Use of the CRY gene to control nematode pests

Abstract

Compositions and methods for conferring nematodic activity to bacteria, plants, plant cells, tissues, and seeds are provided. Furthermore, nematode pest populations, particularly Platylenchus species (Pratylenchus spp.), such as pineapple root-knot nematode (Pratylenchus brachyurus), northern root-knot nematode (Pratylenchus penetrans), scribneri, neglectus nematode (Pratylenchus neglectus), Platylenchus zeae, long-tailed lance nematode (Belonolaimus longicaudatus), Hoplolainus galeatus, Helicotylenchus dihystera, spiral nematode (Helicotylenchus pseudorobustus), or Xiphinema nematode (Xiphinema Methods are provided for killing or controlling populations of Americanum nematodes, root-knot nematodes, false sedge nematodes, soybean cyst nematodes, or lance nematodes. The methods include contacting nematode pests with a pesticidal effective amount of a polypeptide containing a nematode toxin. Furthermore, methods for increasing plant yield by expressing the genes of the present invention in plants are also provided.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology. A method for controlling nematode pests using a novel pest gene is provided.

[0002] Submission of Sequence Listing The sequence listing related to this application is submitted in electronic form through the Patent Center and is hereby incorporated by reference in its entirety into this specification. The name of the "xml" file containing the sequence listing is 230175US02_Sequencelisting.xml. The size of the xml file is 177 KB and it was created on August 21, 2023.

Background Art

[0003] Plant pests are a major cause of the loss of important crops worldwide. Some estimates claim that 40% of the world's crop production is lost due to parasitism by invertebrate pests including nematodes. In addition to losses in agricultural crops, nematode pests are also a burden for vegetable and fruit growers, producers of ornamental flowers, and home gardeners. Most nematodes that parasitize plants feed on roots and are found associated with most plants. Some are endoparasitic and live and feed within tissues such as roots, tubers, buds, seeds, etc. Others are ectoparasitic and feed externally through the plant wall. A single endoparasitic nematode can kill a plant or reduce its productivity. Economically important pests such as root-feeding endoparasites include Meloidogyne species, Rotylenchulus species, Heterodera species, and Pratylenchus species.

[0004] Damage caused by nematodes can dramatically reduce a plant's ability to absorb nutrients and water. Nematodes have the greatest impact on crop productivity when they attack the roots of seedlings immediately after germination. Nematode feeding also creates open wounds that allow for the invasion of a wide variety of plant pathogenic fungi and bacteria. These microbial infections can cause more economic damage than the direct effects of nematode feeding.

[0005] Cyst nematodes are a direct cause of soybean yield losses, as well as indirect losses resulting from the cost of pesticides and the inoptimal use of land for crop rotation. Soybean cyst nematodes (Heterodera glycines) have a negative economic impact that can exceed $1.5 billion annually in North America. Economically significant densities of cyst nematodes typically cause stunted growth in crop plants. Stunted plants have smaller root systems, show signs of mineral deficiency in their leaves, and wither easily.

[0006] Traditional practices for managing nematode infestations include maintaining appropriate fertility and soil pH levels in nematode-infested land; controlling plant diseases that promote nematode invasion, as well as controlling insects and weed pests; using sanitary practices such as tilling, planting, and cultivating nematode-infested fields only after working in non-infested fields; thoroughly cleaning equipment after working in infested fields; not using seeds from plants grown in infested land for planting in non-infested fields unless the seeds have been properly cleaned; and crop rotation in infested fields, alternating between non-host crops such as maize, oats, and alfalfa with host crops, as well as planting resistant or tolerant plant varieties. While many of these may be effective, their implementation is time-consuming and costly. Nematodes are pests that are difficult to control without the use of chemical pesticides or fumigants (e.g., nematicides), and these approaches are subject to many challenges, including nematode resistance and regulatory limitations.

[0007] Due to the devastating damage that nematodes can cause, and the persistent threat of pests developing resistance to current control methods, there is an ongoing need to discover new methods for controlling nematode plant pests that are economically beneficial to farmers, environmentally acceptable, and safe. [Overview of the Initiative] [Means for solving the problem]

[0008] Various embodiments provide novel methods for controlling economically important nematode pests. In particular, transgenic plants and / or plant parts expressing one or more polypeptides of the embodiments have been found to have the ability to inhibit the ability of nematode pests to survive, grow, and reproduce, or to limit damage or loss to nematode-related crop plants. One embodiment further relates to transgenic nematode-resistant plants expressing one or more of the aforementioned proteins, and to methods of using the 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 are highly tolerant or resistant to nematode infestation.

[0009] In one embodiment, a method for controlling nematode pests is provided, comprising contacting the nematode pests with a protein comprising any one of SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof. In another embodiment, the protein may be, for example, Cry21, Cry21Aa, Cry21Aa2, or Cry21Aa1 proteins, as indicated by SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, or their nematodic activity fragments. Cry21, Cry21Aa2, or Cry21Aa1 protein homologs having at least approximately 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% identity with SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, and 53, and any functional fragments thereof.

[0010] According to one embodiment, a method for controlling nematode pests is provided, comprising contacting the nematode pests with a transgenic plant or plant part containing a heterologous nucleic acid molecule that directs the expression of the Cry21 protein of the present invention in the transgenic plant or plant part, wherein the transgenic plant or plant part controls nematode pests more effectively than the same type of plant or plant part that does not express the Cry21 protein.

[0011] In another embodiment, nematode pests are selected from the group consisting of Criconemella, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Meloidogyne, Paratrichodorus, Pratylenchus, Radolpholus, Rotelynchus, Rotylenchulus, Tylenchulus, and Xiphenema. Such nematode pests selected from these genera may be cyst-forming nematodes. In another embodiment, cyst-forming nematodes belong to the genus Heterodera. In yet another embodiment, the nematode pest is Heterodera glycines (soybean cyst nematode). In one embodiment, the nematode pest is Pratylenchus brachyurus, but not SCN and / or both.

[0012] In another embodiment, the transgenic plant or plant part may be alfalfa, apple, apricot, Arabidopsis, artichoke, asparagus, avocado, banana, barley, legumes, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, grains, celery, cherry, citrus fruits, clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, fig, grape, grapefruit, peanut, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, or nut. The transgenic plant or plant part is selected from the group consisting of oats, okra, onions, oranges, ornamental plants or flowers or trees, papaya, parsley, peas, peaches, peanuts, peat, pepper, persimmons, pineapples, plantains, plums, pomegranates, potatoes, pumpkins, radicchio, radishes, rapeseed, raspberries, rice, rye, sorghum, soybeans, spinach, strawberries, sugar beets, sugarcane, sunflowers, sweet potatoes, tangerines, tea, tobacco, tomatoes, climbing plants, watermelons, wheat, yams, and zucchini. In yet another embodiment, the transgenic 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] In yet another embodiment, the Cry21 protein is the Cry21Aa, Cry21Aa1, Cry21Aa2, or Cry21Ba protein. In yet another embodiment, the Cry21 protein comprises an amino acid sequence which is the translation product of a nucleotide sequence whose complement hybridizes under high stringency conditions to SEQ ID NOs: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, or 54.

[0015] In another embodiment, the Cry21 protein of the present invention comprises SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, or its nematode activity 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 to SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof. In another embodiment, the Cry21 protein comprises SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, or Nematode activity homologs 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 to SEQ ID NOs: 1, 7, 9, 13, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof.

[0016] In another embodiment, the transgenic plant of the present invention further comprises or expresses at least one additional pesticide, for example, 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, vegetative 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.

[0017] In another embodiment, a method is provided for conferring nematode resistance to a plant and / or plant part, comprising inserting a heterologous nucleic acid molecule encoding the Cry21 protein into the plant and / or plant part, wherein the plant and / or plant part expresses the Cry21 protein at a nematode inhibitory level to confer 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 Cry21 protein. Such insertion may occur via transformation, gene editing, or breeding.

[0018] In another embodiment, a method is provided for conferring resistance to the pineapple root-knot nematode (Pratylenchus brachyurus) to a plant and / or plant part, comprising inserting a heterologous nucleic acid molecule encoding the Cry21, Cry21Aa1, or Cry21Aa2 protein into the plant and / or plant part, wherein the plant and / or plant part expresses the Cry21 protein at a nematode inhibitory level to confer resistance to the pineapple root-knot nematode (Pratylenchus brachyurus) to the plant and / or plant part, compared to the same type of plant and / or plant part that does not express the Cry21, Cry21Aa1, or Cry21Aa2 protein. Such insertion may occur via transformation, gene editing, or breeding.

[0019] In another embodiment, the Cry21 protein comprises SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, or its pineapple root-knot nematode (Pratylenchus brachyurus) nematode activity 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 to SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof. In another embodiment, the Cry21 protein comprises SEQ ID NOs: 1, 7, 9, 13, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, or pineapple root-knot nematode (Pratylenchus brachyurus) nematode activity homologs 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 to SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof.

[0020] In another embodiment, a method is provided for reducing nematode infectivity to plants and / or plant parts, comprising contacting nematodes with the Cry21 protein, wherein the nematode infectivity is reduced compared to the infectivity of plants and / or plant parts by nematodes that have not been in contact with the Cry21 protein.

[0021] In yet another embodiment, a transgenic soybean plant or plant part thereof is provided, comprising a heterologous nucleic acid molecule encoding the Cry21 protein, wherein the transgenic soybean plant or plant part is resistant to nematode parasitism.

[0022] In another embodiment, a method is provided for producing soybean plants protected from nematode parasitism, comprising transforming soybean plant cells with a nucleic acid molecule encoding the Cry21 protein; and regenerating soybean plants transformed from soybean plant cells, wherein the transformed plants are protected from nematode parasitism.

[0023] In another embodiment, a method is provided for producing soybean plants protected from nematode parasitism, comprising: crossing a first parent soybean plant with a second parent soybean plant (where the first or second parent soybean plant contains a heterologous nucleic acid molecule encoding the Cry21 protein of the present invention); thereby producing a plurality of offspring plants; and selecting transgenic plants protected from nematode parasitism from the plurality of offspring plants.

[0024] Another embodiment provides a method for reducing nematode cyst formation in the roots of a plant that may be infected with nematodes, comprising introducing a nucleic acid molecule capable of instructing the expression of the Cry21 protein into root cells, thereby reducing nematode cyst formation in the plant roots.

[0025] In another aspect of the present invention, a method for reducing nematode cyst formation in the roots of plants susceptible to nematode infection is provided, which comprises introducing into a plant cell a nucleic acid molecule capable of directing the expression of the Cry21 protein therein, thereby reducing nematode cyst formation in the roots of the plant.

[0026] Another aspect is a method of controlling or preventing the growth of nematodes, which comprises providing to a nematode pest a plant material comprising a heterologous DNA capable of directing the expression of the Cry21 protein, wherein the plant inhibits the biological activity of the nematode.

[0027] According to another aspect, a method is provided for providing a grower with a means of controlling nematode pests, the method comprising supplying seeds to the grower, wherein the seeds comprise a heterologous nucleic acid molecule encoding the Cry21 protein and are capable of producing a plant resistant to nematode infestation.

[0028] Another aspect is a method of suppressing the growth of a plant pathogenic nematode population at a location where the growth of a nematode population can be supported, which comprises cultivating at that location a population of transgenic soybean plants comprising a heterologous nucleic acid molecule capable of directing the expression of the Cry21 protein, wherein the growth of the plant pathogenic nematode population is suppressed.

[0029] Another aspect is a method of controlling any one of soybean cyst nematode, peanut root-knot nematode, tobacco root-knot nematode, or false root-knot nematode ( "target pest"), which comprises providing a transgenic soybean plant comprising an expression cassette having any one of SEQ ID NOs: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, or 54 operably linked to a promoter capable of driving the expression of the encoded Cry21 protein to a level sufficient to inhibit the nematode, wherein the growth of the target pest feeding on the soybean plant is reduced as compared to the target pest feeding on a non-transgenic soybean plant not comprising the expression cassette.

[0030] Another embodiment provides a method for improving plant yield in a nematode-infested field, comprising expressing the Cry21 protein in the plants, wherein the plant yield is improved compared to the yield of the same type of plants that do not express the Cry21 protein.

[0031] Another embodiment provides a method for increasing the vitality or yield of transgenic soybean plants exposed to a nematode population, comprising: obtaining transgenic soybean plants by genetically transferring transgenic soybean event into soybean plants, wherein the transgenic soybean event includes a heterologous DNA sequence encoding the Cry21 protein, which confers resistance to nematodes to the transgenic soybean event; and cultivating the transgenic soybean plants or their offspring in a location where nematode parasitism limits the yield of soybean plants that do not contain the heterologous nucleic acid molecule encoding the Cry21 protein, thereby resulting in the transgenic soybean plants having increased vitality or yield compared to control plants.

[0032] Another embodiment provides a method for improving the yield of a soybean field, comprising introducing a nucleic acid molecule capable of instructing the expression of the Cry21 protein into soybean plants to produce transgenic plants; and cultivating multiple transgenic seeds from the transgenic plants in a field to create or result in a soybean field containing multiple transgenic soybean plants having enhanced resistance to nematode parasitism, thereby improving the yield of the soybean field.

[0033] Another embodiment provides a recombinant expression cassette comprising a heterologous promoter sequence operably linked to a nucleic acid molecule encoding the Cry21 protein. Furthermore, the present invention provides a recombinant vector comprising such an expression cassette. Further another embodiment provides a transgenic host cell comprising such an expression cassette. The transgenic host cell according to this embodiment may be a plant cell. Further another embodiment provides a transgenic plant or plant part comprising such a plant cell.

[0034] Another embodiment provides a nematode-killing composition comprising an effective nematode control amount of Cry21 protein and an acceptable agricultural carrier. In another embodiment, the agricultural carrier is a transgenic plant. In yet another embodiment, the transgenic plant is a transgenic soybean plant, and the Cry21 protein is the Cry21 protein having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof. In another embodiment, the Cry21 protein comprises any of SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof.

[0035] Further embodiments provide a method for producing nematode-resistant transgenic plants, comprising: introducing a nucleic acid molecule encoding the Cry21 protein into plant cells to create transgenic plant cells; and regenerating transgenic plants from the transgenic plant cells, wherein the Cry21 protein is expressible in the transgenic plants in amounts effective for controlling nematodes. In another embodiment, the plant is a soybean plant. In another embodiment, the Cry21 protein is a protein having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof. In another embodiment, the Cry21 protein comprises Sequence IDs 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof. In yet another embodiment, the nematode is the target pest. In yet another embodiment, the nematode is Heterodera glycines, Pratylenchus brachyurus, Rotylenchulus reniformis, or a species of the genus Meloidogyne.

[0036] Another embodiment is a method for controlling nematode pests, comprising contacting the nematode pests with a Cry21 protein containing an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 1, 3, 7, 9, 11, 13, 15, 17, 21, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, or 53, or a nematodically effective variant or fragment thereof. Another embodiment includes the method according to claim 40, wherein the nematode pest is selected from any one of the species consisting of Criconemella, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Meloidogyne, Paratrichodorus, Pratylenchus, Radolpholus, Rotelynchus, Rotylenchulus, Tylenchulus, and Xiphenema. Another embodiment includes an expression cassette comprising a nucleic acid encoding a Cry21 protein, which comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 3, 7, 9, 11, 13, 15, 17, 21, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or a nematically effective variant or fragment thereof, wherein the nucleic acid is operably ligated to the nucleic acid in the expression cassette and / or further contained within a plant expression vector.Further embodiments include nucleic acids comprising nucleic acid sequences having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, and 100% sequence identity with respect to any one of sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, or 54.

[0037] Other aspects and embodiments will become apparent to those skilled in the art from the following description and the study of non-limiting examples.

[0038] A brief explanation of sequence listings Sequence ID 1 discloses the amino acid sequence of Axmi296, a Cry21 homolog. Sequence ID 2 discloses the nucleotide sequence of Axmi296, a Cry21 homolog. Sequence ID 3 discloses the amino acid sequence of Axmi018. Sequence ID 4 discloses the nucleotide sequence of Axmi018. Sequence ID 5 discloses the amino acid sequence of Axmi021. Sequence ID 6 discloses the nucleotide sequence of Axmi021. Sequence ID 7 discloses the amino acid sequence of Axmi040. Sequence ID 8 discloses the nucleotide sequence of Axmi040. Sequence ID 9 discloses the amino acid sequence of Axmi049. Sequence ID 10 discloses the nucleotide sequence of Axmi049. Sequence ID 11 discloses the amino acid sequence of Axmi071. Sequence ID 12 discloses the nucleotide sequence of Axmi071. Sequence ID 13 discloses the amino acid sequence of Axmi074. Sequence ID 14 discloses the nucleotide sequence of Axmi074. Sequence ID 15 discloses the amino acid sequence of Axmi088. Sequence ID 16 discloses the nucleotide sequence of Axmi088. Sequence ID 17 discloses the amino acid sequence of Axmi104. Sequence ID 18 discloses the nucleotide sequence of Axmi104. Sequence ID 19 discloses the amino acid sequence of Axmi155. Sequence ID 20 discloses the nucleotide sequence of Axmi155. Sequence ID 21 discloses the amino acid sequence of Axmi169. Sequence ID 22 discloses the nucleotide sequence of Axmi169. Sequence ID 23 discloses the amino acid sequence of Axmi170. Sequence ID 24 discloses the nucleotide sequence of Axmi170. Sequence ID 25 discloses the amino acid sequence of Axmi215. Sequence ID 26 discloses the nucleotide sequence of Axmi215. Sequence ID 27 discloses the amino acid sequence of Axmi218. Sequence ID 28 discloses the nucleotide sequence of Axmi218. Sequence ID 29 discloses the amino acid sequence of Axmi219. Sequence ID 30 discloses the nucleotide sequence of Axmi219. Sequence ID 31 discloses the amino acid sequence of Axmi220. Sequence ID 32 discloses the nucleotide sequence of Axmi220. Sequence ID 33 discloses the amino acid sequence of Axmi227. Sequence ID 34 discloses the nucleotide sequence of Axmi227. Sequence ID 35 discloses the amino acid sequence of Axmi266. Sequence ID 36 discloses the nucleotide sequence of Axmi266. Sequence ID 37 discloses the amino acid sequence of Axmi280. Sequence ID 38 discloses the nucleotide sequence of Axmi280. Sequence ID 39 discloses the amino acid sequence of Axmi287. Sequence ID 40 discloses the nucleotide sequence of Axmi287. Sequence ID 41 discloses the amino acid sequence of Axmi297. Sequence ID 42 discloses the nucleotide sequence of Axmi297. Sequence ID 43 discloses the amino acid sequence of Axmi299. Sequence ID 44 discloses the nucleotide sequence of Axmi299. Sequence ID 45 discloses the amino acid sequence of Axmi340. Sequence ID 46 discloses the nucleotide sequence of Axmi340. Sequence ID 47 discloses the amino acid sequence of Axmi350. Sequence ID 48 discloses the nucleotide sequence of Axmi350. Sequence ID 49 discloses the amino acid sequence of Axmi380. Sequence ID 50 discloses the nucleotide sequence of Axmi380. Sequence ID 51 discloses the amino acid sequence of natural Cry21Aa1. Sequence ID 52 discloses the nucleotide sequence of natural Cry21Aa1. Sequence ID 53 discloses the amino acid sequence of natural Cry21Aa2. Sequence ID 54 discloses the protein sequence of the natural Cry21Aa2. [Brief explanation of the drawing]

[0039] [Figure 1] This shows a plant transformation vector for the expression of Axmi296 in plants. [Figure 2-1] The NEEDLE sequence alignments for Axmi296 and Cry21Aa2 are shown. [Figure 2-2] The NEEDLE sequence alignments for Axmi296 and Cry21Aa2 are shown. [Figure 2-3] The NEEDLE sequence alignments for Axmi296 and Cry21Aa2 are shown. [Figure 3]Figures 3a-3b show the efficacy of Axmi296 against pineapple root-knot nematodes (Pratylenchus brachyurus) in a 60-day greenhouse assay. [Figure 4a-4b] The results show that Axmi296 provides increased yield benefits compared to SCN-unresistant controls in soybean cyst nematode (SCN) field trials. [Figure 4c-d] Figure 4c shows the results from an SCN field trial comparing nematode reduction between the new Cry21 homolog (Axmi296) and wild-type Thorne. Figure 4d shows the results from an SCN field trial comparing yield between the new Cry21 homolog (Axmi296) and wild-type Thorne. [Figure 5] The image shows a field in which Axmi296 plants (left) are growing next to non-resistant control soybean plants (right) in an SCN parasitic field. [Modes for carrying out the invention]

[0040] Before describing the various embodiments of this disclosure, it should be understood that the application of the present invention is not limited to the structural details and component arrangements described below. Other embodiments can be implemented or performed in various ways. It should also be understood that the expressions and terms used herein are for illustrative purposes only and should not be considered limiting.

[0041] Throughout this disclosure, various publications, patents, and published patent specifications are referenced. To the extent permitted, the disclosures of these publications, patents, and published patent specifications are incorporated into this disclosure in their entirety to more fully illustrate the state of the art. Unless otherwise indicated, this disclosure encompasses the prior art of plant breeding, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the scope of the art. For example, Sambrook and Russell,Molecular Cloning: A Laboratory Manual,3rd edition(2001);Current Protocols in Molecular Biology [(FMAusubel,et al.eds.,(1987)];Plant Breeding: Principles and Prospects(Plant Breeding,Vol 1)MD Hayward,N,O.Bosemark,I.Romagosa;Chapman & Hall, (1993.); Colligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995); CURRENT Protocols in Protein Science (John Wiley & Sons, Inc.); and See Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture [RIFreshney, ed. (1987)].

[0042] Unless otherwise specified, technical terms are used according to their conventional usage in the art. Definitions of general terms in molecular biology can be found in Lewin, Genes VII, Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by 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.

[0043] To facilitate understanding of this disclosure, the following definitions are provided:

[0044] The "activity" of the Cry21 protein in this invention means that the Cry21 protein has a toxic effect on nematodes by interfering with or inhibiting feeding and inhibiting the ability of nematode pests to survive, grow, and reproduce (which may or may not cause death of the nematodes), or by limiting nematode-related damage or loss to crop plants.

[0045] As used herein, "and / or" means and includes all possible combinations of one or more of the related enumerated items, as well as the absence of any combination when interpreted as either or.

[0046] "Related to / operatably linked" refers to two nucleic acid sequences that are physically or functionally related. For example, a promoter or regulatory DNA sequence is said to be "related" to an RNA or protein-coding DNA sequence if the two sequences are operatably linked or positioned so that the regulatory DNA sequence affects the expression level of the coding or structural DNA sequence.

[0047] As used herein, the term “contact” refers to the process by which the Cry21 protein of the present invention, or a transgenic plant or plant part expressing the Cry21 protein of an embodiment or aspect thereof, is delivered or administered to target a nematode pest or nematode pest population. Contact describes the physical proximity between the Cry21 protein or the transgenic plant or plant part expressing the Cry21 protein and the target nematode, such that they interact. The transgenic plant or plant part may be brought into contact with the target nematode or nematode population by planting transgenic seeds, transgenic seedlings, cuttings, plant runners, tubers, etc., in a location where the growth of the nematode pest or nematode pest population can be supported.

[0048] A "chimeric gene" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably ligated to, or associated with, a nucleic acid sequence that encodes mRNA or is expressed as a protein, thereby allowing the regulatory nucleic acid sequence to regulate the transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequence of a chimeric gene is usually not operably ligated to the associated nucleic acid sequence, as is the case in nature.

[0049] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. The RNA is then translated within an organism to produce proteins.

[0050] As used herein, the terms “control” or “control” nematodes mean inhibiting, by toxic effect, the ability of nematode pests to survive, grow, feed, and / or reproduce, or limiting nematode-related damage or loss to crop plants. “Control” nematodes may or may not mean killing them.

[0051] "Corresponding to," "corresponding to," or "corresponds to" means that when different Cry21 gene or protein nucleic acid coding sequences or amino acid sequences are aligned with each other, the nucleic acids or amino acids that "correspond" to specific listed positions will align with those positions, but for each nucleic acid coding sequence or amino acid sequence of a particular Cry21, they are not necessarily at these exact numerical positions.

[0052] "Delivering" a toxin means that the toxin comes into contact with a nematode or nematode population, resulting in a toxic effect and control of the nematode or nematode population. Toxins can be delivered in many recognized ways, for example, orally by ingestion by a nematode, or by contact with a nematode, via transgenic plant expression, formulated protein composition, sprayable protein composition, bait matrix, or any other toxin delivery system recognized in the art.

[0053] The term "economic threshold" is defined as the nematode pest population that produces incremental damage equal to the cost of controlling or preventing the damage. This is the level of the nematode population at which the benefits of nematode control equal the cost. In this sense, the economic threshold can be defined as the level of nematode pest damage at which the value of the incremental decrease in crop yield equals the cost of preventing the outbreak. In other words, the economic threshold attempts to determine the point at which controlling a nematode pest population becomes economically feasible. Economic damage to host crops is usually caused by the first generation of nematode offspring and is prevented by transgenic plants expressing the Cry21 protein by reducing the concentration of offspring nematodes in the plant root zone.

[0054] As used herein, “nematode control dose” or interchangeably “biocidal dose” refers to the concentration of Cry21 protein or its functional fragments that, by toxic effect, can inhibit the ability of nematodes to survive, grow, feed, and / or reproduce, or reduce or prevent nematode-related damage or loss in crop plants. “Nematode control dose” may or may not mean killing nematodes.

[0055] Where used herein, "expression cassette" means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, comprising a promoter operably ligated to a nucleotide sequence of interest, which is operably ligated to a termination signal. This also generally includes sequences required for the correct translation of the nucleotide sequence. An expression cassette containing the nucleotide sequence of interest may be a chimera, meaning that at least one of its components is heterologous to at least one of the other components. An expression cassette may be native but obtained in a recombinant form useful for heterologous expression. However, typically, an expression cassette is heterologous with respect to the host; that is, the specific nucleic acid sequence of the expression cassette does not occur naturally in the host cell but must be introduced into the host cell or the ancestor of the host cell by a transformation event. Expression of the nucleotide sequence of an expression cassette may be controlled under the control of a constitutive promoter or an inductive promoter that initiates transcription only when the host cell is exposed to certain specific external stimuli. In multicellular organisms such as plants, the promoter may also be specific to a particular tissue or organ, or to a developmental stage.

[0056] A "gene" is a specific region located within the genome that, in addition to the aforementioned coding nucleic acid sequence, 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. Further elements that may be present are, for example, introns.

[0057] "Nematicides" are defined as toxic biological activities that can control nematodes, preferably by killing them.

[0058] A nucleic acid sequence is "isocoded" with a reference nucleic acid sequence if that nucleic acid sequence encodes a polypeptide that has the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence. For example, the natural coding sequence from a Bacillus species (Bacillus spp.) that encodes the Cry21 protein isocoded with a codon-optimized coding sequence for expression in plants that encode the same Cry21 protein.

[0059] "Isolated" nucleic acid molecules, isolated proteins, or toxins are nucleic acid molecules, proteins, or toxins that exist separately from their natural environment and are therefore not natural products, having been brought about by human hands. Isolated nucleic acid molecules, proteins, or toxins may exist in a purified form or in a non-natural environment, such as recombinant host cells or transgenic plants.

[0060] The term "natural" refers to a coding sequence or gene that is naturally present in the genome of a cell or plant.

[0061] The term “naturally occurring” is used herein to describe an object that can be found in nature as distinct from those artificially produced by humans. For example, a protein or nucleotide sequence present in an organism (including a virus) can be isolated from a natural source, has not been intentionally modified by humans in a laboratory, and is naturally occurring.

[0062] "Plant" refers to any plant at any stage of development, especially seed plants.

[0063] A "plant cell" is a structural and physiological unit of a plant, including a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or it may be part of a higher-order organized unit such as a plant tissue, plant organ, or the whole plant.

[0064] "Plant material" means leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0065] "Plant organs" are clearly and visually structured and differentiated parts of a plant, such as roots, stems, leaves, flower buds, or embryos.

[0066] "Plant part" can be any part of a plant and includes plant cells, plant material, plant organs, or plant tissues.

[0067] As used herein, “plant tissue” means a group of plant cells organized into structural and functional units. This includes any tissue of a plant in a plant body or 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 combination with, or in the absence of, any particular type of plant tissue listed above or otherwise encompassed in this definition is not intended to exclude any other type of plant tissue.

[0068] A "promoter" is an uncoding DNA sequence located upstream of the coding region that contains the RNA polymerase II binding site and initiates DNA transcription. The promoter region may also contain other elements that act as regulators of gene expression.

[0069] A "regulatory element" refers to a sequence involved in controlling the expression of a nucleotide sequence. Regulatory elements include promoters and stop signals that are operably ligated to the target nucleotide sequence. Typically, they contain sequences necessary for the proper translation of the nucleotide sequence.

[0070] As used herein, “resistant” or “resistant” means a transgenic soybean variety in which the majority of nematodes are prevented from surviving and / or reproducing when they attempt to parasitize.

[0071] In the context of two nucleic acid or protein sequences, the term "substantially identical" refers to two or more sequences or subsequences that, when compared and aligned to the greatest extent possible using one of the following sequence comparison algorithms or by visual inspection, have at least 60%, at least 80%, at least 90%, at least 95%, and at least 99% nucleotide or amino acid residue identity. Substantially identical sequences exist over a region of at least about 50 residues, over a region of at least about 100 residues, or sequences are substantially identical over at least about 150 residues. In one embodiment, sequences are substantially identical over the entire length of the coding region. Furthermore, substantially identical nucleic acid or protein sequences perform substantially identical functions.

[0072] In order to determine the percentage of identity between two sequences, a pairwise sequence alignment is generated between these two sequences in the first step. This pairwise alignment in the first step can be generated by various tools known to those skilled in the art, such as the programs "Blast" (Altschul et al. J.Mol.Biol.215:403-410), "Blast2" ("Gap Blast") (Altschul et al., Nucleic Acids Res.25:3389-3402), "Water," "Matcher," and "Needle" from the European Molecular Biology Open Software Suite (EMBOSS, Trends in Genetics 16(6),276 (2000)), or by visual inspection.

[0073] After aligning the two sequences, in a second step, the identity percentage can be determined from the generated alignment. The identity percentage between the two sequences can be calculated from the generated complete alignment, or from regions outside the alignment, for example, from a region of the alignment showing the sequence of the present invention over its full length, or from a region showing the other sequence over its full length, or from a region showing only a portion of the sequence. The alignment region from which the identity percentage value is calculated preferably has a length of at least 100 positions, more preferably at least 150 positions, and most preferably more than 200 positions. To determine the identity percentage, first, the sum over all positions in the alignment region where both sequences show the same residue is calculated, and then this sum is divided by the length of the alignment region, so that the positions with gaps in which the sequences are introduced are either components of the length (length of the alignment region) or subtracted from the length (length of the alignment region - total number of gaps in the alignment region). Then, the obtained value is multiplied by 100 to obtain the identity percentage (identity%).

[0074] In one embodiment, the two sequences are first aligned over their entire length according to the Needleman and Wunsch algorithm (J.Mol.Biol.(1979)48,p.443-453) implemented in the EMBOSS (Trends in Genetics 16(6),276(2000)) program "Needle," preferably using version 6.3.1.2 or later, with the protein sequence aligned using the program's default parameters (gapopen=10.0, gapextend=0.5, and matrix=EBLOSUM62 (EMBOSS version of BLOSUM62 substitution matrix)) and the nucleotide sequence aligned using the default parameters (gapopen=10.0, gapextend=0.5, and matrix=EDNAFULL). Next, the identity percentage (% identity) is determined from the generated complete alignment and calculated as follows: Identity percentage = (total number of positions indicating identical residues in the alignment × 100) / (length of alignment - total number of gaps in the alignment). This value can also be obtained directly from the EMBOSS program "Needle" as a program labeled "Longest Identity" when the parameter option "-nobrief" is applied.

[0075] For protein-coding nucleotide sequences, pairwise alignment is performed over the full length of the coding region of one or more embodiment sequences, from the start codon to the stop codon, excluding introns. Introns present in other sequences may also be removed for pairwise alignment to allow comparison with the sequences of the present invention.

[0076] Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions. The phrase "specifically hybridize" means that, if the sequence is present in a complex mixture (e.g., whole cell) of DNA or RNA, the molecule binds, double-strands, or hybridizes only to a specific nucleotide sequence under stringent conditions. "Substantially binding" refers to complementary hybridization between the probe nucleic acid and the target nucleic acid, encompassing small mismatches that can be adapted by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0077] In the context of nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization conditions" and "stringent 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 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. Generally, highly stringent hybridization and washing conditions are selected so that they are approximately 5°C lower than the thermal melting point (Tm) of a particular sequence at a given ionic strength and pH. Typically, under "stringent conditions," a probe hybridizes to its target subsequence but not to other sequences.

[0078] Nucleic acid molecules are said to be “complements” to another nucleic acid molecule if they exhibit complete complementarity. As used herein, a molecule is said to exhibit “complete complementarity” if all nucleotides of one molecule are complementary to the nucleotides of the other molecule. Two molecules are said to be “minimally complementary” if they can hybridize with each other with sufficient stability to remain annealed to each other, at least under conventional “low stringency” conditions. Similarly, molecules are said to be “complementary” if they can hybridize with each other with sufficient stability to remain annealed to each other, at conventional “high stringency” conditions. Conventional stringency conditions are described in Sambrook, et al., In: Molecular Cloning, A Laboratory Manual, 2nd Edition, 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.

[0079] Tm is the temperature (under a given ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matching probe. Highly stringent conditions are selected to be equal to the Tm of a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids with more than 100 complementary residues on a filter in Southern or Northern blotting is 50% formamide with 1 mg of heparin at 42°C, with hybridization performed overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent washing conditions is 0.2 × SSC washing over 15 minutes at 65°C (see Sambrook below for a description of SSC buffer). Often, a low-stringency wash is performed before a high-stringency wash to remove background probe signal. For example, a moderate stringency wash for double helices exceeding 100 nucleotides is 1×SSC at 45°C for 15 minutes. For example, a low stringency wash for double helices exceeding 100 nucleotides is 4–6×SSC at 40°C for 15 minutes. For short probes (e.g., about 10–50 nucleotides), stringent conditions typically involve a sodium ion concentration of less than about 1.0 M at pH 7.0–8.3, a sodium ion concentration (or other salt) of about 0.01–1.0 M, and a temperature of at least about 30°C. Stringent conditions can also be achieved by adding destabilizers such as formamide. Generally, a signal-to-noise ratio of 2x (or higher) compared to that observed with an unrelated probe in a particular hybridization assay indicates the detection of specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This occurs, for example, when a copy of nucleic acid is made using the maximum codon degeneracy permitted by the genetic code.

[0080] The following is an example of a set of hybridization / washing conditions that can be used to clone a homologous nucleotide sequence that is substantially identical to the reference Cry21 nucleotide sequence of the present invention: the reference nucleotide sequence is preferably washed at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and at 50°C in 2×SSC, 0.1% SDS; more preferably at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and at 50°C in 1×SSC, 0.1% SDS; even more preferably at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and at 50°C in 0.5×SSC, 0.1% SDS; preferably at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM Hybridization with a reference nucleotide sequence is performed under the following conditions: washing in EDTA at 50°C with 0.1×SSC and 0.1% SDS; more preferably, washing in EDTA at 50°C with 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA at 65°C with 0.1×SSC and 0.1% SDS.

[0081] A further indicator that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive or specifically binds to the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein if, for example, the two proteins differ only by conservative substitutions.

[0082] "Synthetic" refers to a nucleotide sequence that contains structural features not present in natural sequences. For example, the Cry21 coding sequence, which is not found naturally in the genus Bacillus and more closely resembles the G+C content and typical codon distribution of dicotyledonous and / or monocotyledonous plant genes, is said to be synthetic.

[0083] "Transformation" is the process of introducing a different nucleic acid into a host cell or organism. In particular, "transformation" refers to the stable integration of DNA molecules into the genome of the target organism.

[0084] "Transformed / transgenic / recombinant" refers to a host organism, such as bacteria or plants, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host genome, or it can exist as an extrachromosomal molecule. Such extrachromosomal molecules can autoreplicate. Transformed cells, tissues, or plants are understood to include not only the final product of the transformation process, but also their genetically modified offspring. "Untransformed," "non-transgenic," or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants, that do not contain heterologous nucleic acid molecules.

[0085] In relation to the present invention, “Cry21 protein” means a Bacillus Cry insecticidal protein (which is a member of the Cry21 class, including, for example, Cry21Aa1, Cry21Aa2, and their homologs). Some structural features that identify a protein as a Cry21 class protein include 1) a size of approximately 130–150 kDa, which corresponds to the specifications for Cry21 proteins described in Crickmore et al., Journal of Invertebrate Pathology 186 (2021), the entire text of which is incorporated herein by reference. Nematode activity “homologous” as used herein means that a specified protein or polypeptide is active against nematodes and has defined relationships with other members of the Cry21 class proteins. This defined relationship may include, but is not limited to, proteins that are at least 60%, at least 70%, at least 80%, or at least 90% identical at the sequence level to another member of the Cry21 class of proteins, while also retaining nematicidal activity.

[0086] As used herein, nucleotides are denoted by the following standard abbreviations based on their bases: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are similarly 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; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0087] The materials and methods of the present invention are useful for killing or controlling nematodes; slowing the growth or reproduction of nematodes; reducing nematode populations; and / or reducing or delaying damage to plants caused by nematode pest infestation. One embodiment provides a method for controlling nematode pests in crop plants such as soybeans by using transgenic crop plants expressing the Cry21 protein.

[0088] Expression of the Cry21 protein in one or more embodiments in transgenic plants or plant parts is effective against nematode pests, for example, but not limited to, species of the genus Meloidogyne (e.g., sweet potato nematode (Meloidogyne incoginita), Java nematode (Meloidogyne javanica), northern nematode (Meloidogyne hapla), and arena nematode (Meloidogyne arenari)), species of the genus Heterodera (e.g., soybean cyst nematode (Heterodera glycines), carrot cyst nematode (Heterodera carotae), sugar beet cyst nematode (Heterodera schachtii), wheat cyst nematode (Heterodera avenae), and clover cyst nematode (Heterodera schachtii). (trifolii), Globodera spp. (e.g., potato cyst nematode (Globodera rostochiensis)), Radopholus spp. (e.g., banana root-leaf nematode (Radopholus similes)), Rotylenculus spp., Pratylenchus spp. (e.g., Pratylenchus neglectans, pineapple root-knot nematode (Pratylenchus brachyurus) and northern root-knot nematode (Pratylenchus penetrans)), Aphelenchoides spp., Helicotylenchus spp.), species of the genera Hopolaimus, Paratricodorus, Longidorus, Nacobbus, Subanguina, Belonlaimus), species of the genera Criconemella, Criconemoides, Ditylenchus, Ditylenchus dipsaci, Dolichodorus, Hemicriconemoides, Hemicycliophora, Hirschmaniella, Hypsoperine, Macroposthonia, Melinius, Punctodera, Quinisulcius This provides a composition that can be used to control species of the genera Scutellonema, Xiphinema, and Tylenchorhynchus.

[0089] One embodiment provides a method for controlling nematode pests, comprising contacting the nematode pests with Cry21 proteins, including SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, and 53, and proteins containing any functional fragments thereof.

[0090] In another embodiment, the nematode is selected from the group consisting of Criconemella, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Meloidogyne, Paratrichodorus, Pratylenchus, Radolpholus, Rotelynchus, Rotylenchulus, Tylenchulus, and Xiphenema. In yet another embodiment, the nematode is a cyst-forming nematode. In yet another embodiment, the nematode belongs to the genus Pratylenchus. In a further embodiment, the nematode is the pineapple root-knot nematode (Pratylenchus brachyurus).

[0091] In another embodiment, the contact step is performed on a plant or plant part transformed with at least one nucleic acid molecule encoding the Cry21 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.

[0092] In another embodiment, the transgenic plant or plant part may be alfalfa, apple, apricot, Arabidopsis, artichoke, asparagus, avocado, banana, barley, legumes, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, grains, celery, cherry, citrus fruits, clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, fig, grape, grapefruit, peanut, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, or nut. The transgenic plant or plant part is selected from the group consisting of oats, okra, onions, oranges, ornamental plants or flowers or trees, papaya, parsley, peas, peaches, peanuts, peat, pepper, persimmons, pineapples, plantains, plums, pomegranates, potatoes, pumpkins, radicchio, radishes, rapeseed, raspberries, rice, rye, sorghum, soybeans, spinach, strawberries, sugar beets, sugarcane, sunflowers, sweet potatoes, tangerines, tea, tobacco, tomatoes, climbing plants, watermelons, wheat, yams, and zucchini. In yet another embodiment, the transgenic plant or plant part is a soybean plant or plant part.

[0093] Another embodiment provides transgenic seeds of one or more embodiments of a transgenic plant, wherein the transgenic seeds contain heterogeneous nucleic acid molecules encoding one or more embodiments of the Cry21 protein.

[0094] Another embodiment also comprises a recombinant vector and expression cassette containing the Cry21 nucleic acid sequence of the present invention. In such a vector, the nucleic acid sequence is contained in an expression cassette containing regulatory elements for the expression of the Cry21 nucleotide sequence in a transgenic host cell capable of expressing the nucleotide sequence. Such regulatory elements typically include a promoter and a termination signal, and preferably also include elements that enable efficient translation of the polypeptide encoded by the nucleic acid sequence of the present invention. The vector containing the nucleic acid sequence can typically be replicated in a specific host cell, e.g., an extrachromosomal molecule, and is therefore used to amplify the nucleic acid sequence of the present invention within a host cell. In one embodiment, the host cell of such a vector is a microorganism such as bacteria, particularly Escherichia coli (E. coli). In another embodiment, the host cell of such a recombinant vector is an endophyte or epiphyte. An example of a host cell of such a vector is a eukaryotic cell, such as a plant cell. Such a plant cell may be a soybean cell or a maize cell. In another embodiment, such a vector is a viral vector and is used to replicate the nucleotide sequence in a specific host cell, e.g., an insect cell or a plant cell. Recombinant vectors are also used to transform transgenic host cells with the nucleotide sequence of the present invention, thereby stably incorporating the nucleotide sequence into the DNA of such transgenic host cells. In one embodiment, such transgenic host cells are prokaryotic cells. In another embodiment, such transgenic host cells are eukaryotic cells such as yeast cells, insect cells, or plant cells. In yet another embodiment, the transgenic host cells are plant cells such as soybean cells or maize cells.

[0095] In further embodiments, the Cry21 nucleotide sequence of the present invention can be modified, for example, by incorporating random mutations in a technique known as in vitro recombination or DNA shuffling to enhance nematode activity. This technique is described in Stemmer et al., Nature 370:389-391 (1994) and U.S. Patent No. 5,605,793, which are incorporated herein by reference. Millions of variant copies of the nucleotide sequence are generated based on the original nucleotide sequence of the present invention, and variants having improved properties (such as increased nematode-killing activity, enhanced stability, or different specificity or range of target nematode pests) are recovered. The method comprises forming a mutagenesized double-stranded polynucleotide from a template double-stranded polynucleotide containing the nucleotide sequence of the present invention, wherein the template double-stranded polynucleotide is cleaved into double-stranded random fragments of a desired size, and the method comprises the steps of: adding one or more single-stranded or double-stranded oligonucleotides to the resulting group of double-stranded random fragments, wherein the oligonucleotides include regions of identity and regions of heterogeneity with respect to the double-stranded template polynucleotide; denaturing the resulting mixture of double-stranded random fragments and oligonucleotides to form single-stranded fragments; and denaturing the resulting group of single-stranded fragments, wherein the single-stranded fragments are identical to the original. A step of incubating with polymerase under conditions that result in annealing in a region of identity and forming pairs of annealed fragments, wherein the region of identity is sufficient for one of the pairs to prime the replication of the other, thereby forming a mutagenic double-stranded polynucleotide; and a step of repeating the second and third steps at least two further cycles, wherein the mixture obtained in the second step of the further cycles comprises mutagenic double-stranded polynucleotides from the third step of the previous cycle, and the further cycles form further mutagenic double-stranded polynucleotides. In one embodiment, the concentration of a single species of double-stranded random fragment in a population of double-stranded random fragments is less than 1% by weight of the total DNA.In a further embodiment, the template double-stranded polynucleotide comprises at least about 100 different polynucleotides. In another embodiment, the size of the double-stranded random fragment is about 5 by ~ 5 kb. In yet another embodiment, the fourth step of the method comprises repeating the second and third steps for at least 10 cycles.

[0096] In further embodiments, the Cry21 nucleotide sequence of the present invention can be modified by deletion of the N-terminus or preferably the C-terminus to encode a functional fragment. The term “functional fragment” refers to a sequence of at least 10, 20, 30, 40, 50, or 60 consecutive amino acids of the Cry21 protein, which is sequence number 1, 7, 9, 13, 21, 23, 33, 35, 39, 41, 51, or 53. A preferred deletion is the removal of the C-terminal crystallization domain downstream of a conserved amino acid motif called “DRIEF” or “DRIE,” which is also shown in Figure 2 and described as “Block 5” in Schnepf et al 1998. Functional fragments with C-terminal deletion of the crystallization domain are expected to produce a toxic core protein independent of proteolytic activation.

[0097] In another embodiment, at least one of the Cry21 nucleotide sequences of the present invention is inserted into a suitable expression cassette including a promoter and a termination signal. Expression of the nucleotide sequence is constitutive, or an inductive promoter is used that initiates transcription in response to various types of stimuli. In a preferred embodiment, the cells on which the toxin is expressed are microorganisms such as viruses, bacteria, or fungi. In one embodiment, a virus such as a baculovirus contains the nucleotide sequence of the present invention in its genome and expresses a large amount of the corresponding insecticidal toxin after infection of a suitable eukaryotic cell suitable for viral replication and nucleotide sequence expression. The insecticidal toxin thus produced is used as an insecticide. Alternatively, a baculovirus modified to contain the nucleotide sequence is used to infect insects in vivo and kill them by the expression of the insecticidal toxin, or by a combination of viral infection and the expression of the insecticidal toxin.

[0098] Bacterial cells are also hosts for the expression of the nucleotide sequences of the present invention. In one embodiment, non-pathogenic symbiotic bacteria that can survive and replicate within plant tissue, so-called endophytes, or non-pathogenic symbiotic bacteria that can colonize the phyllosphere or rhizosphere, so-called epiphytes, 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 Gliocladium are also possible hosts for the expression of the nucleotide sequences of the present invention for the same purpose.

[0099] These genetic manipulation techniques are specific to various available hosts and are known in the art. For example, the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in Escherichia coli (E. coli) by either transcriptional or translational fusion behind the tac or trc promoter. For the expression of operons encoding multiple ORFs, the simplest procedure is to insert the operons into a vector such as pKK223-3 by transcriptional fusion, thereby making the congeneral ribosome binding sites of the heterologous genes available. Techniques for overexpression in Gram-positive species such as Bacillus are also known in the art and can be used in connection with the present invention (Quax et al. In: Industrial Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et al., American Society for Microbiology, Washington (1993)). Alternative systems for overexpression include, for example, the use of yeast vectors, specifically Pichia, Saccharomyces, and Kluyveromyces (Sreekrishna, In: Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, and Skatrud eds., American Society for Microbiology, Washington (1993); Dequin & Bane, Biotechnology L2:173-177 (1994); van den Berg et al., Biotechnology 8:135-139 (1990)).

[0100] In one embodiment, at least one Cry21 protein of the present invention is expressed in a higher organism, such as a plant. In this case, a transgenic plant expressing an effective amount of toxin protects itself from nematode pests. When nematodes begin to feed on such a transgenic plant, they also ingest the expressed Cry21 toxin. This can prevent further feeding of the nematodes within the plant tissue, or it can harm or kill the nematodes, or reduce their ability to reproduce. The nucleotide sequence of the present invention is inserted into an expression cassette and subsequently stably integrated into the plant genome. The plants transformed according to the present invention may be monocots or dicots, and include maize, wheat, barley, rye, sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, chili pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, This includes, but is not limited to, plums, cherries, peaches, nectarines, apricots, strawberries, grapes, raspberries, blackberries, pineapples, avocados, papayas, mangoes, bananas, soybeans, tomatoes, sorghum, sugarcane, sugar beets, sunflowers, rapeseed, clover, tobacco, carrots, cotton, alfalfa, rice, potatoes, eggplants, cucumbers, Arabidopsis thaliana, and woody plants such as conifers and broad-leaved trees.

[0101] Once a desired nucleotide sequence is transformed into a particular plant species, it can be propagated in that species using conventional breeding techniques, or transferred to other varieties of the same species, particularly commercial varieties.

[0102] The nucleotide sequences of the present invention are expressed in transgenic plants, thereby inducing the biosynthesis of the corresponding toxins in the transgenic plants. In this way, transgenic plants with enhanced resistance to nematodes are produced. The nucleotide sequences of the present invention may require modification and optimization for their expression in transgenic plants. In many cases, microbial genes can be expressed at high levels in plants without modification, but low expression in transgenic plants may result from microbial nucleotide sequences that have codons undesirable in plants. It is known in the art that all organisms have specific preferences regarding codon use, and the codons of the nucleotide sequences described in the present invention can be modified to suit plant preferences while maintaining the amino acids encoded thereby. Furthermore, high expression in plants is best achieved from coding sequences having a GC content of at least about 35%, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60%. While preferred gene sequences can be appropriately expressed in both monocotyledonous and dicotyledonous plant species, their preferences have been shown to differ; therefore, sequences can be modified to take into account the specific codon preferences and GC content preferences of monocotyledonous or dicotyledonous plants (Murray et al. Nucl. Acids Res. 17:477-498 (1989)). Furthermore, nucleotide sequences are screened for the presence of malsplice sites that may cause message cleavage. All necessary modifications within the nucleotide sequence, such as those described above, are made using well-known techniques of site-directed mutagenesis, PCR, and synthetic gene construction using methods known in the art.

[0103] In various embodiments, the nucleotide sequences of the present invention can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence can be optimized or synthesized. That is, synthetic sequences or partially optimized sequences can also be used.

[0104] For efficient translation initiation, sequences adjacent to the start codon encoding the start methionine may require modification. For example, they can be modified by encapsulating sequences known to be effective in plants. Joshi has proposed a consensus suitable for plants (NAR 15:6643-6653 (1987)), and Clonetech has proposed further consensus translation initiation factors (1993 / 1994 catalog, p. 210). These consensuses are suitable for use in the nucleotide sequences of the present invention. These sequences are incorporated into a construct containing the nucleotide sequence up to ATG (with the second amino acid left unchanged) (including ATG), or alternatively up to GTC (including ATG) following ATG (potentially with modification of the second amino acid of the transgene).

[0105] As described above, the Cry21 toxin genes of the embodiments can be operably fused to various promoters for expression in plants, including constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preference, and tissue-specific promoters, either as their native sequences or as optimized synthetic sequences, in order to prepare recombinant DNA molecules, i.e., chimeric genes. The choice of promoter varies depending on the temporal and spatial requirements for expression. Thus, expression of the nucleotide sequences encoding the Cry21 protein of the embodiments can be achieved in leaves, stems or stalks, spikes, inflorescences (e.g., spikes, panicles, rachis, etc.), roots, and / or seedlings, but expression in roots is particularly preferred for nematode control. However, protection against multiple types of nematode pests is often required, and therefore, expression in multiple tissues is desirable. Many promoters from dicotyledonous plants have been shown to function in monocotyledonous plants, and vice versa; however, ideally, dicotyledonous promoters are selected for expression in dicotyledonous plants, and monocotyledonous promoters are selected for expression in monocotyledonous plants. Nevertheless, there are no restrictions on the origin of the selected promoters. It is sufficient that they act to drive the expression of the nucleotide sequence of the present invention in the desired cells.

[0106] vector The pest-killing sequences of the present invention may be provided as expression cassettes for expression in target host cells, e.g., plant cells or microorganisms. “Plant expression cassette” means a DNA construct capable of resulting in the expression of a protein from an open reading frame in plant cells. Typically, these contain a promoter and a coding sequence. Often, such constructs also contain a 3' untranslated region. Such constructs may contain a signal sequence or a leader sequence to facilitate the co-translational or post-translational transport of the peptide to specific intracellular structures such as chloroplasts (or other plastids), the endoplasmic reticulum, or the Golgi apparatus.

[0107] A “signal sequence” refers to a sequence known or suspected to result in co-translation or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus and results in some glycosylation. Bacterial insecticidal toxins are often synthesized as protoxins that are proteolytically activated in the intestines of target pests (Chang (1987) Methods Enzymol. 153:507-516). In some embodiments, the signal sequence may be located in a native sequence or may be derived from a sequence of the present invention.

[0108] The “leader sequence” is intended to be any sequence that, when translated, yields an amino acid sequence sufficient to induce the simultaneous translational transport of a peptide chain to intracellular organelles. Therefore, this includes leader sequences that target transport and / or glycosylation by translocation to the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, etc. Accordingly, this specification further provides polypeptides comprising the amino acid sequence of the present invention operably linked to a heterologous leader or signal sequence.

[0109] A "plant transformation vector" refers to a DNA molecule necessary for the efficient transformation of plant cells. Such a molecule may consist of one or more plant expression cassettes, or it may be organized into multiple "vector" DNA molecules. For example, a binary vector is a plant transformation vector that utilizes two non-adjacent DNA vectors that encode all the cis and trans functions essential for the transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).

[0110] A "vector" refers to a nucleic acid construct designed to move between different host cells.

[0111] An "expression vector" refers to a vector having the ability to incorporate, integrate, and express a heterologous DNA sequence or fragment into exogenous cells. A cassette includes a 5' and / or 3' regulatory sequence operably ligated to the sequence of the present invention. "Operatably ligated" means a functional ligation between a promoter and a second sequence, where the promoter sequence initiates and mediates the transcription of the DNA sequence corresponding to the second sequence. Generally, operably ligated means that the ligated nucleic acid sequences are adjacent, and if it is necessary to ligate the coding regions of two proteins, it means they are adjacent and within the same reading frame. In some embodiments, a nucleotide sequence is operably ligated to a heterologous promoter that can direct the expression of the nucleotide sequence in a host cell such as a microbial host cell or a plant host cell. A cassette may further include at least one additional gene that is co-transformed into an organism. Alternatively, additional genes may be provided on multiple expression cassettes.

[0112] In various embodiments, the nucleotide sequences of the present invention are operably linked to heterologous promoters, such as plant promoters.

[0113] Such expression cassettes include multiple restriction sites for inserting pest-killing sequences so that they are under transcriptional control of the regulatory region.

[0114] The expression cassette comprises, in the 5' to 3' direction of transcription, a transcription and translation initiation region (i.e., a promoter), the DNA sequence of the present invention, and a translation and transcription termination region (i.e., a termination region) that functions in the plant. The promoter may be natural or analogous, or exotic or heterologous, to the host plant and / or the DNA sequence of the present invention. Furthermore, the promoter may be a natural sequence or a synthetic sequence. If the promoter is “natural” or “homologous” to the host plant, the promoter is intended to be found in the natural plant into which the promoter is introduced. If the promoter is exotic or heterologous to the DNA sequence of the present invention, the promoter is intended not to be a naturally occurring or naturally occurring promoter of the operably linked DNA sequence of the present invention. The promoter may be inducible or constitutive. It may be naturally occurring, composed of a selection of various naturally occurring promoters, or partially or completely synthesized. Guidance for promoter design is provided by promoter structure studies such as Harley and Reynolds (1987) Nucleic Acids Res. 15:2343-2361. Furthermore, the promoter's position relative to transcription initiation can be optimized. See, for example, Roberts et al. (1979) Proc. Natl. Acad. Sci. USA, 76:760-764. Many promoters suitable for use in plants are well known in the art.

[0115] For example, suitable constitutive promoters for use in plants include: promoters derived from plant viruses such as the peanut chlorotic streak kalimovirus (PClSV) promoter (U.S. Patent No. 5,850,019); the 35S promoter derived 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 (U.S. Patent No. 5,563,328) and the full-length transcription promoter derived from fig mosaic virus (FMV) (U.S. Patent No. 5,378,619); promoters derived from genes such as comeactin (McElroy et al. (1990) Plant Cell 2:163-171 and U.S. Patent No. 5,641,876); and ubiquitin (Christensen et al. al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689) and Grefen et al. (2010) Plant J, 64:355-365; pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730 and U.S. Patent No. 5,510,474); Corn H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. 231:276-285 and Atanassova et al. (1992) Plant J.2(3):291-300); Brassica napus ALS3 of napus (International Publication No. 97 / 41228); plant ribulose diphosphate carboxylase / oxygenase (RuBisCO) small subunit gene; circovirus (Australian Patent No. 689311) or cassava leaf vein mosaic virus (CsVMV, U.S. Patent No. 7,053,205); soybean-derived promoters (Pbdc6 or Pbdc7, described in International Publication No. 2014 / 150449, or the ubiquitin 3 promoter described in U.S. Patents No. 7,393,948 and 8,395,021); and promoters of various Agrobacterium genes (see U.S. Patents No. 4,771,002, 5,102,796, 5,182,200, and 5,428,147).

[0116] Suitable inducible promoters for use in plants include: a copper-responsive ACE1-derived promoter (Mett et al. (1993) PNAS 90:4567-4571); a maize In2 gene promoter responsive to benzenesulfonamide herbicide toxicity reducers (Hershey et al. (1991) Mol.Gen.Genetics 227:229-237 and Gatz et al. (1994) Mol.Gen.Genetics 243:32-38); and a Tn10-derived Tet repressor promoter (Gatz et al. (1991) Mol.Gen.Genet. 227:229-237). Another inducible promoter for use in plants is one that allows plants to respond to inducers to which they do not normally respond. An exemplary inducible promoter of this type is an inducible promoter derived from a steroid hormone gene, whose transcriptional activity is induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421), or a 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., 24:265-273). Other inducible promoters for use in plants are described in European Patent No. 332104, PCT International Publication No. 93 / 21334 and PCT International Publication No. 97 / 06269, which are incorporated herein by reference in their entirety. Promoters composed of parts of other promoters, and partially or completely synthesized promoters may also be used. For example, see Ni et al. (1995) Plant J.7:661-676 and PCT International Publication No. 95 / 14098, which describe such promoters for use in plants.

[0117] In one embodiment of the present invention, the nematicidal protein of the present invention can be expressed using promoter sequences specific to a particular region or tissue of a plant, such as a seed-specific promoter (Datla, R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), particularly the napine promoter (European Patent Application Publication No. 255378 A1), the phaseolin promoter, the glutenin promoter, the helanchinin promoter (International Publication No. 92 / 17580), the albumin promoter (International Publication No. 98 / 45460), the oleosin promoter (International Publication No. 98 / 45461), the SAT1 promoter, or the SAT3 promoter (PCT / US98 / 06978).

[0118] Inducible promoters can also be advantageously selected from phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI), PR1 family genes, nopaline synthase (nos) and vspB promoters (U.S. Patent No. 5,670,349, Table 3), HMG2 promoter (U.S. Patent No. 5,670,349), apple β-galactosidase (ABG1) promoter, and apple aminocyclopropane hydrocarbon synthase (ACC synthase) promoter (International Publication No. 98 / 45445). Multiple promoters may be used in the constructs of the present invention, including sequentially.

[0119] A promoter may contain one or more enhancer elements, or may be modified to contain them. In some embodiments, a promoter may contain multiple enhancer elements. A promoter containing enhancer elements yields a higher level of transcription compared to a promoter that does not contain them. Suitable enhancer elements for use in plants include the PClSV enhancer element (U.S. Patent No. 5,850,019), the CaMV 35S enhancer element (U.S. Patents No. 5,106,739 and 5,164,316), and the FMV enhancer element (Maiti et al. (1997) Transgenic Res. 6:143-156); the translational activator of tobacco mosaic virus (TMV) described in International Publication No. 87 / 07644, or the translational activator of tobacco etch virus (TEV) described, for example, in Carrington & Freed 1990, J. Virol. 64:1590-1597, or introns such as the adh1 intron of maize or intron 1 of rice actin. See also PCT International Publication No. 96 / 23898, International Publication No. 2012 / 021794, International Publication No. 2012 / 021797, International Publication No. 2011 / 084370, and International Publication No. 2011 / 028914.

[0120] In many cases, such constructs may contain 5' and 3' untranslated regions. Such constructs may contain signal sequences or leader sequences to facilitate the co-translational or post-translational transport, or secretion, of the peptide of interest to specific intracellular structures such as chloroplasts (or other plastids), the endoplasmic reticulum, or the Golgi apparatus. For example, a construct can be engineered to contain a signal peptide that facilitates the transfer of the peptide to the endoplasmic reticulum.

[0121] The "3' untranslated region" refers to a polynucleotide located downstream of the coding sequence. Polyadenylation signal sequences and other sequences encoding regulatory signals that can influence the addition of polyadenylate tracts to the 3' end of the mRNA precursor are the 3' untranslated region. The "5' untranslated region" refers to a polynucleotide located upstream of the coding sequence.

[0122] Other upstream or downstream untranslated elements include enhancers. Enhancers are polynucleotides that act to increase the expression of the promoter region. Enhancers are well known in the art and include, but are not limited to, the SV40 enhancer region and the 35S enhancer element.

[0123] The termination region may be native to the transcription start region, native to the operably ligated target DNA sequence, native to the plant host, or derived from another source (i.e., exogenous or heterologous to the promoter, target DNA sequence, plant host, or any combination thereof). Convenient termination regions, such as those for octopine synthase and nopalin synthase, are available from the A. tumefaciens Ti plasmid. Guerineau et al.(1991)Mol.Gen.Genet.262:141-144;Proudfoot(1991)Cell 64:671-674;Sanfacon et al.(1991)Genes Dev.5:141-149;Mogen et al.(1990)Plant Cell 2:1261-1272;Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0124] Where appropriate, genes can be optimized to increase their expression in transformed host cells (synthetic DNA sequences). That is, genes can be synthesized using codons preferred by the host cell to improve expression, or using codons at codon usage frequencies preferred by the host. Expression of open reading frames of synthetic DNA sequences in cells results in the production of polypeptides of the present invention. Synthetic DNA sequences may be useful for simply removing undesirable restriction endonuclease sites, for facilitating DNA cloning strategies, for modifying or eliminating any potential codon bias, for modifying or improving GC content, for removing or altering alternative reading frames, and / or for modifying or removing intron / exon splice recognition sites, polyadenylation sites, Shine-Dalgarno sequences, undesirable promoter elements, etc., that may be present in native DNA sequences. Generally, the GC content of genes increases. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion on the use of host-preferred codons. 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 (incorporated herein by reference).

[0125] It is also possible to introduce other modifications to the DNA sequence using the synthetic DNA sequence, such as the introduction of an intron sequence or the creation of a DNA sequence that is expressed as a protein fusion with an organelle-targeting sequence, such as a chloroplast-transfer peptide, an apoplast / vacuole-targeting peptide, or a peptide sequence that results in the retention of the resulting peptide in the endoplasmic reticulum. Thus, in one embodiment, the nematicidal protein is targeted to chloroplasts for expression. In this way, if the expression cassette encoding the nematicidal protein is not directly inserted into the chloroplast genome, the expression cassette further comprises a nucleic acid encoding a transport peptide for inducing the nematicidal protein to chloroplasts. Such transport peptides are known in the art. For example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem.Biophys.Res.Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481.

[0126] For information on chloroplast-targeting pest-killing genes, see, for example, U.S. Patent No. 5,380,831 (incorporated herein by reference).

[0127] Plant transformation The methods of the embodiments include introducing a nucleotide construct into a plant. By “introducing,” it is intended that the nucleotide construct be presented to the plant in such a manner that the construct can access the inside of the plant's cells. The methods of the present invention do not require the use of a specific method to introduce the nucleotide construct into the plant, but only require that the nucleotide construct can access the inside of at least one plant cell. Methods for introducing nucleotide constructs into plants are known in the art and include, but are not limited to, stable transformation, transient transformation, gene editing, and virus-mediated methods.

[0128] A “transgenic plant,” “transformed plant,” or “stable transformed” plant, cell, or tissue refers to a plant in which an exogenous nucleic acid sequence or DNA fragment has been incorporated into or integrated into a plant cell. These nucleic acid sequences may be exogenous or not present in non-transformed plant cells, or may be endogenous or may be present in non-transformed plant cells.

[0129] "Heterogeneous" generally refers to nucleic acid sequences that are added to a cell or a portion of its natural genome, not endogenously, but through means such as infection, transfection, microinjection, electroporation, or microprojection.

[0130] The transgenic plants of the present invention express one or more of the novel toxin sequences disclosed herein. In some embodiments, the protein or nucleotide sequences of the present invention are advantageously combined in plants with other genes encoding proteins or RNA that confer useful agricultural properties to such plants. Among the genes encoding proteins or RNA that confer useful agricultural properties to transgenic plants, examples include DNA sequences encoding proteins that confer resistance to one or more herbicides, and DNA encoding RNA that confers resistance to specific insects, resistance to specific diseases, or provides control of nematodes or insects. Such genes are described in particular in PCT Patent Application International Publication No. 91 / 02071 and International Publication No. 95 / 06128, and in U.S. Patent No. 7,923,602 and U.S. Patent Application Publication No. 20100166723, which are each incorporated herein by reference in their entirety. In various embodiments, the transgenic plant further includes one or more additional genes for insect resistance (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 the transgenic plant may include any genes that confer the desired agricultural trait.

[0131] DNA sequences encoding proteins that confer resistance to specific herbicides to transformed plant cells and plants include the bar gene or PAT gene that confer resistance to glufosinate herbicides, or Streptomyces ceralicolor as described in International Publication No. 2009 / 152359. Coelicolor) gene, gene encoding appropriate EPSPS that confer resistance to EPSPS-targeting herbicides such as glyphosate and its salts (U.S. Patent Nos. 4,535,060, 4,769,061, 5,094,945, 4,940,835, 5,188,642, 4,971,908, 5,145,783, 5,310,667, 5,312,910, 5,627,061, and 5,633,435), gene encoding glyphosate-N-acetyltransferase (e.g.) U.S. Patent Nos. 8,222,489, 8,088,972, 8,044,261, 8,021,857, 8,008,547, 7,999,152, 7,998,703, 7,863,503, 7,714,188, 7,709,702, 7,666,644, 7,666,643, 7,531,339, 7,527,955, and 7,405,074), genes encoding glyphosate oxidoreductase (e.g., U.S. Patent Nos. 5,463,Examples include HPPD inhibitor resistance genes described in International Publication No. 2004 / 055191, International Publication No. 199638567, U.S. Patent No. 6791014, International Publication No. 2011 / 068567, International Publication No. 2011 / 076345, International Publication No. 2011 / 085221, International Publication No. 2011 / 094205, International Publication No. 2011 / 068567, International Publication No. 2011 / 094199, International Publication No. 2011 / 094205, International Publication No. 2011 / 145015, International Publication No. 2012 / 056401, and International Publication No. 2014 / 043435).

[0132] Among the DNA sequences encoding appropriate EPSPS that confer resistance to herbicides targeting EPSPS, more specifically, include genes encoding plant EPSPS, particularly maize EPSPS, in particular maize EPSPS containing two mutations, especially a mutation at amino acid position 102 and a mutation at amino acid position 106 (International Publication No. 2004 / 074443), described in U.S. Patent No. 6,566,587 and hereinafter referred to as "double mutant maize EPSPS" or "2mEPSPS", or genes encoding EPSPS isolated from Agrobacterium, described in Sequence ID No. 2 and Sequence ID No. 3 of U.S. Patent No. 5,633,435 and also referred to as "CP4".

[0133] Among the DNA sequences encoding appropriate EPSPS that confer resistance to herbicides targeting EPSPS, more specifically, genes encoding EPSPS, GRG23, or ACE5 from Arthrobacter globiformis, as well as mutants GRG23 ACE1, GRG23 ACE2, or GRG23 ACE3, in particular mutants or variants of GRG23 described in International Publication No. 2008 / 100353, such as GRG23(ace3)R173K, sequence number 29 in International Publication No. 2008 / 100353.

[0134] In the case of DNA sequences encoding EPSPS, or more specifically, the genes described above, it is advantageous that sequences encoding transit peptides, particularly those described in U.S. Patent No. 5,510,471 or No. 5,633,448, are located prior to the sequences encoding these enzymes.

[0135] Exemplary herbicide resistance traits that can be combined with the nucleic acid sequences of the present invention further include at least one ALS (acetolactate synthase) inhibitor (International Publication No. 2007 / 024782); the mutant Arabidopsis ALS / AHAS gene (U.S. Patent No. 6,855,533); a gene encoding 2,4-D-monooxygenase that confers resistance to 2,4-D (2,4-dichlorophenoxyacetic acid) by metabolism (U.S. Patent No. 6,153,401); and a gene encoding dicamba monooxygenase that confers resistance to dicamba (3,6-dichloro-2-methoxybenzoic acid) by metabolism (U.S. Patent Publication No. 2008 / 0119361 and U.S. Patent Publication No. 2008 / 0120739).

[0136] In various embodiments, the nucleic acids of the present invention are stacked with one or more herbicide resistance genes, including one or more HPPD inhibitor herbicide resistance genes and / or one or more genes that are resistant to glyphosate and / or glufosinate.

[0137] More specifically, among the DNA sequences encoding proteins related to resistance characteristics to insects, Bt proteins are widely described in the literature and are well known to those skilled in the art. Proteins extracted from bacteria such as Photorhabdus are also examples (International Publication No. 97 / 17432 and International Publication No. 98 / 08932).

[0138] More specifically, among DNA sequences encoding proteins intended to confer novel properties of resistance to insects, there are Bt Cry or VIP proteins that are widely described in the literature and well known to those skilled in the art. These include Cry1F protein or hybrids derived from Cry1F protein (e.g., hybrid Cry1A-Cry1F proteins described in U.S. Patents No. 6,326,169, 6,281,016, and 6,218,188, or their toxic fragments), Cry1A type protein or its toxic fragments, preferably Cry1Ac protein or hybrids derived from Cry1Ac protein (e.g., hybrid Cry1Ab-Cry1Ac protein described in U.S. Patent No. 5,880,275), or Cry1Ab or Bt2 protein or its insecticidal fragment described in European Patent No. 451878, and those described in International Publication No. 2002 / 057664. Cry2Ae, Cry2Af, or Cry2Ag proteins or their toxic fragments, Cry1A.105 protein or its toxic fragment as described in International Publication No. 2007 / 140256 (SEQ ID NO: 7), VIP3Aa19 protein with NCBI accession number ABG20428, VIP3Aa20 protein with NCBI accession number ABG20429 (SEQ ID NO: 2 in International Publication No. 2007 / 142840), VIP3A protein produced in Wata Event COT202 or COT203 (International Publication Nos. 2005 / 054479 and 2005 / 054480, respectively), Cry proteins as described in International Publication No. 2001 / 47952, Estruch VIP3Aa protein or its toxic fragments as described in Proc Natl Acad Sci US A.28; 93(11):5389-94 and U.S. Patent No. 6,291,156, the genus Xenorhabdus (as described in International Publication No. 98 / 50427), the genus Serratia (especially S. entomophila (S.This includes insecticidal proteins derived from *Entomophila* or strains of the genus *Photorhabdus*, such as the *Photorhabdus* Tc protein described in International Publication No. 98 / 08932 (e.g., Waterfield et al., 2001, Appl Environ Microbiol. 67(11):5017-24; French-Constant and Bowen, 2000, Cell Mol Life Sci.; 57(5):828-33). Also included herein are any variants or variants of any of these proteins that differ from any of the above sequences, particularly the sequences of their toxic fragments, by several (1 to 10, preferably 1 to 5) amino acids, or are fused to a transport peptide such as a plastid transport peptide or another protein or peptide.

[0139] In various embodiments, the nucleic acids of the present invention can be combined in plants with one or more genes that confer desirable traits such as herbicide resistance, insect resistance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency, improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality, stress tolerance, and improved reproduction.

[0140] Particularly useful transgenic events that can be combined with the gene of the present invention in the same plant species (for example, by crossbreeding, or by retransforming plants containing another transgenic event with the chimeric gene of the present invention) include: event BPS-CV127-9 (soybean, herbicide resistant, deposited as NCIMB number 41603, described in International Publication 2010 / 080829); and event DAS21606-3 / 1606 (soybean, herbicide resistant, deposited as PTA-11028). (as described in International Publication 2012 / 033794); Event DAS-44406-6 / pDAB8264.44.06.1 (Soybean, herbicide resistant, deposited as PTA-11336, as described in International Publication 2012 / 075426); Event DAS-14536-7 / pDAB8291.45.36.2 (Soybean, herbicide resistant, deposited as PTA-11335, as described in International Publication 2012 / 075429); Event DAS68416 (Soybean, herbicide resistant, ATCC) Deposited as PTA-10442, described in International Publication 2011 / 066384 or International Publication 2011 / 066360); Event DP-305423-1 (Soybean, Quality trait, Not deposited, described in US Patent Application Publication 2008-312082 or International Publication 2008 / 054747); Event DP-356043-5 (Soybean, Herbicide resistance, ATCC) Deposited as PTA-8287, described in U.S. Patent Publication No. 2010-0184079 or International Publication No. 2008 / 002872); Event FG72 (Soybean, herbicide resistant, deposited as PTA-11041, described in International Publication No. 2011 / 063413); Event LL27 (Soybean, herbicide resistant, deposited as NCIMB 41658, described in International Publication No. 2006 / 108674 or U.S. Patent Publication No. 2008-320616); Event LL55 (Soybean, herbicide resistant, deposited as NCIMB 41660, described in International Publication No. 2006 / 108675 or U.S. Patent Publication No. 2008-196127);Event MON87701 (Soybean, insect control, deposited as ATCC PTA-8194, described in U.S. Patent Publication No. 2009-130071 or International Publication 2009 / 064652); Event MON87705 (Soybean, quality trait - herbicide resistance, deposited as ATCC PTA-9241, described in U.S. Patent Publication No. 2010-0080887 or International Publication 2010 / 037016); Event MON87708 (Soybean, herbicide resistance, deposited as ATCC PTA-9670, described in International Publication 2011 / 034704); Event MON87712 (Soybean, yield, deposited as PTA-10296, described in International Publication 2012 / 051199); Event MON87754 (Soybean, quality trait, ATCC Deposited as PTA-9385, described in International Publication 2010 / 024976); Event MON87769 (Soybean, Quality Trait, ATCC) Deposited as PTA-8911, described in U.S. Patent Application Publication 2011-0067141 or International Publication 2009 / 102873); Event MON89788 (Soybean, Herbicide Resistance, ATCC) Deposited as PTA-6708, described in U.S. Patent Application Publication No. 2006-282915 or International Publication Brochure 2006 / 130436); Event SYHT0H2 / SYN-000H2-5 (Soybean, Herbicide Resistant, Deposited as PTA-11226, described in International Publication Brochure 2012 / 082548); Event EE-GM3 / FG72 (Soybean, Herbicide Resistant, ATCC Accession Number PTA-11041) (Optionally stacked with Event EE-GM1 / LL27 or Event EE-GM2 / LL55) Public release 2011 / 063413A2 pamphlet); Event DAS-68416-4 (Soybeans, herbicide resistance, ATCC accession number PTA-10442, International release 2011 / 066360A1 pamphlet); Event DAS-68416-4 (Soybeans, herbicide resistance, ATCC accession number PTA-10442, International release 2011 / 066384A1 pamphlet); Event DAS-21606-3 (Soybeans, herbicide resistance, ATCC accession number PTA-11028, International release 2012 / 033794A2 pamphlet);Event MON-87712-4 (Soybean, Quality Traits, ATCC Accession Number PTA-10296, International Publication 2012 / 051199A2 brochure); Event DAS-44406-6 (Soybean, Stack Herbicide Resistance, ATCC Accession Number PTA-11336, International Publication 2012 / 075426A1 brochure); Event DAS-14536-7 (Soybean, Stack Herbicide Resistance, ATCC Accession Number PTA-11335, International Publication 2012 / 075429A1 brochure); Event SYN-000H2-5 (Soybean, Herbicide Resistance, ATCC Accession Number PTA-11226, International Publication 2012 / 082548A2 brochure); Examples include BENT 8264.44.06.1 (Soybean, Stack Herbicide Resistant, Accession Number PTA-11336, International Publication 2012 / 075426A2); ENT 8291.45.36.2 (Soybean, Stack Herbicide Resistant, Accession Number PTA-11335, International Publication 2012 / 075429A2); ENT SYHT0H2 (Soybean, ATCC Accession Number PTA-11226, International Publication 2012 / 082548A2); and ENT pDAB8264.42.32.1 (Soybean, Stack Herbicide Resistant, ATCC Accession Number PTA-11993, International Publication 2013 / 010094A1).

[0141] Furthermore, this specification provides a method for producing soybean plants or seeds, comprising combining a nucleotide sequence encoding SEQ ID NOs: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54, or any functional fragment thereof, with another SCN resistance locus / gene (for example, by combining a soybean plant or seed containing a nucleotide sequence encoding SEQ ID NOs: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54, or any functional fragment thereof, with another SCN resistance locus / gene present within the same soybean plant / seed), and sowing seeds containing a nucleotide sequence encoding SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, and the other SCN resistance locus / gene. In one embodiment, the plant, cell, or seed of the present invention contains one or more other SCN resistance loci / genes occurring in soybeans in order to obtain a combination of different SCN resistance sources in the soybean plant, cell, or seed of the present invention.Several soybean SCN resistance loci or genes are known, and one or more of them can be combined in the same plant, cell, or seed with plants containing sequence numbers 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54, or any functional fragment thereof, for example, resistance sources PI 88788, PI 548402 (Peking), PI Examples include any one SCN resistance gene / locus derived from 437654 (Hartwig or CystX®), or any combination thereof, or one or more of the endogenous SCN resistance loci / genes rhg1, rhg1-b, rhg2, rhg3, Rhg4, Rhg5, qSCN11, cqSCN-003, cqSCN-005, cqSCN-006, cqSCN-007, or any SCN resistance loci identified on any one of 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. 129(12):2295-2311; Kim and Diers 2013,Crop Science 53:775-785;Kazi et al.2010,Theor.Appl.Gen.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. 91:574-581).Furthermore, in one embodiment, the plant or seed of the present invention is an SCN-resistant source PI 548316, PI 567305, PI 437654, PI 90763, PI 404198B, PI 88788, PI 468916, PI 567516C, PI 209332, 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, PI 22897, or PI It is combined with one or more SCN resistance loci in soybeans obtained from any one of the 494182 genes.

[0142] Transformation of plant cells can be achieved by one of several techniques known in the art. The pesticide genes of the present invention can be modified to obtain or enhance expression in plant cells. Typically, constructs expressing such proteins contain a promoter that drives gene transcription, as well as a 3' untranslated region that enables transcription termination and polyadenylation. The composition of such constructs is well known in the art. In some cases, it may be useful to manipulate the gene so that the resulting peptide is secreted or targeted within the plant cell. For example, the gene can be manipulated to contain a signal peptide that facilitates the translocation of the peptide into the endoplasmic reticulum. It may also be preferable to manipulate the plant expression cassette to contain introns so that intron mRNA processing is required for expression.

[0143] Typically, a plant expression cassette is inserted into a plant transformation vector. This plant transformation vector may consist of one or more DNA vectors required to achieve plant transformation. For example, it is common practice in the art to use plant transformation vectors composed of multiple consecutive DNA segments. These vectors are often referred to in the art as "binary vectors." Agrobacterium-mediated transformation most frequently uses binary vectors and vectors with helper plasmids, where the size and complexity of the DNA fragments required to achieve efficient transformation are extremely large, and separating the functions into separate DNA molecules is advantageous. A binary vector typically contains a plasmid vector with cis-acting sequences (such as left and right boundaries) necessary for T-DNA transfer, a selection marker engineered for expression in plant cells, and a "target gene" (a gene engineered for expression in plant cells from which the production of transgenic plants is desired). Sequences necessary for bacterial replication are also present on this plasmid vector. The cis-acting sequences are positioned to enable efficient introduction into plant cells and expression within them. For example, the selection marker gene and the pest-killing gene are located between the left and right boundary sequences. Often, the second plasmid vector contains a trans-acting factor that mediates the introduction of T-DNA from Agrobacterium into plant cells. This plasmid often contains pathogenic functions (Vir genes) that enable infection of plant cells by Agrobacterium, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451), as well as DNA transfer via cleavage at the boundary sequence and vir-mediated DNA transfer. Several Agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation.The second plasmid vector is not required to transform plants by other methods such as microprojection, microinjection, electroporation, or polyethylene glycol.

[0144] Generally, 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 the application of appropriate selection at the maximum threshold level (dependent on the selection marker gene) to recover transformed plant cells from the non-transformed cell population. Explants are typically transferred to a fresh supply of the same medium and cultured periodically. The transformed cells are then placed on a regenerating medium supplemented with the maximum threshold level of the selector, after which they differentiate into shoots. The shoots are then transferred to a selective rooting medium, and rooted shoots or plantlets are recovered. 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) 745 Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured periodically. General descriptions of techniques and methods for producing 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. Since transformed material contains many cells, both transformed and untransformed cells are present in any fragment or cell population of the target callus or tissue. The ability to kill untransformed cells and allow transformed cells to proliferate leads to the cultivation of transformed plants. Often, the ability to remove untransformed cells limits the rapid recovery of transformed plant cells and the success of producing transgenic plants.

[0145] Transformation protocols and protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell being transformed, i.e., monocots or dicots. The creation of transgenic plants may be carried out by one of several methods, including but not limited to microinjection, electroporation, direct gene transfer, introduction of heterologous DNA into plant cells by Agrobacterium (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA attached to particles, ballistic particle acceleration, aerosol beam transformation (U.S. Patent Publication No. 20010026941, U.S. Patent No. 4,945,050, International Publication No. 91 / 00915, U.S. Patent Publication No. 2002015066), Lec1 transformation, and various other non-particle direct mediation methods for introducing DNA.

[0146] Methods for chloroplast transformation are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; and Svab and Maliga (1993) EMBO J.12:601-606. This method relies on the delivery of DNA containing a selection marker by particle gun and the targeting of the DNA to the plastid genome by homologous recombination. Furthermore, plastid transformation can be achieved by transactivation of silent transgenes on plastids by tissue-preferential expression of nuclear-encoded plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0147] After integrating heterologous foreign DNA into plant cells, untransformed cells are isolated and propagated from this selection process by applying appropriate selection at the maximum threshold level in culture medium to kill untransformed cells and periodically transferring them to fresh medium. Cells transformed with plasmid vectors are identified and propagated by sequential passaging and challenge with appropriate selection. The presence of the incorporated heterologous gene of interest in the genome of the transgenic plant can then be confirmed using molecular and biochemical methods.

[0148] Transformed cells can be grown into plants according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be cultivated and pollinated with the same or different transformed strains, and the resulting hybrids may have the constitutive expression of the desired phenotypic traits. Two or more generations may be cultivated to ensure that the expression of the desired phenotypic traits is stably maintained and inherited, and then seeds may be harvested to confirm that the expression of the desired phenotypic traits has been achieved. In this way, the present invention provides transformed seeds (also referred to as "transgenic seeds") having the nucleotide constructs of the present invention stably incorporated into the genome, for example, the expression cassettes of the present invention.

[0149] Evaluation of plant transformation After introducing heterologous foreign DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods, such as the analysis of nucleic acids, proteins, and metabolites associated with the integrated gene.

[0150] PCR analysis is a rapid method for screening transformed cells, tissues, or shoots for the presence of incorporated genes in an early stage before transplantation into soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). PCR is performed using oligonucleotide primers specific to the target gene or a background of Agrobacterium vectors.

[0151] Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, cited above). Generally, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated on an agarose gel, and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed, for example, with a radiolabeled 32P target DNA fragment to confirm the integration of the transgene into the plant genome, according to standard techniques (Sambrook and Russell, 2001, cited above).

[0152] In Northern blotting, RNA is isolated from a specific tissue of a transformant, fractionated on a formaldehyde agarose gel, and blotted onto a nylon filter according to a standard procedure routinely used in the art (Sambrook and Russell, 2001, cited above). Subsequently, the expression of RNA encoded by the pesticide gene is tested by hybridizing the filter with a radioactive probe derived from the pesticide gene using a method known in the art (Sambrook and Russell, 2001, cited above).

[0153] Western blotting and biochemical assays can be performed on transgenic plants to confirm the presence of proteins encoded by pest-killing genes using antibodies that bind to one or more epitopes present on nematode proteins, following standard procedures (Sambrook and Russell, 2001, cited above). Mass spectrometry is currently a widely accepted method of choice used in many biological studies to quantify low-abundance proteins and peptides. Mass spectrometry assays have been developed for each target protein of interest and are used to quantify gene expression in transformed tissues. References: (J Proteome Res. 2009 Feb;8(2):787-797. Target protein quantification by mass spectrometry: methods and applications. Sheng Pan,1,*Ruedi Aebersold,2,3 Ru Chen,4 John Rush,5 David R. Goodlett,6 Martin W. McIntosh,7 Jing Zhang,1 and Teresa A. Brentnall4.)

[0154] Pest-killing activity in plants In another embodiment, transgenic plants can be produced that express nematodetic proteins having pesticidal activity against nematode pests. For example, the methods described above can be used to produce transgenic plants, although the method by which the transgenic plant cells are produced is not important to the present invention. Methods known or described in the art, such as Agrobacterium-mediated transformation, particulate gun transformation, and non-particulate-mediated methods, may be used at the discretion of the experimenter. Plants expressing nematodetic proteins can be isolated by common methods described in the art, such as callus transformation, selection of transformed calluses, and regeneration of fertile plants from such transgenic calluses. In such processes, any gene can be used as a selection marker, insofar as its expression in plant cells confers the ability to identify or select transformed cells.

[0155] Many markers have been developed for use in plant cells, including those for resistance to chloramphenicol, aminoglycoside G418, and hygromycin. Other genes encoding products involved in chloroplast metabolism can also be used as selection markers. For example, genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone may find specific applications. Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314 (bromoxynyl-resistant nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res. 18:2188 (AHAS imidazolinone-resistant gene). Furthermore, the genes disclosed herein are useful as markers for evaluating the transformation of bacterial or plant cells. Methods for detecting the presence of transgenes in plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, reproductive bodies, embryos, or their offspring are well known in the art. In one embodiment, the presence of a transgene is detected by testing its pesticidal activity against nematode pests.

[0156] Fertile plants expressing nematicidal proteins can be tested for nematicidal activity against nematode pests, and plants exhibiting optimal activity have been selected for further breeding. A standard greenhouse method has been developed to evaluate nematode resistance in new soybean varieties. See Niblack, TL et al. A standard greenhouse method for assessing soybean cyst nematode resistance in soybean: SCE08 (Standardized Cyst Evaluation 2008). Plant Health Prog. 10, 33 (2009) (incorporated herein by reference). Using a modification of this standard protocol, nematicidal activity in fertile plants expressing nematicidal proteins can be demonstrated. See Kahn, TW, Duck, NB, McCarville, MT et al. A Bacillus thuringiensis Cry protein controls soybean cyst nematode in transgenic soybean plants. Nat Commun. 12, 3380 (2021). https: / / doi.org / 10.1038 / s41467-021-23743-3 (incorporated herein by reference).

[0157] The present invention 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, maize, sorghum, wheat, sunflower, tomato, cruciferous plants, peppers, potatoes, cotton, rice, soybeans, sugar beets, sugarcane, tobacco, barley, and rapeseed, Brassica sp., alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oat, vegetables, ornamental plants, and conifers.

[0158] Use in pest control General methods for using strains or variants thereof containing the nucleotide sequence of the present invention for genetic engineering purposes as pest control or pesticides of other organisms are known in the art. See, for example, U.S. Patent No. 5,039,523 and European Patent Application Publication No. 0480762A2.

[0159] Microorganisms can be genetically modified to contain nucleotide sequences encoding SEQ ID NOs: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54, or variants or fragments thereof having nematicidal activity, and the proteins can be used to protect crops and products from pests. In one embodiment of the present invention, whole cells of a toxin (pesticide)-producing organism, i.e., undissolved cells, are treated with a reagent that extends the activity of the toxin produced in the cells when the cells are applied to an environment of the target pest.

[0160] Alternatively, the pesticide may be produced by introducing a pesticide gene into a cell host. The expression of the pesticide gene directly or indirectly leads to the intracellular production and maintenance of the pesticide. In one embodiment of the present invention, these cells are then treated under conditions that extend the activity of the toxin produced in the cells when the cells are applied to an environment of the target pest. The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides can then be formulated according to the prior art for application to environments inhabited by the target pest, such as soil, water, and plant leaves. See, for example, European Patent No. 0192319 and the references cited therein. Alternatively, for example, cells expressing the gene of the present invention can be formulated so that the resulting substance can be applied as a pesticide.

[0161] The active ingredients of the present invention are typically applied in the form of compositions and can be applied to the crop area or plant being treated, either simultaneously or sequentially with other compounds. These compounds may be fertilizers, herbicides, cryoprotectants, surfactants, detergents, pesticide soaps, dormant spray oils, polymers, and / or sustained-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, virucidates, fungicides, amoebicides, pesticides, mycicides, bactericidal agents, nematicides, molluscicidal agents, or mixtures of some of these preparations, together with, if necessary, further agriculturally acceptable carriers, surfactants, or application-enhancing adjuvants conventionally used in formulation technology. Suitable carriers and adjuvants may be solid or liquid and correspond to substances commonly used in formulation technology, such as natural or recycled inorganic substances, solvents, dispersants, wetting agents, tackifiers, binders, or fertilizers. Similarly, the formulation may be prepared as an edible "bait," or it may be formed as a pest "trap" that allows the target pest of the pesticide to ingest or consume it.

[0162] Methods of applying the pesticide composition of the present invention, which contains the active ingredient of the present invention, or at least one of the nematicidal proteins disclosed herein as SEQ ID NOs: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54, or effective nematicidal variants or fragments thereof, include foliar application, seed treatment (coating), and soil treatment. The number of applications and the amount applied depend on the intensity of infestation by the corresponding pest.

[0163] The compositions can be formulated in dosage forms such as powder, dust, pellets, granules, spray, emulsion, colloid, or solution, and cell cultures containing polypeptides can be prepared by conventional means such as drying, freeze-drying, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration. In all such compositions containing at least one such pest control polypeptide, the polypeptide may be present at a concentration of about 1% to about 99% by weight.

[0164] Nematode pests can be killed or reduced in number in a given area by the method of the present invention, or can be applied preventively to an environmental area to prevent parasitism by susceptible pests. Preferably, the pests ingest or come into contact with a pesticidal amount of polypeptide.

[0165] The described pest-killing compositions may be prepared by formulating bacterial cells, crystals and / or spore suspensions, or isolated protein components with a desired agriculturally acceptable carrier. The compositions may be formulated before administration by appropriate means such as lyophilization, freeze-drying, or drying, or in an aqueous carrier, medium, or suitable diluent such as physiological saline or other buffer. The formulated compositions may be in the form of a powder or granular material, a suspension in oil (vegetable oil or mineral oil), or a water or oil / water emulsion, or a wettable powder, or in combination with any other carrier material suitable for agricultural use. Suitable agricultural carriers may be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" encompasses all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc., commonly used in pest-killing technology, which are well known to those skilled in the art. Formulations can be prepared by mixing with one or more solid or liquid adjuvants and by various means, for example, by using conventional formulation techniques, homogeneously mixing, blending and / or grinding the pesticide composition with suitable adjuvants. Suitable formulations and methods of application are described in U.S. Patent No. 6,468,523, which is incorporated herein by reference.

[0166] Methods to increase plant yield A method for increasing plant yield is provided. The method comprises providing a plant or plant cells expressing a polynucleotide encoding a nematicidal polypeptide sequence disclosed herein, and cultivating the plant or its seeds in a field infested with (or susceptible to infestation by) a nematode pest having nematicidal activity in which the polypeptide is nematicidal. In some embodiments, the Cry21 polypeptide described herein has nematicidal activity against Pratylenchus spp., and the field is infested with Pratylenchus spp. In various embodiments, Pratylenchus spp. is pineapple root-knot nematode (Pratylenchus brachyurus) or Pratylenchus Zeae. In further embodiments, the nematode is root-knot nematode, lesion nematode, soybean cyst nematode, or Lance nematode or target pest.

[0167] As defined herein, the term “yield” refers to the quality and / or quantity of biomass produced by a plant. By “biomass,” any plant product being measured is intended. An increase in biomass product is any improvement in the yield of a measured plant product. Increases in plant yield have several commercial applications. For example, an increase in plant leaf biomass can increase the yield of leafy vegetables for human or animal consumption. In addition, an increase in leaf biomass can be used to increase the production of plant-derived pharmaceutical or industrial products. An increase in yield can include, but is not limited to, an increase 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 a plant that does not express the pesticide proteins disclosed herein. In certain ways, plant yield increases as a result of improved nematode resistance in plants expressing the nematode proteins disclosed herein. The expression of nematicidal proteins results in a reduction in the ability of pests to parasitize or feed on plants. In various embodiments, the expression of nematicidal proteins results in improved plant stress management, including improved root development (e.g., improved root or root hair growth), improved yield, faster sprouting, increased stress tolerance and / or improved recovery from stress, increased mechanical strength, improved drought resistance, reduced fungal disease infection, and improved plant health, compared to plants that do not express the nematicidal proteins of the present invention.

[0168] Plants may also be treated with one or more chemical compositions comprising one or more herbicides, insecticides, or fungicides. Examples of such chemical compositions include: Fruit / vegetable herbicides: atrazine, bromacil, diuron, glyphosate, linuron, metrivudine, simazine, trifluralin, fluazifop, glufosinate, halosulfuron-gowan, paraquat, propizamide, cethoxydim, butafenacil, halosulfuron, indadiphram.

[0169] Fruit / vegetable insecticides: Aldicarb, Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Cyfluthrin / Beta-Cyfluthrin, Esfenvalerate, Lambda-Cyhalotrin, Acequinosyl, Bifenazate, Methoxyfenozide, Novalon, Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim, Spirodiclofen, Gamma-Cyhalotrin, Spiromesifen, Spinosad, Linaki Cypir, thiadipir, triflumulon, spirotetramat, imidacloprid, flubendiamide, thiodicarb, metaflumizone, sulfoxaflor, cyflumetofen, cyanopyrafen, clothianidin, thiamethoxam, spinotoram, thiodicarb, flonicamide, methiocarb, emamectin benzoate, indoxacarb, phenamifos, pyriproxyfen, fenbutane oxide; fruit / vegetable fungicides: ametoctrazine, azo Xystrobin, Benciavaricarb, Boscalid, Captan, Carbendazim, Chlorothalonil, Copper, Cyazofamide, Cyflufenamid, Cymoxanil, Cyproconazole, Cyprodinil, Difenoconazole, Dimethomorph, Dithianone, Phenamidon, Fenhexamide, Fluazinam, Fludioxonil, Fluopicolid, Fluopyram, Fluoxastrobin, Fluxapiroxad, Forpet, Fosetil, Iprodione, Iprovarica Lube, isopyrazam, kresoximmethyl, mancozeb, mandipropamide, metalaxyl / mephenoxam, methylam, metraphenone, mycrobutanil, penconazole, penthiopyrad, picoxystrobin, propamocarb, propiconazole, propineb, proquinazide, prothioconazole, pyraclostrobin, pyrimethanil, quinoxyfen, spiroxamine, sulfur, tebuconazole, thiophanate-methyl, trifloxystrobin.

[0170] Grain herbicides: 2,4-D, amidosulfuron, bromoxynil, carfentrazone-E, chlorotoluron, chlorsulfuron, clodinahop-P, clopyralide, dicamba, diclohop-M, diflufenican, phenoxaprop, floraslam, flucarbazone-NA, flufenacet, flupyrosulfuron-M, fluroxypyr, flurutamon, glyphosate, iodosulfuron, ioxinil, isoproturon, MCPA, mesosulfuron, metosulfuron, pendimethalin, pinoxadene, propoxycarbazone, prosulfocarb, pyroximam, sulfosulfuron, thifensulfuron, tralcoxidime, triasulfuron, tribenulon, trifluralin, tritosulfuron.

[0171] Grain fungicides: Azoxystrobin, bixafen, boscalid, carbendazim, chlorothalonil, cyflufenamide, cyproconazole, cyprodinil, dimoxystrobin, epoxyconazole, fenpropidine, fenpropimorph, fluopyram, fluoxastrobin, fluquinconazole, fluxapyroxad, isopyrazam, kresoximmethyl, metconazole, metraphenone, penthiopyrad, picoxystrobin, prochloraz, propiconazole, proquinazide, prothioconazole, pyraclostrobin, quinoxyfen, spiloxamine, tebuconazole, thiophanate-methyl, trifloxystrobin.

[0172] Grain insecticides: Dimethoate, lambda-cyhalothrin, deltamethrin, alpha-cypermethrin, beta-cyfluthrin, bifenthrin, imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinotefuran, chlorpyrifos, pyrimicarb, methiocarb, sulfoxaflor. Corn herbicides: Atrazine, alachlor, bromoxynil, acetochlor, dicamba, clopyralid, (S-)dimethenamide, glufosinate, glyphosate, isoxaflutol, (S-)metholchlor, mesotrione, nicosulfuron, primisulfuron, limsulfuron, sulcotrione, horamsulfuron, topramezon, tembotrione, saflufenacil, thiencarbazone, flufenacet, pyroxasulfone.

[0173] Corn insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumulon, Linaxipir, Deltamethrin, Thiodicarb, Beta-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Tebupyrimfos, Ethiprol, Thiadipir, Thiacloprid, A Cetamiprid, dinotefuran, avermectin; Maize fungicides: azoxystrobin, bixafen, boscalid, cyproconazole, dimoxystrobin, epoxyconazole, fenitropan, fluopyram, fluoxastrobin, fluxapiroxad, isopyrazam, metconazole, penthiopyrad, picoxystrobin, propiconazole, prothioconazole, pyraclostrobin, tebuconazole, trifloxystrobin.

[0174] Rice herbicides: Butachlor, propanil, azimsulfuron, bensulfuron, cyhalofop, dimuron, fentrazamide, imazosulfuron, mefenacet, oxadiclomefone, pyrazosulfuron, pyributilob, quinchlorac, thiobencarb, indanophan, flufenacet, fentrazamide, halosulfuron, oxadiclomefone, benzobicyclon, pyrifthalide, penoxulam, bispyrivac, oxaziargyl, ethoxysulfuron, pretilachlor, mesotrione, tefuryltrione, oxadiazone, phenoxaprop, pyrimisulfan.

[0175] Rice insecticides: Diazinon, Phenobucarb, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Clothianidin, Ethiprol, Flubendiamide, Linaxipr, Deltamethrin, Acetamiprid, Thiamethoxam, Thiazipr, Spinosad, Spinotram, Emamectin benzoate, Cypermethrin, Chlorpyrifos, Etofenprox, Carbofuran, Benfuracarb, Sulfoxaflor.

[0176] Rice fungicides: Azoxystrobin, carbendazim, carpropamide, diclocimet, difenoconazole, edifenfos, ferimzon, gentamicin, hexaconazole, himexazole, ipropenfos (IBP), isoprothiolane, isothianil, kasugamycin, mancozeb, metminostrobin, orysastrobin, penciclon, probenazole, propiconazole, propineb, pyroquilon, tebuconazole, thiophanate-methyl, thiadinil, tricyclazole, trifloxystrobin, validamycin.

[0177] Cotton herbicides: Diuron, fluomethron, MSMA, oxyfluorfen, promethrin, trifluralin, carfentrazone, cretodym, fluazihop-butyl, glyphosate, norflurazone, pendimethalin, pyrithiobac sodium, trifloxysulfuron, tepraloxidim, glufosinate, flumioxazine, tidiazulon; Cotton insecticides: Acephate, aldicarb, chlorpyrifos, cypermethrin, deltamethrin, abamectin, acetamiprid, emamectin benzoate, imidacloprid, indoxacarb, lambda-cyhalothrin, spinosad, thiodicarb, gamma-cyhalothrin, spiromesifen, pyridaryl, flonicamide Flubendiamide, triflumulone, linaxipill, beta-cyfluthrin, spirotetramat, clothianidin, thiamethoxam, thiacloprid, dinotefuran, flubendiamide, thiadipyr, spinosad, spinotoram, gamma-cyhalothrin, 4-[(6-chloropyridine-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, thiodicarb, avermectin, flonicamide, pyridaryl, spiromesifen, sulfoxaflor.

[0178] Cotton fungicides: Azoxystrobin, bixafen, boscalid, carbendazim, chlorothalonil, copper, cyproconazole, difenoconazole, dimoxystrobin, epoxyconazole, phenamidon, fluazinam, fluopyram, fluoxastrobin, fluxapyroxad, iprodione, isopyrazam, isothianil, mancozeb, maneb, metominostrobin, penthiopyrad, picoxystrobin, propineb, prothioconazole Pyraclostrobin, Quintozene, Tebuconazole, Tetraconazole, Thiophanate-methyl, Trifloxystrobin; Soybean herbicides: Alachlor, Bentazone, Trifluralin, Chlorimulon-ethyl, Chloransrum-methyl, Phenoxaprop, Homesaphen, Fluazihop, Glyphosate, Imazamox, Imazakine, Imazetapir, (S-) Metrolchlor, Metrivudine, Pendimethalin, Tepraloxidim, Glufosinate.

[0179] Soybean insecticides: Lambda-cyhalothrin, Methomyl, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinotefuran, Flubendiamide, Linaxipil, Thiazipil, Spinosad, Spinotram, Emamectin benzoate, Fipronil, Ethiprol, Deltamethrin, Beta-cyfluthrin, Gamma and Lambda-cyhalothrin, 4-[(6-chloropyridine-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, Spirotetramat, Spirodiclofen, Triflumulon, Flonicamide, Thiodicarb, Beta-cyfluthrin.

[0180] Soybean fungicides: Azoxystrobin, bixafen, boscalid, carbendazim, chlorothalonil, copper, cyproconazole, difenoconazole, dimoxystrobin, epoxyconazole, fluazinam, fluopyram, fluoxastrobin, flutriafor, fluxapiroxad, isopyrazam, iprodione, isothianil, mancozeb, maneb, metconazole, metminostrobin, mycrobutanil, penthiopyrad, picoxystrobin, propiconazole, propineb, prothioconazole, pyraclostrobin, tebuconazole, tetraconazole, thiophanate-methyl, trifloxystrobin.

[0181] Sugar beet herbicides: Chloridazon, Desmedifam, Etofmesart, Fenmedifam, Trialate, Clopyralid, Fluazihop, Renasil, Metamitron, Kimmelac, Cycloxidim, Triflusulfuron, Tepraloxidim, Quizalohop; Sugar beet insecticides: Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinotefuran, Deltamethrin, Beta-Cyfluthrin, Gamma / Lambda-Cyhalothrin, 4-[(6-Chloropyridine-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, Tefluthrin, Linaxipyr, Thiazipyr, Fipronil, Carbofuran.

[0182] Canola herbicides: Clopyralid, diclofop, fluazifop, glufosinate, glyphosate, metazachlor, trifluralin, etametsulfuron, kinmelac, quizalofop, cretodym, tepraloxidim; Canola fungicides: Azoxystrobin, bixafen, boscalid, carbendazim, cyproconazole, difenoconazole, dimoxystrobin, epoxyconazole, fluazinam, fluopyram, fluoxastrobin, flusilazole, fluxapyroxad, iprodione, isopyrazam, mepicort chloride, metconazole, metminostrobin, paclobutrazol, penthiopyrad, picoxystrobin, prochloraz, prothioconazole, pyraclostrobin, tebuconazole, thiophanate-methyl, trifloxystrobin, vinclozolin.

[0183] Canola insecticides: Carbofuran, thiacloprid, deltamethrin, imidacloprid, clothianidin, thiamethoxam, acetamiprid, dinotefuran, beta-cyfluthrin, gamma and lambda-cyhalothrin, tau-fluvalinate, ethiprole, spinosad, spinotoram, flubendiamide, linaxipyr, thiadipyr, 4-[(6-chloropyridine-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one.

[0184] The following embodiments are provided as examples, not as limitations. [Examples]

[0185] Example 1. Expression of the nematode-causing Cry21 gene in soybeans Soybean events expressing novel Cry21 homologs (SEQ ID NO: 2 (Axmi296) and SEQ ID NO: 54 (Cry21Aa2), respectively) were developed by Agrobacterium-mediated transformation of Thorne soybean plants using a construct containing a 4-hydroxyphenylpyruvate dioxygenase protein (HPPD) inhibitor-resistant herbicide gene (described in International Publication No. 2014 / 043435) and the Cry21 coding sequence. Wild-type Thorne soybeans served as a non-nematode-resistant control. When expressed in soybean plants, Cry21 reduces the number of pineapple root-knot nematodes (Pratylenchus brachyurus) proliferating in the roots compared to wild-type plants. This particular Cry21 (SEQ ID NO: 2) gene shares approximately 65% ​​sequence homology with Cry21Aa. The alignment of Cry21 presented in this application with known Cry genes, including Cry21s, represents a potentially novel or different mechanism of action that can be combined with other nematode resistance genes to control nematodes.

[0186] Figure and table showing plant transformation vectors for Axmi296 (SEQ ID NO: 2) expression in plants. Figure 1 shows a plant transformation vector for the expression of Axmi296 in plants.

[0187] Table 1 shows a description of the genetic elements of the construct.

[0188] Table 2 lists references regarding the genetic elements of the constructs.

[0189] [Table 1-1]

[0190] [Table 1-2]

[0191] [Table 2-1]

[0192] [Table 2-2]

[0193] Figure 2 shows the NEEDLE sequence alignments of Axmi296 and Cry21Aa2. The "DRIE" motif, which separates the N-terminal domain from the C-terminal crystalline domain, is indicated by "*". Identical amino acids are shaded in black, and the consensus sequence (Cons) is shown below the aligned sequence. Overall, there is 66% sequence identity between Axmi296 and Cry21Aa2 (74% sequence identity if gaps are ignored). The internal data suggest that both of these genes are active against nematodes, and this is not limited by theory, but the following alignments suggest that the two proteins likely utilize the same mechanism of action against plant nematodes.

[0194] Example 2. Soybean transformation Soybean transformation was achieved using methods well known in the art, such as the method using Agrobacterium tumefaciens-mediated transformed soybean half-seed explants, essentially using the method described in Paz et al. (2006), Plant Cell Rep. 25:206. Transformants were identified using tenbotrione as a selection marker. The appearance of green shoots was observed and recorded as an indicator of resistance to the herbicides isoxaflutol or tenbotrione. Resistant transgenic shoots showed normal greening comparable to wild-type soybean shoots not treated with isoxaflutol or tenbotrione, while wild-type soybean shoots treated with the same amount of isoxaflutol or tenbotrione were completely bleached. This indicates that the presence of HPPD protein enables resistance to HPPD inhibitory herbicides such as isoxaflutol or tenbotrione.

[0195] Transplant resistant green shoots into a rooting medium or graft them. After an acclimatization period, move the rooted plantlets to a greenhouse. In the transformation medium, select plants containing the transgene.

[0196] Example 3. Greenhouse and field trials Overall, the soybean events of the novel Cry21 homolog and Cry21Aa2 were tested in greenhouses and in eight field trials against RKN, SCN, and pineapple root-knot nematodes (Pratylenchus brachyurus) in the Midwestern United States and Brazil. Exemplary field trial results are shown below.

[0197] Figures 3a and 3b show the effectiveness of Axmi296 against pineapple root-knot nematode (Pratylenchus brachyurus). Greenhouse data show increased mortality of pineapple root-knot nematode (Pratylenchus brachyurus) compared to Thorne WT (wild type, non-resistant control), and a slight advantage over soybean Cry14 (known resistant control).

[0198] Figures 4a and 4b show that Axmi296 yields increased yield benefits compared to SCN-unresistant controls in SCN field trials (three independent locations).

[0199] Figure 4c shows the results of a field trial in which the novel Cry21 homolog (Axmi296) reduced root SCN cysts by 20% compared to wild-type Thorne.

[0200] Figure 4d shows the results of a field trial in an SCN-infested field, in which the novel Cry21 homolog (Axmi296) resulted in an average yield increase of approximately 2% compared to wild-type Thorne.

[0201] Figure 5 shows an Axmi296 plant (left) next to a non-resistant control soybean plant (right) in an SCN parasitic field.

[0202] Table 3 shows novel Cry21 homologs and further Cry21 homologous sequences. Protein sequence identity to the nearest Cry holotype is determined by pairwise alignment according to Crickmore et al., 2021.

[0203] [Table 3]

[0204] Table 4 shows the sequence listing of the novel Cry21 homolog sequences disclosed herein.

[0205] [Table 4-1]

[0206] [Table 4-2]

[0207] [Table 4-3]

[0208] [Table 4-4]

[0209] [Table 4-5]

[0210] [Table 4-6]

[0211] [Table 4-7]

[0212] Table 4-8

[0213] Table 4-9

[0214] Table 4-10

[0215] Table 4-11

[0216] Table 4-12

[0217] Table 4-13

[0218] Table 4-14

[0219] Table 4-15

[0220] Table 4-16

[0221] Table 4-17

[0222] Table 4-18

[0223] Table 4-19

[0224] Table 4-20

[0225] Table 4-21

[0226] Table 4-22

[0227] Table 4-23

[0228] Table 4-24

[0229] Table 4-25

[0230] Table 4-26

[0231] Table 4-27

[0232] Table 4-28

[0233] Table 4-29

[0234] Table 4-30

[0235] Table 4-31

[0236] Table 4-32

[0237] Table 4-33

[0238] Table 4-34

[0239] Table 4-35

[0240] Table 4-36

[0241] Table 4-37

[0242] Table 4-38

[0243] Table 4-39

[0244] Table 4-40

[0245] Table 4-41

[0246] Table 4-42

[0247] Table 4-43

[0248] Table 4-44

[0249] Table 4-45

[0250] Table 4-46

[0251] Table 4-47

[0252] Table 4-48

[0253] [Table 4-49]

[0254] [Table 4-50]

[0255] [Table 4-51]

[0256] All publications and patent applications referenced herein represent the level of skill of those skilled in the art to which the present invention pertains. All publications and patent applications are incorporated herein by reference as is specifically and individually indicated, as if each individual publication or patent application were incorporated by reference.

[0257] Although the aforementioned invention has been described in some detail with reference to figures and examples to clarify its understanding, it will be clear that certain modifications and alterations can be made within the scope of the attached claims.

Claims

1. A method for controlling nematode pests, comprising contacting the nematode pests with a Cry21 protein containing an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of Sequence IDs 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, or 53, or a variant or fragment thereof that is effective in killing nematodes.

2. The method according to claim 1, wherein the nematode pest is selected from any one of the following species: Criconemella, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Meloidogyn, Paratrichodorus, Platylenchus, Radolpholus, Rotelynchus, Rotylenchulus, Tylenchulus, and Xiphinema.

3. The method according to claim 1 or 2, wherein the nematode pest is the target pest and / or the pineapple root-knot nematode (Pratylenchus brachyurus).

4. The method according to any one of claims 1 to 3, wherein the nematically effective variants or fragments include at least 10, 20, 30, 40, 50, or 60 consecutive amino acids of SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, or 53.

5. A nucleic acid encoding a protein according to any one of claims 1 to 4.

6. A plant or plant part expressing the nucleic acid described in claim 5 in its cells.

7. The aforementioned plants or plant parts include: Criconemella, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Meloidogyn, and Paratrichodus. The plant or plant part according to claim 6, which can be parasitized by nematode pests from any one of the species consisting of odorus, Platylenchus, Radolphorus, Rotelynchus, Rotylenchulus, Tylenchulus, and Xiphinema.

8. The plant or plant part according to claim 6 or 7, wherein the plant or plant part can be parasitized by target pests and / or pineapple root-knot nematode (Pratylenchus brachyurus).

9. The plant or plant part according to any one of claims 6 to 8, wherein the plant is dicotyledonous or monocotyledonous.

10. The aforementioned plants include alfalfa, apples, apricots, Arabidopsis thaliana, artichokes, asparagus, avocados, bananas, barley, legumes, beets, blackberries, blueberries, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrots, cassava, cauliflower, grains, celery, cherries, citrus fruits, clementines, coffee, corn, cotton, cucumbers, eggplants, endive, eucalyptus, figs, grapes, grapefruit, peanuts, ground cherries, kiwifruit, lettuce, leeks, lemons, limes, pine, maize, mangoes, melons, millet, mushrooms, and nuts. A plant or plant part according to any one of claims 6 to 9, selected from the group consisting of genetically modified plants selected from the group consisting of oats, okra, onions, oranges, ornamental plants or flowers or trees, papaya, parsley, peas, peaches, peanuts, peat, pepper, persimmons, pineapples, plantains, plums, pomegranates, potatoes, pumpkins, radicchio, radishes, rapeseed, raspberries, rice, rye, sorghum, soybeans, spinach, strawberries, sugar beets, sugarcane, sunflowers, sweet potatoes, tangerines, tea, tobacco, tomatoes, climbing plants, watermelons, wheat, yams, and zucchini.

11. The plant or plant part according to any one of claims 6 to 10, wherein the plant or plant part is a soybean, a Brassica, or a maize.

12. The plant or plant part according to any one of claims 6 to 11, wherein the plant is a transgenic plant or a transgenic plant part.

13. An expression cassette comprising nucleic acids encoding the Cry21 protein, which include amino acid sequences having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, and 100% sequence identity with any one of sequence numbers 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, and 53, or nematically effective variants or fragments thereof.

14. The expression cassette according to claim 13, wherein the nucleic acid is operably linked to the nucleic acid.

15. The expression cassette according to claim 13 or 14, wherein the expression cassette is located within a plant expression vector.

16. A cell comprising an expression cassette according to any one of claims 13 to 15.

17. A cell comprising an expression cassette according to any one of claims 13 to 15 and another expression cassette containing a second Cry gene.

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

19. A nematically effective fusion protein containing any one of sequence numbers 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, or 53.

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

21. A method for controlling the pineapple root-knot nematode (Pratilenchus brachyurus), comprising the expression of Cry21, Cry21Aa, Cry21Aa2, or Cry21Aa1 proteins in a plant, wherein the proteins come into contact with the pineapple root-knot nematode (Pratilenchus brachyurus) pest, thereby controlling the pineapple root-knot nematode (Pratilenchus brachyurus) pest.

22. The method according to claim 21, wherein the Cry21, Cry21Aa, Cry21Aa2, or Cry21Aa1 protein comprises an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, or 53, or a nematically effective variant or fragment thereof.

23. The method according to claim 21 or 22, wherein the plant is a soybean plant.

24. nucleic acids encoding proteins, comprising amino acid sequences having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of sequence numbers 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, or 53, or nematically effective variants or fragments thereof.

25. The nucleic acid according to claim 24, wherein the nucleic acid comprises a nucleic acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, and 100% sequence identity with respect to any one of sequence numbers 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, or 54.

26. The nucleic acid according to claim 24 or 25, wherein the nucleic acid sequence includes any one of sequence numbers 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, or 54.

27. A plant, plant cell, or plant part expressing the nucleic acid described in any one of claims 24 to 26 within its cells.

28. The plant, plant cell, or plant part according to claim 27, wherein the plant is a soybean.

29. An expression cassette comprising the nucleic acid according to any one of claims 24 to 26.

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

31. The expression cassette according to claim 29 or 30, wherein the promoter is a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

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

33. A cell comprising an expression cassette according to any one of claims 29 to 32.

34. The cell according to claim 23, wherein the cell is a bacterial cell, a plant cell, a yeast cell, or an insect cell.

35. A vector comprising an expression cassette according to any one of claims 29 to 31.

36. A cell comprising the vector described in claim 35.

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

38. A plant comprising an expression cassette according to any one of claims 29 to 32, wherein the promoter can drive the expression of the protein to a level sufficient to inhibit a target pest and / or pineapple root-knot nematode (Pratilenchus brachyurus), and the proliferation of the target pest and / or pineapple root-knot nematode (Pratilenchus brachyurus) on the plant is reduced compared to the proliferation of the target pest and / or pineapple root-knot nematode (Pratilenchus brachyurus) on the plant compared to the proliferation of the target pest and / or pineapple root-knot nematode (Pratilenchus brachyurus) on the plant.

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

40. A method for controlling nematode pests, comprising contacting the nematode pests with a Cry21 protein containing an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of Sequence IDs 1, 3, 7, 9, 11, 13, 15, 17, 21, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, or 53, or a variant or fragment thereof that is effective in controlling nematodes.

41. The method according to claim 40, wherein the nematode pest is selected from any one of the following species: Criconemella, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Meloidogyn, Paratrichodorus, Platylenchus, Radolpholus, Rotelynchus, Rotylenchulus, Tylenchulus, and Xiphinema.

42. An expression cassette comprising nucleic acids encoding the Cry21 protein, which include amino acid sequences having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, and 100% sequence identity with any one of sequence numbers 1, 3, 7, 9, 11, 13, 15, 17, 21, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and 53, or nematically effective variants or fragments thereof.

43. The expression cassette according to claim 42, wherein the nucleic acid is operably linked to the nucleic acid.

44. The expression cassette according to claim 42 or 43, wherein the expression cassette is located within a plant expression vector.

45. The nucleic acid according to claim 42, wherein the nucleic acid comprises a nucleic acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, and 100% sequence identity with respect to any one of sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, or 54.

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