Use of novel genes for controlling nematode pests

Abstract

Compositions and methods for conferring nematicidal activity upon bacteria, plants, plant cells, tissues, and seeds are provided. Methods for killing or controlling populations of nematode pests, such as cyst nematode species, for example, *Heterodera glycines* (soybean cyst nematode), root-knot nematodes, kidney-shaped nematodes, short-bodied nematode species, or lanceolate nematode populations, are also provided. These methods further include contacting the nematode pest with a pest-killing amount of a polypeptide containing a nematicidal toxin. Further methods include increasing plant yield by expressing genes of the embodiments in plants.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 593,568, filed on October 27, 2023, the entire contents of which are hereby incorporated by reference. Technical Field

[0002] The embodiments described herein relate to the field of molecular biology. Methods for controlling nematode pests using novel pest genes are provided. Submission of sequence list

[0003] The sequence list associated with this application was submitted electronically via the Patent Centre and is hereby incorporated in its entirety by reference. The name of the "xml" file containing the sequence list is PF230140US01_SEQLISTING_St26.xml. The xml file is 30 KB in size and was created on September 23, 2024. Background Technology

[0004] Plant pests are a major contributor to crop losses worldwide. Some estimates claim that invertebrate pests, including nematodes, cause up to 40% of global crop yield losses. Beyond field crop losses, nematode pests are also a burden on vegetable and fruit growers, ornamental flower producers, and home gardeners. Nematodes that infect plants (most of which are root feeders) are associated with a wide range of plant species. Some nematodes are endoparasites, living and feeding within the tissues of roots, tubers, buds, seeds, etc. Others are ectoparasites, feeding from the outside through the plant wall. A single endoparasitic nematode can kill a plant or reduce its productivity. Endoparasitic root feeders include economically important pests such as root-knot nematodes (Meloidogyne), kidney-shaped nematodes (Rotylenchulus), cyst nematodes (Heterodera), and root-rot nematodes (Pratylenchus).

[0005] Nematode damage can severely reduce plant nutrient and water uptake. Nematodes have the greatest impact on crop productivity when they attack the roots of seedlings immediately after seed germination. Nematode feeding also creates open wounds, providing entry points for a variety of plant-pathogenic fungi and bacteria. These microbial infections can be more economically destructive than the direct effects of nematode feeding.

[0006] Soybean cyst nematodes are a direct cause of soybean yield loss, as well as an indirect cause of losses due to the cost of pesticides and suboptimal use of land for crop rotation. The negative economic impact of soybean cyst nematodes (Heterodera glycines) in North America may exceed $1 billion annually. Economically damaging cyst nematode densities typically lead to crop stunting. Stunted plants have smaller root systems, leaves exhibiting mineral deficiency symptoms, and are prone to wilting.

[0007] Due to the potentially devastating damage caused by nematodes, there is an ongoing need to discover new methods for controlling nematode plant pests that offer economic benefits to growers and are environmentally acceptable and safe. Summary of the Invention

[0008] Various embodiments provide novel methods for controlling economically important nematode pests. Transgenic plants and / or plant parts expressing the polypeptides of the embodiments herein have been found to inhibit the survival, growth, and reproduction of nematode pests, or limit nematode-related damage or loss to crop plants. Another embodiment further relates to transgenic nematode-resistant plants expressing any one or more of the proteins described herein, and methods for using transgenic plants alone or in combination with other nematode control strategies to confer maximum nematode control efficiency with minimal environmental impact. Plants and plant parts expressing the proteins described herein exhibit high tolerance or resistance to nematode infestation.

[0009] One embodiment provides a method for controlling a nematode pest, the method comprising contacting the nematode pest with a protein comprising any of SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof. In another embodiment, the protein may be a Cry5-like protein, such as that shown by SEQ ID NO: 2, 4, 6, 8 or its nematode-active fragment. In yet another embodiment, Cry5-like protein homologs having at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% identity with SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof are further described. Another embodiment provides a Cry5-like sequence comprising the consensus sequence SEQ ID NO: 9.

[0010] One embodiment provides a method for controlling a nematode pest, the method comprising contacting the nematode pest with a transgenic plant or plant part containing a heterologous nucleic acid molecule that directs the expression of a Cry5-like protein of one or more embodiments in the transgenic plant or plant part, wherein the transgenic plant or plant part controls the nematode pest compared to the same type of plant or plant part that does not express the Cry5-like protein.

[0011] In another embodiment, the nematode pest is selected from the group comprising: *Criconemella*, *Ditylenchus*, *Globodera*, *Helicotylenchus*, cyst nematodes, *Longidorus*, root-knot nematodes, *Paratrichodorus*, short-bodied nematodes, *Radolpholus*, *Rotelynchus*, kidney-shaped nematodes, *Tylenchulus*, and *Xiphinema*. Such nematode pests selected from these genera can be cyst-forming nematodes. In another embodiment, these cyst-forming nematodes belong to the genus *Heterodera*. In yet another embodiment, the nematode pest is *Heterodera glycines* (soybean cystnematode) or *Rotylenchulus reniformis*.

[0012] Another embodiment provides a transgenic plant or plant part selected from the group consisting of: alfalfa, apple, apricot, Arabidopsis, artichoke, asparagus, avocado, banana, barley, legume, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, cereal, celery, cherry, citrus, Clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, fig, grape Grapes, grapefruit, groundnuts, ground cherries, kiwifruit, lettuce, leeks, lemons, limes, pine trees, corn, mangoes, melons, millet, mushrooms, nuts and oats, okra, onions, oranges, ornamental plants or flowers or trees, papayas, parsley, peas, peaches, peanuts, peat, peppers, persimmons, pineapples, plantains, plums, pomegranates, potatoes, pumpkins, radishes, rapeseed, raspberries, rice, rye, sorghum, soybeans, spinach, strawberries, sugar beets, sugarcane, sunflowers, sweet potatoes, tangerines, tea, tobacco, tomatoes, vines, watermelons, wheat, yams, and zucchini. In yet another embodiment, the genetically modified plant or plant part is a soybean plant or plant part.

[0013] In another embodiment, the plant part is a root. In yet another embodiment, the root is a soybean root.

[0014] Another embodiment provides a Cry5-like protein comprising an amino acid sequence that is the translation product of a nucleotide sequence, the complementary sequence of which hybridizes with SEQ ID NO: 1, 3, 5 or 7 under highly stringent conditions.

[0015] In another embodiment, the Cry5-like protein comprises SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof, or a nematode active homolog having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2, 4, 6, and 8 and any functional fragment thereof. In yet another embodiment, the Cry5-like sequence comprises the consensus sequence SEQ ID NO: 9. In another embodiment, the transgenic plant of one or more embodiments further comprises or expresses at least one additional pest control agent, such as, but not limited to, patatin, Bacillus thuringiensis insecticidal protein, Bacillus thuringiensis nematicidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, Bacillus sphearicus insecticidal protein, VIP3, and / or RNAi molecules targeting nematode pests. In another embodiment, the Bacillus thuringiensis nematicidal protein is selected from the group consisting of Cry5, Cry6, Cry13, Cry14, Cry21, and Cry55.

[0016] Another embodiment provides a method for conferring nematode resistance to a plant and / or a plant part, the method comprising inserting a heterologous nucleic acid molecule encoding a Cry5-like protein into the plant and / or plant part, wherein the plant and / or plant part expresses the Cry5-like protein at a nematode-inhibiting level, thereby conferring nematode resistance to the plant and / or plant part compared to the same type of plant and / or plant part that does not express the Cry5-like protein. Such insertion can occur via transformation, gene editing, or through breeding.

[0017] Another embodiment provides a method for conferring resistance to soybean cyst nematode or kidney-shaped nematode in plants and / or plant parts, the method comprising inserting a heterologous nucleic acid molecule encoding a Cry5-like protein into the plant and / or plant part, wherein the plant and / or plant part expresses the Cry5-like protein at a nematode-inhibiting level, thereby conferring resistance to soybean cyst nematode or kidney-shaped nematode in the plant and / or plant part compared to the same type of plant and / or plant part that does not express the Cry5-like protein. Such insertion can occur via transformation, gene editing, or through breeding.

[0018] Another embodiment provides a Cry5-like protein comprising SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof, or a soybean cyst nematode or kidney-shaped nematode homolog thereof having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof. In another embodiment, the Cry5-like protein comprises SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof, or an active homolog of *Solanum lyratum* or *Nematodeus reniformis* having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof. In another embodiment, the Cry5-like sequence comprises the consensus sequence SEQ ID NO: 9.

[0019] Another embodiment provides a method for reducing the infectivity of nematodes to plants and / or plant parts, the method comprising contacting the nematode with a Cry5-like protein, wherein the infectivity of the nematode to the plant and / or plant parts is reduced compared to the infectivity of the nematode to the plant and / or plant parts that has not been contacted with the Cry5-like protein.

[0020] Another embodiment provides a transgenic soybean plant or a plant part thereof comprising a heterologous nucleic acid molecule encoding a Cry5-like protein, wherein the transgenic soybean plant or plant part is resistant to nematode infection.

[0021] Another embodiment provides a method for producing soybean plants resistant to nematode infestation, the method comprising transforming soybean plant cells with a nucleic acid molecule encoding a Cry5-like protein; and regenerating the transformed soybean plant from the soybean plant cells, wherein the transformed plant is resistant to nematode infestation.

[0022] Another embodiment provides a method for producing soybean plants resistant to nematode infestation, the method comprising hybridizing a first parent soybean plant with a second parent soybean plant, wherein the first or second parent soybean plant contains a heterologous nucleic acid molecule encoding a Cry5-like protein of one or more embodiments, thereby producing a plurality of progeny plants; and selecting transgenic plants resistant to nematode infestation from the plurality of progeny plants.

[0023] Another embodiment provides a method for reducing nematode cyst development on the roots of plants susceptible to nematode infection, the method comprising introducing a nucleic acid molecule capable of directing the expression of a Cry5-like protein into the cells of the root, thereby reducing nematode cyst development on the roots of the plant.

[0024] Another embodiment provides a method for reducing nematode cyst development on the roots of plants capable of being infected by nematodes, the method comprising introducing a nucleic acid molecule capable of driving the expression of a Cry5-like protein, such as a protein having a consensus sequence as shown in SEQ ID NO: 9, into plant cells, for example, in the roots, thereby reducing nematode cyst development on the roots of the plant.

[0025] Another embodiment provides a method for controlling or preventing nematode growth, the method comprising providing plant material to the nematode pest containing heterologous DNA capable of directing the expression of a Cry5-like protein, wherein the plant inhibits nematode biological activity.

[0026] Another embodiment provides a method for providing growers with means of controlling nematode pests, the method comprising supplying growers with seeds containing a heterologous nucleic acid molecule encoding a Cry5-like protein, and wherein the seeds are capable of producing plants resistant to nematode infection.

[0027] Another embodiment provides a method for suppressing the growth of a plant-pathogenic nematode population in a location capable of supporting its growth, the method comprising growing a transgenic soybean plant population containing a heterologous nucleic acid molecule capable of directing the expression of a Cry5-like protein in the location, wherein the growth of the plant-pathogenic nematode population is suppressed.

[0028] Another embodiment provides a method for controlling any of soybean cyst nematodes, disease nematodes, root-knot nematodes, or kidney nematodes (“target pests”), the method comprising providing a transgenic soybean plant containing an expression cassette having any of SEQ ID NO: 1, 3, 5, or 7 operably linked to a promoter capable of driving the expression of an encoded Cry5-like protein to a level sufficient to inhibit the nematodes, wherein the proliferation of target pests feeding on the soybean plant is reduced compared to target pests feeding on non-transgenic soybean plants that do not contain the expression cassette.

[0029] Another embodiment provides a method for increasing plant yield in a nematode-infested field, the method comprising expressing a Cry5-like protein in the plant, wherein the plant yield is increased compared to the yield of the same type of plant that does not express the Cry5-like protein.

[0030] Another embodiment provides a method for increasing the vigor or yield of a transgenic soybean plant exposed to a nematode population, the method comprising: introducing a transgenic soybean event into a soybean plant to obtain a transgenic soybean plant, wherein the transgenic soybean event contains a heterologous DNA sequence encoding a Cry5-like protein that confers resistance to the nematode; and growing the transgenic soybean plant or its progeny at a location where nematode infection has limited yield in soybean plants that do not contain the heterologous nucleic acid molecule encoding the Cry5-like protein, thereby giving the transgenic soybean plant increased vigor or yield compared to a control plant.

[0031] Another embodiment provides a method for increasing the yield of a soybean field, the method comprising: introducing a nucleic acid molecule capable of directing the expression of a Cry5-like protein into a soybean plant to produce a transgenic plant; and cultivating multiple transgenic seeds from the transgenic plant in a field to produce or form a soybean field containing multiple transgenic soybean plants with enhanced resistance to nematode infection, thereby increasing the yield of the soybean field.

[0032] Another embodiment provides a recombinant expression cassette comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule encoding a Cry5-like protein. Further embodiments provide a recombinant vector comprising such an expression cassette. Still further, another embodiment provides a transgenic host cell comprising such an expression cassette. The transgenic host cell according to the embodiments may be a plant cell. Even further still, another embodiment provides a transgenic plant or plant part comprising such a plant cell.

[0033] Another embodiment provides a nematicidal composition comprising a nematode-controlled effective amount of a Cry5-like protein and an acceptable agricultural vector. In another embodiment, the agricultural vector is a transgenic plant. In another embodiment, the transgenic plant is a transgenic soybean plant, and the Cry5-like protein is a Cry5-like protein having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof. In another embodiment, the Cry5-like protein comprises SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof. Another embodiment provides a Cry5-like sequence comprising the consensus sequence SEQ ID NO: 9.

[0034] Another embodiment provides a method for producing a nematode-resistant transgenic plant, the method comprising introducing a nucleic acid molecule encoding a Cry5-like protein into a plant cell to create a transgenic plant cell; regenerating the transgenic plant from the transgenic plant cell, wherein the Cry5-like protein is expressed in an effective amount in the transgenic plant to control nematodes. According to another embodiment, the plant is a soybean plant. In another embodiment, the Cry5-like protein is a protein having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 2, 4, 6, 8, and any functional fragment thereof. In yet another embodiment, the Cry5-like protein comprises SEQ ID NO: 2, 4, 6, 8, and any functional fragment thereof. A further embodiment provides a Cry5-like sequence comprising the consensus sequence SEQ ID NO: 9.

[0035] In another embodiment, the nematode is a target pest. In yet another embodiment, the nematode is a species of the genus *Soybean cyst nematode*, *Brief-body nematode*, *Kidney-shaped nematode*, or *Root-knot nematode*. A brief explanation of sequence listings

[0036] SEQ ID NO: 1 discloses a Cry5-like nucleotide sequence.

[0037] SEQ ID NO: 2 discloses a Cry5-like amino acid sequence.

[0038] SEQ ID NO: 3 discloses a Cry5-like truncated nucleotide sequence.

[0039] SEQ ID NO: 4 discloses a Cry5-like truncated amino acid sequence.

[0040] SEQ ID NO: 5 discloses the nucleotide sequence of a Cry5-like homolog.

[0041] SEQ ID NO: 6 discloses the amino acid sequence of a Cry5-like homolog.

[0042] SEQ ID NO: 7 discloses a truncated nucleotide sequence of a Cry5-like homolog.

[0043] SEQ ID NO: 8 discloses a truncated amino acid sequence of a Cry5-like homolog.

[0044] SEQ ID NO: 9 discloses the consensus amino acid sequence of a Cry-5-like protein.

[0045] SEQ ID NO: 10 discloses the amino acid sequence of Cry5Ba1 mentioned in Figure 2. Attached Figure Description

[0046] Figure 1 Plant transformation vectors for expressing SEQ ID NO: 2 in plants are described.

[0047] Figure 2a The NEEDLE sequence alignment of the novel Cry5-like protein SEQ ID NO:2 and Cry5Ba1 was depicted.

[0048] Figure 2b The NEEDLE sequence alignment of the novel Cry5-like protein SEQ ID NO: 2 and its homology SEQ ID NO: 6 was depicted.

[0049] Figure 3 A bar graph summarizing results from root digging field trials is presented, in which sporangium counting analysis shows that soybean plants expressing the novel Cry5-like sequence had 34% fewer SCN females (SCN sporangia) on their roots compared to wild-type Thorne soybean plants without the novel Cry5-like sequence.

[0050] Figure 4 Bar graphs depict the results of field trials summarizing the yield of soybean plants expressing the novel Cry5-like sequence, which on average was 6% higher than wild-type Thorne soybean at three independent trial sites.

[0051] Figure 5 This is a photograph depicting a soybean plant (right) expressing Cry5-like protein next to a non-resistant control soybean plant (left) in a field infected with SCN.

[0052] Figure 6A bar graph summarizing greenhouse results for kidney-shaped nematodes (Kidney-shaped Nematodes) shows that soybean plants expressing the novel Cry5-like sequence had approximately 75% fewer kidney-shaped nematodes in their roots compared to wild-type Thorne soybean plants without the novel Cry5-like sequence. Detailed Implementation

[0053] Before explaining the various embodiments disclosed herein, it should be understood that the application of the embodiments herein is not limited to the details of the construction and arrangement of the components set forth in the following description. Other embodiments may be practiced or performed in various ways. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered restrictive.

[0054] Throughout this disclosure, references have been made to various publications, patents, and published patent specifications. Where permitted, the disclosures of these publications, patents, and published patent specifications have been incorporated herein by reference in their entirety to provide a more comprehensive description of the prior art. Unless otherwise stated, this disclosure covers conventional techniques in plant breeding, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the scope of the art. See, for example, Sambrook and Russell, *Molecular Cloning: A Laboratory Manual*, 3rd edition (2001); *Current Protocols in Molecular Biology* [(edited by F.M. Susubel et al., (1987)]; *Plant Breeding: Principles and Prospects* (*Plant Breeding*, Volume 1); MD Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall (1993); edited by Coligan, Dunn, Ploegh, Speicher, and Wingfeld (1995); *CURRENT Protocols in Protein Science* (John Wiley & Sons, Inc.); *Methods in Enzymology* (Academic Press). Inc. (Academic Publishing Company): PCR 2: A Practical Approach (edited by MJ MacPherson, BD Flames and GR Taylor (1995)), Harlow and Lane (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by RI Freshney (1987)).

[0055] Unless otherwise stated, technical terms are used in accordance with their conventional usage in the art. Definitions of commonly used terms in molecular biology can be found in the following literature: Lewin, Genes VII, Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Wiley-Interscience, 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, VCH Publishers, Inc., 1995; Ausubel et al. (1987) Current Protocols in Molecular Biology, Green Publishing; Sambrook and Russell. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition.

[0056] For ease of understanding this disclosure, the following definitions are provided:

[0057] The “activity” of Cry5-like proteins means that Cry5-like proteins are toxic to nematodes by disrupting or preventing feeding, inhibiting the ability of nematode pests to survive, grow and reproduce (which may or may not cause nematode death), or limiting nematode-related damage or loss to crop plants.

[0058] As used herein, “and / or” means and covers any and all possible combinations of one or more of the listed items, as well as the absence of combinations when interpreted in an alternative (or) context.

[0059] "Association / operational linkage" refers to two nucleic acid sequences that are physically or functionally related. For example, if a promoter or regulatory DNA sequence is operably linked or positioned such that the regulatory DNA sequence will affect the expression level of a coding or structural DNA sequence, then these two sequences are said to be "associated" with a DNA sequence encoding RNA or a protein.

[0060] As used herein, the term "contact" refers to the process of delivering or applying the Cry5-like protein of the examples or a transgenic plant or plant part expressing the Cry5-like protein of the examples to a target nematode pest or nematode population. Contact describes the physical proximity of the Cry5-like protein or a transgenic plant or plant part expressing the Cry5-like protein to the target nematode, causing them to interact. Contact between the transgenic plant or plant part and the target nematode or nematode population can be achieved by planting transgenic seeds, seedlings, cuttings, shoots, tubers, etc., in a location capable of supporting the growth of the nematode pest or nematode population.

[0061] A chimeric gene is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to or associated with a nucleic acid sequence encoding mRNA or expressed as a protein, such that the regulatory nucleic acid sequence can regulate the transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequence of a chimeric gene is typically not operatively linked to an associated nucleic acid sequence found in nature.

[0062] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. RNA can then be translated into proteins in an organism.

[0063] As used herein, the term "to control or controlling" nematodes refers to the ability of a nematode pest to survive, grow, feed, and / or reproduce through toxic effects, or to limit nematode-related damage or loss in crop plants. "Controlling" nematodes may or may not mean killing nematodes.

[0064] "Corresponding to" or "corresponds to" means that when the nucleic acid coding sequences or amino acid sequences of different Cry5-like genes or proteins are compared with each other, the nucleic acid or amino acid "corresponding to" certain listed positions is the nucleic acid or amino acid that is compared with these positions but is not necessarily at these precise numerical positions relative to the corresponding nucleic acid coding sequence or amino acid sequence of a specific Cry5-like protein.

[0065] "Delivery" of toxins means that the toxin comes into contact with nematodes or nematode populations, thereby resulting in toxic effects and control of the nematodes or nematode populations. Toxins can be delivered in many recognized ways, such as orally via nematode ingestion or through contact with nematodes via transgenic plant expression, formulated protein compositions, sprayable protein compositions, bait substrates, or any other toxin delivery system recognized in the art.

[0066] The term "economic threshold" is defined as the level of a nematode population that produces incremental damage equal to the cost of controlling or preventing that damage. It is the level of the nematode population at which the benefits of nematode control are equal to its costs. In this respect, the economic threshold can be defined as the level of nematode damage at which the incremental reduction in crop yield equals the cost of preventing its occurrence. In other words, the economic threshold attempts to determine the point at which controlling a nematode population becomes economically feasible. Economic damage to host crops is typically caused by the first generation of progeny nematodes and is prevented by transgenic plants expressing Cry5-like proteins by reducing the concentration of progeny nematodes in the plant's root zone.

[0067] As used herein, “effective control dose for nematodes” or, alternatively, “biotoxic dose”, refers to the concentration of a Cry5-like protein or a functional fragment thereof that has the ability to inhibit the survival, growth, feeding, and / or reproduction of nematodes through toxic effects, or to reduce or prevent nematode-related damage or loss in crop plants. “Effective control dose for nematodes” may or may not mean killing nematodes.

[0068] As used herein, an "expression cassette" refers to a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, comprising a promoter operatively linked to the target nucleotide sequence, which in turn is operatively linked to a termination signal. It typically also contains the sequence required for the correct translation of the nucleotide sequence. An expression cassette containing the target nucleotide sequence can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of the other components. An expression cassette can also be naturally occurring but obtained in a recombinant form suitable for heterologous expression. The expression cassette can be heterologous with respect to the host, meaning that the specific nucleic acid sequence of the expression cassette is not naturally present in the host cell and must have been introduced into the host cell or the progenitor cell of the host cell through a transformation event. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive or inducible promoter that initiates transcription only when the host cell is exposed to certain external stimuli. In the case of multicellular organisms (such as plants), the promoter can also be specific to a particular tissue or organ or developmental stage.

[0069] A "gene" is a defined region within the genome that, in addition to the coding nucleic acid sequence described above, contains other (primarily regulatory) nucleic acid sequences responsible for controlling the expression of the coding portion (i.e., transcription and translation). Genes may also contain other 5' and 3' untranslated sequences and termination sequences. Other elements that may be present include, for example, introns.

[0070] "Nematode killer" is defined as the ability to control the toxic biological activity of nematodes and may include killing nematodes.

[0071] When a nucleic acid sequence encodes a polypeptide that has the same amino acid sequence as a polypeptide encoded by a reference nucleic acid sequence, the nucleic acid sequence is "equicoded" with the reference nucleic acid sequence. For example, the natural coding sequence of a Cry5-like protein from a species of Bacillus spp. is codon-optimized to encode the same Cry5-like protein in a plant.

[0072] "Isolated" nucleic acid molecules, or isolated proteins or toxins, are nucleic acid molecules, proteins, or toxins that exist through artificial manipulation away from their natural environment and are therefore not natural products. Isolated nucleic acid molecules, proteins, or toxins may exist in purified form or may exist in non-natural environments (such as, for example, recombinant host cells or transgenic plants).

[0073] The term "natural" refers to coding sequences or genes that are naturally present in the genome of cells or plants.

[0074] The term "naturally occurring" is used in this document to describe objects that can be found in nature and are distinct from artificially produced objects. For example, a protein or nucleotide sequence present in an organism (including viruses) that can be isolated from natural sources and has not been intentionally modified in a laboratory is naturally occurring.

[0075] "Plant" refers to any plant at any stage of development, including seed plants.

[0076] A plant cell is a structural and physiological unit of a plant, which contains a protoplast and a cell wall. Plant cells can exist as isolated single cells or cultured cells, or as part of higher tissue units such as, for example, plant tissues, plant organs, or the whole plant.

[0077] "Plant material" means leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0078] "Plant organs" are unique and distinctly structured and differentiated parts of a plant, such as roots, stems, leaves, flower buds, or embryos.

[0079] "Plant part" can be any part of a plant and includes plant cells, plant material, plant organs, or plant tissues.

[0080] As used herein, “plant tissue” means a group of plant cells organized into structural and functional units. This includes any plant tissue in situ or in a culture. The term includes, but is not limited to, the whole plant, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in the presence or absence of any particular type of plant tissue as listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[0081] A promoter is a non-translated DNA sequence located upstream of the coding region that contains a binding site for RNA polymerase II and initiates DNA transcription. The promoter region may also include other elements that act as regulators of gene expression.

[0082] "Regulatory elements" are sequences that participate in controlling the expression of nucleotide sequences. Regulatory elements include promoters that are operatively linked to the target nucleotide sequence, and termination signals. They may also encompass sequences required for the correct translation of nucleotide sequences.

[0083] As used in this article, “resistant” or “resistance” refers to genetically modified soybean varieties that prevent most nematodes from surviving and / or reproducing when they attempt to infect the plant.

[0084] In the context of two nucleic acid or protein sequences, the term "substantially identical" means two or more sequences or subsequences that have at least 60%, 80%, 90%, 95%, and at least 99% nucleotide or amino acid residue identity when compared and aligned against the maximum correspondence measured by visual inspection using one of the following sequence comparison algorithms. Substantially identical identity can be present in sequence regions of at least about 50 residues, regions of at least about 100 residues, or regions of at least about 150 residues. In one embodiment, the sequences are substantially identical over the entire length of the coding region. Furthermore, substantially identical nucleic acid or protein sequences perform substantially the same function.

[0085] To determine the percentage of identity between two sequences (“identity percentage”), in the first step, a pairwise sequence alignment between the two sequences is generated. This pairwise alignment in the first step can be generated by various tools known to those skilled in the art, such as, for example, the program “Blast” (Altschul et al. J. Mol. Biol. [Journal of Molecular Biology] 215:403-410), “Blast2” (“gap Blast”) (Altschul et al., Nucleic Acids Res. [Nucleic Acids Research] 25:3389-3402.), programs from the European Molecular Biology Open Software Suite (EMBOSS, Trends in Genetics [Trends in Genetics] 16 (6), 276 (2000)), such as “Water”, “Matcher”, or “Needle”, or generated by visual inspection.

[0086] After aligning two sequences, in the second step, an identity percentage value can be determined based on the resulting alignment. The identity percentage between the two sequences can be calculated based on the resulting full alignment, or based on a region in the alignment (e.g., a region in the alignment that displays one or more embodiments of the sequence over its full length, or a region that displays another sequence over its full length), or based on a region that displays only a portion of the sequence. The alignment region used to calculate the identity percentage value has a length of at least 100 positions, at least 150 positions, or more than 200 positions. To determine the identity percentage, first, the sum of all positions where the two sequences display the same residues in the alignment region is calculated, and then this sum is divided by the length of the alignment region, where the positions where the sequences have introduced vacancies are a component of the length (length of the alignment region), or subtracted from the length (length of the alignment region - total number of vacancies in the alignment region). The resulting value is then multiplied by 100 to obtain the identity percentage (%identity).

[0087] In one embodiment, the two sequences are aligned to full length according to the algorithm of Needleman and Wunsch (J. Mol. Biol. [Journal of Molecular Biology] (1979) 48, pp. 443-453), as implemented in the program "Needle" from EMBOSS (Trends in Genetics 16 (6), 276 (2000), preferred version 6.3.1.2 or later), using the program default parameters for protein sequences (gapopen = 10.0, gapextend = 0.5 and matrix = EBLOSUM62 (the EMBOSS version replacing BLOSUM62) and the default parameters for nucleotide sequences (gapopen = 10.0, gapextend = 0.5 and matrix = EDNAFULL). The percentage of identity (% identity) is then determined based on the resulting full alignment and calculated as follows: Percentage of identity = (Sum of positions showing identical residues in the alignment × 100) / (Alignment length - total number of vacancies in the alignment). This value can also be obtained directly from the EMBOSS program "Needle" as the program marked "longest identity" when the parameter option "-nobrief" is applied.

[0088] For nucleotide sequences encoding proteins, pairwise alignment can be performed over the full length of the coding region (from the start codon to the stop codon, excluding introns) of one or more embodiments. Introns present in another sequence can also be removed for pairwise alignment to allow comparison with sequences from one or more embodiments.

[0089] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions. The phrase "specific hybridization" refers to the fact that, under stringent conditions, a molecule binds, doubles, or hybridizes only with a specific nucleotide sequence present in a complex mixture (e.g., total cellular) DNA or RNA. "Substantially binding" refers to complementary hybridization between the probe nucleic acid and the target nucleic acid, and includes slight mismatches that can be adjusted by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0090] In the context of nucleic acid hybridization experiments (such as DNA and RNA hybridization), "strict hybridization conditions" and "strict hybridization washing conditions" are sequence-dependent and vary under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. Extensive guidelines on nucleic acid hybridization can be found in the following literature: Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes part I chapter 2; "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Typically, highly stringent hybridization and washing conditions are chosen to be higher than the melting point (T0) of the specific sequence at defined ionic strengths and pH values. m The temperature is approximately 5°C lower. Typically, under “strict conditions,” the probe will hybridize with its target sequence but not with other sequences.

[0091] If nucleic acid molecules exhibit perfect complementarity, they are called “complements” of another nucleic acid molecule. As used herein, molecules are said to exhibit “perfect complementarity” when every nucleotide of one molecule is complementary to a nucleotide of another molecule. If two molecules can hybridize with each other under conditions sufficient to allow them to remain annealed to each other under at least conventional “low-strict” conditions, they are called “minimally complementary.” Similarly, if molecules can hybridize with each other under conditions sufficient to allow them to remain annealed to each other under conventional “high-strict” conditions, they are called “complementary.” The standard stringent conditions are described by Sambrook et al., in Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and Haymes et al., in Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985), which are incorporated herein by reference in their entirety.

[0092] T m This is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at defined ionic strength and pH). Very stringent conditions are chosen to ensure Ti of the target sequence with the specific probe. mThe same applies. An example of stringent hybridization conditions for complementary nucleic acids with more than 100 complementary residues hybridizing on a filter membrane in DNA or RNA blotting is overnight hybridization at 42°C with 50% formamide and 1 mg heparin. An example of highly stringent wash conditions is 0.15 M NaCl, 72°C, for approximately 15 minutes. An example of stringent wash conditions is 0.2×SSC wash buffer, 65°C, for 15 minutes (see Sambrook below for a description of the SSC buffer). Typically, a low-stringent wash is performed before a high-stringent wash to remove background probe signal. For example, for duplexes exceeding 100 nucleotides, an example of a medium-stringent wash is 1×SSC, 45°C, for 15 minutes. For example, for duplexes exceeding 100 nucleotides, an example of a low-stringent wash is 4–6×SSC, 40°C, for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentrations (or other salts) at pH 7.0 to 8.3, and temperatures typically at least about 30°C. Stringent conditions can also be achieved by adding a destabilizing agent (such as formamide). Generally, in a specific hybridization assay, a signal-to-noise ratio that is twice (or higher) than that observed with unrelated probes indicates that specific hybridization has been detected. If nucleic acids that do not hybridize with each other under stringent conditions encode substantially identical proteins, then these nucleic acids remain substantially identical. This occurs, for example, when nucleic acid copies are generated using the maximum codon degeneracy allowed by the genetic code.

[0093] The following are examples of hybridization / washing conditions that can be used to clone homologous nucleotide sequences substantially identical to the reference Cry5-like nucleotide sequences of one or more embodiments: Hybridization of the reference nucleotide sequence with the reference nucleotide sequence at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, followed by washing at 50°C in 2×SSC, 0.1% SDS; hybridization at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, followed by washing at 50°C in 1×SSC, 0.1% SDS; hybridization at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, followed by washing at 50°C in 0.5×SSC, 0.1% SDS; hybridization at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, followed by washing at 50°C in 0.5×SSC, 0.1% SDS; hybridization at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA. Hybridize in EDTA and wash at 50°C in 0.1×SSC, 0.1% SDS; or hybridize at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA and wash at 65°C in 0.1×SSC, 0.1% SDS.

[0094] A further indication that two nucleic acid sequences or proteins are substantially identical is the presence of immune cross-reactivity or specific binding between the protein encoded by the first nucleic acid and the protein encoded by the second nucleic acid. Thus, one protein is typically substantially identical to the second protein, for example, where the two proteins differ only due to conserved substitutions.

[0095] "Synthetic" refers to a nucleotide sequence that contains structural features not found in natural sequences. For example, Cry5-like coding sequences that are more similar to the G+C content and normal codon distribution of genes in dicotyledonous and / or monocotyledonous plants and are not naturally found in Bacillus are called synthetic.

[0096] "Transformation" is the process of introducing heterologous nucleic acids into host cells or organisms. In particular, "transformation" refers to the stable integration of DNA molecules into the genome of the target organism.

[0097] "Transformed / GMO / Recombinant" refers to a host organism, such as bacteria or plants, in which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or it can exist as an extrachromosomal molecule. Such an extrachromosomal molecule can replicate automatically. Transformed cells, tissues, or plants should be understood to encompass not only the final products of the transformation process but also their transgenic progeny. "Non-transformed," "non-GMO," or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants, that do not contain a heterologous nucleic acid molecule.

[0098] Generally, "Cry5-like protein" refers to the Bacillus Cry insecticidal protein that shows the highest global identity with the Cry5 sequence (see Table 3), but is not a member of any known Cry5 class Cry5A, Cry5B, Cry5C, Cry5D, and Cry5E (based on the definition described in Crickmore et al., Journal of Invertebrate Pathology 186 (2021) and the content of https: / / www.bpprc-db.org in April 2023). Therefore, in one embodiment, a Cry5-like protein would be a protein showing at least 60% global sequence identity with SEQ ID NO: 2, 4, 6, or 8, but less than 75% identity with any of the Cry5 orthotype specimen sequences Cry5Aa1, Cry5Ba1, Cry5Ca1, Cry5Da1, and Cry5Ea1 as provided at https: / / www.bpprc-db.org.

[0099] As used herein, a nematode-active "homolog" means that the indicated protein or polypeptide is active against nematodes and has a defined relationship with another member of the Cry5-like protein class. This defined relationship may include, but is not limited to: 1) a protein that is at least 60%, 70%, 80%, or 90% identical at the sequence level to another member of the Cry5-like protein class while retaining nematode-killing activity. One embodiment provides a homolog encoded by the nucleotide sequence SEQ ID NO: 5 disclosed in SEQ ID NO: 6. The consensus sequences of SEQ ID NO: 2 and SEQ ID NO: 6 are disclosed in SEQ ID NO: 9.

[0100] As used herein, nucleotides are represented by their bases using the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are also represented 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).

[0101] The materials and methods of the embodiments can be used to kill or control nematodes; delay the growth or reproduction of nematodes; reduce nematode populations; and / or reduce or delay damage to plants caused by nematode pests. Further embodiments provide a method for controlling nematode pests in crop plants (such as soybeans) by using transgenic crop plants expressing a Cry5-like protein.

[0102] The expression of the Cry5-like protein in transgenic plants or plant parts of the examples produces compositions that can be used to control nematode pests, such as, but not limited to, target pests, species of the genus *Meloidogyne* (e.g., *Meloidogyne incoginita* and *Meloidogyne javanica*, *Meloidogyne hapla*, *Meloidogyne arenari*), species of the genus *Heterodera* (e.g., *Heterodera carotae*, *Heteroderaschachtii*, *Heterodora avenae*, and *Heterodoratrifolii*), species of the genus *Globodera* (e.g., *Globodera rostochiensis*), and species of the genus *Radix* (e.g., *Radix Radopholus*). Species of the genera *Similes*, *Pratylenchus* (e.g., *Pratylenchus reniformis*), *Pratylenchus* (e.g., *Pratylenchus neglectans*, *Pratylenchus brachyurus*, and *Pratylenchus penetrans*), *Aphelenchoides*, *Hoplolaimus*, *Pratylenchus*, *Nacobbus*, *Subanguina*, *Belonlaimus*, *Criconemoides*, *Ditylenchus*, and *Ditylenchus*. Species of the genera *dipsaci*, *Dolichodorus*, *Hemicriconemoides*, *Hemicycliophora*, *Hirschmaniella*, *Hypsoperine*, *Macroposthonia*, *Melinius*, *Punctodera*, *Quinisulcius*, *Scutellonema*, *Draconis*, and *Tylenchorhynchus*.

[0103] One embodiment provides a method for controlling nematode pests, the method comprising contacting the nematode pest with a Cry5-like protein comprising SEQ ID NO: 2, 4, 6, 8 and any functional fragment thereof.

[0104] In another embodiment, the nematode is selected from the group consisting of: *Small ring nematode*, *Stem nematode*, *Golden nematode*, *Spiral nematode*, *Cyclophorus nematode*, *Long needle nematode*, *Root-knot nematode*, *Pseudo-spinous nematode*, *Short-bodied nematode*, *Perforating nematode*, *Coilia nematode*, *Kidney-shaped nematode*, *Paddy-sharp nematode*, and *Strombus nematode*. In yet another embodiment, the nematode is a cyst-forming nematode. In yet another embodiment, the nematode belongs to the genus *Cyclophorus*. In still another embodiment, the nematode is *Soybean cyst nematode* or *Kidney-shaped nematode*.

[0105] In another embodiment, the contact step is performed using a plant or plant part transformed with at least one nucleic acid molecule encoding a Cry5-like protein. In yet another embodiment, the plant or plant part is a soybean plant or plant part. In yet another embodiment, the soybean plant part is a soybean root.

[0106] In another embodiment, the genetically modified plant or plant part is selected from the group consisting of: alfalfa, apple, apricot, Arabidopsis thaliana, artichoke, asparagus, avocado, banana, barley, legume, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, cereal, celery, cherry, citrus, Clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, fig, grape, grapefruit, peanut, ground cherry, kiwi, lettuce, leek. Lemon, lime, pine, corn, mango, melon, millet, mushroom, nuts and oats, okra, onion, orange, ornamental plants or flowers or trees, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, red chicory, radish, rapeseed, raspberry, rice, rye, sorghum, soybean, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, orange, tea, tobacco, tomato, vine, watermelon, wheat, yam, and zucchini. In yet another embodiment, the genetically modified plant or plant part is a soybean plant or plant part.

[0107] Another embodiment covers transgenic seeds of the transgenic plant of the embodiments, wherein the transgenic seeds contain a heterologous nucleic acid molecule encoding one or more Cry5-like proteins of the embodiments. Another embodiment provides a recombinant vector and expression cassette containing a Cry5-like nucleic acid sequence of the embodiments. In such a vector, the nucleic acid sequence may be contained in an expression cassette containing regulatory elements for expressing the Cry5-like nucleotide sequence in a transgenic host cell capable of expressing the nucleotide sequence. Such regulatory elements typically contain promoters and termination signals, and may also contain elements that allow efficient translation of the polypeptide encoded by the nucleic acid sequence of the embodiments. Vectors containing nucleic acid sequences are generally capable of replicating in a specific host cell (e.g., an extrachromosomal molecule) and are therefore used to amplify the nucleic acid sequence of the embodiments in host cells. In one embodiment, the host cell used for such a vector is a microorganism, such as bacteria, for example, *Escherichia coli* (E. coli). In another embodiment, the host cell used for such a recombinant vector is an endophytic or epiphytic bacterium. An example of a host cell used for such a vector is a eukaryotic cell, such as a plant cell. Such a plant cell may be a soybean cell or a corn cell. In another embodiment, such a vector is a viral vector and is used to replicate a nucleotide sequence in a specific host cell (e.g., an insect cell or a plant cell). The recombinant vector is also used to transform the nucleotide sequence of the embodiment into a transgenic host cell, whereby the nucleotide sequence is stably integrated into the DNA of such a transgenic host cell. In one embodiment, such a transgenic host cell is a prokaryotic cell. In another embodiment, such a transgenic host cell is a eukaryotic cell, such as a yeast cell, insect cell, or plant cell. In yet another embodiment, the transgenic host cell is a plant cell, such as a soybean cell or a corn cell.

[0108] In another embodiment, for example, the Cry5-like nucleotide sequence of the embodiment can be modified by incorporating random mutations in a technique known as in vitro recombination or DNA shuffling to increase nematode activity. This technique is described in Stemmer et al., Nature [Nature] 370:389-391 (1994) and U.S. Patent No. 5,605,793, which are incorporated herein by reference. Millions of mutant copies of the nucleotide sequence are generated based on the original nucleotide sequence of the embodiment, and variants with improved properties, such as increased nematode-killing activity, enhanced stability, or different specificity or range of target nematode pests, are recovered. This method encompasses the formation of mutagenic double-stranded polynucleotides from a template double-stranded polynucleotide containing the nucleotide sequence of an embodiment, wherein the template double-stranded polynucleotide has been cleaved into double-stranded random fragments of desired size, and includes the step of adding one or more single-stranded or double-stranded oligonucleotides to the resulting population of double-stranded random fragments, wherein the oligonucleotides contain identity regions and heterologous regions with the double-stranded template polynucleotide; denaturing the resulting mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resulting population of single-stranded fragments with a polymerase under conditions that cause the single-stranded fragments to anneal in the identity regions to form annealed fragment pairs, wherein the identity regions are sufficient to cause one member of a pair to initiate the replication of the other member, thereby forming a mutagenic double-stranded polynucleotide; and repeating the second and third steps for at least two additional cycles, wherein the resulting mixture in the second step of the additional cycles contains the mutagenic double-stranded polynucleotide from the third step of the previous cycle, and the additional cycles form additional mutagenic double-stranded polynucleotides. In one embodiment, the concentration of a single double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the total DNA. In another embodiment, the template double-stranded polynucleotide contains at least about 100 polynucleotides. In yet another embodiment, the size of the double-stranded random fragment is about 5 bp to 5 kb. In yet another embodiment, the fourth step of the method includes repeating steps two and three for at least 10 cycles.

[0109] In another embodiment, the Cry5-like nucleotide sequence of the embodiment can be modified by N- or C-terminal deletion to encode a functional fragment. The term "functional fragment" refers to a sequence of amino acids for a Cry5-like protein, wherein these sequence of amino acids are at least 10, 20, 30, 40, 50, or 60 consecutive amino acids of SEQ ID NO: 2 or 6. Deletion will remove the C-terminal crystalline domain downstream of the conserved amino acid motif referred to as "DRIEF" or "DRIE," which also... Figure 2a and 2bHighlighted in the image, and previously described as “block 5” in Schnepf et al. (1998), functional fragments with a C-terminal deletion of a crystalline domain are expected to produce toxic core proteins that are independent of proteolytic activation. Exemplary functional fragments with a C-terminal crystalline domain downstream of a conserved “DRIEF” domain are encoded by SEQ ID NO: 3 and SEQ ID NO: 7, and contain the amino acid sequences SEQ ID NO: 4 and 8, respectively.

[0110] In another embodiment, at least one nucleotide sequence from the Cry5-like nucleotide sequence of the embodiment is inserted into a suitable expression cassette containing a promoter and a termination signal. Expression of the nucleotide sequence is constitutive or uses an inducible promoter that initiates transcription in response to various types of stimuli. In one embodiment, the cell in which the toxin is expressed is a microorganism, such as a virus, bacteria, or fungus. In another embodiment, a virus (such as a baculovirus) contains the nucleotide sequence of the embodiment in its genome and expresses a large amount of the corresponding insecticidal toxin after infecting suitable eukaryotic cells suitable for viral replication and expression of the nucleotide sequence. The resulting insecticidal toxin is used as an insecticide. Alternatively, a baculovirus engineered to contain the nucleotide sequence is used to infect insects in vivo and kill the insects by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin.

[0111] Bacterial cells also serve as hosts for expressing the nucleotide sequences of the embodiments. In one embodiment, non-pathogenic symbiotic bacteria (so-called endophytes) capable of living and replicating within plant tissues, or non-pathogenic symbiotic bacteria (so-called epiphytes) capable of colonizing the leaf or rhizosphere, are used. Such bacteria include those belonging to the genera *Agrobacterium*, *Alcaligenes*, *Azospirillum*, *Azotobacter*, *Bacillus*, *Clavibacter*, *Enterobacter*, *Erwinia*, *Flavobacter*, *Klebsiella*, *Pseudomonas*, *Rhizobium*, *Serratia*, *Streptomyces*, and *Xanthomonas*. Symbiotic fungi (such as Trichoderma and Gluconobacterium) are also possible hosts for expressing the nucleotide sequences of the present invention for the same purpose.

[0112] Techniques for these gene manipulations are specific to the different available hosts and are known in the art. For example, expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coli via transcriptional or translational fusion after a tac or trc promoter. For the expression of operons encoding multiple ORFs, the simplest procedure is to insert the operon into a vector (such as pKK223-3) during a transcriptional fusion, thereby allowing the use of the homologous ribosome binding site of the heterologous gene. Techniques for overexpression in Gram-positive species (such as Bacillus) are also known in the art and can be used in the context of the examples (Quax et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics, ed. Baltz et al., American Society for Microbiology, Washington (1993)). Alternative systems for overexpression include those that rely on yeast vectors and include those using Pichia, Saccharomyces, and Kluyveromyces (Sreekrishna, in: Industrial microorganisms: basic and applied molecular genetics, edited by Baltz, Hegeman, and Skautrud, American Society for Microbiology, Washington (1993); Dequin and Bane, Biotechnology L2:173-177 (1994); van den Berg et al., Biotechnology 8:135-139 (1990)).

[0113] In one embodiment, at least one Cry5-like protein of the embodiment is expressed in a higher organism (e.g., a plant). In this case, the transgenic plant expressing an effective amount of the toxin protects itself from nematode pests. When nematodes begin to feed on such a transgenic plant, they also ingest the expressed Cry5-like toxin. This may prevent nematodes from further feeding in plant tissues, may harm or kill nematodes, or may reduce nematode reproductive capacity. The nucleotide sequence of the embodiment is inserted into an expression cassette, which is then stably integrated into the plant genome. The plants transformed according to the embodiments can be monocotyledonous or dicotyledonous plants, and include, but are not limited to, corn, wheat, barley, rye, sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis thaliana, and woody plants (such as conifers and deciduous trees).

[0114] Once the desired nucleotide sequence has been transformed into a specific plant species, it can be propagated in that species or transferred to other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The nucleotide sequence of the example is expressed in a transgenic plant, thereby inducing the biosynthesis of the corresponding toxin in the transgenic plant. In this way, transgenic plants with enhanced resistance to nematodes are generated. For expression in transgenic plants, the nucleotide sequence of the example may require modification and optimization. Although in many cases genes from microorganisms can be expressed at high levels in plants without modification, low expression in transgenic plants may be due to the microbial nucleotide sequence having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon use, and the codons of one or more of the nucleotide sequences described in the examples can be changed to conform to plant preferences while maintaining the amino acids thereby encoded. Furthermore, high expression in plants is achieved by coding sequences having a GC content of at least about 35% (greater than about 45%, greater than about 50%, and greater than about 60%). Although gene sequences can be adequately expressed in both monocot and dicot species, they can be modified to account for specific codon preferences and GC content in monocot or dicot species, as these have been shown to differ (Murray et al., Nucl. Acids Res. 17:477-498 (1989)). Furthermore, nucleotide sequences are screened for inappropriate splicing sites that could lead to information truncation. All necessary modifications to the nucleotide sequence, such as those described above, are performed using methods known in the art, employing well-known techniques such as site-directed mutagenesis, PCR, and synthetic gene construction.

[0115] In various embodiments, the nucleotide sequences of the embodiments can be optimized for expression in any plant. It should be recognized that all or any part of the gene sequence can be optimized or synthetic. That is, synthetic or partially optimized sequences can also be used.

[0116] For translation to be effectively initiated, the sequence adjacent to the start methionine encoding the start codon may need to be modified. For example, they can be modified by including sequences known to be effective in plants. Joshi proposed consensus sequences suitable for plants (NAR 15:6643-6653 (1987)), and Clonetech proposed further consensus translation initiators (1993 / 1994 catalog, page 210). Further patent publication WO 2022 / 261348 discloses methods and compositions for altering protein accumulation (all the foregoing references are incorporated herein by reference in their entirety). These consensus sequences apply to the nucleotide sequences of the examples. The sequences are incorporated into constructs containing nucleotide sequences up to and including ATG (while keeping the second amino acid unmodified).

[0117] The Cry5-like toxin gene of the embodiments, whether as its natural sequence or as the optimized synthetic sequence described above, can be operatively fused into a variety of promoters for expression in plants, including constitutive, inducible, time-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 of expression. Therefore, expression of the nucleotide sequence encoding the Cry5-like protein of the embodiments can be achieved in leaves, stems or stalks, spikes, inflorescences (e.g., spike tips, panicles, rachis, etc.), roots, and / or seedlings, but for nematode control, expression in roots is also possible. However, in many cases, protection against more than one type of nematode pest is sought, thus requiring expression in multiple tissues. Although many promoters from dicotyledons have proven operable in monocotyledons and vice versa, ideally, dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters are selected for expression in monocotyledons. However, there are no restrictions on the source of the selected promoter; it is acceptable as long as it can drive the expression of the nucleotide sequence of the embodiment in the desired cells.

[0118] carrier

[0119] The biohazard-killing sequences in the embodiments can be provided in expression cassettes for expression in target host cells (e.g., plant cells or microorganisms). A “plant expression cassette” refers to a DNA construct capable of causing protein expression from an open reading frame in plant cells. Typically, these contain a promoter and a coding sequence. Typically, such constructs will also contain a 3' untranslated region. Such constructs may contain signal sequences or leader sequences to facilitate co-translation or post-translational transport of the peptide to certain intracellular structures such as chloroplasts (or other plastids), endoplasmic reticulum, or Golgi apparatus.

[0120] "Signal sequence" refers to a sequence known or suspected of causing co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, resulting in some form of glycosylation. Bacterial insecticidal toxins are often synthesized as prototoxins, which are activated by proteolysis in the gut of the target pest (Chang (1987) Methods Enzymol. [Enzymological Methods] 153:507-516). In some embodiments, the signal sequence is located in a natural sequence or may be derived from the sequence of the embodiment.

[0121] "Lead sequence" refers to any amino acid sequence that, during translation, generates a sequence sufficient to trigger co-translational transport of the peptide chain to subcellular organelles. Therefore, this includes leader sequences for targeted transport and / or glycosylation via the endoplasmic reticulum, vacuoles, plastids (including chloroplasts), mitochondria, etc. Accordingly, this document further provides a polypeptide comprising an amino acid sequence operatively linked to a heterologous leader sequence or signal sequence.

[0122] "Plant transformation vector" refers to the DNA molecule necessary for the efficient transformation of plant cells. Such a molecule can consist of one or more plant expression cassettes and can be organized into more than one "vector" DNA molecule. For example, a binary vector is a plant transformation vector that uses two discontinuous DNA vectors to encode all the necessary cis and trans-acting functions for transforming plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).

[0123] A "vector" is a nucleic acid construct designed for transfer between different host cells.

[0124] "Expression vector" refers to a vector capable of incorporating, integrating, and expressing a heterologous DNA sequence or fragment in exogenous cells. The cassette will include 5' and / or 3' regulatory sequences operatively linked to the sequences of the embodiment. "Operably linked" means a functional link between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Typically, operatively linked means that the linked nucleic acid sequences are adjacent and located within the same reading frame when two protein-coding regions need to be joined. In some embodiments, the nucleotide sequence is operatively linked to a heterologous promoter capable of directing the expression of the nucleotide sequence in a host cell, such as a microbial host cell or a plant host cell. Additionally, the cassette may contain at least one additional gene to be co-transformed into an organism. Alternatively, one or more additional genes may be provided on multiple expression cassettes.

[0125] In various embodiments, the nucleotide sequence of the embodiment is operatively linked to a heterologous promoter, such as a plant promoter.

[0126] Such expression cassettes provide multiple restriction sites for inserting harmful organism-killing sequences that will be regulated by transcription in the regulatory region.

[0127] The expression cassette will include, in a 5'-3' transcriptional orientation, a transcription and translation initiation region (i.e., the promoter), the DNA sequence of the example, and a transcription and translation termination region (i.e., the termination region). The promoter may be natural or similar, foreign or heterologous, for the plant host and / or the DNA sequence of the example. Additionally, the promoter may be a natural sequence or an alternatively synthesized sequence. When a promoter is "natural" or "homologous" for the plant host, this means that the promoter is present in the natural plant in which it is introduced. When a promoter is foreign or heterologous for the DNA sequence of the example, this means that the promoter is not natural or naturally occurring for the operatively linked DNA sequence of the example. The promoter may be inducible or constitutive. It may be naturally occurring, may consist of portions of various naturally occurring promoters, or may be partially or completely synthetic. Promoter design guidelines are provided through studies of promoter structure, such as those by Harley and Reynolds (1987) Nucleic Acids Res. [Nucleic Acid Research] 15:2343-2361. Additionally, the position of the promoter relative to the transcription start point can be optimized. See, for example, Roberts et al. (1979) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 76:760-764. Many suitable promoters for plants are well known in the art.

[0128] For example, suitable constitutive promoters for use in plants include: promoters from plant viruses, such as the peanut chlorosis virus (PClSV) promoter (US Patent No. 5,850,019); the 35S promoter from cauliflower mosaic virus (CaMV) (Odell et al. (1985) Nature 313:810-812); the 35S promoter described in Kay et al. (1987) Science 236:1299-1302; the promoter of the Chlorella virus methyltransferase gene (US Patent No. 5,563,328) and the full-length transcript promoter from Scrophularia mosaic virus (FMV) (US Patent No. 5,378,619); and promoters from genes such as rice actin (McElroy et al. (1990) Plant Cell). 2:163-171 and US Patent 5,641,876); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. [Plant Molecular Biology] 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. [Plant Molecular Biology] 18:675-689) and Grefen et al. (2010) Plant J [Plant Journal], 64:355-365; pEMU (Last et al. (1991) Theor. Appl. Genet. [Theoretical and Applied Genetics] 81:581-588); MAS (Velten et al. (1984) EMBO J. [Journal of the European Society for Molecular Biology] 3:2723-2730 and US Patent 5,510,474); maize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. [Molecular Genetics and Genomics]) 231:276-285 and Atanassova et al. (1992) Plant J. [Plant Journal] 2(3):291-300); European rapeseed (Brassica napus) ALS3 (PCT application WO 97 / 41228); plant ribulose-dicarboxylase / oxygenase (RuBisCO) small subunit gene; porcine circovirus (AU 689 311) or cassava vein mosaic virus (CsVMV, US 7,053,205; promoters from soybean (Pbdc6 or Pbdc7 as described in WO / 2014 / 150449, or ubiquitin 3 promoters as described in U.S. Patent Nos. 7,393,948 and 8,395,021); and promoters of various Agrobacterium genes (see U.S. Patent Nos. 4,771,002, 5,102,796, 5,182,200 and 5,428,147).

[0129] Suitable inducible promoters for use in plants include: copper-responsive promoters from the ACE1 system (Mett et al. (1993) PNAS 90:4567-4571); promoters of the corn In2 gene responsive to the benzyl sulfonamide herbicide safener (Hershey et al. (1991) Mol. Gen. Genetics 227:229-237 and Gatz et al. (1994) Mol. Gen. Genetics 243:32-38); and promoters of the Tet repressor protein from Tn10 (Gatz et al. (1991) Mol. Gen. Genet. 227:229-237). Another type of inducible promoter for use in plants is an inducible promoter responsive to inducers that plants do not normally respond to. Exemplary inducible promoters of this type are inducible promoters derived from steroid hormone genes whose transcriptional activity is induced by glucocorticoids (Schena et al. (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 88:10421) or the recent application of the chimeric transcription activator XVE (for use in estrogen receptor-based inducible plant expression systems activated by estradiol) (Zuo et al. (2000) Plant J [Plant Journal], 24:265-273). Other inducible promoters for use in plants are described in EP 332104, PCT WO 93 / 21334 and PCT WO 97 / 06269, which are incorporated herein by reference in their entirety. Promoters composed of partially and partially or fully synthesized promoters from other promoters may also be used. See, for example, Ni et al. (1995) Plant J. 7:661-676 and PCT WO95 / 14098 describing such promoters for use in plants.

[0130] In one embodiment, a promoter sequence specific to a specific region or tissue of the plant can be used to express the nematicidal protein of the embodiment, such as a seed-specific promoter (Datla, R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), especially the napin promoter (EP 255378 A1), phaseolin promoter, glutenin promoter, helianthinin promoter (WO 92 / 17580), albumin promoter (WO 98 / 45460), oleosin promoter (WO 98 / 45461), SAT1 promoter, or SAT3 promoter (PCT / US98 / 06978).

[0131] Alternatively, inducible promoters advantageously selected from the following can be used: phenylalanine ammonia-lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, protease inhibitor (PI), PR1 family genes, carmine synthase (nos) and vspB promoters (US 5 670 349, Table 3), HMG2 promoter (US 5 670 349), apple β-galactosidase (ABG1) promoter, and apple aminocyclopropane carboxylic acid synthase (ACC synthase) promoter (WO 98 / 45445). Multiple promoters can be used in the constructs of the examples, including sequentially.

[0132] Promoters may contain or be modified to include one or more enhancer elements. In some embodiments, a promoter may contain multiple enhancer elements. Promoters containing enhancer elements provide higher levels of transcription compared to promoters that do not contain enhancer elements. Suitable enhancer elements for use in plants include the PClSV enhancer element (US Patent No. 5,850,019), the CaMV 35S enhancer element (US Patent Nos. 5,106,739 and 5,164,316), and the FMV enhancer element (Maiti et al. (1997) Transgenic Res. 6:143-156); transcriptional activators of tobacco mosaic virus (TMV) as described in application WO 87 / 07644 or, for example, of tobacco etch virus (TEV) as described by Carrington and Freed 1990, J. Virol. 64:1590-1597, or introns such as the adh1 intron of corn or the intron 1 of rice actin. See also PCT WO 96 / 23898, WO 2012 / 021794, WO 2012 / 021797, WO 2011 / 084370 and WO 2011 / 028914.

[0133] Typically, such constructs may contain 5' and 3' untranslated regions. These constructs may also contain signal or leader sequences to facilitate co-translation or post-translational transport of the target peptide to certain intracellular structures, such as chloroplasts (or other plastids), the endoplasmic reticulum, or the Golgi apparatus, or to promote the secretion of the target peptide. For example, constructs may be engineered to contain a signal peptide to facilitate peptide translocation to the endoplasmic reticulum.

[0134] The "3' untranslated region" refers to a polynucleotide located downstream of the coding sequence. The polyadenylation signal sequence and other sequences encoding regulatory signals that can affect the addition of polyadenylation nucleotides to the 3' end of the mRNA precursor constitute the 3' untranslated region. The "5' untranslated region" refers to a polynucleotide located upstream of the coding sequence.

[0135] Other upstream or downstream non-translational elements include enhancers. Enhancers are polynucleotides that increase the expression of promoter regions. Enhancers are well known in the art and include, but are not limited to, SV40 enhancer regions and 35S enhancer elements.

[0136] Termination regions can be natural with respect to transcription initiation regions, natural with respect to the target DNA sequence to which operative linking is possible, natural with respect to the plant host, or derived from another source (i.e., foreign or heterologous with respect to the promoter, target DNA sequence, plant host, or any combination thereof). Convenient termination regions can be obtained from Ti-plasmids of Agrobacterium tumefaciens, such as octopaline synthase and carmine synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. [Molecular Genetics and Genomics] 262:141-144; Proudfoot (1991) Cell [Cell] 64:671-674; Sanfacon et al. (1991) Genes Dev. [Genes and Development] 5:141-149; Mogen et al. (1990) Plant Cell [Plant Cell] 2:1261-1272; Munroe et al. (1990) Gene [Genes] 91:151-158; Ballas et al. (1989) Nucleic Acids Res. [Nucleic Acid Research] 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. [Nucleic Acid Research] 15:9627-9639.

[0137] Where appropriate, one or more genes can be optimized to increase expression in transformed host cells (synthetic DNA sequences). That is, genes can be synthesized using host-preferred codons to improve expression, or genes can be synthesized using codons at the frequency of host-preferred codon usage. Expression of the open reading frames of the synthetic DNA sequence in cells results in the production of the polypeptides of the examples. Synthetic DNA sequences can be used to simply remove unwanted restriction endonuclease sites to favor DNA cloning strategies, alter or remove any potential codon bias, alter or improve GC content, remove or alter altered reading frames, and / or alter or remove intron / exon splicing recognition sites, polyadenylation sites, Shine-Delgarno sequences, unwanted promoter elements, etc., that may be present in the native DNA sequence. Typically, the GC content of the gene will be increased. For a discussion of host-preferred codon usage, see, for example, Campbell and Gowri (1990) Plant Physiol. [Plant Physiology] 92:1-11. Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, U.S. Patent Publication No. 20090137409, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

[0138] It is also possible that the synthetic DNA sequence can be used to introduce other modifications into the DNA sequence, such as introducing intron sequences, producing DNA sequences that express as protein fusions with organelle-targeting sequences, such as chloroplast transport peptides, apoplast / vacuole-targeting peptides, or peptide sequences that cause the resulting peptides to be retained in the endoplasmic reticulum. Thus, in one embodiment, the nematicide protein is targeted to the chloroplast for expression. In this way, without directly inserting the expression cassette encoding the nematicide protein into the chloroplast genome, the expression cassette will additionally contain nucleic acids encoding a transport peptide to guide the nematicide protein to the chloroplast. Such transport peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. [Journal of Plant Molecular Biology] 9:104-126; Clark et al. (1989) J. Biol. Chem. [Journal of Biochemistry] 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. [Plant Physiology] 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. [Biochemical and Biophysical Research Communications] 196:1414-1421; and Shah et al. (1986) Science [Science] 233:478-481. The pest-killing gene will be targeted to the chloroplast; see, for example, U.S. Patent No. 5,380,831, which is incorporated herein by reference.

[0139] Plant transformation

[0140] The methods of the embodiments involve introducing nucleotide constructs into plants. "Introduction" means presenting the nucleotide construct to a plant in a manner that allows the construct to enter the interior of a plant cell. The methods of the embodiments do not require the use of a specific method for introducing nucleotide constructs into plants; they only require that the nucleotide construct enter the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art, including but not limited to stable transformation methods, transient transformation methods, gene editing, and virus-mediated methods.

[0141] "Transgenic plant," "transformed plant," or "stable transformed" plant, cell, or tissue refers to a plant in which exogenous nucleic acid sequences or DNA fragments have been incorporated or integrated into the plant cell. These nucleic acid sequences include those that are exogenous or not present in untransformed plant cells, as well as those that may be endogenous or present in untransformed plant cells.

[0142] "Heterogeneous" usually refers to nucleic acid sequences that are not endogenous to the cell or part of the natural genome in which they reside and have been added to the cell through infection, transfection, microinjection, electroporation, microprojection, etc.

[0143] The transgenic plants of the embodiments express one or more of the novel toxin sequences disclosed herein. In some embodiments, the protein or nucleotide sequences of the embodiments are advantageously combined in the plant with other genes encoding proteins or RNA that confer useful agronomical characteristics to such plants. Among the genes encoding proteins or RNA that confer useful agronomical characteristics to the transformed plants, reference may be made to DNA sequences encoding proteins that confer resistance to one or more herbicides, other proteins that confer resistance to certain insects, those proteins that confer resistance to certain diseases, DNA sequences encoding RNA that provides nematode or insect control, etc. Such genes are described in published PCT patent applications WO 91 / 02071 and WO 95 / 06128, and U.S. Patent 7,923,602 and U.S. Patent Application Publication No. 20100166723, each of which is incorporated herein by reference in its entirety. In various embodiments, the transgenic plant further comprises one or more additional insect resistance genes (e.g., Cry1, such as members of the Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, and Cry1F families; Cry2, such as members of the Cry2A family; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc.). Those skilled in the art will understand that transgenic plants can contain any gene that confers the desired agronomic trait.

[0144] Among the DNA sequences encoding proteins that confer tolerance to certain herbicides in transformed plant cells and plants, references may be made to the bar or PAT genes or Streptomyces coelicolor genes that confer tolerance to glufosinate herbicides as described in WO 2009 / 152359, and genes encoding appropriate EPSPS that confer tolerance to herbicides targeting EPSPS (such as glyphosate and its salts) (US 4,535,060, US 4,769,061, US 5,094,945, US 4,940,835, US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5,312,910, US 5,627,061, US 4,535,060, US 5,312,910, US 5,627,061, US 5,310,667, US 5,312,910, US 5,627,061 ...312,910, US 5,627,061, US 5,312,910, US 5,312,910, US 5,312,91 Genes encoding glyphosate-n-acetyltransferase (e.g., US 8,222,489, US 8,088,972, US 8,044,261, US 8,021,857, US 8,008,547, US 7,999,152, US 7,998,703, US 7,863,503, US 7,714,188, US 7,709,702, US 7,666,644, US 7,666,643, US 7,531,339, US 7,527,955 and US 7,405,074), genes encoding glyphosate oxidoreductase (e.g., US 5,463,175), or genes encoding HPPD inhibitor tolerance proteins (e.g., WO HPPD inhibitor tolerance genes described in WO 2004 / 055191, WO 199638567, US 6791014, WO2011 / 068567, WO 2011 / 076345, WO 2011 / 085221, WO 2011 / 094205, WO 2011 / 068567, WO2011 / 094199, WO 2011 / 094205, WO 2011 / 145015, WO 2012 / 056401 and WO 2014 / 043435).

[0145] Among the DNA sequences encoding suitable EPSPS that confer tolerance to herbicides targeting EPSPS, more particular reference will be made to the gene encoding plant EPSPS, especially corn EPSPS, particularly the corn EPSPS containing two mutations (specifically a mutation at amino acid position 102 and a mutation at amino acid position 106) described in U.S. Patent No. 6,566,587 (hereinafter referred to as double mutant corn EPSPS or 2mEPSPS), or the gene encoding EPSPS isolated from Agrobacterium and described by Sequence ID No. 2 and Sequence ID No. 3 of U.S. Patent No. 5,633,435 (also known as CP4).

[0146] In the DNA sequence encoding suitable EPSPS that confers tolerance to herbicides targeting EPSPS, reference will be made to EPSPS GRG23 from Arthrobacter globiformis, and also to mutants GRG23 ACE1, GRG23 ACE2, or GRG23 ACE3, as well as GRG23 mutants or variants as described in WO 2008 / 100353, such as the gene for GRG23(ace3)R173K in SEQ ID No. 29 of WO 2008 / 100353.

[0147] In the case of DNA sequences encoding EPSPS and the aforementioned genes, the sequences encoding these enzymes are preceded by sequences encoding transport peptides, particularly the sequences of “optimized transport peptides” described in U.S. Patent Nos. 5,510,471 or 5,633,448.

[0148] Exemplary herbicide tolerance traits that can be combined with the nucleic acid sequences of the embodiments further include at least one ALS (acetyllactate synthase) inhibitor (WO 2007 / 024782); a mutant Arabidopsis ALS / AHAS gene (US Patent No. 6,855,533); a gene encoding a 2,4-D-monooxygenase that confers tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid) through metabolism (US Patent No. 6,153,401); and a gene encoding a dicamba monooxygenase that confers tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid) through metabolism (US 2008 / 0119361 and US 2008 / 0120739).

[0149] In various embodiments, the nucleic acid of the embodiment is superimposed with one or more herbicide tolerance genes, including one or more HPPD inhibitor herbicide tolerance genes, and / or one or more genes tolerant to glyphosate and / or glufosinate.

[0150] In the DNA sequences encoding proteins involved in insect resistance, Bt proteins, which are widely described in the literature and well known to those skilled in the art, will be mentioned. Proteins extracted from bacteria such as *Bacillus luminifera* (WO 97 / 17432 and WO 98 / 08932) will also be mentioned.

[0151] In such DNA sequences encoding target proteins that confer novel insect resistance properties, the Bt Cry or VIP proteins, which are widely described in the literature and well known to those skilled in the art, will be mentioned. These include Cry1F protein or hybrids derived from Cry1F protein (e.g., hybrid Cry1A-Cry1F protein or its toxic fragments as described in US 6,326,169, US 6,281,016, US 6,218,188); Cry1A type protein or its toxic fragments; Cry1Ac protein or hybrids derived from Cry1Ac protein (e.g., hybrid Cry1Ab-Cry1Ac protein as described in US 5,880,275); or Cry1Ab or Bt2 protein or its insecticidal fragments as described in EP 451878; Cry2Ae, Cry2Af or Cry2Ag protein or its toxic fragments as described in WO 2002 / 057664; and Cry1A.105 protein (SEQ ID No.) as described in WO 2007 / 140256. 7) or its toxic fragments; VIP3Aa19 protein of NCBI accession number ABG20428; VIP3Aa20 protein of NCBI accession number ABG20429 (SEQ ID No. 2 in WO2007 / 142840); VIP3A protein produced in the COT202 or COT203 cotton events (WO2005 / 054479 and WO 2005 / 054480, respectively); Cry protein as described in WO 2001 / 47952; VIP3Aa protein or its toxic fragments as described in Estruch et al. (1996), Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences] 28;93(11):5389-94 and US6,291,156; VIP3Aa protein or its toxic fragments derived from pathogenic bacilli (as described in WO 98 / 50427), Serratia (particularly from Serratia tinctoria (S. tumefaciens)). Insecticidal proteins of *Epiphyta entomophila* or *Epiphyta* species, such as the Tc protein from *Epiphyta* as described in WO 98 / 08932 (e.g., Waterfield et al., 2001, *ApplEnviron Microbiol*. 67(11):5017-24; French-Constant and Bowen, 2000, *Cell Mol Life Sci*. 57(5):828-33). Furthermore, this document includes any variants or mutants of any of these proteins that differ from any of the aforementioned sequences in some (1-10 or 1-5) amino acids, such as in the sequence of their toxic fragments, or in their fusion with transport peptides such as plasmid transport peptides or another protein or peptide.

[0152] In various embodiments, the nucleic acids of the embodiments may be combined in plants with one or more genes that confer desired traits, such as herbicide tolerance, insect tolerance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency, improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality, stress tolerance, improved regeneration, etc.

[0153] Useful transgenic events that can be combined with the genes of the current embodiment in plants of the same species (e.g., by hybridization or by retransforming a plant containing another transgenic event with the chimeric gene of the embodiment) include event BPS-CV127-9 (soybean, herbicide tolerant, deposited at NCIMB 41603, described in WO 2010 / 080829); event DAS21606-3 / 1606 (soybean, herbicide tolerant, deposited at PTA-11028, described in WO 2012 / 033794); event DAS-44406-6 / pDAB8264.44.06.1 (soybean, herbicide tolerant, deposited at PTA-11336, described in WO2012 / 075426); and event DAS-14536-7. / pDAB8291.45.36.2 (soybean, herbicide tolerance, deposited as PTA-11335, described in WO 2012 / 075429), event DAS68416 (soybean, herbicide tolerance, deposited as ATCC PTA-10442, described in WO 2011 / 066384 or WO 2011 / 066360); event DP-305423-1 (soybean, quality traits, not deposited, described in USA 2008-312082 or WO 2008 / 054747); event DP-356043-5 (soybean, herbicide tolerance, deposited as ATCC PTA-8287, described in USA 2010-0184079 or WO Event FG72 (soybean, herbicide tolerance, deposited as PTA-11041, described in WO 2011 / 063413), Event LL27 (soybean, herbicide tolerance, deposited as NCIMB41658, described in WO 2006 / 108674 or USA 2008-320616), Event LL55 (soybean, herbicide tolerance, deposited as NCIMB 41660, described in WO 2006 / 108675 or USA 2008-196127), Event MON87701 (soybean, insect control, deposited as ATCC PTA-8194, described in USA). 2009-130071 or WO2009 / 064652); Event MON87705 (Soybean, quality trait - herbicide tolerance, deposited as ATCC PTA-9241, described in USA 2010-0080887 or WO 2010 / 037016); Event MON87708 (Soybean, herbicide tolerance, deposited as ATCC PTA-9670, described in WO 2011 / 034704);Event MON87712 (soybean, yield, deposited as PTA-10296, described in WO 2012 / 051199), Event MON87754 (soybean, quality traits, deposited as ATCC PTA-9385, described in WO 2010 / 024976); Event MON87769 (soybean, quality traits, deposited as ATCC PTA-8911, described in USA2011-0067141 or WO 2009 / 102873); Event MON89788 (soybean, herbicide tolerance, deposited as ATCCPTA-6708, described in USA 2006-282915 or WO 2006 / 130436); Event SYHT0H2 / Events SYN-000H2-5 (soybean, herbicide tolerance, deposited as PTA-11226, described in WO 2012 / 082548), EE-GM3 / FG72 (soybean, herbicide tolerance, ATCC accession number PTA-11041), optionally superimposed with event EE-GM1 / LL27 or event EE-GM2 / LL55 (WO 2011 / 063413 A2); DAS-68416-4 (soybean, herbicide tolerance, ATCC accession number PTA-10442, WO 2011 / 066360A1); and DAS-68416-4 (soybean, herbicide tolerance, ATCC accession number PTA-10442, WO 2011 / 066384) are also mentioned. A1); Event DAS-21606-3 (Soybean, herbicide tolerance, ATCC Registry No. PTA-11028, WO 2012 / 033794 A2); Event MON-87712-4 (Soybean, quality traits, ATCC Registry No. PTA-10296, WO2012 / 051199 A2); Event DAS-44406-6 (Soybean, superimposed herbicide tolerance, ATCC Registry No. PTA-11336, WO 2012 / 075426 A1); Event DAS-14536-7 (Soybean, superimposed herbicide tolerance, ATCC Registry No. PTA-11335, WO 2012 / 075429 A1); Event SYN-000H2-5 (Soybean, herbicide tolerance, ATCC Registry No. PTA-11226, WO 2012 / 082548) A2); Event 8264.44.06.1 (Soybean, superimposed herbicide tolerance, accession number PTA-11336, WO 2012075426 A2); Event 8291.45.36.2 (Soybean, superimposed herbicide tolerance, accession number PTA-11335, WO 2012075429 A2);Event SYHT0H2 (soybean, ATCC accession number PTA-11226, WO 2012 / 082548A2); Event pDAB8264.42.32.1 (soybean, superimposed herbicide tolerance, ATCC accession number PTA-11993, WO 2013 / 010094 A1).

[0154] Furthermore, this document provides a method for producing soybean plants or seeds comprising a combination of a nucleotide sequence encoding SEQ ID NO: 1, 3, 5, 7 or any functional fragment thereof with another SCN resistance locus / gene, such as by combining soybean plants or seeds comprising a nucleotide sequence encoding SEQ ID NO: 1, 3, 5, 7 or any functional fragment thereof with another SCN resistance locus / gene present in the same soybean plant / seed, and planting seeds comprising a nucleotide sequence encoding SEQ ID NO: 1, 3, 5, 7 or any functional fragment thereof and said other SCN resistance locus / gene. In one embodiment, the plant, cell, or seed of the embodiment contains one or more other SCN resistance loci / genes present in soybean to obtain a combination of different SCN resistance sources in the soybean plant, cell, or seed of the embodiment. Several soybean SCN resistance loci or genes are known, and one or more of these loci or genes can be combined in the same plant, cell, or seed with plants containing SEQ ID NO: 1, 3, 5, 7 or any functional fragment thereof, such as those from resistance sources PI 88788, PI 548402 (Peking), PI Any of the SCN resistance genes / locus 437654 (Hartwig or CYSTX) or any combination thereof, or one or more of the natural SCN resistance loci / genes rhg1, rhg1-b, rhg2, rhg3, Rhg4, Rhg5, qSCN11, cqSCN-003, cqSCN-005, cqSCN-006, cqSCN-007, or any SCN resistance locus identified on soybean chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof (Kim et al. 2016, Theor. Appl. Genet. [Theoretical and Applied Genetics] 129(12):2295-2311; Kim and Diers 2013, Crop Science [Crop Science]). 53:775-785; Kazi et al. 2010, Theor. Appl. Gen. [Theoretical and Applied Genetics] 120(3):633-644; Glover et al. 2004, Crop Science 44(3):936-941; www.soybase.org; Concibido et al. 2004, Crop Science 44:1121-1131; Webb et al. 1995, Theor. Appl. Genet. [Theoretical and Applied Genetics] 91:574-581).In one embodiment, the plant or seed of the embodiment is combined with one or more SCN resistance loci in soybean, which are obtained from SCN resistance sources PI 548316, PI567305, PI 437654, PI 90763, PI 404198B, PI 88788, PI 468916, PI 567516C, PI209332, PI 438489B, PI 89772, Peking, PI 548402, PI 404198A, PI 561389B, PI 629013, PI 507471, PI 633736, PI 507354, PI 404166, PI 437655, PI 467312, PI 567328, PI22897 or PI Any one of 494182.

[0155] Transformation of plant cells can be achieved using one of several techniques known in the art. The pest-killing genes of the embodiments can be modified to obtain or enhance expression in plant cells. Typically, constructs expressing such proteins will contain a promoter driving gene transcription, as well as a 3' untranslated region allowing transcription termination and polyadenylation. The organization of such constructs is well known in the art. In some cases, it may be useful to engineer genes so that the resulting peptides are secreted or otherwise targeted within plant cells. For example, genes can be engineered to contain signal peptides to promote peptide translocation to the endoplasmic reticulum. Plant expression cassettes can be engineered to contain introns such that intron mRNA processing is required for expression.

[0156] Typically, a plant expression cassette is inserted into a plant transformation vector. This transformation vector can consist of one or more DNA vectors required to achieve plant transformation. For example, plant transformation vectors consisting of more than one contiguous DNA segment are commonly used in the art. These vectors are commonly referred to in the art as “binary vectors.” Binary vectors, as well as vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation, where the size and complexity of the DNA segments required for efficient transformation are considerable, and it is advantageous to segregate functions onto different DNA molecules. Binary vectors typically contain a plasmid vector containing cis-acting sequences (such as left and right borders) required for T-DNA transfer, an engineered selection marker that can be expressed in plant cells, and a “target gene” (a gene engineered for expression in plant cells, with the intention of generating transgenic plants). The plasmid vector also contains sequences required for bacterial replication. The arrangement of the cis-acting sequences allows for efficient transfer to and expression in plant cells. For example, the selection marker gene and the pest-killing gene are located between the left and right borders. Typically, the second plasmid vector contains a trans-acting factor that mediates the transfer of T-DNA from Agrobacterium to plant cells. This plasmid typically contains virulence functions (Vir genes) that allow Agrobacterium to infect plant cells and transfer DNA via cleavage at the boundary sequence and vir-mediated DNA transfer, as understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. A second plasmid vector is not essential for transforming plants via other methods such as microspraying, microinjection, electroporation, polyethylene glycol, etc.

[0157] Typically, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying an appropriately selected maximum threshold level (depending on the selective marker gene) to recover the transformed plant cells from a cluster of untransformed cell masses. Typically, explants are transferred to a fresh supply of the same medium and cultured routinely. Subsequently, after being placed on a regeneration medium supplemented with a maximum threshold level of selectant, the transformed cells differentiate into shoots. The shoots are then transferred to a selective rooting medium to recover the rooted shoots or plantlets. The transgenic plantlets then grow into mature plants and produce fertile seeds (e.g., Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Typically, explants are transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods used to generate transgenic plants can be found in Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Because transformed material contains numerous cells; both transformed and untransformed cells are present in any patch of target callus or tissue or cell population. The ability to kill untransformed cells and allow transformed cells to proliferate produces transformed plant cultures. Typically, the ability to remove untransformed cells limits the rapid recovery of transformed plant cells and the successful generation of transgenic plants.

[0158] Transformation protocols, and protocols for introducing nucleotide sequences into plants, can vary depending on the type of plant or plant cell targeted for transformation (i.e., monocot or dicot). Transgenic plants can be generated by one of several methods, including but not limited to microinjection, electroporation, direct gene transfer, introduction of heterologous DNA into plant cells via Agrobacterium (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous DNA adhering to particles, ballistic particle acceleration, aerosol micelle transformation (US Publication No. 20010026941; US ​​Patent No. 4,945,050; International Publication No. WO 91 / 00915; US Publication No. 2002015066), Lec1 transformation, and various other non-particle-directly mediated methods for DNA transfer.

[0159] Methods for transforming chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:913-917; Svab and Maliga (1993) EMBO J. [Journal of the European Society for Molecular Biology] 12:601-606. These methods rely on particle gun delivery of selectively labeled DNA and targeting the DNA to the plastid genome via homologous recombination. Alternatively, plastid transformation can be accomplished by transactivating silent plastid-carrying transgenes through tissue-biased expression of a nuclear-encoded, plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 91:7301-7305.

[0160] After the heterologous DNA is integrated into plant cells, an appropriate maximum selection threshold level is applied to the culture medium to kill untransformed cells, and the presumed transformed cells that survived this selection treatment are isolated and proliferated by periodically transferring them to fresh culture medium. Cells transformed with plasmid vectors are identified and proliferated through continuous passage and stimulation with appropriate selection. The presence of the target heterologous gene integrated into the genome of the transgenic plant can then be confirmed using molecular and biochemical methods.

[0161] The transformed cells can be grown into plants in a conventional manner. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same or different transformed lines, and the resulting hybrids with constitutive expression of the desired phenotypic trait can be identified. Two or more generations can be grown to ensure that the expression of the desired phenotypic trait is stably maintained and inherited, and the seeds are then harvested to ensure that the expression of the desired phenotypic trait has been achieved. In this way, transformed seeds (also known as “transgenic seeds”) are provided having one or more nucleotide constructs of the embodiments stably incorporated into their genome, such as the expression cassettes of the embodiments.

[0162] Evaluation of plant transformation

[0163] After introducing heterologous DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods, such as analyzing nucleic acids, proteins and metabolites associated with the integrated gene.

[0164] PCR analysis is a rapid method for screening the presence of genes incorporated into transformed cells, tissues, or shoots at an early stage before transplantation into soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Spring Harbor Laboratory Press, Cold Spring Harbor, New York). PCR is performed using oligonucleotide primers specific to the target gene or the Agrobacterium vector background.

[0165] Plant transformation can be confirmed by DNA blot analysis of genomic DNA (Sambrook and Russell, 2001, ibid.). Typically, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel, and transferred to a nitrocellulose or nylon membrane. Then, following standard techniques (Sambrook and Russell, 2001, ibid.), DNA is blotted using, for example, radiolabeled... 32 P-target DNA fragment detection membranes, or "imprints," are used to confirm the integration of introduced genes into the plant genome.

[0166] In RNA blot analysis, RNA was isolated from specific tissues of the transformant according to standard procedures routinely used in the art (Sambrook and Russell, 2001, ibid.), fractionated in formaldehyde agarose gel, and blotted onto a nylon filter membrane. The expression of RNA encoded by the nemesis gene was then tested by hybridizing the filter membrane with a radioactive probe derived from the nemesis gene, using methods known in the art (Sambrook and Russell, 2001, ibid.).

[0167] Transgenic plants can be subjected to Western blotting, biochemical assays, etc., and the presence of proteins encoded by pest-killing genes can be confirmed by standard procedures (Sambrook and Russell, 2001, ibid.) using antibodies that bind to one or more epitopes present on nematicides.

[0168] Pesticide activity in plants

[0169] In another embodiment, transgenic plants expressing nematicidal proteins with pest-killing activity against nematode pests can be generated. The methods described above by way of example can be used to generate transgenic plants, but the manner in which transgenic plant cells are generated is not definitive. Methods known or described in the art, such as Agrobacterium-mediated transformation, gene gun transformation, and non-particle-mediated methods, can be used according to the experimenter's judgment. Plants expressing nematicidal proteins can be isolated using common methods described in the art, such as by transforming callus, selecting transformed callus, and regenerating fertile plants from such transgenic callus. In such methods, any gene can be used as a selectable marker, provided that its expression in plant cells confers the ability to identify or select transformed cells.

[0170] Many markers for plant cells have been developed, such as those for resistance to chloramphenicol, aminoglycoside G418, and hygromycin. Other genes encoding products involved in chloroplast metabolism can also be used as selective markers. For example, genes providing resistance to plant herbicides such as glyphosate, bromobenzonitrile, or imidazolinone can have specific uses. Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314 (bromobenzonitrile resistant nitrile hydrolase gene); and Sathasivan et al. (1990) Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene). Furthermore, the genes disclosed herein can be used as markers for assessing bacterial or plant cell transformation. Methods for detecting the presence of transgenes in plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, or their progeny are well known in the art. In one embodiment, the presence of transgenes is detected by testing for pest-killing activity against nematode pests.

[0171] The nematicidal activity of fertile plants expressing nematicidal proteins against nematode pests can be tested, and plants exhibiting the best activity can be selected for further breeding. Methods for determining pest activity are available in the art. Typically, the proteins are mixed and used for feeding assays. See, for example, Marrone et al. (1985) J. of Economic Entomology 78:290-293. Methods for testing the nematicidal efficiency of plants expressing such nematicidal proteins are known in the art and have been described, for example, in Kahn et al. (2021) Nature Communications, 12(1), 3380 (incorporated hereby by reference).

[0172] One or more embodiments can be used to transform any plant species, including but not limited to monocots and dicots. Examples of target plants include, but are not limited to, corn, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and rapeseed, Brassica species, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia nut, apricot, oats, vegetables, ornamental plants, and conifers.

[0173] Applications in pest control

[0174] General methods for employing strains containing the nucleotide sequence or variants thereof of the embodiments in pest control or in the engineering of other organisms as pesticides are known in the art. See, for example, U.S. Patent Nos. 5,039,523 and EP 0480762 A2.

[0175] Microorganisms can be genetically modified to contain nucleotide sequences encoding SEQ ID NO: 2, 4, 6, 8 or their nematicidal active variants or fragments, and proteins can be used to protect crops and products from pests. In one embodiment, when cells are applied to an environment of one or more target pests, the intact (i.e., unlyzed) cells of an organism that produces toxins (pesticides) are treated with an agent that prolongs the activity of the toxins produced in the cells.

[0176] Alternatively, pest control agents can be produced by introducing a pest-killing gene into a cell host. Expression of the pest-killing gene directly or indirectly leads to the intracellular production and maintenance of the pest control agent. In one embodiment, when cells are applied to an environment containing one or more target pests, these cells are then treated under conditions that prolong the activity of the toxins produced in the cells. The resulting product retains the toxicity of the toxin. These naturally encapsulated pest control agents can then be formulated according to conventional techniques for use in environments containing target pests, such as soil, water, and plant leaves. See, for example, EPA 0192319 and the references cited therein. Alternatively, cells expressing the genes of the embodiments can be formulated to allow the resulting material to be applied as a pest control agent.

[0177] The active ingredients in the embodiments are typically applied in the form of a composition and may be applied simultaneously or sequentially with other compounds to the crop area or plant to be treated. These compounds may be fertilizers, herbicides, cryoprotectants, surfactants, detergents, biocidal soaps, dormant oils, polymers, and / or timed release or biodegradable carrier formulations that allow for long-term administration to the target area after a single application of the formulation. They may also be selective herbicides, chemical insecticides, viricides, microbial agents, amoebics, pest control agents, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these formulations, if desired, together with other agriculturally acceptable carriers, surfactants, or application-promoting adjuvants commonly used in the field of formulations. Suitable carriers and adjuvants may be solid or liquid and correspond to substances commonly used in formulation technology, such as natural or recycled minerals, solvents, dispersants, wetting agents, thickeners, binders, or fertilizers. Similarly, preparations can be made into edible "bait" or pest "traps" to allow target pests to ingest or consume pest-killing preparations.

[0178] Methods of applying the active ingredient or agrochemical composition of the embodiments containing at least one nematicidal protein disclosed herein as SEQ ID NO: 2, 4, 6, 8 or a nematicidal effective variant or fragment thereof include foliar application, seed coating, and soil application. The amount and proportion of application depend on the intensity of the corresponding pest infestation.

[0179] The composition can be formulated as a powder, dust, pill, granule, spray, emulsion, colloid, solution, or the like, and can be prepared by conventional methods such as dehydration, freeze-drying, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of cell cultures containing the polypeptide. In all such compositions containing at least one such biocidal polypeptide, the polypeptide may be present at a concentration of about 1% to about 99% by weight.

[0180] The methods described in the examples can kill or reduce the number of nematode pests in a given area, or can be applied preventively to environmental areas to prevent the infestation of susceptible pests. Pests can ingest or come into contact with a biocidally effective amount of the peptide.

[0181] The described biocidal composition can be prepared by formulating suspensions of bacterial cells, crystals, and / or spores, or isolated protein components having a desired agriculturally acceptable carrier. The composition can be formulated prior to application by suitable methods, such as lyophilization, freeze-drying, dehydration, or in an aqueous carrier, medium, or suitable diluent such as saline or other buffer. The formulated composition can be in the form of powder or granular material, or in the form of a suspension in oil (vegetable or mineral oil), or in the form of an oil or oil / water emulsion, or as a wettable powder, or in combination with any other carrier material suitable for agricultural applications. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" encompasses all adjuvants, inert components, dispersions, surfactants, thickeners, binders, etc., commonly used in biocidal formulation techniques; these are well known to those skilled in the art in biocidal formulation. The formulation can be mixed with one or more solid or liquid adjuvants and prepared by various means, such as homogenizing, blending, and / or grinding the biocidal composition with a suitable adjuvant using conventional formulation techniques. Suitable formulations and methods of application are described in U.S. Patent No. 6,468,523, which is incorporated herein by reference.

[0182] Methods for increasing plant yield

[0183] Methods for increasing plant yields are provided. These methods include providing a plant or plant cell expressing a polynucleotide encoding a nematicidal polypeptide sequence disclosed herein, and growing the plant or its seeds in a field infested (or susceptible to) a nematode pest, the polypeptide having nematicidal activity against that nematode pest. In some embodiments, the Cry5-like polypeptide described herein has nematicidal activity against a species of the genus *Soybean cyst nematode*, and the field is infested with said *Soybean cyst nematode* species. In various embodiments, the *Soybean cyst nematode* species is *Soybean cyst nematode*. In another embodiment, the nematode is a root-rot nematode, a disease nematode, *Soybean cyst nematode*, or a spear nematode, or a target pest.

[0184] As used herein, the plant term “yield” refers to the quality and / or quantity of biomass produced by a plant. “Biomass” means any measured plant product. An increase in biomass production means any improvement in the yield of a measured plant product. Increased plant yield has several commercial applications. For example, increasing the biomass in plant leaves can increase the yield of leafy green vegetables for human or animal consumption. Additionally, increased leaf biomass can be used to increase the production of plant-derived pharmaceuticals or industrial products. Increased yield can include any statistically significant increase, including but not limited to increases of at least 1%, at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, or more, compared to plants that do not express the pest-killing proteins described herein. Increased plant yield is due to improved nematode resistance in plants expressing the nematicidal proteins disclosed herein. Expression of nematicidal proteins leads to a reduction in the ability of pests to infect or consume the plant. In various embodiments, compared with plants that do not express the nematicidal protein of the embodiments, the expression of the nematicidal protein leads to improved root development (e.g., improved root or root hair growth), improved yield, faster emergence, improved plant stress management (including increased stress tolerance and / or improved stress recovery), increased mechanical strength, improved drought resistance, reduced fungal disease infection, and improved plant health.

[0185] Plants can also be treated with one or more chemical compositions, including one or more herbicides, insecticides, or fungicides. Exemplary chemical compositions include:

[0186] A. Fruit / vegetable herbicides: Atrazine, chlorpyrifos, diuron, glyphosate, linuron, cypermethrin, simazine, trifluralin, pyrifluquinazon, glufosinate, chlorpyrifos, paraquat, pendimethalin, haloxyfop-methyl, flupropargyl, chlorpyrifos, and triazine indomethacin.

[0187] B. Fruit / Vegetable Insecticides: Aldicarb, Bacillus thuringiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Delmethrin, Abamectin, Cypermethrin / β-cypermethrin, Centrifugerin, Lambda-cyhalothrin, Acaricide, Bifenazate, Mefenoxam, Flufenoxam, Cyclofenazate, Thiamethoxam, Dinotefuran, Pyrimethanil, Spirodiclofen, γ-Cypermethrin, Spirodiclofen, Spinosad, Chlorpyrifos, Chlorpyrifos The following are listed: chlorantraniliprole, bromocyanamide, chlorfenapyr, spirotetramat, imidacloprid, flufenoxuron, thiamethoxam, cyfluthrin, flupyradifurone, dicofol, cyanopyr, thiamethoxam, sinomethrin, sinomethrin, thiamethoxam, flufenoxuron, cypermethrin, emamectin benzoate, indoxacarb, fenpropathrin, pyriproxyfen, and benzoyl tin oxide.

[0188] C. Fruit / Vegetable Fungicides: Azoxystrobin, Azoxystrobin, Benzyl sulfide, Cyazofamid, Captan, Carbendazim, Chlorothalonil, Copper, Cyazofamid, Cyclosulfuron, Cyclosulfuron, Cyclosulfuron, Azoxystrobin, Difenoconazole, Dimethomorph, Dichlorophenoxyacetic acid, Anthraquinone, Imidacloprid, Cyclosulfuron, Fluazinam, Fluopyram, Fluopyram, Flupyraclostrobin, Fluopyram, Captan Tris(ethylphosphonic acid), iprodione, propiconazole, pyraclostrobin, azoxystrobin, mancozeb, dimethomorph, metalaxyl / metalaxyl-M, mancozeb, benomyl, cyproconazole, tebuconazole, pyraclostrobin, azoxystrobin, propiconazole, propineb, propineb, propoxyquin, prothioconazole, azoxystrobin, pyrimethanil, quinoxalic acid, spirocycline, sulfur, tebuconazole, thiophanate-methyl, and azoxystrobin.

[0189] Cereal herbicides: 2,4-D, pyrimisulfuron, bromosulfuron-methyl, chlorpyrifos-E, chlorpyrifos, clodinafop-P, dichloropyridinic acid, dicamba, quizalofop-M, pyrfluthrin, quizalofop-methyl, flusulfuron-methyl, fluoxetine-NA, fluthiamethoxam, flupyrimisulfuron-M, fluroxypyr, furazolidone, glyphosate, iodosulfuron-methyl, iodosulfuron-methyl, isoproturon, MCPA, mesosulfuron-methyl, mesosulfuron-methyl, pendimethalin, clodinafop-methyl, bensulfuron-methyl, sulfadiazine, sulfonylsulfuron-methyl, sulfadiazine, sulfadiazine, sulfadiazine, thifensulfuron-methyl, oxadiazine, bensulfuron-methyl, trifluralin, and trifluralin.

[0190] Grain fungicides: azoxystrobin, bifenthrin, boscalid, carbendazim, chlorothalonil, cycloflufenoxam, cyclopyrrolidone, azoxystrobin, ether amine, flutriafol, benzyl benzoate, butyl morpholine, flupyradifurone, fluquinazole, fluazinam, fluazinam, fluazinam, pyraclostrobin, ether amine, tebuconazole, benomyl, pyraclostrobin, azoxystrobin, prochloraz, propiconazole, propoxyquin, prothioconazole, azoxystrobin ester, quinoxalic acid, spirocyclohexane, tebuconazole, thiophanate-methyl, and azoxystrobin.

[0191] Cereal insecticides: dimethoate, lambda-cyhalothrin, deltamethrin, α-cyhalothrin, β-cyhalothrin, bifenthrin, imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, dinotefuran, chlorpyrifos, pirimicarb, methiocarb, and flonicamid.

[0192] Corn herbicides: atrazine, metolachlor, bromobenzonitrile, acetochlor, dicamba, dichloropyridine, (S) dimethoprim, glyphosate, glyphosate, isoxaflutole, (S-) metolachlor, mesosulfuron, nicosulfuron, flusulfuron, sulfadiazine, sulfadiazine, formamide sulfadiazine, benzoxazine, cyclosulfuron, sulfadiazine, thiamethoxam, fluthiamethoxam, and pyroxasulfon.

[0193] Insecticides for corn: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-cyhalothrin, Heptamethrin, Terbufos, Thiamethoxam, Thiamethoxam, Spirodiclofen, Fipronil, Lufenuron, Cypermethrin, Chlorfenapyr, Deltamethrin, Thiamethoxam, β-Cypermethrin, Cypermethrin, Bifenthrin, Lufenuron, Butylpyridinium, Ethylbutazone, Brofenoxam, Thiamethoxam, Acetamiprid, Dinotefuran, and Abamectin.

[0194] Fungicides for corn: azoxystrobin, bifenthiophanate-methyl, boscalid, cyclopyrrolidone, fenamidoxime, flutriafol, seed coat ester, fluopyram, flupyraclostrobin, fluopyram, pyraclostrobin, pyraclostrobin, fenamidoxime, pyraclostrobin, pyraclostrobin, pyraclostrobin, propiconazole, prothioconazole, azoxystrobin, tebuconazole, and oxadiazon.

[0195] Rice herbicides: butachlor, propargite, tetrazole sulfidon, bensulfuron-methyl, cyhalofop-butyl, chlorpyrifos, tetrazole sulfidon, imazalil, benzylthiamethoxam, oxadiazon, pyrimisulfuron, barnyardgrass, quinclorac, quizalofop-p-ethyl, indicarb, fluthiamethoxam, tetrazole sulfidon, chlorpyrifos, benzo[a]cyclohexane, cyclohexyl-methyl, penoxsulam, bispyribac-sodium, propyzoxystrobin, ethoxysulfuron, pretilachlor, mesotrione, tebufenozide, oxadiazon, quizalofop-p-ethyl, and pyrimisulfan.

[0196] Rice insecticides: diazinon, tebufenozide, carbofuran, thiamethoxam, dinotefuran, fipronil, imidacloprid, isoprocarb, thiamethoxam, cyclonepropalin, thiamethoxam, acetamiprid, flufenoxuron, chlorantraniliprole, deltamethrin, acetamiprid, thiamethoxam, brofenoxuron, spinosad, snotomum, emamectin benzoate, cypermethrin, chlorpyrifos, permethrin, carbofuran, carbofuran, and flupyradifurone.

[0197] Rice fungicides: azoxystrobin, carbendazim, cyproconazole, diclofenac, difenoconazole, cymoxanil, azoxystrobin, gentamicin, hexaconazole, oxamyl, isoprothiolane (IBP), isoprothiolane, isothiazinam, kasugamycin, mancozeb, fenoxystrobin, oxadiazon, pendimethalin, thiamethoxam, propiconazole, propineb, quinclorac, tebuconazole, thiophanate-methyl, thiamethoxam, tricyclazole, oxadiazon, and jinggangmycin.

[0198] Cotton herbicides: diuron, fenflur, MSMA, ethoxyflufen, prochloraz, trifluralin, cyclohexane, clethodim, butylpyrrolidone, glyphosate, pyrazosulfuron, pendimethalin, pyrimisulfuron, trifluralin, pyrazosulfuron, cyclohexane, propyzoxystrobin, and thidium uranium.

[0199] Insecticides for cotton: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Acetamiprid, Emamectin, Imidacloprid, Indoxacarb, Lambda-cyhalothrin, Spinosad, Thiamethoxam, Gamma-cyhalothrin, Spirodiclofen, Pyridalyl, Flupyridine, Flufenoxuron, Chlorantraniliprole, β-carbamate - Cypermethrin, Spirotetramat, Thiamethoxam, Thiamethoxam, Thiamethoxam, Dinotefuran, Flufenoxam, Bromnipotentiamide, Sponsauce, Ethyl Sponsauce, γ-Cypermethrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one), Thiamethoxam, Abamectin, Flupyradifurone, Acetaminophen, Spirodiclofen, and Flupyradifurone.

[0200] Cotton fungicides: azoxystrobin, bifenthrin, cyprodinil, carbendazim, chlorothalonil, copper, cyclohexane, difenoconazole, fenpropathrin, flutriafol, imidacloprid, fluazinam, fluopyram, flupyraclostrobin, fluopyram, iprodione, pyraclostrobin, isothiazine, mancozeb, mancozeb, fenoxystrobin, pyraclostrobin, pyridaben, propineb, prothioconazole, azoxystrobin, pentachloronitrobenzene, tebuconazole, flufenoxuron, thiophanate-methyl, and azoxystrobin.

[0201] Soybean herbicides: metolachlor, bentazon, trifluralin, chlorpyrifos, chlorpyrifos-methyl, quizalofop-p-ethyl, flusulfanilamide, pyrfluthrin, glyphosate, metolachlor, methoxyfenozide, metolachlor, methoxyfenozide, (S-)-metolachlor, cypermethrin, pendimethalin, pyranofurone, and glufosinate.

[0202] Soybean insecticides: λ-cyhalothrin, methomyl, imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, dinotefuran, flufenoxuron, chlorantraniliprole, spinosad, snutomom, emamectin benzoate, fipronil, acetamiprid, deltamethrin, β-cyhalothrin, γ and λ-cyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, spirotetramat, spirodiclofen, chlorfenapyr, flufenoxuron, thiamethoxam, and β-cyhalothrin.

[0203] Soybean fungicides: azoxystrobin, bifenthrin, cyazofamid, carbendazim, chlorothalonil, copper, cyclohexane, difenoconazole, fenpropathrin, flutriafol, fluazinam, fluopyram, flupyraclostrobin, fenpyroxacin, fluopyram, pyraclostrobin, iprodione, isothiazine, mancozeb, mancozeb, tebuconazole, fenoxystrobin, pyrimethanil, cyazofamid, cyazofamid, cyazofamid, propiconazole, propineb, prothioconazole, thiophanate-methyl, tebuconazole, flufenoxuron, thiophanate-methyl, and azoxystrobin.

[0204] Beet herbicides: chlorpyrifos, betaine, betaine furfur, betaine nitrate, chlorpyrifos, dichloropyridinic acid, pyrfluthrin, cyclopyridinium, benzoate, quinacrine, thiamethoxam, flusulfuron, pyranofurone, and quizalofop-p-ethyl.

[0205] Beetroot insecticides: imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, dinotefuran, deltamethrin, β-cyhalothrin, γ / λ cyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, heptaflupyr, chlorantraniliprole, cyyaxypyr, fipronil, and carbofuran.

[0206] Canola herbicides: dichloropyridine, quizalofop-P-ethyl, pyrfluthrin, glyphosate, pyrazosulfuron, trifluralin, benzoyl permethrin, quizalofop-P-ethyl, clethodim, and pyranofop-P-ethyl.

[0207] Canola fungicides: azoxystrobin, bifenthrin, cyazofamid, carbendazim, cyclohexane, difenoconazole, fenpropathrin, flutriafol, fluazinam, fluopyram, flupyraclostrobin, flusilazole, fluopyram, iprodione, pyraclostrobin, mepiquat chloride, tebuconazole, fenoxystrobin, paclobutrazol, pyraclostrobin, cyazofamid, prochloraz, prothioconazole, azoxystrobin, tebuconazole, thiophanate-methyl, azoxystrobin, and vinclozolin.

[0208] Canola insecticides include: carbofuran, thiamethoxam, deltamethrin, imidacloprid, thiamethoxam, acetamiprid, dinotefuran, β-cyhalothrin, γ and λ cyhalothrin, tau-fluvaleriate, acetamiprid, spinosad, snutomom, flufenoxuron, chlorantraniliprole, brofenoxuron and 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one.

[0209] The following examples are provided in an illustrative rather than restrictive manner. Example

[0210] Example 1. Expression of Cry5-like genes in soybeans used to kill nematodes.

[0211] A soybean event expressing a Cry5-like protein (SEQ ID NO: 2) was developed via Agrobacterium-mediated Thorne soybean plant transformation using a construct containing a gene encoding a gene for herbicide-resistant 4-hydroxyphenylpyruvate dioxygenase protein (HPPD) inhibitor (described in WO 2014043435) and a Cry5-like coding sequence. See also: Figure 1Table 1 depicts the plant transformation vectors used to express SEQ ID NO: 2 in plants; Table 1 shows the genetic elements of the Cry5-like transformation vectors or constructs; and Table 2 shows the references for the transformation vectors or constructs described in Table 1. Wild-type Thorne soybean served as a non-nematode resistance control. When Cry5-like was expressed in soybean plants, it reduced the number of soybean cyst nematodes (Heterodera glycines) that reproduce in the roots compared to wild-type plants. This particular Cry5-like (SEQ ID NO: 1) gene shares approximately 44.9% sequence identity with Cry5Ba1 (Figure 2).

[0212] Having less than 45% NEEDLE default identity with any Cry orthotype specimen sequence, SEQ ID NO:2 is not a member of any known Cry class (based on the definition described in Crickmore et al., Journal of Invertebrate Pathology 186 (2021) and the Cry orthotype specimen sequence available at https: / / www.bpprc-db.org in May 2023), but exhibiting the highest global identity with the Cry5 orthotype specimen sequence (see Table 3), and is therefore described as Cry5-like in the context of this embodiment of the application.

[0213] Figure 2a The NEEDLE sequence alignment of the novel Cry5-like protein SEQ ID NO:2 and Cry5Ba1 is shown. The “DRIEF” motif separating the N-terminal domain from the C-terminal crystal domain is indicated by “…”. “” indicates the sequence. Identical amino acids are indicated by black shading. Overall, there is 44.9% sequence identity between SEQ ID NO: 2 and Cry5Ba1.

[0214] Figure 2b The NEEDLE sequence alignment of the novel Cry5-like protein SEQ ID NO:2 and its homolog SEQ ID NO:6 is shown. The “DRIEF” motif, separating the N-terminal domain from the C-terminal crystal domain, is represented by… The same amino acids are indicated by black shading. The consensus sequence (Cons) is given below the aligned sequence and is referred to as SEQ ID NO:9. Overall, there is 93.7% sequence identity between Cry5-like SEQ ID NO:2 and Cry5-like homolog SEQ ID NO:6.

[0215] Table 1: Description of Cry5-like genetic elements in transformation vectors or constructs

[0216]

[0217] Table 2: References for the transformation vectors or constructs described in Table 1

[0218]

[0219] Table 3: Protein sequence identity of Cry5-like proteins with the closest Cry holotype specimen, determined by pairwise alignment, according to Crickmore et al., 2021.

[0220]

[0221] Example 2: Soybean Conversion

[0222] Soybean transformation was achieved using methods well-known in the art, such as the described method of transformation of soybean hemi-seed explants mediated by Agrobacterium tumefaciens, which essentially follows the method described by Paz et al. (2006), Plant Cell Reports 25:206. Cyclosulfonyl was used as a selection marker to identify transformants. The appearance of green shoots was observed and recorded as an indicator of tolerance to the herbicides isoxaflutole or cyclosulfonyl. Tolerant transgenic shoots would show normal greening comparable to wild-type soybean shoots not treated with isoxaflutole or cyclosulfonyl, while wild-type soybean shoots treated with the same amount of isoxaflutole or cyclosulfonyl would be completely bleached. This indicates that the presence of HPPD proteins enables tolerance to HPPD inhibitor herbicides, such as isoxaflutole or cyclosulfonyl.

[0223] Tolerant green shoots were transferred to rooting media or transplanted. After the acclimatization period, the rooted plantlets were transferred to a greenhouse. The transgenic plants were then sprayed with an HPPD inhibitor herbicide supplemented with ammonium methyl sulfate rapeseed oil (e.g., cyclosulfuron at a rate of 100 g AI / ha, or mesotrione at a rate of 300 g AI / ha). Ten days after application, symptoms caused by the herbicide application were evaluated and compared with those observed in wild-type plants under the same conditions.

[0224] Example 3: Greenhouse and Field Trials

[0225] Overall, the soybean event SEQ ID NO:2 was tested in greenhouses and eight field trials in the Midwestern United States and Brazil in 2022 for RKN (root-knot nematode), SCN (soybean cyst nematode), shortest-tailed short-bodied nematode, and kidney-shaped nematode.

[0226] Field trial results

[0227] Early visual observations (approximately 40 days after planting) indicated that plants expressing the novel Cry5-like sequence were slightly healthier (and slightly larger) than plants without the novel Cry5-like sequence.

[0228] Figure 3 The results of a root-digging field trial are shown from sporangium counting analysis, which showed that soybean plants expressing the novel Cry5-like sequence had 34% fewer SCN females (SCN sporangia) on their roots compared to wild-type Thorne soybean plants without the novel Cry5-like sequence.

[0229] Figure 4 Field trial results of the yield of soybean plants expressing the novel Cry5-like sequence are shown, which on average were 6% higher than wild-type Thorne soybean at three independent test sites.

[0230] Figure 5 This is a photograph depicting a soybean plant (right) expressing Cry5-like protein next to a non-resistant control soybean plant (left) in a field infected with SCN.

[0231] Figure 6 Greenhouse results for kidney-shaped nematodes (Kidney-shaped Nematodes) are shown, indicating that soybean plants expressing the novel Cry5-like sequence had approximately 75% fewer kidney-shaped nematodes in their roots compared to wild-type Thorne soybean plants without the novel Cry5-like sequence.

[0232] All publications and patent applications mentioned in this specification indicate the level of skill of a person skilled in the art to which the embodiments pertain. All publications and patent applications are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

[0233] Although the foregoing embodiments have been described in detail by way of illustration and examples for purposes of clarity, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

Claims

1. A method for controlling nematode pests, the method comprising contacting the nematode pest with a Cry5-like protein, the Cry5-like protein comprising an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 2, 4, 6, 8 or their nematicidal effective variants or fragments and / or containing amino acids having a consensus sequence as shown in SEQ ID NO:

9.

2. The method of claim 1, wherein the nematode pest is selected from any one of the following species: small ring nematode, stem nematode, golden nematode, spiral nematode, cyst nematode, long needle nematode, root-knot nematode, pseudo-spinous nematode, short-bodied nematode, perforating nematode, coiled nematode, kidney-shaped nematode, pad-shaped nematode, and dagger nematode.

3. The method according to claims 1-2, wherein the nematode pest is a target pest and / or soybean cyst nematode or kidney-shaped nematode.

4. The method of claims 1-3, wherein the effective nematicide variant or fragment thereof comprises at least 10, 20, 30, 40, 50 or 60 consecutive amino acids of SEQ ID NO: 2, 4, 6 and 8 and / or contains amino acids of the consensus sequence shown in SEQ ID NO:

9.

5. A nucleic acid that encodes a protein as described in any one of claims 1-4.

6. A plant or plant part that expresses the nucleic acid as claimed in claim 5 in its cells.

7. The plant or plant part as claimed in claim 6, wherein the plant or plant part is capable of being infected by a nematode pest, the nematode pest being any one of the following species: small ring nematode, stem nematode, golden nematode, spiral nematode, cyst nematode, long needle nematode, root-knot nematode, pseudo-spinous nematode, short-bodied nematode, perforating nematode, coiling nematode, kidney-shaped nematode, pad-shaped nematode, and dagger nematode.

8. The plant or plant part as described in claims 6-7, wherein the plant or plant part is capable of being infected by the target pest and / or soybean cyst nematode or kidney-shaped nematode.

9. The plant or plant part as described in claims 6-8, wherein the plant is a dicotyledonous plant or a monocotyledonous plant.

10. The plant or plant part as described in claims 6-9, wherein the plant is selected from the group consisting of: alfalfa, apple, apricot, Arabidopsis thaliana, artichoke, asparagus, avocado, banana, barley, legume, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, cereal, celery, cherry, citrus, Clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, fig, grape, grapefruit, peanut, ground ivy, etc. Cherries, kiwis, lettuce, leeks, lemons, limes, pine trees, corn, mangoes, melons, millet, mushrooms, nuts and oats, okra, onions, oranges, ornamental plants or flowers or trees, papayas, parsley, peas, peaches, peanuts, peat, peppers, persimmons, pineapples, plantains, plums, pomegranates, potatoes, pumpkins, red chicory, radishes, rapeseed, raspberries, rice, rye, sorghum, soybeans, soybeans, spinach, strawberries, sugar beets, sugarcane, sunflowers, sweet potatoes, oranges, tea, tobacco, tomatoes, vines, watermelons, wheat, yams, and zucchini.

11. The plant or plant part as described in claims 6-10, wherein the plant or plant part is soybean, Brassica, or maize.

12. The plant or plant part as described in claims 6-11, wherein the plant is a transgenic plant or a transgenic plant part.

13. An expression cassette comprising: a nucleic acid encoding a Cry5-like protein comprising an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 2, 4, 6, 8 or their nematicidal effective variants or fragments thereof; and / or a nucleic acid encoding a protein containing a consensus sequence as shown in SEQ ID NO:

9.

14. The expression cassette of claim 13, wherein the nucleic acid is operatively ligated to the nucleic acid.

15. The expression cassette as claimed in claims 13-14, wherein the expression cassette is in a plant expression vector.

16. A cell comprising the expression cassette as described in claims 13-15.

17. A cell comprising an expression cassette as described in claims 13-15 and another expression cassette comprising another cry gene.

18. The cell of claim 16 or 17, wherein the cell is a bacterial or plant cell.

19. A nematicide-effective fusion protein comprising any one of SEQ ID NO: 2, 4, 6, 8 and / or a protein containing a consensus sequence as shown in SEQ ID NO:

9.

20. The fusion protein of claim 19, wherein another portion of the fusion protein is derived from a protein from Bacillus thuringiensis, synthesized from the protein, or substantially equivalent to the protein.

21. A method for controlling soybean cyst nematode or kidney-shaped nematode, the method comprising expressing a Cry5-like protein in a plant, wherein the protein comes into contact with the soybean cyst nematode or kidney-shaped nematode pest, thereby controlling the soybean cyst nematode or kidney-shaped nematode pest.

22. The method of claim 21, wherein the Cry5-like protein comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 2, 4, 6, 8 or their nematicidal effective variants or fragments and / or contains amino acids with a consensus sequence as shown in SEQ ID NO:

9.

23. The method of claims 21 and 22, wherein the plant is a soybean plant.

24. A nucleic acid encoding a protein comprising an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 2, 4, 6, 8 or their nematicidal effective variants or fragments, and / or a nucleic acid encoding a protein containing a consensus sequence as shown in SEQ ID NO:

9.

25. The nucleic acid of claim 24, wherein the nucleic acid comprises a nucleic acid sequence having a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 1, 3, 5, or 7, and / or encoding a nucleic acid containing a protein having a consensus sequence as shown in SEQ ID NO:

9.

26. The nucleic acid of claims 24 and 25, wherein the nucleic acid sequence comprises any one of SEQ ID NO: 1, 3, 5 or 7, and / or a nucleic acid encoding a protein containing a consensus sequence as shown in SEQ ID NO:

9.

27. A plant, plant cell, or plant part expressing the nucleic acid as described in claims 24-26 in its cells.

28. The plant, plant cell, or plant part as described in claim 27, wherein the plant is a soybean plant.

29. An expression cassette comprising the nucleic acid as described in claims 24-26.

30. The expression cassette of claim 29, wherein the nucleic acid is operatively linked to a promoter.

31. The expression cassette of claim 30, wherein the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

32. The expression cassette according to claims 29-31, wherein the promoter is a plant promoter.

33. A cell comprising the expression cassette as described in claims 29-32.

34. The cell of claim 33, wherein the cell is a bacterial cell, plant cell, yeast cell, or insect cell.

35. A carrier comprising the expression cassette as described in claims 29-31.

36. A cell comprising the carrier as described in claim 35.

37. The cell of claim 36, wherein the cell is a bacterial cell, plant cell, yeast cell, or insect cell.

38. A plant comprising an expression cassette as described in claims 29-32, wherein the promoter is capable of driving the expression of the protein to a level sufficient to inhibit target pests and / or soybean cyst nematodes or kidney-shaped nematodes, wherein the proliferation of target pests and / or soybean cyst nematodes or kidney-shaped nematodes feeding on the plant is reduced compared to the proliferation of target pests and / or soybean cyst nematodes or kidney-shaped nematodes feeding on a control plant not containing the expression cassette.

39. The plant of claim 38, wherein the plant is a soybean plant.

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