2005/096704, all of which are hereby incorporated 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 to the chloroplast (Comai et. al. (1988) J. Biol. Chem. 263: 15104-15109; van den Broeck, et. al. (1985) Nature 313: 358-363; U.S. Pat. No. 5,639,949) or a secretion signal sequence from aleurone cells (Koehler &Ho, Plant Cell 2: 769-783 (1990) ) . Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et. al. (1990) Plant Molec. Biol. 14: 357-368) . In one embodiment, the signal sequence selected includes the known cleavage site, and the fusion constructed takes into account any amino acids after the cleavage site (s) , which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. These construction techniques are well known in the art and are equally applicable to any cellular compartment.

It will be recognized that the above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.

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

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 and the like) any vector is suitable and linear DNA containing only the construction of interest can be used. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al., Biotechnology 4: 1093-1096 (1986) ) . For both direct gene transfer and Agrobacterium-mediated transfer, transformation is usually (but not necessarily) undertaken with a selectable marker that may be a positive selection (Phosphomannose Isomerase) , provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (glyphosate or glufosinate) . However, the choice of selectable marker is not critical to the invention.

Agrobacterium-mediated transformation is a commonly used method for transforming plants because of its high efficiency of transformation and because of its broad utility with many different species. Agrobacterium-mediated transformation typically involves transfer of the binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain that may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) Plant Cell 5: 159-169) . The transfer of the recombinant binary vector to Agrobacterium can be accomplished by a triparental mating procedure using Escherichia coli carrying the recombinant binary vector, a helper E. coli strain that carries a plasmid that is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation (

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

Dicots as well as monocots may be transformed 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 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996) ; Chan et al. (Plant Mol Biol 22 (3) : 491-506, 1993) , Hiei et al. (Plant J 6 (2) : 271-282, 1994) , which disclosures are incorporated by reference herein. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14 (6) : 745-50, 1996) or Frame et al. (Plant Physiol 129 (1) : 13-22, 2002) , which disclosures are incorporated by reference herein. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225) . The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711) . Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hagen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

Transformation of a plant by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is regenerated on selection medium carrying an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders.

As discussed previously, another method for transforming plants, plant parts and plant cells involves propelling inert or biologically active particles at plant tissues and cells. See, e.g., US Patent Nos. 4,945,050; 5,036,006 and 5,100,792. Generally, this method involves propelling inert or biologically active particles at the plant cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the nucleic acid of interest. Alternatively, a cell or cells can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., a dried yeast cell, a dried bacterium or a bacteriophage, each containing one or more nucleic acids sought to be introduced) also can be propelled into plant tissue.

In other embodiments, a polynucleotide encoding a Cry protein of the disclosure can be directly transformed into the plastid genome. A major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter. Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al. (1994) Proc. Nati. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation) . The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin or streptomycin can be utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45) . The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub, J.M., and Maliga, P. (1993) EMBO J. 12, 601-606) . Substantial increases in transformation frequency can be obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-cletoxifying enzyme aminoglycoside-3'-adenyltransf erase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917) . Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089) . Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the disclosure. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10%of the total soluble plant protein. In one embodiment, a polynucleotide of the disclosure can be inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Thus, plants homoplastic for plastid genomes containing a nucleotide sequence of the disclosure can be obtained, which are capable of high expression of the polynucleotide.

Methods of selecting for transformed, transgenic plants, plant cells or plant tissue culture are routine in the art and can be employed in the methods of the disclosure provided herein. For example, a recombinant vector of the disclosure also can include an expression cassette comprising a nucleotide sequence for a selectable marker, which can be used to select a transformed plant, plant part or plant cell. As used herein, “selectable marker” means a nucleotide sequence that when expressed imparts a distinct phenotype to the plant, plant part or plant cell expressing the marker and thus allows such transformed plants, plant parts or plant cells to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like) , or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., the R-locus trait) . Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

Examples of selectable markers include, but are not limited to, a nucleotide sequence encoding neo or nptII, which confers resistance to kanamycin, G418, and the like (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 (Hinchee et al. (1988) Biotech. 6: 915-922) ; a nucleotide sequence encoding a nitrilase such as bxn from Klebsiella ozaenae that 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 imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP Patent Application No. 154204) ; a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (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 a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI) ) that confers an ability to metabolize mannose (US Patent Nos. 5,767,378 and 5,994,629) ; a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyl tryptophan; or a nucleotide sequence encoding hph that confers resistance to hygromycin. One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette of this disclosure.

Additional selectable markers include, but are not limited to, a nucleotide sequence encoding β-glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., “Molecular cloning of the maize R-nj allele by transposon-tagging with Ac” 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson &Appels eds., Plenum Press 1988) ) ; a nucleotide sequence encoding β-lactamase, an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75: 3737-3741) ; a nucleotide sequence encoding xylE that 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 in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129: 2703-2714) ; a nucleotide sequence encoding β-galactosidase, an enzyme for which there are chromogenic substrates; a nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection (Ow et al. (1986) Science 234: 856-859) ; a nucleotide sequence encoding aequorin which may be employed in calcium-sensitive bioluminescence detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126: 1259-1268) ; or a nucleotide sequence encoding green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14: 403-406) . One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette of this disclosure.

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

Additionally, the genetic properties engineered into the transgenic seeds and plants, plant parts, or plant cells of the disclosure described above can be passed on by sexual reproduction or vegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling.

A polynucleotide therefore can be introduced into the plant, plant part or plant cell in any number of ways that are well known in the art, as described above. Therefore, no particular method for introducing one or more polynucleotides into a plant is relied upon, rather any method that allows the one or more polynucleotides to be stably integrated into the genome of the plant can be used. Where more than one polynucleotides is to be introduced, the respective polynucleotides can be assembled as part of a single nucleic acid molecule, or as separate nucleic acid molecules, and can be located on the same or different nucleic acid molecules. Accordingly, the polynucleotides can be introduced into the cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol.

In some embodiments, the disclosure provides a method of controlling a pest comprising contacting the pest 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: 1-3, wherein the pest is selected from the group consisting of Helicoverpa armigera (Cotton bollworm/Old World bollworm) , Mythimna separata (Oriental armyworm) , Athetis lepigone (Two-spotted armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , Agrotis ipsilon (Black cutworm) , Conogethes punctiferalis (Yellow peach moth) , Chilo suppressalis (Striped stem borer) , Sesamia inferens (Pink stem borer) , and Ostrinia furnacalis (Asian corn borer) . In other embodiments, the composition is a formulation for topical application to a plant. In still other embodiments, the composition is a transgenic plant. In further embodiments, the composition is a combination of a formulation topically applied to a transgenic plant. In some embodiments, the formulation comprises the first Cry protein of the disclosure when the transgenic plant comprises the second pest control agent. In other embodiments, the formulation comprises the second pest control agent when the transgenic plant comprises the first Cry protein of the disclosure.

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

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 macrocyclic lactones, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas and juvenile hormone mimics. In other embodiments, the chemical pesticide is selected from the group consisting of abamectin, acephate, acetamiprid, amidoflumet (S-1955) , avermectin, azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin, carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole, fenothicarb, fenoxycarb, fenpropathrin, fenproximate, fenvalerate, fipronil, flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701) , flufenoxuron, fonophos, halofenozide, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde, methamidophos, methidathion, methomyl, methoprene, methoxychlor, monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron (XDE-007) , oxamyl, parathion, parathion-methyl, permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060) , sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin, trichlorfon and triflumuron, aldicarb, oxamyl, fenamiphos, amitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad. In still other embodiments, the chemical pesticide is selected from the group consisting of cypermethrin, cyhalothrin, cyfluthrin and beta-cyfluthrin, esfenvalerate, fenvalerate, tralomethrin, fenothicarb, methomyl, oxamyl, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, avermectin, emamectin, endosulfan, ethiprole, fipronil, flufenoxuron, triflumuron, diofenolan, pyriproxyfen, pymetrozine and amitraz.

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

In further embodiments, the second pest control agent is a Vip3 vegetative insecticidal protein selected from the group consisting of Vip3Aa1, Vip3Aa2, Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vip3Aa8, Vip3Aa9, Vip3Aa10, Vip3Aa11, Vip3Aa12, Vip3Aa13, Vip3Aa14, Vip3Aa15, Vip3Aa16 , Vip3Aa17, Vip3Aa18, Vip3Aa19, Vip3Aa20, Vip3Aa21, Vip3Aa22, Vip3Aa2 , Vip3Aa24, Vip3Aa25, Vip3Aa26, Vip3Aa27, Vip3Aa28, Vip3Aa29, Vip3Aa30, Vip3Aa31, Vip3Aa32, Vip3Aa33 , Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38, Vip3Aa39, Vip3Aa40, Vip3Aa41, Vip3Aa42, Vip3Aa43, Vip3Aa44, Vip3Ab1, Vip3Ab2, Vip3Ac1, Vip3Ad1, Vip3Ad2, Vip3Ae1, Vip3Af1, Vip3Af2, Vip3Af3, Vip3Ag1, Vip3Ag2, Vip3Ag3 HM117633, Vip3Ag4, Vip3Ag5, Vip3Ah1, Vip3Ba1, Vip3Ba2, Vip3Bb1, Vip3Bb2 and Vip3Bb3.

In still further embodiments, the first Cry protein of the disclosure and the second pest control agent are co-expressed in a transgenic plant. This co-expression of more than one pesticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary. Alternatively, a plant, Parent 1, can be genetically engineered for the expression of the Cry protein of the disclosure. A second plant, Parent 2, can be genetically engineered for the expression of a second pest control agent. By crossing Parent 1 with Parent 2, progeny plants are obtained which express all the genes introduced into Parents 1 and 2.

In further embodiments, the disclosure provides a method of producing a pest-resistant (e.g., an insect-resistant) transgenic plant, comprising, introducing into a plant a polynucleotide, a chimeric gene, a recombinant vector, an expression cassette or a nucleic acid molecule comprising a nucleotide sequence that encodes a Cry protein of the disclosure, wherein the nucleotide sequence is expressed in the plant, thereby conferring to the plant resistance to a pest, and producing an insect-resistant transgenic plant, wherein the pest is selected from the group consisting of Helicoverpa armigera (Cotton bollworm/Old World bollworm) , Mythimna separata (Oriental armyworm) , Athetis lepigone (Two-spotted armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , Agrotis ipsilon (Black cutworm) , Conogethes punctiferalis (Yellow peach moth) , Chilo suppressalis (Striped stem borer) , Sesamia inferens (Pink stem borer) , and Ostrinia furnacalis (Asian corn borer) . In some embodiments, the Agrotis ipsilon (Black cutworm) is from a population of Agrotis ipsilon (Black cutworm) from China. In some embodiments, the introducing is achieved by transforming the plant. In other embodiments, the introducing is achieved by crossing a first plant comprising the chimeric gene, recombinant vector, expression cassette or nucleic acid molecule of the disclosure with a different second plant.

In some embodiments, the disclosure encompasses a method of providing a farmer with a means of controlling a pest, the method comprising supplying or selling to the farmer plant material such as a seed, the plant material comprising a polynucleotide, chimeric gene, expression cassette or a recombinant vector capable of expressing a Cry protein of the disclosure in a plant grown from the seed, as described above, wherein the pest is selected from the group consisting of Helicoverpa armigera (Cotton bollworm/Old World bollworm) , Mythimna separata (Oriental armyworm) , Athetis lepigone (Two-spotted armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , Agrotis ipsilon (Black cutworm) , Conogethes punctiferalis (Yellow peach moth) , Chilo suppressalis (Striped stem borer) , Sesamia inferens (Pink stem borer) , and Ostrinia furnacalis (Asian corn borer) . In some embodiments, the Agrotis ipsilon (Black cutworm) is from a population of Agrotis ipsilon (Black cutworm) from China.

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

EXAMPLES

Example 1. Activity of Cry Proteins Against Noctuidae, Crambidae, and Pyralidae pests

Cry proteins comprising the amino acid sequence of SEQ ID NOs: 1-3 were tested in an artificial diet bioassay against a China population of each of Helicoverpa armigera (Cotton bollworm/Old World bollworm) , Mythimna separata (Oriental armyworm) , Athetis lepigone (Two-spotted armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , Agrotis ipsilon (Black cutworm) , Conogethes punctiferalis (Yellow peach moth) , Chilo suppressalis (Striped stem borer) , Sesamia inferens (Pink stem borer) , and Ostrinia furnacalis (Asian corn borer) . These Cry proteins have been previously described as shown in Table 1.

Table 1. References for Cry Protein Disclosures.

Cry Protein	SEQ ID NO:	Publication No.
A BT29 protein	1	WO2018111553
A BT29-BT22 chimeric protein	2	WO2018111553
A BT29-Cry1FA chimeric protein	3	WO2018111553

Essentially, an equal amount of protein in solution was applied to the surface of an artifical insect diet in multi-well plates. After the diet surface dried, 24 larvae were added to each well. The plates were sealed and maintained at ambient laboratory conditions with regard to temperature, lighting and relative himidity. A positive-control group consisted of larvae exposed to a known active Cry protein. Negative control groups consisted of larvae exposed to insect diet treated with only the buffer solution. Percent mortality was assessed after about 3-4 days. For each protein, the experiment was repeated at least two times.

Results of the bioassay are shown in Table 2, where a “-” means 0%mortality, a “+/-” means 1-9%mortality (this category also includes 0%mortality with strong larval growth inhibition) , a “+” means 10-24%mortality, a “++” means 25-74%mortality, and a “+++” 75-100%mortality. Also shown in Table 2 is an indication of the activity of the Cry proteins against four North American species of pest insects in the family Noctuidae that includes black cutworm (Agrotis ipsilon) , fall armyworm (Spodoptera frugiperda) , corn earworm (Helicoverpa zea) and European corn borer (Ostrinia nubilalis) . For these four insect species, activity is represented simply as a “+” or “-” with no percent mortality indicated based on published data. Cells marked with “nt” represent that the protein was not tested against that pest species or no published information indicate that the Cry protein has been tested against that pest species.

Table 2. Results of bioassays with Cry Proteins.

Example 2. Expression and Activity of Cry Proteins in Maize Plants

Transgenic maize plants are prepared using the Cry proteins of the disclosure. Transformation of immature maize embryos is performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798 803. Briefly, Agrobacterium strain LBA4404 (pSB1) is 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 which is operably linked to a terminator and the second expression cassette comprises a plant expressible promoter operably linked to a selectable marker which is operably linked to a terminator. Expression of the selectable marker allows for identification of transgenic plants on selection media. Both expression cassettes are cloned into a suitable vector for Agrobacterium-mediated maize transformation. The transformed Agrobacterium strain is grown on YEP (yeast extract (5 g/L) , peptone (10g/L) , NaCl (5g/L) , 15g/l agar, pH 6.8) solid medium for 2-4 days at 28℃. Approximately 0.8 X 10 ⁹ Agrobacterium cells are suspended in LS-inf media supplemented with 100 μM As. Bacteria are pre-induced in this medium for approximately 30-60 minutes.

Immature embryos from an inbred maize line are excised from 8-12 day old ears into liquid LS-inf +100 μM As. Embryos are rinsed once with fresh infection medium. Agrobacterium solution is then added and embryos are vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos are then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between approximately 20 and 25 embryos per petri plate are transferred to LSDc medium supplemented with cefotaxime (250 mg/l) and silver nitrate (1.6 mg/l) and cultured in the dark at approximately 28℃ for 10 days.

Immature embryos, producing embryogenic callus are transferred to LSD1M0.5S medium. The cultures are selected on this medium for approximately 6 weeks with a subculture step at about 3 weeks. Surviving calli are transferred to Reg1 medium supplemented with mannose. Following culturing in the light (16 hour light/8 hour dark regiment) , green tissues are then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets are transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill. ) containing Reg3 medium and grown in the light. After about 2-3 weeks, plants are tested for the presence of the selectable marker gene and the Bt cry gene by PCR. Positive plants from the PCR assay are transferred to a greenhouse for further evaluation.

Transgenic plants are evaluated for copy number (determined by Taqman analysis) , protein expression level (determined by ELISA) , and efficacy against the insect species in Example 1 in bioassays. Specifically, plant tissue (leaf or silks) is excised from single copy events (V3-V4 stage) and infested with neonate larvae of the pests in Example 1, then incubated at room temperature for 5 days.

Example 3. Expression and Activity of Cry Proteins in Rice Plants

Transgenic rice plants are prepared using the Cry proteins of the disclosure. Methods of rice transformation are known in the art, such as the protocols set forth in Hiei et al. (Plant Journal 1994, 6 (2) : 271-282) and Zaidi et al. (Mol Biotechnol 2009, 43: 232-242) .

Transgenic plants are evaluated for copy number (determined by Taqman analysis) , protein expression level (determined by ELISA) , and efficacy against insect species in Example 1 in bioassays. Specifically, plant tissue (leaf or tiller) is excised from single copy events and infested with neonate larvae of the pests in Example 1, then incubated at room temperature for 4 days.

Results of the transgenic plant tissue bioassay from Example 2 and Example 3 are expected to confirm that the Cry proteins of the disclosure, when expressed in transgenic plants, are toxic to the pests in Example 1.

Example 4: Expression and Activity of Cry Proteins in Soybean Plants

Transgenic soy plants are prepared using the Cry proteins of the disclosure. Methods of soy transformation are known in the art, such as the protocols set forth in US Patent Publication No. US2004034889. Binary vectors for soybean transformation are constructed with a soybean appropriate promoter driving the expression of Cry proteins of the disclosure. The genes encoding the Cry proteins of the disclosure may be codon-optimized for soybean expression based upon the predicted amino acid sequence of their coding regions. Agrobacterium binary transformation vectors containing an expression cassette comprising a Cry protein coding sequence are constructed by also adding a transformation selectable marker gene. The selectable marker coding sequences may also be codon-optimized for expression in soybean.

Transgenic plants are evaluated for copy number (determined by Taqman analysis) , protein expression level (determined by ELISA) , and efficacy against insect species in Example 1 in bioassays. Specifically, plant tissue (leaf) is excised from single copy events and infested with neonate larvae of the pests in Example 1, then incubated at room temperature for 4 days.

Results of the transgenic plant tissue bioassay from Example 4 are expected to confirm that the Cry proteins of the disclosure, when expressed in transgenic plants, are toxic to the pests in Example 1.

Claims

A method of inhibiting the growth or killing a pest, comprising contacting said pest with a Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of Mythimna separata (Oriental armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , and Sesamia inferens (Pink stem borer) .
A method for controlling a pest population, comprising contacting the pest population with an insecticidally-effective amount of a Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of Mythimna separata (Oriental armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , and Sesamia inferens (Pink stem borer) .
The method according to claim 1 or 2, wherein said pest or pest population is further contacted with a second insecticidal protein different than the Cry protein.
The method of claim 3, wherein the second insecticidal protein is selected from the group consisting of a second Cry protein, a Vip protein, a protease inhibitor, a lectin, an alpha-amylase, and a peroxidase.
The method according to any of claims 1 to 4, wherein said contacting step is carried out with a microorganism or a plant expressing said protein, or insecticidal fragment thereof.
The method according to claim 5, wherein said plant is stably transformed with a DNA sequence encoding said protein, or insecticidal fragment thereof.
The method according to claim 6, wherein said plant is a monocotyledonous or dicotyledonous plant.
The method according to claim 7, wherein the monocotyledonous plant is a corn or rice plant.
The method according to claim 7, wherein the dicotyledonous plant is a soybean plant.
A method for protecting a plant from a pest, comprising expressing in the plant or cell thereof, an insecticidally-effective amount of a Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of Mythimna separata (Oriental armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , and Sesamia inferens (Pink stem borer) .
A method for controlling a pest population, comprising contacting the pest population with an insecticidally-effective amount of a Cry protein comprising the amino acid sequence of any of SEQ ID NOs: 1-3, or an insecticidal fragment thereof, wherein the pest is selected from the group consisting of Mythimna separata (Oriental armyworm) , Spodoptera litura (Common cutworm/Oriental leafworm) , and Sesamia inferens (Pink stem borer) .