Control of ostrinia furnacalis

Abstract

The present invention provides a method for controlling Ostrinia furnacalis (Ostrinia furnacalis) and protecting a crop, in particular corn, from economic damage caused by the Ostrinia furnacalis (Ostrinia furnacalis). The invention further relates to the use of a plant 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 Asiatic Ostrinia furnacalis.

Description

[0001] This application is a divisional application of the invention patent application filed on January 14, 2020, with application number 202010050555.2 and invention title "Control of Asian Corn Borer". Invention Field

[0002] This invention relates to methods for controlling or combating the genus Ostrinia, particularly the Asian corn borer (Ostrinia furnacalis) (Lepidoptera, Crambidae) (also known as the Asian corn borer), by using harmful organism-killing proteins and nucleic acid molecules encoding them, especially by using the Cry protein of Bacillus thuringiensis (“Bt”) and the cry gene encoding said Cry protein. Background of the Invention

[0003] 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 harmless to plants and other non-target organisms. Therefore, compositions containing Bacillus thuringiensis strains or their insecticidal proteins can be used as environmentally acceptable insecticides to control agricultural insect pests or insect vectors of a wide variety of human or animal diseases.

[0004] Cry proteins from Bacillus thuringiensis exhibit potent insecticidal activity against lepidopteran, dipteran, and coleopteran pests. These proteins also show activity against pests in the orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson, J. 1993. The Bacillus Thuringiensis family tree. Advanced Engineered Pesticides. Marcel Dekker, Inc., New York, NY). These proteins were initially classified as Cry I to Cry VI, primarily based on their insecticidal activity. The main categories are Lepidoptera-specific (I), Lepidoptera and Diptera-specific (II), Coleoptera-specific (III), Diptera-specific (IV), and Nematode-specific (V) and (VI). These proteins are further classified into subfamilies; within each family, more closely related proteins are assigned grouping letters, such as CryIA, CryIB, CryIC, etc. Within each group, more closely related proteins are given names such as CryIC(a), CryIC(b), etc. The terms “Cry toxin” and “δ-endotoxin” are used interchangeably with the term “Cry protein.” Current nomenclature of Cry proteins and genes is based on 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 system, each toxin was assigned a unique name that combined the first level (Arabic numerals), the second level (uppercase letters), the third level (lowercase letters), and the fourth level (another set of Arabic numerals). In the current classification, Roman numerals have been replaced with Arabic numerals in the first level. For example, "CryIA(a)" under the old nomenclature is now "Cry1Aa" under the current nomenclature.According to Ibrahim et al. (2010, Bioeng. Bugs, 1:31-50), Cry toxins can still be classified into six main categories based on their insect host specificity, including: Group 1 – Lepidoptera (e.g., Cry1, Cry9, and Cry15); Group 2 – Lepidoptera and Diptera (e.g., Cry2); Group 3 – Coleoptera (Cry3, Cry7, and Cry8); Group 4 – Diptera (Cry4, Cry10, Cry11, Cry16, Cry17, Cry19, and Cry20); Group 5 – Lepidoptera and Coleoptera (Cry1I); and Group 6 – Nematodes (Cry6). The Cry1I, Cry2, Cry3, Cry10, and Cry11 toxins (73-82 kDa) are distinctive because they appear to be naturally truncated versions of the larger Cry1 and Cry4 proteins (130-140 kDa).

[0005] Cry proteins are globular protein molecules that accumulate as protoxins in crystalline form during the spore-forming stage of Bt. Upon ingestion by pests, these crystals typically dissolve, releasing the protoxins. The size of the protoxins can range, for example, 130-140 kDa for many lepidopteran active Cry proteins (e.g., Cry1 and Cry9), and 60-80 kDa for coleopteran active Cry3 and lepidopteran / diptera active Cry2. After the crystals are dissolved by susceptible insects, the released protoxins are processed in the insect's 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, 130-140 kDa Cry prototoxins are typically activated by proteolytic removal of a 25-30 amino acid N-terminal peptide and approximately half of the remaining protein from the C-terminus, resulting in a mature Cry toxin of approximately 60-70 kDa. Prototoxins of 60-80 kDa (e.g., Cry2 and Cry3) are also processed, but to a different degree than the larger prototoxins. Smaller prototoxins typically remove the same or more amino acids from the N-terminus compared to larger prototoxins, but fewer amino acids 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 Cry proteins are quite toxic to specific target insects, but many have narrow activity profiles.

[0006] Cry proteins typically possess 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 α-helices and is involved in 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 “pizzaner” configuration (de Maagd et al., 2001, ibid.). Domains II and III are involved in receptor recognition and binding and are therefore considered determinants of toxin specificity.

[0007] Many commercially valuable plants, including common crops, are vulnerable to plant pests, including insect and nematode pests, leading to significant declines in crop yield and quality. For example, plant pests are a major contributor to the loss of important crops worldwide. In the United States alone, approximately $8 billion is lost annually due to invertebrate pests, including insects. Insect pests are also a burden for vegetable and fruit growers, ornamental flower producers, and home gardeners.

[0008] Insect pests are primarily controlled through intensive application of chemical pesticides, which are active by inhibiting insect growth, preventing feeding or reproduction, or causing death. Biological pest control agents, such as Bacillus thuringiensis strains expressing biotoxins (e.g., Cry proteins), have also been applied to crop plants with satisfactory results, thus providing alternatives or complements to chemical pesticides. Genes encoding some of these Cry proteins have been isolated, and their expression in heterologous hosts (e.g., transgenic plants) has shown to provide another tool for controlling economically important insect pests. Most Cry proteins are active against a very limited spectrum of insect pests. And typically, activity against one insect species cannot predict activity against different insect species.

[0009] The Asian corn borer (Asian corn borer) is most common throughout Asia and Southeast Asia. It thrives in tropical regions where its host crops are continuously cultivated year-round. While corn is its primary food source, it also eats and damages bell peppers, cotton, hops, millet, pearl millet, foxtail millet, sugarcane, sorghum, and ginger. Additionally, it can be found on many wild plants, such as wormwood, Job's tears, knotweed, wild sugarcane, Johnson's grass, and para grass.

[0010] The Asian corn borer can be controlled with chemical pesticides and is susceptible to certain Bt Cry proteins. Therefore, good insect control can be achieved; however, like other pests, the Asian corn borer has developed high levels of resistance to insecticides and certain Cry proteins. For example, the Asian corn borer has shown resistance to Cry1Ie, Cry1Ah, Cry1Ab, Cry1F, and others. Therefore, there is a need for new control methods that use pesticide proteins that can target the Asian corn borer, particularly populations that have become resistant to chemical pesticides and especially those resistant to existing Cry proteins. To date, there have been no reports of controlling the Asian corn borer using the Cry protein of this invention or a modified Cry protein. Invention Overview

[0011] This invention provides a method for controlling the Asian corn borer and protecting crops (particularly maize) from economic damage caused by the Asian corn borer. The invention further relates to the use of plants, particularly monocotyledonous plants, especially maize (Zea mays), stably transformed with nucleic acid molecules encoding the Cry protein of this invention (alone or in combination with other insecticidal proteins), for controlling or combating the Asian corn borer. The invention also further relates to the use of insecticidal formulations comprising the Cry protein of this invention for protecting plants from Asian corn borer infestation. The invention also relates to plants, particularly monocotyledonous plants, especially maize plants, which are susceptible to Asian corn borer infestation, and to plants transformed with expressible nucleic acid molecules encoding the Cry protein of this invention to combat or control Asian corn borer pest populations.

[0012] According to the present invention, a method is provided to combat and / or control insects of the genus Stem borer, particularly the Asian corn borer (Asian corn borer), by contacting these insects with a Cry protein or an insecticidal fragment thereof comprising an amino acid sequence of any one of SEQ ID NO: 1-5.

[0013] Furthermore, according to the present invention, the contact step can be carried out using an insecticidal composition comprising: the Cry protein of the present invention or an insecticidal fragment thereof, and an acceptable agricultural carrier. Additionally, insect contact can be with plants stably transformed with an expressible nucleic acid molecule encoding the Cry protein of the present invention, particularly monocotyledonous plants, especially maize plants, thereby enabling the transformed plants to express the Cry protein of the present invention or an insecticidal fragment thereof in an effectively controlled manner with varying insect loads.

[0014] Furthermore, plants infested by the Asian corn borer, especially monocotyledonous plants, particularly corn plants, are protected from ongoing economic damage from this insect by stable conversion with the gene encoding the Cry protein of this invention.

[0015] Brief description of sequences in a sequence list

[0016] SEQ ID NO: 1 is the amino acid sequence of the Cry1Gb-Cry1If-Cry1Gb (BT29-BT22) hybrid Cry protein.

[0017] SEQ ID NO: 2 is the amino acid sequence of the Cry1Bb-Cry1Ca-Cry1Ac (TIC860) hybrid Cry protein.

[0018] SEQ ID NO: 3 is the amino acid sequence of the Cry1Be-Cry1Ka-Cry1Ab (TIC867) hybrid Cry protein.

[0019] SEQ ID NO: 4 is the amino acid sequence of the Cry1Be-Cry1Ka-Cry1Be (TIC867-23) hybrid Cry protein.

[0020] SEQ ID NO: 5 is the amino acid sequence of the Cry1Be-Cry1Ca-Cry1Ab (TIC868) hybrid protein. Invention Details

[0021] This description is not intended to be a detailed list of all the different ways in which the invention can be practiced, or of all the features that can be added to the invention. For example, a feature exemplified with respect to one embodiment may be incorporated into other embodiments, and a feature exemplified with respect to a particular embodiment may be removed from that embodiment. Therefore, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. Furthermore, given this disclosure, numerous variations and additions to the various embodiments presented herein will be clearly apparent to those skilled in the art without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention, and not to exhaustively describe all permutations, combinations, and variations thereof.

[0022] 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 pertains. The terminology used in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0023] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “plant” refers to one or more plants and includes their equivalents known to those skilled in the art, etc.

[0024] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives (“or”).

[0025] The term "about" is used herein to mean approximately, roughly, around, or near. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundary to be higher or lower than the stated value. Typically, the term "about" is used herein to change a value to be higher or lower than the stated value by a variation of up or down (higher or lower) by 20% (preferably 10%). With respect to temperature, the term "about" means ± 1°C, preferably ± 0.5°C. When the term "about" is used in the context of this invention (e.g., in conjunction with temperature or molecular weight values), precise values ​​(i.e., no "about") are preferred.

[0026] "Controlling" insects means suppressing the ability of insect pests to survive, grow, feed, or reproduce through toxic effects, or limiting insect-related damage or loss in crop plants, or protecting the yield potential of crops when grown in the presence of insect pests. "Controlling" insects may or may not mean killing insects, although it preferably means killing insects.

[0027] When used in this specification, the terms “comprising” or “including” expressly indicate the presence of the stated features, integers, steps, operations, elements, or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0028] As used herein, the transitional phrase “consistently of…” (and grammatical variations) means that the scope of the claim is to be interpreted as covering the material or steps explicitly described in the claim, as well as those that do not substantially alter the fundamental and novel features of the claimed invention. Therefore, when used in the claims of this invention, the term “consistently of…” is not intended to be interpreted as equivalent to “comprising (including)”.

[0029] As used herein, the term "Cry protein" refers to an insecticidal protein that can occur in crystalline form in Bacillus thuringiensis or related bacteria. The term "Cry protein" can refer to the protoxin form or any of its insecticidal fragments or toxins.

[0030] "Delivery" of a composition or toxic protein means that the composition or toxic protein comes into contact with an insect, which promotes the oral uptake of the composition or toxic protein, thereby resulting in a toxic effect and control over the insect. The composition or toxic protein can be delivered in many recognized ways, including but not limited to transgenic plant expression, formulated protein compositions, sprayable protein compositions, bait substrates, or any other protein delivery system recognized in the art.

[0031] "Effective control of insect populations" means the concentration of such toxic proteins that inhibit the ability of insects to survive, grow, feed, or reproduce through toxic effects, or limit insect-related damage or loss in crop plants, or protect the yield potential of crops when grown in the presence of insect pests. "Effective control of insect populations" may or may not mean killing insects, although it preferably means killing insects.

[0032] In this paper, a “gene” is defined as a genetic unit containing one or more polynucleotides, which occupies a specific location on a chromosome or plasmid and contains genetic instructions about a specific characteristic or trait in an organism.

[0033] As used herein, "pesticide," "insecticide," etc., refer to the ability of the Cry protein of the present invention to control pest-like organisms, or to control the amount of Cry protein in pest-like organisms as defined herein. Therefore, the pest-killing Cry protein can kill or inhibit the ability of pest-like organisms (e.g., insect pests) to survive, grow, feed, or reproduce.

[0034] Nucleotides are designated in this document using the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Similarly, amino acids are designated using 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).

[0035] This invention is based on the results of a toxicity assay performed by feeding Asian corn borers (Asian corn borers) an artificial diet containing purified Cry toxins derived from Bacillus thuringiensis, and surprisingly, it showed that certain Cry proteins are toxic to the Asian corn borer (see Example 1). Therefore, these active Bt proteins can be used to provide maximum protection against this important pest and can prevent or reduce the development of insect resistance to Bt insecticides in the field.

[0036] The “Cry protein” of the present invention may be naturally occurring or engineered, and encompasses the full-length protein (protoxin) having the amino acid sequence shown in any one of SEQ ID NO: 1-5 in the sequence listing, as well as any of its insecticidal fragments.

[0037] The present invention also includes polynucleotides as fragments of polynucleotides encoding the Cry protein protoxin. "Fragment" means a portion of the nucleotide sequence encoding the Cry protein. A fragment of the nucleotide sequence may encode a biologically active portion of the Cry protein, a so-called "toxin fragment," or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. Nucleic acid molecules comprising fragments of the nucleotide sequence encoding the Cry protein contain at least approximately 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 with respect to SEQ ID NO: 1), depending on the intended use. "Consecutive" nucleotides refer to nucleotide residues that are adjacent to each other. Some fragments of the nucleotide sequence of the present invention will encode toxin fragments that retain the biological activity of the Cry protein and thus retain insecticidal activity. "Retaining insecticidal activity" means that the fragment will possess 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, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al., (1988) Biochem. J. 252:199-206; Marrone et al., (1985) J. of Economic Entomology 78:290-293; and U.S. Patent No. 5,743,477, all of which are incorporated herein by reference in their entirety.

[0038] The toxic fragments of the Cry protein of the present invention will encode at least approximately 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 protein of the present invention.

[0039] As used herein, a Cry protein that is “toxic” to insect pests means that the Cry protein functions as an orally active insect control agent to kill the insect pest, or that the Cry protein can disrupt or prevent insect feeding or cause growth inhibition of the insect pest, either or without causing the death of the insect. When the Cry protein of the present invention is delivered to an insect or when the insect comes into oral contact with the Cry protein, the result is typically the death of the insect, or a slowing of the insect’s growth, or the cessation of feeding by the insect, thus providing the insect with a source of the toxic Cry protein.

[0040] In some embodiments, the present invention provides a method for inhibiting or killing the Asian corn borer pest, the method comprising contacting the Asian corn borer pest with a Cry protein or an insecticidal fragment thereof comprising an amino acid sequence of any one of SEQ ID NO: 1-5.

[0041] In some embodiments, the present invention provides a method for controlling populations of the Asian corn borer pest, the method comprising contacting the pest population with an insecticidal amount of a Cry protein or an insecticidal fragment thereof comprising an amino acid sequence comprising any one of SEQ ID NO: 1-5.

[0042] In a further embodiment of the invention, the Asian corn borer pest or pest population is further contacted with a second insecticidal protein that has an amino acid sequence different from that of the Cry protein comprising any one of SEQ ID NO: 1-5. In other embodiments, the second insecticidal protein is selected from the group consisting of: Cry protein, Vip protein, protease inhibitors, lectins, α-amylase, and peroxidase.

[0043] In other embodiments of the invention, the contact step (wherein the Cry protein of the invention comes into contact with the Asian corn borer pest) is performed using a microorganism or plant expressing the protein or an insecticidal fragment thereof. In other embodiments, the plant is stably transformed with a nucleic acid molecule encoding the Cry protein of the invention 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 plant, or the dicotyledonous plant is a soybean plant.

[0044] In some embodiments, the present invention provides a method for protecting plants from the Asian corn borer pest, the method comprising expressing an insecticidally effective amount of a Cry protein or an insecticidal fragment thereof comprising any one of the amino acid sequences in SEQ ID NO: 1-5 in the plant or its cells. In other embodiments, the plant is a monocotyledonous or dicotyledonous plant. In still other embodiments, the monocotyledonous plant is a corn plant, or the dicotyledonous plant is a soybean plant.

[0045] For it to be effective against the Asian corn borer, the Cry protein is first orally ingested by the insect. However, the Cry protein can be delivered to the insect in many recognized ways. Methods of orally delivering the protein to the insect include, but are not limited to, (1) providing the protein in a transgenic plant, wherein the insect consumes (ingests) one or more parts of the transgenic plant, thereby ingesting the polypeptide expressed in the transgenic plant; (2) providing the protein in a formulated protein composition that can be applied to or incorporated into, for example, an insect growth medium; (3) providing the protein in a protein composition that can be applied to a surface, for example, sprayed onto the surface of a plant part, and then ingested by the insect when it consumes one or more of the sprayed plant parts; (4) a bait matrix; or (5) any other protein delivery system recognized in the art. Therefore, any method of orally delivering the toxic Cry protein of the present invention can be used in the method of the present invention. In some particular embodiments, the Cry protein of the present invention is orally delivered to the insect, wherein the insect ingests one or more parts of the transgenic plant.

[0046] In other embodiments, the Cry protein of the present invention is orally delivered to an insect, wherein the insect ingests one or more portions of a plant sprayed with a composition comprising the Cry protein of the present invention. Delivery of the compositions of the present invention to a plant surface can be carried out using any method known to those skilled in the art for applying compounds, compositions, formulations, etc., to a plant surface. Some non-limiting examples of delivery to or contact with a plant or its portions include spraying, dusting, sprinkling, dispersing, misting, atomizing, broadcasting, soaking, soil injection, soil incorporation, saturation (e.g., root, soil treatment), impregnation, irrigation, wrapping, leaf or stem penetration, lateral application, or seed treatment, and combinations thereof. These and other procedures for contacting a plant or its portions with a compound, composition, or formulation are well known to those skilled in the art.

[0047] In some embodiments of the invention, the insecticidal Cry protein of the invention is expressed in higher organisms (e.g., plants). In this case, the transgenic plants expressing an effective amount of the insecticidal protein protect themselves from plant pests such as insect pests. When the Asian corn borer larva begins to feed on such a transgenic plant, it ingests the expressed insecticidal Cry protein. This can prevent the insect from further biting into the plant tissue, or even harm or kill the insect. A polynucleotide encoding the Cry protein of the invention is inserted into an expression cassette, which is then stably integrated into the plant genome. In other embodiments, the polynucleotide is included in a non-pathogenic self-replicating virus. The plants that can be transformed according to the present invention can be monocotyledonous or dicotyledonous plants, and include, but are not limited to, corn, soybean, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, rapeseed, alfalfa, tobacco, peanut, vegetables (including sweet potato, green bean, pea, chicory, lettuce, cabbage, cauliflower, kale, turnip, carrot, eggplant, cucumber, radish, spinach, potato, tomato, asparagus, onion, garlic, melon, pepper, celery, squash, pumpkin, dense zucchini), 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 thaliana) and woody plants (e.g., conifers and deciduous trees). Preferably, the plants used in this invention are crop plants, such as corn, soybean, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, sugar beet, sugarcane, tobacco, barley, rapeseed, etc. Once the desired polynucleotide has been transformed into a specific plant species, it can be propagated within that species or transferred to other varieties of the same species (especially commercial varieties) using conventional breeding techniques.

[0048] The polynucleotide encoding the Cry protein of the present invention is expressed in transgenic plants, thereby inducing the biosynthesis of the Cry protein (in protoxin or toxin form) encoded in said transgenic plants. In this way, transgenic plants with enhanced yield protection in the presence of Asian corn borer population stress are produced. For expression in transgenic plants, the nucleotide sequence encoding the Cry protein may require modification and optimization. Although genes from microbial organisms can be expressed at high levels in plants without modification in many cases, 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 specific preferences for codon usage, and the codons of the nucleotide sequences described in this invention can be varied to conform to plant preferences while retaining the amino acids thereby encoded. Furthermore, high expression in plants (e.g., maize plants) is best achieved from coding sequences having a GC content of at least about 35%, or at least about 45%, or at least about 50%, or at least about 60%. Microbial nucleotide sequences with low GC content may be poorly expressed in plants due to the presence of ATTTA motifs that can destabilize information and AATAAA motifs that can cause inappropriate polyadenylation. While some gene sequences can be adequately expressed in both monocot and dicot species, sequences can be modified to address specific codon and GC content preferences for monocots or dicots, as these preferences have been shown to differ (Murray et al., Nucl. Acids Res. 17:477-498 (1989)). Additionally, nucleotide sequences can be screened for the presence of irregular splicing sites that may cause information truncation. All necessary modifications within the nucleotide sequences (e.g., those described above) are performed using well-known site-directed mutagenesis, PCR, and synthetic gene construction techniques (using methods described, for example, in U.S. Patent Nos. 5,625,136, 5,500,365, and 6,013,523).

[0049] For effective translation initiation, the sequence adjacent to the starting methionine may need to be modified. For example, they can be modified by including sequences known to be effective in plants. Joshi has proposed suitable common sequences for plants (NAR 15:6643-6653 (1987)). These common sequences are suitable for use with the nucleotide sequences of the present invention. The sequence is incorporated into the construct containing the nucleotide sequence up to and including ATG (while leaving the second amino acid unmodified), or alternatively up to and including GTC following ATG (with the possibility of modifying the second amino acid of the transgene).

[0050] The polynucleotide sequence encoding the Cry protein of the present invention can be operatively fused to a wide variety of promoters (including constitutive promoters, inducible promoters, time-regulated promoters, developmentally regulated promoters, chemically regulated promoters, tissue-preferred promoters, and tissue-specific promoters) for expression in plants to prepare recombinant DNA molecules, i.e., chimeric genes. The choice of promoter will vary depending on the temporal and spatial requirements of expression and also on the target species. Therefore, expression of the nucleotide sequence of the present invention in leaves, in culms or stems, in spikes, in inflorescences (e.g., spikes, panicles, rachis, etc.), in roots, or in seedlings is preferred. However, in many cases, protection against more than one type of insect pest is sought, and therefore expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to function in monocotyledons and vice versa, it is ideal to select promoters from dicotyledons for expression in dicotyledons and promoters from monocotyledons for expression in monocotyledons. However, there are no restrictions on the origin of the chosen promoter; it is sufficient that they function in driving the expression of the nucleotide sequence in the desired cells.

[0051] Suitable constitutive promoters include, for example, the CaMV 35S promoter (SEQ ID NO: 1546; Odell et al., Nature 313:810-812, 1985); the Arabidopsis thaliana At6669 promoter (SEQ ID NO: 1652; see PCT publication WO04081173A2); 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. Plant100:456-462, 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 cyclic protein (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 (SEQ ID NO: 1535; see also PCT application number IL / 2005 / 000627); and synthetic super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Patent Nos. 5,659,026, 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, and 5,608,142. Tissue-specific or tissue-preferred promoters useful for expressing the novel cry protein coding sequence of the present invention in plants (particularly corn) are those that direct expression in roots, pith, leaves, or pollen. Suitable tissue-specific promoters include, but are not limited to, leaf-specific promoters [e.g., by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol.].105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J.3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993, as described], 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), legume globulin (Ellis et al., Plant Mol. Biol. 10: 203-214, 1988), gluten (rice) (Takaiwa et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa et al., FEBS Letts. 221: 43-47, 1987), zein (Matzke et al., Plant Mol. Biol, 14(3), 323-32, 1990), napA (Stalberg et al., Planta 199: 515-519, 1996), wheat SPA (Albanietal, Plant Cell, 9: 171-184, 1997), sunflower oil protein (Cummins et al., 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 gliadin (EMB03:1409-15, 1984), barley ltrl promoter, barley B1, C, D gliadin (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 gliadin NRP33, rice globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), rice α-globulin REB / OHP-1 (Nakase et al., Plant Mol. Biol. 33:513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR gene family (Plant J 12:235-46, 1997), sorghum γ-sorghum gliadin (Plant Mol. Biol. 33:513-68 ... [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 olein (Wu et al., J. Biochem., 123:386, 1998)], flower-specific promoters [e.g., AtPRP4, chalcone synthase (chsA) (Van der Meer et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217:240-245, [1989), aptala-3], plant reproductive tissues [e.g., OsMADS promoter (US Patent Application 2007 / 0006344)].

[0052] The nucleotide sequence can also be expressed under the regulation of a chemically regulated promoter. This makes the Cry protein of the present invention synthesized only when crop plants are treated with inducing chemicals. Examples of such chemically regulated techniques for gene expression are described in detail in published application EP 0 332 104 and U.S. Patent No. 5,614,395. In one embodiment, the chemically regulated promoter is the tobacco PR-1a promoter.

[0053] Another class of promoters useful in this invention are wound-inducible promoters. Numerous promoters expressed at wound sites and also at sites of infection by plant pathogens have been described. Ideally, such promoters should be locally active only at the site of insect invasion, and in this way, the insecticidal protein accumulates only in the cells where it is necessary to synthesize the insecticidal protein to kill the invading insect pest. Examples of this type of promoter include those described in the following literature: 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).

[0054] Non-limiting examples of promoters useful in inducing tissue-specific expression patterns in this invention include green tissue-specific promoters, root-specific promoters, stem-specific promoters, or flower-specific promoters. Promoters suitable for expression in green tissues include a number of promoters regulating genes involved in photosynthesis, many of which have been cloned from monocots and dicots. 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 U.S. Patent No. 5,466,785). Another promoter useful in this invention is the stem-specific promoter described in U.S. Patent No. 5,625,136, which naturally drives the expression of the maize trpA gene.

[0055] In addition to selecting a suitable promoter, constructs for expressing insecticidal toxins in plants also require a suitable transcription terminator to be operatively linked downstream of a heteronucleotide sequence. Several such terminators are available and are 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 invention.

[0056] Numerous other sequences can be incorporated into the expression cassettes described in this invention. These include sequences that have shown enhanced expression, such as intron sequences (e.g., from Adhl and Bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0057] It may be preferred to target the expression of the nucleotide sequences of the present invention to different cellular locations in plants. In some cases, targeting in the cytosol may be desirable, while in others, targeting in some subcellular organelles may be preferred. Any mechanism for targeting gene products (e.g., in plants) can be used to practice the present invention, and such mechanisms are known to exist in plants and the sequences controlling these mechanisms have been characterized in considerable detail. Sequences that induce the targeting of gene products to other cellular compartments have been characterized. The N-terminal sequence can be responsible for targeting the target protein to any cellular compartment, such as vacuoles, mitochondria, peroxisomes, proteosomes, endoplasmic reticulum, chloroplasts, starch grains, pollen makers, apoplasts, or the cell wall of plants (e.g., Unger et al., Plant Molec. Biol. 13: 411-418 (1989); Rogers et al., (1985) Proc. Natl. Acad. Sci. USA 82:6512-651; U.S. Patent No. 7,102,057; WO 2005 / 096704, all of which are incorporated herein by reference). Optionally, the signal sequence can be an N-terminal signal sequence from waxy, an N-terminal signal sequence from γ-zein, a starch-binding domain, a C-terminal starch-binding domain, or a chloroplast targeting sequence (which imports mature proteins into chloroplasts) (Comai et al., (1988) J. Biol. Chem. 263: 15104-15109; van den Broeck et al., (1985) Nature 313: 358-363; US Patent No. 5,639,949) or secretory signal sequences from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, the N-terminal sequence, together with the C-terminal sequence, is responsible for vacuolar targeting of the gene product (Shinshi et al., (1990) Plant Molec. Biol. 14:357-368). In one embodiment, the selected signal sequence includes a known cleavage site, and the constructed fusion takes into account any amino acid following said cleavage site that is 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 by replacing some amino acids within the transgenic sequence. These construction techniques are well known in the art and are equally applicable to any cell compartment.

[0058] It will be recognized that the cell-targeting mechanisms described above can be used not only in conjunction with their homologous promoters but also with heterologous promoters in order to influence specific cell-targeting targets under the transcriptional regulation of promoters with expression patterns different from those of the promoter from which the target signal originates.

[0059] Procedures for transforming plants are well known in the art and have been described throughout the literature. Non-limiting examples of methods for plant transformation include transformation via bacterial-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, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrochemical, chemical, physical (mechanical), or biological mechanism, including any combination thereof, that results in the introduction of nucleic acids into plant cells. General guidelines known in the field regarding various plant transformation methods include Miki et al. (“Procedures for Introducing Foreign DNA into Plants”, in Methods in Plant Molecular Biology and Biotechnology, edited by Glick, BR and Thompson, JE (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0060] For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA boundary sequence are suitable, while for direct gene transfer (e.g., particle bombardment, etc.), any vector is suitable and linear DNA containing only the target construct can be used. In the case of direct gene transfer, transformation or co-transformation with a single DNA species 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) performed with a selective marker, which can be positive selection (mannose phosphate isomerase) providing resistance to antibiotics (kanamycin, hygromycin, or methotrexate) or herbicides (glyphosate or glufosinate). However, the choice of selective marker is not critical to this invention.

[0061] Agrobacterium-mediated transformation is a widely used method for transforming plants due to its high transformation efficiency and its broad availability to many different species. Agrobacterium-mediated transformation typically involves the transfer of a binary vector carrying the target exogenous DNA to a suitable Agrobacterium strain, which can depend on a complement of the vir gene carried by the host Agrobacterium strain on a co-resident Ti plasmid or on the chromosome (Uknes et al., (1993) Plant Cell 5:159-169). The transfer of recombinant binary vectors to Agrobacterium can be accomplished via a triparental mating procedure using Escherichia coli carrying the recombinant binary vector and a helper Escherichia coli strain carrying a plasmid capable of moving the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium via nucleic acid transformation (Höfgen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[0062] Both dicotyledonous and monocotyledonous plants can be transformed using Agrobacterium. Agrobacterium-mediated transformation methods for rice include those well-known for rice transformation, such as those described in any of the following publications: 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), the disclosures of which are incorporated herein by reference as if fully described. 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 contents of which are incorporated herein by reference as if fully described. As an example, the methods are further described in the following references: B. Jenes et al., Techniques for Gene Transfer, in Transgenic Plants, Vol.1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press (1993) 128-143; and Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225. Preferably, the nucleic acid or construct to be expressed is cloned into a vector suitable for transforming *Agrobacterium tumefaciens*, such as pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). *Agrobacterium* transformed with such a vector can then be used in known ways for the transformation of plants (e.g., plants used as model organisms such as *Arabidopsis thaliana*) or crop plants (e.g., tobacco plants)), for example by immersing crushed or chopped leaves in a solution of *Agrobacterium* and then culturing them in a suitable culture medium.Plant transformation using *Agrobacterium tumefaciens* has been described, for example, by Hagen and Willmitzer in *Nucl. Acid Res.* (1988) 16, 9877, or especially from FF White, Vectors for Gene Transfer in Higher Plants, in *Transgenic Plants*, Vol. 1, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press, 1993, pp. 15–38.

[0063] Plant transformation via recombinant Agrobacterium typically involves co-culturing Agrobacterium with explants from plants, following methods well-known in the art. Transformed tissues are regenerated on selective media carrying antibiotic or herbicide resistance markers between the boundaries of binary plasmid T-DNA.

[0064] As previously discussed, another method for transforming plants, plant parts, and plant cells involves advancing inert or biologically active particles into plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792. Typically, this method involves advancing inert or biologically active particles into plant cells under conditions that are effective for penetrating the outer surface of the cell and providing incorporation into the cell interior. When using inert particles, the carrier can be introduced into the cell by coating the particles with a carrier containing the target nucleic acid. Alternatively, the cell can be surrounded by the carrier, such that the carrier is carried into the cell by trailing the particles. Biologically active particles (e.g., dried yeast cells, dried bacteria, or bacteriophages, each containing one or more nucleic acids to be introduced) can also be advanced into plant tissues.

[0065] In other embodiments, the polynucleotide encoding the Cry protein of the present invention can be directly transformed into the plastid genome. A key advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without significant modifications, and that plastids can express multiple open reading frames under the control of a single promoter. Plastid transformation techniques are described in detail in U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application No. WO 95 / 16783, and in McBride et al., (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing a region of cloned plastid DNA flanking a selective marker (alongside the target gene) into a suitable target tissue, for example, by using biological projectiles or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation). Flanking regions of 1 to 1.5 kb (called target sequences) promote homologous recombination with the plastome genome, thus allowing substitution 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 could be used as selectivity markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, JM and Maliga, P. (1992) Plant Cell 4, 39-45). The presence of cloning sites between these markers allows for the creation of plastome-targeting vectors for the introduction of exogenous genes (Staub, JM and Maliga, P. (1993) EMBO J. 12, 601-606). Significantly increased transformation frequencies can be achieved by replacing recessive rRNA or r-protein antibiotic resistance genes with a dominant selective marker (bacterial aadA gene, which encodes spectinomycin detoxification enzyme, aminoglycoside-3'-adenosyltransferase) (Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA90, 913-917). Previously, this marker has been successfully used for high-frequency transformation of the plastid genome in the green alga *Chlamydomonas reinhardtii* (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Other selective markers useful for plastid transformation are known in the art and are included within the scope of this invention.Typically, approximately 15-20 cell division cycles are required after transformation to reach the isotopic state. Plastid expression (where genes are inserted into the circular plasmid genome, which contains thousands of copies in each plant cell, via homologous recombination) leverages the enormous copy number advantage of genes expressed more readily than those expressed nuclearly, allowing expression levels exceeding 10% of total soluble plant proteins to be easily achieved. In one embodiment, the polynucleotides of the invention can be inserted into a plasmid-targeting vector and transformed into the plasmid genome of the desired plant host. Thus, plants homologous with respect to the plasmid genome containing the nucleotide sequences of the invention can be obtained, capable of high expression of the polynucleotides.

[0066] Methods for selecting transformed transgenic plants, plant cells, or plant tissue cultures are conventional in the art and can be used in the methods of the present invention provided herein. For example, the recombinant vector of the present invention may also include an expression cassette containing a nucleotide sequence relating to a selective marker that can be used to select transformed plants, plant parts, or plant cells. As used herein, “selective marker” means a nucleotide sequence that, when expressed, confers a different phenotype on plants, plant parts, or plant cells expressing the marker, and thus allows such transformed plants, plant parts, or plant cells to be distinguished from those without the marker. Such nucleotide sequences may encode selective or screening markers, depending on whether the marker confers a trait that can be selected by chemical means, such as by using a selection reagent (e.g., antibiotics, herbicides, etc.), or whether the marker is simply a trait that can be identified by observation or testing, such as by screening (e.g., R-locus trait). Of course, many examples of suitable selective markers are known in the art and can be used in the expression cassettes described herein.

[0067] Examples of selective markers include, but are not limited to: nucleotide sequences encoding neo or nptII, which confer resistance to kanamycin, G418, etc. (Potrykus et al., (1985) Mol. Gen. Genet. 199:183-188); nucleotide sequences encoding bar, which confer resistance to phosphinic acid; nucleotide sequences encoding altered 5-enolpyruvate-shikimate-3-phosphate (EPSP) synthase, which confer resistance to glyphosate (Hinchee et al., (1988) Biotech.6:915-922); and nucleotide sequences encoding nitrile hydrolases (e.g., bxn from Klebsiella ozaenae), which confer resistance to bromobenzonitrile (Stalker et al., (1988) Science). Nucleotide sequences encoding altered acetyllactone synthase (ALS) confer resistance to imidazolinone, sulfonylureas, or other chemicals that inhibit ALS (European Patent Application No. 154204); nucleotide sequences encoding methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al., (1988) J. Biol. Chem. 263:12500-12508); nucleotide sequences encoding maltose dehalogenase confer resistance to maltose; nucleotide sequences encoding mannose-6-phosphate isomerase (also known as phospmannose isomerase (PMI)) confer the ability to metabolize mannose (US Patent Nos. 5,767,378 and 5,994,629); nucleotide sequences encoding altered anthranilate synthase confer resistance to 5-methyltryptophan; or nucleotide sequences encoding hph confer resistance to hygromycin. Those skilled in the art can select appropriate selective markers for use in the expression box of the present invention.

[0068] Other selective markers include, but are not limited to: nucleotide sequences encoding β-glucuronidase, or uidA (GUS), which encodes an enzyme for a variety of known chromogenic substrates; nucleotide sequences encoding R-lobe products that regulate the production of anthocyanin pigments (red) 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)); and nucleotide sequences encoding β-lactamases (an enzyme for a variety of known chromogenic substrates, such as PADAC, chromogenic cephalosporins) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA). 75:3737-3741); nucleotide sequence encoding xylE (which encodes catechol dioxygenase) (Zukowsky et al., (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); nucleotide sequence encoding tyrosinase, which is an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone (which then condenses to form melanin) (Katz et al., (1983) J. Gen. Microbiol.129:2703-2714); nucleotide sequence encoding β-galactosidase (an enzyme for which a chromogenic substrate is present); nucleotide sequence encoding luciferase (lux), which allows for bioluminescent detection (Ow et al., (1986) Science 234:856-859); nucleotide sequence encoding jellyfish luminescent protein, which can be used in calcium-sensitive bioluminescent detection (Prasher et al., (1985) Biochem). Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al., (1995) Plant Cell Reports 14:403-406). Those skilled in the art can select suitable selective markers for use in the expression cassette of the present invention.

[0069] Furthermore, as is well known in the art, complete transgenic plants can be regenerated from transformed plant cells, plant tissue cultures, or cultured protoplasts using any of a wide variety of known techniques. Plant regeneration from plant cells, plant tissue cultures, or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); and Vasil IR (eds.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984) and Vol. II (1986)).

[0070] Furthermore, the genetic characteristics described above, which have been modified into the transgenic seeds and plants, plant parts, or plant cells of this invention, can be transmitted through sexual reproduction or vegetative growth, and thus can be maintained and propagated in offspring plants. Typically, maintenance and propagation utilize agricultural methods known to have been developed for specific purposes (e.g., harvesting, sowing, or cultivation).

[0071] Therefore, polynucleotides can be introduced into plants, plant parts, or plant cells in any number of ways well-known in the art, as described above. Thus, regardless of a specific method for introducing one or more polynucleotides into a plant, any method that allows said one or more polynucleotides to be stably integrated into the plant genome can be used. When more than one polynucleotide is to be introduced, the respective polynucleotides can be assembled as part of a single nucleic acid molecule, or assembled as separate nucleic acid molecules, and can be located on the same or different nucleic acid molecules. Therefore, said polynucleotides can be introduced into target cells in a single transformation event, in separate transformation events, or, for example, in a plant, as part of a breeding program.

[0072] In some embodiments, the present invention provides a method for controlling the Asian corn borer, the method comprising contacting the Asian corn borer with a composition comprising a first insecticidal protein and a second pest control agent different from the first insecticidal protein, wherein the first insecticidal protein is a Cry protein comprising the amino acid sequence of any one of SEQ ID NO: 1-5. 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 a further embodiment, the composition is a combination of formulations for topical application to a transgenic plant. In some embodiments, when the transgenic plant contains the second pest control agent, the formulation comprises the first Cry protein of the present invention. In other embodiments, when the transgenic plant contains the first Cry protein of the present invention, the formulation comprises the second pest control agent.

[0073] In some embodiments, the second pest control agent may be an agent selected from the group consisting of: chemical pest control agents such as insecticides, Bacillus thuringiensis (Bt) insecticidal proteins, Xenorhabdus insecticidal proteins, Photorhabdus insecticidal proteins, Brevibacillus laterosporus insecticidal proteins, Bacillus sphaericus insecticidal proteins, protease inhibitors (both serine and cysteine ​​types), lectins, α-amylase, peroxidase, cholesterol oxidase, and double-stranded RNA (dsRNA) molecules.

[0074] In other embodiments, the second pest control agent is a chemical pest control agent selected from the group consisting of: pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrolides, γ-aminobutyric acid (GABA) antagonists, insecticidal ureas, and juvenile hormone mimics. In other embodiments, the chemical pest control agent is selected from the group consisting of: abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, bifenazate, buprofezin, and carbofuran. Bofuran, 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, fenthion othiocarb, fenoxycarb, fenpropathrin, fenpyroximate, fenvalerate, fipronil, flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophosHalofenozide, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde, methamidophos, methidathion, methomyl, methoprene, methoxychlor, monocrotophos, methoxyfenozide, nithiazin, and novalura. ron), 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, triflumuron, aldicarb, fenamiphos, amitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor, etoxazole, fenazaquin.Fenbutatin oxide, cypermethrin, abamectin, hexythiazox, propargite, pyridaben, and tebufenpyrad. In other embodiments, the chemical pest control agent is selected from the group consisting of: cypermethrin, lambda-cyhalothrin, deltamethrin and lambda-cyhalothrin, S-cypermethrin, fenvalerate, tetrabromopropylate, benzalkonium chloride, methomyl, fenvalerate, thiamethoxam, thiamethoxam, imidacloprid, thiamethoxam, indoxacarb, spinosad, abamectin, avermectin, emamectin thiophanate-methyl, endosulfan, acetamiprid, fipronil, flufenoxuron, chlorfenapyr, benzyl ether, pyriproxyfen, pymetrozine, and amitraz.

[0075] In another embodiment, 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 stage insecticidal proteins (VIPs), and any insecticidal chimeras of the aforementioned 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, C ry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Gb, Cry1Gc, Cry1Ha, Cry1Hb, Cry1Hc, Cry1Ia, Cry1Ib, Cry1Ic, Cry1Id, Cry1Ie, Cry1If, Cry1Ig, Cr y1Ja, Cry1Jb, Cry1Jc, Cry1Jd, Cry1Ka, Cry1La, Cry1Ma, Cry1Na, Cry1Nb, Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry 2Ah, Cry2Ai, Cry2Aj, Cry2Ak, Cry2Al, Cry2Ba, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry4Aa, Cry4Ba, Cry4Ca, Cry4Cb, Cry4Cc, Cry5Aa, Cry5 Ab, Cry5Ac, Cry5Ad, Cry5Ba, Cry5Ca, Cry5Da, Cry5Ea, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry7Ac, Cry7Ba, Cry7Bb, Cry7Ca, Cry7Cb, Cry7D a, 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.

[0076] In a further embodiment, the second pest control agent is a Vip3 vegetative-stage 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, and Vip3Aa25. Vip3Aa26, Vip3Aa27, Vip3Aa28, Vip3Aa29, Vip3Aa30, Vip3Aa31, Vip3Aa32, Vip 3Aa33, Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38, Vip3Aa39, Vip3Aa4 0. 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.

[0077] In a further embodiment, the first Cry protein and the second pest control agent of the present invention are co-expressed in transgenic plants. This co-expression of more than one pest-killing active ingredient in the same transgenic plant can be achieved by genetically modifying the plant to include and express all the necessary genes. Alternatively, a plant "parent 1" can be genetically modified to express the Cry protein of the present invention. A second plant "parent 2" can be genetically modified to express the second pest control agent. By crossing "parent 1" with "parent 2", offspring plants expressing all the genes introduced into "parent 1" and "parent 2" are obtained.

[0078] In a further embodiment, the present invention provides a method for producing transgenic plants resistant to pests (e.g., insect-resistant), the method comprising introducing a polynucleotide, chimeric gene, recombinant vector, expression cassette, or nucleic acid molecule containing a nucleotide sequence encoding the Cry protein of the present invention into a plant, wherein the nucleotide sequence is expressed in the plant, thereby conferring resistance to the Asian corn borer pest and producing an insect-resistant transgenic plant. In some embodiments, the introduction is achieved by transforming the plant. In other embodiments, the introduction is achieved by hybridizing a first plant containing the chimeric gene, recombinant vector, expression cassette, or nucleic acid molecule of the present invention with a different second plant.

[0079] In some embodiments, the present invention includes a method for providing farmers with means of controlling lepidopteran pests, the method comprising supplying or selling to farmers plant material, such as seeds, the plant material containing a polynucleotide, chimeric gene, expression cassette, or recombinant vector capable of expressing the Cry protein of the present invention in plants grown from the seeds, as described above.

[0080] The embodiments of the present invention can be better understood by referring to the following examples. The foregoing and following description of the embodiments of the present invention, as well as the various embodiments, are not intended to limit the claims, but are merely illustrative. Therefore, it will be understood that the claims are not limited to the specific details of these embodiments. Those skilled in the art will recognize that other embodiments of the invention can be practiced without departing from the spirit and scope of this disclosure, which is defined by the appended claims. Example

[0081] Example 1. Activity of Cry protein against Asian corn borer

[0082] In an artificial dietary bioassay, Cry proteins containing the amino acid sequences SEQ ID NO: 1-5 were tested against the Chinese population (CN-ACB; Asian corn borer) of the Asian corn borer (a crop pest belonging to the family Ceratopodidae). These Cry proteins have been previously described as shown in Table 1.

[0083] Table 1. References regarding publicly available information about the Cry protein

[0084] Essentially, equal amounts of the protein in solution were applied to the surface of an artificial insect diet in a porous plate. After the diet surface dried, CN-ACB larvae were added to each well. The plates were sealed and maintained under ambient laboratory conditions (related to temperature, light, and relative humidity). The positive control group consisted of larvae exposed to the known active CN-ACB Cry protein. The negative control group consisted of larvae exposed to an insect diet treated only with the buffer solution and larvae on an untreated insect diet; i.e., diets alone. Mortality was assessed after approximately 3–4 days and scored relative to the control.

[0085] The results of the CN-ACB bioassay are shown in column 3 of Table 2, where “-” indicates no activity compared to the control group, “+ / -” indicates 0-10% activity compared to the control group (this category also includes 0% mortality with strong larval growth inhibition), “+” indicates 10-25% activity compared to the control group, “++” indicates 25-75% activity compared to the control group, and “+++” indicates 75-100% activity compared to the control group. Table 2 also shows an indication of the activity of the Cry protein against a North American strain (NA-ECB; European corn borer (Ostrinia nubilalis)) of the European corn borer (a species in the same genus as the Asian corn borer). For this insect species, activity is simply expressed as “+” or “-”, without indicating the percentage of mortality (based on published data). Cells marked “nt” indicate that no publicly available information suggests that the Cry protein has been tested against NA-ECB. These results clearly demonstrate that the bioactivity or lack of bioactivity against the North American population of the European corn borer does not accurately predict the bioactivity of the same Cry protein against the Chinese population of the closely related species (Asian corn borer).

[0086] Table 2. Results of bioassays for *Stemona stolonifer* using Cry protein.

[0087] Example 2. Expression and activity of Cry protein in corn plants.

[0088] Transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798-803. Briefly, Agrobacterium strain LBA4404 (pSB1) was transformed using an expression vector containing two expression cassettes. The first cassette contained a plant-expressible promoter operably linked to a Cry protein-coding sequence (which is operably linked to a terminator), and the second cassette contained a plant-expressible promoter operably linked to a selective marker (which is operably linked to a terminator). Expression of the selective marker allowed for the identification of transgenic plants on selective media. Both expression cassettes were cloned into suitable vectors for Agrobacterium-mediated transformation of rice or maize. The transformed Agrobacterium strain was grown at 28°C on YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), 15 g / L agar, pH 6.8) solid medium for 2–4 days. Approximately 0.8 × 10 9 One Agrobacterium cell was suspended in LS-inf medium supplemented with 100 μM As. The bacteria were pre-induced in this medium for approximately 30–60 minutes.

[0089] Immature embryos from inbred corn lines were excised from 8-12 day old ears and placed in liquid LS-inf + 100 μM As. The embryos were rinsed once with fresh infection medium. Then, Agrobacterium solution was added, and the embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred to LSAs medium with the scutellaria side up and cultured in the dark for two to three days. Subsequently, approximately 20 to 25 embryos / plate were 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°C for 10 days.

[0090] Immature embryos producing embryogenic callus were transferred to LSD1M0.5S medium. The cultures were selected on this medium for approximately 6 weeks, including a subculturing step of approximately 3 weeks. Surviving callus tissue was transferred to Reg1 medium supplemented with mannose. After culturing in light (16-hour light / 8-hour dark regime), green tissue was transferred to Reg2 medium without growth regulators and incubated for approximately 1–2 weeks. Plantlets were then transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and allowed to grow in light. After approximately 2–3 weeks, the plants were tested by PCR for the presence of selective marker genes and the Bt cry gene. Positive plants from the PCR assay were transferred to a greenhouse for further evaluation.

[0091] The transgenic plants were evaluated based on copy number (determined by Taqman analysis), protein expression levels (determined by ELISA), and efficacy against the target insect species (in the leaf excision bioassay). Specifically, plant tissue (leaves or silks) was excised from single-copy events (V3-V4 stage) and inoculated with newborn larvae of the Asian corn borer, followed by incubation at room temperature for 5 days. The results of the transgenic plant tissue bioassay confirmed that the Cry protein of this invention is toxic to the Asian corn borer when expressed in transgenic plants.

Claims

1. A method for inhibiting or killing the Asian corn borer pest, the method comprising contacting the Asian corn borer with a Cry protein or an insecticidal fragment thereof comprising an amino acid sequence of any one of SEQ ID NO: 1-5.

2. A method for controlling populations of Asian corn borer pests, the method comprising contacting the pest population with an insecticidal amount of a Cry protein or an insecticidal fragment thereof comprising an amino acid sequence comprising any one of SEQ ID NO: 1-5.

3. The method according to claim 1 or 2, wherein the Asian corn borer pest or pest population is further contacted with a second insecticidal protein that has an amino acid sequence different from that of the Cry protein comprising any one of SEQ ID NO: 1-5.

4. The method according to claim 3, wherein the second insecticidal protein is selected from the group consisting of: Cry protein, Vip protein, protease inhibitors, lectins, α-amylase and peroxidase.

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

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

7. The method according to claim 6, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.

8. The method according to claim 7, wherein the monocotyledonous plant is a maize plant, or the dicotyledonous plant is a soybean plant.

9. A method for protecting plants from the Asian corn borer pest, the method comprising expressing an insecticidal amount of a Cry protein or an insecticidal fragment thereof comprising any one of SEQ ID NO: 1-5 in the plant or its cells.

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