Use of CRY gene for controlling nematode pests

Abstract

Compositions and methods for conferring nematicidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Further provided are methods for killing or controlling a population of nematode pests, in particular Pratylenchus species, the present invention relates to a population of nematodes, such as Pratylenchus brachycaeus, Pratylenchus punctatus, Pratylenchus schriberi, Pratylenchus neglectus, Pratylenchus zeae, Blonolaimus longicaudatus, Hoplolaimus galeae, Helicida dihysteria, Helicida pseudomonas, or Xiphinea americana, a root-knot nematode, a reniform nematode, a soybean cyst nematode, or a spear nematode population, for example, a population of nematodes, such as Pratylenchus brachycaeus, Pratylenchus sp., Pratylenchus sp. The methods comprise contacting the nematode pest with a pesticidally effective amount of a polypeptide comprising a nematicidal toxin. Further included are methods of increasing plant yield by expressing a gene of an embodiment in a plant.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of molecular biology. Methods for controlling nematode pests using novel pest genes are provided. SUBMISSION OF SEQUENCE LISTING

[0002] The sequence listing associated with this application is filed in electronic format via Patent Center and is hereby incorporated by reference in its entirety. The name of the “xml” file containing the sequence listing is 230175US02_Sequencelisting.xml. The size of the xml file is 177 KB, and the xml file was generated on August 21, 2023. BACKGROUND

[0003] Plant pests are a major factor in the loss of important crops worldwide. Some estimates claim that 40% of crop production is lost globally due to infestation by invertebrate pests, including nematodes. In addition to losses in field crops, nematode pests are also a burden to vegetable and fruit growers, ornamental flower producers, and home gardeners. Nematodes that infect plants, most of which are root feeders, are associated with most plants. Some nematodes are endoparasitic, living and feeding within tissues of roots, tubers, shoots, seeds, and the like. Other nematodes are ectoparasitic, feeding externally through the plant wall. A single endoparasitic nematode can kill a plant or reduce its productivity. Endoparasitic root feeders include economically important pests such as root knot nematodes (Meloidogyne species), reniform nematodes (Rotylenchulus species), cyst nematodes (Heterodera species), and root-lesion nematodes (Pratylenchus species).

[0004] Damage by nematodes can severely reduce nutrient and water uptake by plants. Nematodes have their greatest impact on crop productivity when they attack the roots of seedlings immediately after germination. Nematode feeding also creates open wounds that provide entry for a variety of plant pathogenic fungi and bacteria. These microbial infections can be more economically devastating than the direct effects of nematode feeding.

[0005] Cyst nematodes are a cause of direct loss of soybean yield and indirect losses due to the cost of killing pests and non-optimal use of land for crop rotation. The annual negative economic impact of the soybean cyst nematode (Heterodera glycines) in North America can exceed $1.5 billion. Cyst nematodes at economically damaging densities typically cause crop plants to be stunted. Stunted plants have smaller root systems, leaf symptoms of mineral deficiency, and are prone to wilt.

[0006] Traditional practices for managing nematode infestations include maintaining proper fertility and soil pH levels on nematode-infested land; controlling plant diseases that foster nematode infestation, as well as insect and weed pests; using sanitation measures such as tilling, planting, and cultivating of nematode-infested fields only after working on non-infested fields; thoroughly cleaning equipment after working on infested fields; not using seeds of plants grown on infested land for planting on non-infested fields unless properly cleaned; and crop rotation of infested fields, alternating host crops with non-host crops such as corn, oats, and alfalfa, and planting resistant or tolerant plant varieties. While many of these practices can be effective, they are both time-consuming and expensive to implement. Nematodes are difficult to control without the use of chemical pesticides or fumigants (e.g., nematicides), and these methods are accompanied by many challenges, such as nematode resistance, regulatory restrictions, etc.

[0007] Due to the devastating harm that nematodes can cause and the continued threat of pest resistance to current control measures, there is a constant need to discover new methods for controlling nematode plant pests that provide economic benefit to growers and are environmentally acceptable and safe. SUMMARY

[0008] Various embodiments provide new methods for controlling economically important nematode pests. In particular, transgenic plants and / or plant parts expressing one or more polypeptides of the embodiments are found to be capable of inhibiting the ability of nematode pests to survive, grow, and reproduce, or to limit nematode-related damage or loss to crop plants. One embodiment further relates to transgenic nematode-resistant plants expressing any one or more of the proteins, and methods of using the transgenic plants alone or in combination with other nematode control strategies to impart 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] According to one aspect, a method for controlling nematode pests is provided, the method comprising contacting the nematode pest with a protein comprising any one of SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof. In another aspect, the protein may be a Cry21, Cry21Aa, Cry21Aa2, or Cry21Aa1 protein, for example as demonstrated by SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 or their nematode-active fragments. Cry21, Cry21Aa2, or Cry21Aa1 protein homologs having at least about 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, or any functional fragment thereof.

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

[0011] On the other hand, this nematode pest is selected from the group consisting of: *Criconemella*, *Ditylenchus*, *Globodera*, *Helicotylenchus*, cyst nematodes, *Longidorus*, root-knot nematodes, *Paratrichodorus*, short-bodied nematodes, *Radolpholus*, *Rotelynchus*, kidney-shaped nematodes, *Tylenchulus*, and *Xiphinema*. Such nematode pests selected from these genera can be cyst-forming nematodes. On the other hand, these cyst-forming nematodes belong to the genus *Heterodera*. On yet another hand, this nematode pest is the soybean cyst nematode (*Heterodera glycines*, *soybean cyst nematode*). In one embodiment, the nematode pest is the shortest-tailed, short-bodied nematode (Pratylenchus brachyurus), but not SCN and / or both.

[0012] On the other hand, the genetically modified plant or plant part was selected from the following groups: alfalfa, apple, apricot, Arabidopsis, artichoke, asparagus, avocado, banana, barley, legumes, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, cereal, celery, cherry, citrus, Clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, fig, grape, and grape. Grapefruit, groundnut, ground cherry, kiwi, lettuce, leeks, lemon, lime, pine, corn, mango, melon, millet, mushroom, nuts and oats, okra, onion, orange, ornamental plants or flowers or trees, papaya, parsley, peas, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soybean, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, vine, watermelon, wheat, yam, and zucchini. On the other hand, the genetically modified plant or plant part is a soybean plant or plant part.

[0013] On the other hand, the plant part is the root. On yet another hand, the root is the soybean root.

[0014] In another aspect, the Cry21 protein is Cry21Aa, Cry21Aa1, Cry21Aa2, or rCry21Ba protein. In yet another aspect, the Cry21 protein contains an amino acid sequence that is the translation product of a nucleotide sequence, the complementary sequence of which hybridizes under highly stringent conditions with SEQ ID NO: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, or 54.

[0015] In another aspect, the Cry21 protein of the present invention comprises SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, or its nematode active 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 with SEQ ID NO: 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 NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, or a nematode active homolog having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1, 7, 9, 13, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof.

[0016] In another aspect, the transgenic plants of the present invention further comprise or express at least one additional pest control agent, such as, but not limited to, patatin, Bacillus thuringiensis insecticidal protein, Bacillus thuringiensis nematode-killing protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, Bacillus sphearicus insecticidal protein, VIP3, and / or RNAi molecules targeting nematode pests. In another aspect, the Bacillus thuringiensis nematode-killing protein is selected from the group consisting of Cry5, Cry6, Cry13, Cry14, Cry21, and Cry55.

[0017] According to another aspect, a method for conferring nematode resistance to plants and / or plant parts is provided, the method comprising inserting a heterologous nucleic acid molecule encoding a Cry21 protein into the plant and / or plant part, wherein the plant and / or plant part expresses the Cry21 protein at a nematode-inhibiting level, thereby conferring nematode resistance to the plant and / or plant part compared to the same type of plant and / or plant part that does not express the Cry21 protein. Such insertion can occur via transformation, gene editing, or through breeding.

[0018] According to another aspect, a method is provided for conferring resistance to the shortest-tailed nematode in plants and / or plant parts, the method comprising inserting a heterologous nucleic acid molecule encoding a 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-inhibiting level, so as to confer resistance to the shortest-tailed nematode in 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 can occur via transformation, gene editing, or through breeding.

[0019] On the other hand, the Cry21 protein comprises SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, or its shortest-tailed nematode active homolog that has at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 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 NO: 1, 7, 9, 13, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof, or an active homolog of the shortest-tailed nematode nematode with at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof.

[0020] According to another aspect, a method for reducing the infectivity of nematodes to plants and / or plant parts is provided, the method comprising contacting the nematode with the Cry21 protein, wherein the infectivity of the nematode to the plant and / or plant parts is reduced compared to the infectivity of the nematode to the plant and / or plant parts that has not been contacted with the Cry21 protein.

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

[0022] On the other hand, a method is provided for producing soybean plants resistant to nematode infestation, the method comprising transforming soybean plant cells with a nucleic acid molecule encoding a Cry21 protein; and regenerating the transformed soybean plant from the soybean plant cells, wherein the transformed plant is resistant to nematode infestation.

[0023] According to another aspect, a method for producing soybean plants resistant to nematode infestation is provided, the method comprising hybridizing a first parent soybean plant with a second parent soybean plant, wherein the first or second parent soybean plant contains a heterologous nucleic acid molecule encoding the Cry21 protein of the present invention, thereby producing a plurality of progeny plants; and selecting transgenic plants resistant to nematode infestation from the plurality of progeny plants.

[0024] On the other hand, a method is provided to reduce nematode cyst development on the roots of plants that can be infected by nematodes, the method comprising introducing a nucleic acid molecule capable of directing the expression of the Cry21 protein into the cells of the root, thereby reducing nematode cyst development on the roots of the plant.

[0025] In another aspect of the invention, a method for reducing nematode cyst development on the roots of plants susceptible to nematode infection is provided, the method comprising introducing a nucleic acid molecule capable of directing the expression of the Cry21 protein to the roots into plant cells, thereby reducing nematode cyst development on the roots of the plant.

[0026] On the other hand, a method for controlling or preventing nematode growth is provided, which includes providing the nematode pest with plant material containing heterologous DNA capable of directing the expression of the Cry21 protein, wherein the plant inhibits the nematode's biological activity.

[0027] On the other hand, a method is provided for providing growers with a means of controlling nematode pests, the method comprising supplying growers with seeds containing a heterologous nucleic acid molecule encoding the Cry21 protein, and wherein the seeds are capable of producing plants resistant to nematode infection.

[0028] On the other hand, a method is provided for suppressing the growth of a plant-pathogenic nematode population in a location capable of supporting its growth, the method comprising growing a transgenic soybean plant population containing a heterologous nucleic acid molecule capable of directing the expression of the Cry21 protein in the location, wherein the growth of the plant-pathogenic nematode population is suppressed.

[0029] On the other hand, a method is provided for controlling any of soybean cyst nematodes, disease nematodes, root-knot nematodes, or kidney nematodes (“target pests”), the method comprising providing a transgenic soybean plant containing an expression cassette having any of SEQ ID No: 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 an encoded Cry21 protein to a level sufficient to inhibit nematodes, wherein the proliferation of target pests feeding on the soybean plant is reduced compared to target pests feeding on non-transgenic soybean plants that do not contain the expression cassette.

[0030] On the other hand, a method for increasing plant yield in nematode-infested fields is provided, which includes expressing the Cry21 protein in the plant, wherein the plant yield is increased compared to the yield of the same type of plant that does not express the Cry21 protein.

[0031] On the other hand, a method is provided for increasing the vigor or yield of transgenic soybean plants exposed to nematode populations, the method comprising: infiltrating a transgenic soybean event into a soybean plant to obtain a transgenic soybean plant, wherein the transgenic soybean event contains a heterologous DNA sequence encoding a Cry21 protein, the Cry21 protein conferring resistance to nematodes on the transgenic soybean event; and growing the transgenic soybean plant or its progeny at a location where nematode infection has limited yield in soybean plants that do not contain the heterologous nucleic acid molecule encoding the Cry21 protein, thereby giving the transgenic soybean plant increased vigor or yield compared to a control plant.

[0032] On the other hand, a method for increasing the yield of soybean fields is provided, which includes: introducing a nucleic acid molecule that can guide the expression of the Cry21 protein into soybean plants to produce transgenic plants; and cultivating multiple transgenic seeds from the transgenic plants in the field to produce or form a soybean field containing multiple transgenic soybean plants with enhanced resistance to nematode infection, thereby increasing the yield of the soybean field.

[0033] On the other hand, a recombinant expression cassette is provided, comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule encoding the Cry21 protein. Further, the invention provides a recombinant vector comprising such an expression cassette. Even further, another aspect provides a transgenic host cell comprising such an expression cassette. The transgenic host cell according to this aspect can be a plant cell. Even further still, another aspect provides a transgenic plant or plant part comprising such a plant cell.

[0034] In another aspect, a nematicidal composition is provided, comprising a nematode-controlled effective amount of Cry21 protein and an acceptable agricultural vector. In yet another aspect, the agricultural vector is a transgenic plant. In yet another aspect, the transgenic plant is a transgenic soybean plant, and the Cry21 protein is a Cry21 protein having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof. In yet another aspect, the Cry21 protein comprises SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof.

[0035] Another aspect provides a method for producing a nematode-resistant transgenic plant, the method comprising introducing a nucleic acid molecule encoding a Cry21 protein into a plant cell to create a transgenic plant cell; regenerating a transgenic plant from the transgenic plant cell, wherein the Cry21 protein is expressed in an effective amount in the transgenic plant to control nematodes. According to another aspect, the plant is a soybean plant. In another aspect, the Cry21 protein is a protein having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof. In yet another aspect, the Cry21 protein comprises SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, and any functional fragment thereof. In yet another aspect, the nematode is a target pest. On the other hand, the nematode is a species of the genus *Soybean cyst nematode*, *Shortest-tailed short-bodied nematode*, *Rotylenchulus reniformis*, or *Root-knot nematode*.

[0036] Another aspect includes a method for controlling nematode pests, the method comprising contacting the nematode pest with a Cry21 protein, the Cry21 protein comprising 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 NO: 1, 3, 7, 9, 11, 13, 15, 17, 21, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 or an effective nematode variant or fragment thereof. Another aspect includes the method of claim 40, wherein the nematode pest is selected from any one of the following species: small ring nematodes, stem nematodes, golden nematodes, spiral nematodes, cyst nematodes, long needle nematodes, root-knot nematodes, pseudo-spinous nematodes, short-bodied nematodes, perforating nematodes, coiled nematodes, kidney-shaped nematodes, pad-shaped nematodes, and dagger nematodes. On the other hand, it includes an expression cassette containing a nucleic acid encoding a Cry21 protein, the Cry21 protein containing an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 3, 7, 9, 11, 13, 15, 17, 21, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 or their nematicidal effective variants or fragments thereof, and wherein the nucleic acid is operatively ligated in the expression cassette and / or further in a plant expression vector. Another embodiment includes a nucleic acid comprising a nucleic acid sequence having a sequence identity of 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% with any one of SEQ ID NO: 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 non-limiting examples. A brief description of sequence listings

[0038] SEQ ID NO:1 discloses the amino acid sequence of Cry21 homolog Axmi296.

[0039] SEQ ID NO:2 discloses the nucleotide sequence of Cry21 homolog Axmi296.

[0040] SEQ ID NO:3 discloses the amino acid sequence of Axmi018.

[0041] SEQ ID NO:4 discloses the nucleotide sequence of Axmi018.

[0042] SEQ ID NO:5 discloses the amino acid sequence of Axmi021.

[0043] SEQ ID NO:6 discloses the nucleotide sequence of Axmi021.

[0044] SEQ ID NO:7 discloses the amino acid sequence of Axmi040.

[0045] SEQ ID NO:8 discloses the nucleotide sequence of Axmi040.

[0046] SEQ ID NO:9 discloses the amino acid sequence of Axmi049.

[0047] SEQ ID NO:10 discloses the nucleotide sequence of Axmi049.

[0048] SEQ ID NO:11 discloses the amino acid sequence of Axmi071.

[0049] SEQ ID NO:12 discloses the nucleotide sequence of Axmi071.

[0050] SEQ ID NO:13 discloses the amino acid sequence of Axmi074.

[0051] SEQ ID NO:14 discloses the nucleotide sequence of Axmi074.

[0052] SEQ ID NO:15 discloses the amino acid sequence of Axmi088.

[0053] SEQ ID NO:16 discloses the nucleotide sequence of Axmi088.

[0054] SEQ ID NO:17 discloses the amino acid sequence of Axmi104.

[0055] SEQ ID NO:18 discloses the nucleotide sequence of Axmi104.

[0056] SEQ ID NO:19 discloses the amino acid sequence of Axmi155.

[0057] SEQ ID NO:20 discloses the nucleotide sequence of Axmi155.

[0058] SEQ ID NO:21 discloses the amino acid sequence of Axmi169.

[0059] SEQ ID NO:22 discloses the nucleotide sequence of Axmi169.

[0060] SEQ ID NO:23 discloses the amino acid sequence of Axmi170.

[0061] SEQ ID NO:24 discloses the nucleotide sequence of Axmi170.

[0062] SEQ ID NO:25 discloses the amino acid sequence of Axmi215.

[0063] SEQ ID NO:26 discloses the nucleotide sequence of Axmi215.

[0064] SEQ ID NO:27 discloses the amino acid sequence of Axmi218.

[0065] SEQ ID NO:28 discloses the nucleotide sequence of Axmi218.

[0066] SEQ ID NO:29 discloses the amino acid sequence of Axmi219.

[0067] SEQ ID NO:30 discloses the nucleotide sequence of Axmi219.

[0068] SEQ ID NO:31 discloses the amino acid sequence of Axmi220.

[0069] SEQ ID NO:32 discloses the nucleotide sequence of Axmi220.

[0070] SEQ ID NO:33 discloses the amino acid sequence of Axmi227.

[0071] SEQ ID NO:34 discloses the nucleotide sequence of Axmi227.

[0072] SEQ ID NO:35 discloses the amino acid sequence of Axmi266.

[0073] SEQ ID NO:36 discloses the nucleotide sequence of Axmi266.

[0074] SEQ ID NO:37 discloses the amino acid sequence of Axmi280.

[0075] SEQ ID NO:38 discloses the nucleotide sequence of Axmi280.

[0076] SEQ ID NO:39 discloses the amino acid sequence of Axmi287.

[0077] SEQ ID NO:40 discloses the nucleotide sequence of Axmi287.

[0078] SEQ ID NO:41 discloses the amino acid sequence of Axmi297.

[0079] SEQ ID NO:42 discloses the nucleotide sequence of Axmi297.

[0080] SEQ ID NO:43 discloses the amino acid sequence of Axmi299.

[0081] SEQ ID NO:44 discloses the nucleotide sequence of Axmi299.

[0082] SEQ ID NO:45 discloses the amino acid sequence of Axmi340.

[0083] SEQ ID NO:46 discloses the nucleotide sequence of Axmi340.

[0084] SEQ ID NO:47 discloses the amino acid sequence of Axmi350.

[0085] SEQ ID NO:48 discloses the nucleotide sequence of Axmi350.

[0086] SEQ ID NO:49 discloses the amino acid sequence of Axmi380.

[0087] SEQ ID NO:50 discloses the nucleotide sequence of Axmi380.

[0088] SEQ ID NO:51 discloses the amino acid sequence of natural Cry21Aa1.

[0089] SEQ ID NO:52 discloses the nucleotide sequence of natural Cry21Aa1.

[0090] SEQ ID NO:53 discloses the amino acid sequence of natural Cry21Aa2.

[0091] SEQ ID NO:54 discloses the protein sequence of the natural Cry21Aa2. Attached Figure Description

[0092] Figure 1 Plant transformation vectors for expressing Axmi296 in plants are described.

[0093] Figure 2 The NEEDLE sequence alignment of Axmi296 and Cry21Aa2 was depicted.

[0094] Figure 3aand 3b The effectiveness of Axmi296 against the shortest-tailed nematode was demonstrated in a 60-day greenhouse assay.

[0095] Figure 4a and 4b The results depict the increased yield benefits provided by Axmi296 relative to the non-resistant SCN control in a soybean cyst nematode (SCN) field trial.

[0096] Figure 4c The results of the SCN field trial, which compared nematode reduction between the novel Cry21 homolog (Axmi296) and the wild-type Thorne, are described.

[0097] Figure 4d The results of the SCN field trial, which compared the yield of the novel Cry21 homolog (Axmi296) with that of wild-type Thorne, are described.

[0098] Figure 5 A field photograph depicts Axmi296 plants (left) growing next to non-resistant soybean control plants (right) in a field infected with SCN. Detailed Implementation

[0099] Before explaining the various embodiments disclosed herein, it should be understood that the application of the invention is not limited to the details of the construction and arrangement of the components set forth in the following description. Other embodiments may be practiced or performed in various ways. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting.

[0100] Throughout this disclosure, references have been made to various publications, patents, and published patent specifications. Where permitted, the disclosures of these publications, patents, and published patent specifications have been incorporated herein by reference in their entirety to provide a more comprehensive description of the prior art. Unless otherwise stated, this disclosure covers conventional techniques in plant breeding, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the scope of the art. See, for example, Sambrook and Russell, *Molecular Cloning: A Laboratory Manual*, 3rd edition (2001); *Current Protocols in Molecular Biology* [(edited by F.M. Susubel et al., (1987)]; *Plant Breeding: Principles and Prospects* (*Plant Breeding*, Volume 1); MD Hayward, N. O. Bosemark, I. Romagosa; Chapman and Hall, (1993); edited by Coligan, Dunn, Ploegh, Speicher, and Wingfeld (1995); *CURRENT Protocols in Protein Science* (John Wiley & Sons, Inc.); *Methods in Enzymology* (Academic Press, Inc.): PCR 2: A Practical Approach [PCR2: Practical Approach] (edited by MJ MacPherson, BD Flames and GR Taylor (1995)), edited by Harlow and Lane (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture [edited by RI Freshney (1987)].

[0101] Unless otherwise stated, technical terms are used in accordance with their conventional usage in the art. Definitions of commonly used terms in molecular biology can be found in the following literature: Lewin, Genes VII, Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Wiley-Interscience, 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, VCH Publishers, Inc., 1995; Ausubel et al. (1987) Current Protocols in Molecular Biology, Green Publishing; Sambrook and Russell. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition.

[0102] For ease of understanding this disclosure, the following definitions are provided:

[0103] The “activity” of the Cry21 protein of the present invention means that the Cry21 protein has a toxic effect on nematodes by disrupting or preventing feeding, inhibiting the ability of nematode pests to survive, grow and reproduce (which may or may not cause nematode death), or limiting nematode-related damage or loss to crop plants.

[0104] As used herein, “and / or” means and covers any and all possible combinations of one or more of the listed items, as well as the absence of combinations when interpreted in an alternative (or) context.

[0105] "Association / operational linkage" refers to two nucleic acid sequences that are physically or functionally related. For example, if a promoter or regulatory DNA sequence is operably linked or positioned such that the regulatory DNA sequence will affect the expression level of a coding or structural DNA sequence, then these two sequences are said to be "associated" with a DNA sequence encoding RNA or a protein.

[0106] As used herein, the term "contact" refers to the process of delivering or applying the Cry21 protein of the present invention, or a transgenic plant or plant part expressing the Cry21 protein of an embodiment or aspect thereof, to a target nematode pest or nematode population. Contact describes the physical proximity of the Cry21 protein, or a transgenic plant or plant part expressing the Cry21 protein, to a target nematode, such that they interact. Contact between a transgenic plant or plant part and a target nematode or nematode population can be achieved by planting transgenic seeds, seedlings, cuttings, shoots, tubers, etc., in a location capable of supporting the growth of the nematode pest or nematode population.

[0107] A chimeric gene is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to or associated with a nucleic acid sequence encoding mRNA or expressed as a protein, such that the regulatory nucleic acid sequence can regulate the transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequence of a chimeric gene is typically not operatively linked to an associated nucleic acid sequence found in nature.

[0108] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. RNA is then translated in an organism to produce proteins.

[0109] As used herein, the term "to control or controlling" nematodes refers to the ability of a nematode pest to survive, grow, feed, and / or reproduce through toxic effects, or to limit nematode-related damage or loss in crop plants. "Controlling" nematodes may or may not mean killing nematodes.

[0110] "Corresponding to" means that when the nucleic acid coding sequences or amino acid sequences of different Cry21 genes or proteins are compared with each other, the nucleic acid or amino acid "corresponding to" certain listed positions is the nucleic acid or amino acid that is compared with these positions but is not necessarily at these precise numerical positions relative to the corresponding nucleic acid coding sequence or amino acid sequence of a specific Cry21.

[0111] "Delivery" of toxins means that the toxin comes into contact with nematodes or nematode populations, thereby resulting in toxic effects and control of the nematodes or nematode populations. Toxins can be delivered in many recognized ways, such as orally via nematode ingestion or through contact with nematodes via transgenic plant expression, formulated protein compositions, sprayable protein compositions, bait substrates, or any other toxin delivery system recognized in the art.

[0112] The term "economic threshold" is defined as the level of a nematode population that produces incremental damage equal to the cost of controlling or preventing that damage. It is the level of the nematode population at which the benefits of nematode control are equal to its costs. In this respect, the economic threshold can be defined as the level of nematode damage at which the incremental reduction in crop yield equals the cost of preventing its occurrence. In other words, the economic threshold attempts to determine the point at which controlling a nematode population becomes economically feasible. Economic damage to host crops is typically caused by the first generation of progeny nematodes and is prevented by transgenic plants expressing the Cry21 protein by reducing the concentration of progeny nematodes in the plant's root zone.

[0113] As used herein, “effective control dose for nematodes” or, alternatively, “biotoxic dose”, refers to the concentration of Cry21 protein or a functional fragment thereof that has the ability to inhibit the survival, growth, feeding, and / or reproduction of nematodes through toxic effects, or to reduce or prevent nematode-related damage or loss in crop plants. “Effective control dose for nematodes” may or may not mean killing nematodes.

[0114] As used herein, an "expression cassette" refers to a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, comprising a promoter operatively linked to the target nucleotide sequence, which in turn is operatively linked to a termination signal. It typically also contains the sequence required for the correct translation of the nucleotide sequence. An expression cassette containing the target nucleotide sequence can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of the other components. An expression cassette can also be naturally occurring but already obtained in a recombinant form suitable for heterologous expression. However, typically, the expression cassette is heterologous with respect to the host, meaning that the specific nucleic acid sequence of the expression cassette is not naturally present in the host cell and must have been introduced into the host cell or the progenitor cell of the host cell through a transformation event. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive or inducible promoter that initiates transcription only when the host cell is exposed to certain external stimuli. In the case of multicellular organisms (such as plants), the promoter can also be specific to a particular tissue or organ or developmental stage.

[0115] A "gene" is a defined region within the genome that, in addition to the coding nucleic acid sequence described above, contains other (primarily regulatory) nucleic acid sequences responsible for controlling the expression of the coding portion (i.e., transcription and translation). Genes may also contain other 5' and 3' untranslated sequences and termination sequences. Other elements that may be present include, for example, introns.

[0116] "Nematodes" are defined as toxic biological activities that can control nematodes (preferably by killing them).

[0117] When a nucleic acid sequence encodes a polypeptide that has the same amino acid sequence as a polypeptide encoded by a reference nucleic acid sequence, the nucleic acid sequence is "equicoded" with the reference nucleic acid sequence. For example, the natural coding sequence of the Cry21 protein from a species of Bacillus spp. is codon-optimized to be expressed in plants that encode the same Cry21 protein.

[0118] "Isolated" nucleic acid molecules, or isolated proteins or toxins, are nucleic acid molecules, proteins, or toxins that exist through artificial manipulation away from their natural environment and are therefore not natural products. Isolated nucleic acid molecules, proteins, or toxins may exist in purified form or may exist in non-natural environments (such as, for example, recombinant host cells or transgenic plants).

[0119] The term "natural" refers to coding sequences or genes that are naturally present in the genome of cells or plants.

[0120] The term "naturally occurring" is used in this document to describe objects that can be found in nature and are distinct from artificially produced objects. For example, a protein or nucleotide sequence present in an organism (including viruses) that can be isolated from natural sources and has not been intentionally modified in a laboratory is naturally occurring.

[0121] "Plant" refers to any plant at any stage of development, especially seed plants.

[0122] A plant cell is a structural and physiological unit of a plant, which contains a protoplast and a cell wall. Plant cells can exist as isolated single cells or cultured cells, or as part of higher tissue units such as, for example, plant tissues, plant organs, or the whole plant.

[0123] "Plant material" means leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0124] "Plant organs" are unique and distinctly structured and differentiated parts of a plant, such as roots, stems, leaves, flower buds, or embryos.

[0125] "Plant part" can be any part of a plant and includes plant cells, plant material, plant organs, or plant tissues.

[0126] As used herein, “plant tissue” means a group of plant cells organized into structural and functional units. This includes any plant tissue in situ or in a culture. The term includes, but is not limited to, the whole plant, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in the presence or absence of any particular type of plant tissue as listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[0127] A promoter is a non-translated DNA sequence located upstream of the coding region that contains a binding site for RNA polymerase II and initiates DNA transcription. The promoter region may also include other elements that act as regulators of gene expression.

[0128] "Regulatory elements" are sequences that participate in controlling the expression of nucleotide sequences. Regulatory elements include promoters that are operatively linked to the target nucleotide sequence, and termination signals. They typically also encompass the sequences required for the correct translation of the nucleotide sequence.

[0129] As used in this article, “resistant” or “resistance” refers to genetically modified soybean varieties that prevent most nematodes from surviving and / or reproducing when they attempt to infect the plant.

[0130] In the context of two nucleic acid or protein sequences, the term "substantially identical" means two or more sequences or subsequences that have at least 60%, at least 80%, at least 90%, at least 95%, and at least 99% nucleotide or amino acid residue identity when compared and aligned against the maximum correspondence measured by visual inspection using one of the following sequence comparison algorithms. Substantially identical sequence identity is present in a sequence region of at least about 50 residues, a region of at least about 100 residues, or the sequences are substantially identical over at least about 150 residues. In one embodiment, the sequences are substantially identical over the entire length of the coding region. Furthermore, substantially identical nucleic acid or protein sequences perform substantially the same function.

[0131] To determine the percentage of identity between two sequences (“identity percentage”), in the first step, a pairwise sequence alignment between the two sequences is generated. This pairwise alignment in the first step can be generated by various tools known to those skilled in the art, such as, for example, the program “Blast” (Altschul et al. J. Mol. Biol. [Journal of Molecular Biology] 215:403-410), “Blast2” (“gap Blast”) (Altschul et al., Nucleic Acids Res. [Nucleic Acids Research] 25:3389-3402.), programs from the European Molecular Biology Open Software Suite (EMBOSS, Trends in Genetics [Trends in Genetics] 16 (6), 276 (2000)), such as “Water”, “Matcher”, or “Needle”, or generated by visual inspection.

[0132] After aligning two sequences, in the second step, an identity percentage value can be determined based on the resulting alignment. The identity percentage between the two sequences can be calculated based on the resulting full alignment, or based on a region in the alignment (e.g., a region in the alignment that displays the sequence of the present invention along its full length, or a region that displays another sequence along its full length), or based on a region that displays only a portion of the sequence. The alignment region used to calculate the identity percentage value preferably has a length of at least 100 positions, more preferably at least 150 positions, and most preferably greater than 200 positions. To determine the identity percentage, first, the sum of all positions where the two sequences display the same residues in the alignment region is calculated, and then this sum is divided by the length of the alignment region, where the positions where the sequences have introduced vacancies are a component of the length (length of the alignment region), or (length of the alignment region - total number of vacancies in the alignment region) is subtracted from the length. The obtained value is then multiplied by 100 to obtain the identity percentage (% identity).

[0133] In one embodiment, the two sequences are aligned to full length according to the algorithm of Needleman and Wunsch (J. Mol. Biol. [Journal of Molecular Biology] (1979) 48, pp. 443-453), as implemented in the program "Needle" from EMBOSS (Trends in Genetics 16 (6), 276 (2000), preferred version 6.3.1.2 or later), using the program default parameters for protein sequences (gapopen = 10.0, gapextend = 0.5 and matrix = EBLOSUM62 (the EMBOSS version replacing BLOSUM62) and the default parameters for nucleotide sequences (gapopen = 10.0, gapextend = 0.5 and matrix = EDNAFULL). The percentage of identity (% identity) is then determined based on the resulting full alignment and is calculated as follows: Percentage of identity = (Sum of positions showing identical residues in the alignment × ... 100) / (length of the alignment - total number of empty spaces in the alignment). When the parameter option "-nobrief" is applied, this value can also be obtained directly from the EMBOSS program "Needle" as the program marked "longest identity".

[0134] For nucleotide sequences encoding proteins, pairwise alignment should be performed over the full length of the coding region (from the start codon to the stop codon, excluding introns) of one or more embodiments of the sequence. Introns present in another sequence may also be removed for pairwise alignment to allow comparison with the sequence of the present invention.

[0135] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions. The phrase "specific hybridization" refers to the fact that, under stringent conditions, a molecule binds, doubles, or hybridizes only with a specific nucleotide sequence present in a complex mixture (e.g., total cellular) DNA or RNA. "Substantially binding" refers to complementary hybridization between the probe nucleic acid and the target nucleic acid, and includes slight mismatches that can be adjusted by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0136] In the context of nucleic acid hybridization experiments (such as DNA and RNA hybridization), "strict hybridization conditions" and "strict hybridization washing conditions" are sequence-dependent and vary under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. Extensive guidelines on nucleic acid hybridization can be found in the following literature: Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes part I chapter 2; and "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Typically, highly stringent hybridization and washing conditions are chosen to be approximately 5°C lower than the thermal melting point (Tm) of the specific sequence at defined ionic strengths and pH. Typically, under "stringent conditions," the probe will hybridize with its target sequence but not with other sequences.

[0137] If nucleic acid molecules exhibit perfect complementarity, they are called “complements” of another nucleic acid molecule. As used herein, molecules are said to exhibit “perfect complementarity” when every nucleotide of one molecule is complementary to a nucleotide of another molecule. If two molecules can hybridize with each other under conditions sufficient to allow them to remain annealed to each other under at least conventional “low-strict” conditions, they are called “minimally complementary.” Similarly, if molecules can hybridize with each other under conditions sufficient to allow them to remain annealed to each other under conventional “high-strict” conditions, they are called “complementary.” The standard stringent conditions are described by Sambrook et al. in *Molecular Cloning: A Laboratory Manual*, 2nd 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.

[0138] Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at defined ionic strength and pH). Choose very stringent conditions to match the Tm of a specific probe. An example of stringent hybridization conditions for hybridizing complementary nucleic acids with more than 100 complementary residues on a filter membrane in DNA or RNA blotting is overnight hybridization at 42°C with 50% formamide and 1 mg heparin. An example of highly stringent wash conditions is 0.15 M NaCl, 72°C, for approximately 15 minutes. An example of stringent wash conditions is 0.2×SSC wash buffer, 65°C, for 15 minutes (see Sambrook below for a description of the SSC buffer). Typically, a low-stringent wash is performed before a high-stringent wash to remove background probe signal. For example, for duplexes of more than 100 nucleotides, an example of a medium-stringent wash is 1×SSC, 45°C, for 15 minutes. For example, for duplexes of more than 100 nucleotides, an example of a low-stringent wash is 4–6×SSC, 40°C, for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentrations (or other salts) at pH 7.0 to 8.3, and temperatures typically at least about 30°C. Stringent conditions can also be achieved by adding a destabilizing agent (such as formamide). Generally, in a specific hybridization assay, a signal-to-noise ratio that is twice (or higher) than that observed with unrelated probes indicates that specific hybridization has been detected. If nucleic acids that do not hybridize with each other under stringent conditions encode substantially identical proteins, then these nucleic acids remain substantially identical. This occurs, for example, when nucleic acid copies are generated using the maximum codon degeneracy allowed by the genetic code.

[0139] The following are examples of hybridization / washing conditions that can be used to clone homologous nucleotide sequences substantially identical to the reference Cry21 nucleotide sequence of this invention: The reference nucleotide sequence is preferably hybridized at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, and washed at 50°C in 2×SSC, 0.1% SDS; more ideally, hybridized at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, and washed at 50°C in 1×SSC, 0.1% SDS; more ideally, hybridized again at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, and washed at 50°C in 0.5×SSC, 0.1% SDS; preferably, hybridized at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA. Hybridization in EDTA, followed by washing in 0.1×SSC and 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA at 50°C, followed by washing in 0.1×SSC and 0.1% SDS at 65°C.

[0140] A further indication that two nucleic acid sequences or proteins are substantially identical is the presence of immune cross-reactivity or specific binding between the protein encoded by the first nucleic acid and the protein encoded by the second nucleic acid. Thus, one protein is typically substantially identical to the second protein, for example, where the two proteins differ only due to conserved substitutions.

[0141] "Synthetic" refers to a nucleotide sequence that contains structural features not found in the natural sequence. For example, the Cry21 coding sequence, which is more similar to the G+C content and normal codon distribution of genes in dicotyledonous and / or monocotyledonous plants and is not naturally found in Bacillus, is called synthetic.

[0142] "Transformation" is the process of introducing heterologous nucleic acids into host cells or organisms. In particular, "transformation" refers to the stable integration of DNA molecules into the genome of the target organism.

[0143] "Transformed / GMO / Recombinant" refers to a host organism, such as bacteria or plants, in which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or it can exist as an extrachromosomal molecule. Such an extrachromosomal molecule can replicate automatically. Transformed cells, tissues, or plants should be understood to encompass not only the final products of the transformation process but also their transgenic progeny. "Non-transformed," "non-GMO," or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants, that do not contain a heterologous nucleic acid molecule.

[0144] In the context of this invention, "Cry21 protein" refers to Bacillus Cry insecticidal proteins (i.e., members of the Cry21 class, including, for example, but not limited to, Cry21Aa1, Cry21Aa2, and their homologs). Some structural features that identify a protein as a Cry21 class protein include: 1) a size of about 130-150 kDa; and falling within the specifications of Cry21 proteins as described in Crickmore et al., Journal of Invertebrate Pathology 186 (2021), which is incorporated herein by reference in its entirety. As used herein, a nematode-active "homolog" means that the indicated protein or polypeptide is active against nematodes and has a defined relationship with other members of the Cry21 class. Such a defined relationship may include, but is not limited to: 1) a protein that is at least 60%, or at least 70%, or at least 80%, or at least 90% identical at the sequence level to another member of the Cry21 class while retaining nematode-killing activity.

[0145] As used herein, nucleotides are represented by their bases using the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are also represented by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0146] The materials and methods of this invention can be used to kill or control nematodes; delay the growth or reproduction of nematodes; reduce nematode populations; and / or reduce or delay damage to plants caused by nematode pests. One embodiment provides a method for controlling nematode pests in crop plants (such as soybeans) by using transgenic crop plants expressing the Cry21 protein.

[0147] Expression of the Cry21 protein in one or more embodiments in transgenic plants or plant parts produces compositions that can be used to control nematode pests, such as, but not limited to, target pests, species of the genus *Meloidogyne* (e.g., *Meloidogyne incoginita* and *Meloidogyne javanica*, *Meloidogyne hapla*, *Meloidogyne arenari*), species of the genus *Heterodera* (e.g., *Heterodera carotae*, *Heteroderaschachtii*, *Heterodora avenae*, and *Heterodoratrifolii*), species of the genus *Globodera* (e.g., *Globodera rostochiensis*), and species of the genus *Radix* (e.g., *Radix Radopholus*). Species of the genera *Similes*, *Nematoda*, *Pratylenchus* (e.g., *Pratylenchus neglectans*, *Pratylenchus penetrans*), *Aphelenchoides*, *Hoplolaimus*, *Nematoda*, *Nacobbus*, *Subanguina*, *Belonlaimus*, *Criconemoides*, *Ditylenchus dipsaci*, *Dolichodorus*, and *Hymenolepis*. Species of the genera *Hemicriconemoides*, *Hemicycliophora*, *Hirschmaniella*, *Hypsoperine*, *Macroposthonia*, *Melinius*, *Punctodera*, *Quinisulcius*, *Scutellonema*, *Draconis*, and *Tylenchorhynchus*.

[0148] One embodiment provides a method for controlling nematode pests, the method comprising contacting the nematode pest with a Cry21 protein comprising SEQ ID No: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof.

[0149] In another embodiment, the nematode is selected from the group consisting of: *Nematoda*, *Steel Nematoda*, *Golden Nematoda*, *Spiral Nematoda*, *Cystic Nematoda*, *Longneedle Nematoda*, *Root-knot Nematoda*, *Pseudo-spinous Nematoda*, *Brutal Nematoda*, *Perforator Nematoda*, *Coilia*, *Kidney Nematoda*, *Paddy-sharp Nematoda*, and *Draconis Nematoda*. In yet another embodiment, the nematode is a cyst-forming nematode. In yet another embodiment, the nematode belongs to the genus *Brutal Nematoda*. In yet another embodiment, the nematode is the shortest-tailed, short-bodied nematode.

[0150] In another embodiment, the contact step is performed using 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.

[0151] In another embodiment, the genetically modified plant or plant part is selected from the group consisting of: alfalfa, apple, apricot, Arabidopsis thaliana, artichoke, asparagus, avocado, banana, barley, legume, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, cereal, celery, cherry, citrus, Clementine, coffee, corn, cotton, cucumber, eggplant, endive, eucalyptus, fig, grape, grapefruit, peanut, ground cherry, kiwi, lettuce, leek. Lemon, lime, pine, corn, mango, melon, millet, mushroom, nuts and oats, okra, onion, orange, ornamental plants or flowers or trees, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, red chicory, radish, rapeseed, raspberry, rice, rye, sorghum, soybean, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, orange, tea, tobacco, tomato, vine, watermelon, wheat, yam, and zucchini. In yet another embodiment, the genetically modified plant or plant part is a soybean plant or plant part.

[0152] Another embodiment provides a transgenic seed of a transgenic plant of one or more embodiments, wherein the transgenic seed contains a heterologous nucleic acid molecule encoding the Cry21 protein of one or more embodiments.

[0153] Another embodiment also covers recombinant vectors and expression cassettes containing the Cry21 nucleic acid sequence of the present invention. In such vectors, the nucleic acid sequence is contained in an expression cassette that includes regulatory elements for expressing the Cry21 nucleotide sequence in a transgenic host cell capable of expressing the nucleotide sequence. Such regulatory elements typically include promoters and termination signals, and preferably also include elements that allow efficient translation of the polypeptide encoded by the nucleic acid sequence of the present invention. Vectors containing the nucleic acid sequence are generally capable of replicating in specific host cells (such as extrachromosomal molecules) and are therefore used to amplify the nucleic acid sequence of the present invention in host cells. In one embodiment, the host cell for such a vector is a microorganism, such as bacteria, particularly Escherichia coli (E. coli). In another embodiment, the host cell for such a recombinant vector is an endophytic or epiphytic bacterium. An example of a host cell for such a vector is a eukaryotic cell, such as a plant cell. Such a plant cell could be a soybean cell or a corn 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). The recombinant vector is also used to transform the nucleotide sequence of the present invention into a transgenic host cell, whereby the nucleotide sequence is stably integrated into the DNA of such a transgenic host cell. In one embodiment, such a transgenic host cell is a prokaryotic cell. In another embodiment, such a transgenic host cell is a eukaryotic cell, such as a yeast cell, insect cell, or plant cell. In yet another embodiment, the transgenic host cell is a plant cell, such as a soybean cell or corn cell.

[0154] In another embodiment, for example, the Cry21 nucleotide sequence of the present invention can be modified by incorporating random mutations in a technique known as in vitro recombination or DNA shuffling to increase nematode activity. This technique is described in Stemmer et al., Nature [Nature] 370:389-391 (1994) and U.S. Patent No. 5,605,793, which are incorporated herein by reference. Millions of mutant copies of the nucleotide sequence are generated based on the original nucleotide sequence of the present invention, and variants with improved properties, such as increased nematode-killing activity, enhanced stability, or different specificity or range of target nematode pests, are recovered. This method encompasses the formation of mutagenic double-stranded polynucleotides from a template double-stranded polynucleotide containing the nucleotide sequence of the present invention, wherein the template double-stranded polynucleotide has been cleaved into double-stranded random fragments of desired size, and includes the steps of adding one or more single-stranded or double-stranded oligonucleotides to the resulting population of double-stranded random fragments, wherein the oligonucleotides contain identity regions and heterologous regions with the double-stranded template polynucleotide; denaturing the resulting mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resulting population of single-stranded fragments with a polymerase under conditions that cause the single-stranded fragments to anneal in the identity regions to form annealed fragment pairs, wherein the identity regions are sufficient to cause one member of a pair to initiate the replication of the other member, thereby forming a mutagenic double-stranded polynucleotide; and repeating the second and third steps for at least two additional cycles, wherein the resulting mixture in the second step of the additional cycles contains the mutagenic double-stranded polynucleotide from the third step of the previous cycle, and the additional cycles form additional mutagenic double-stranded polynucleotides. In embodiments, the concentration of a single double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the total DNA. In another embodiment, the template double-stranded polynucleotide contains at least about 100 polynucleotides. In yet another embodiment, the size of the random double-stranded fragment is about 5 to 5 kb. In yet another embodiment, the fourth step of the method includes repeating steps two and three for at least 10 cycles.

[0155] In another embodiment, the Cry21 nucleotide sequence of the present invention can be modified by N- or preferably C-terminal deletion to encode a functional fragment. The term "functional fragment" refers to a sequence of amino acids for the Cry21 protein, wherein these sequence of amino acids are at least 10, 20, 30, 40, 50, or 60 consecutive amino acids of SEQ ID NO: 1, 7, 9, 13, 21, 23, 33, 35, 39, 41, 51, or 53. A preferred deletion would remove the C-terminal crystalline domain downstream of the conserved amino acid motif referred to as "DRIEF" or "DRIE," which also... Figure 2Highlighted in the image, and previously described as “block 5” in Schnepf et al. (1998), this functional fragment with a C-terminal deletion of a crystalline domain is expected to produce toxic core proteins that are independent of proteolytic activation.

[0156] In another embodiment, at least one nucleotide sequence from the Cry21 nucleotide sequence of the present invention is inserted into a suitable expression cassette containing a promoter and a termination signal. Expression of the nucleotide sequence is constitutive or uses an inducible promoter that initiates transcription in response to various types of stimuli. In a preferred embodiment, the cell in which the toxin is expressed is a microorganism, such as a virus, bacteria, or fungus. In an 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 infecting suitable eukaryotic cells suitable for viral replication and expression of the nucleotide sequence. The resulting insecticidal toxin is used as an insecticide. Alternatively, baculoviruses engineered to contain the nucleotide sequence are used to infect insects in vivo and kill the insects by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin.

[0157] Bacterial cells are also hosts used to express the nucleotide sequences of the present invention. In embodiments, non-pathogenic symbiotic bacteria (so-called endophytes) capable of living and replicating within plant tissues, or non-pathogenic symbiotic bacteria (so-called epiphytes) capable of colonizing the leaf or rhizosphere, are used. Such bacteria include *Agrobacterium*, *Alcaligenes*, *Azospirillum*, *Azotobacter*, *Bacillus*, *Clavibacter*, *Enterobacter*, *Erwinia*, *Flavobacter*, *Klebsiella*, *Pseudomonas*, *Rhizobium*, *Serratia*, *Streptomyces*, and *Xanthomonas*. Symbiotic fungi (such as Trichoderma and Gluconobacterium) are also possible hosts for expressing the nucleotide sequences of the present invention for the same purpose.

[0158] Techniques for these gene manipulations are specific to the different available hosts and are known in the art. For example, expression vectors pKK223-3 and pKK223-2 can be used in *E. coli* to express heterologous genes following a tac or trc promoter via transcriptional or translational fusion. For the expression of operons encoding multiple ORFs, the simplest procedure is to insert the operon into a vector (such as pKK223-3) during transcriptional fusion, thereby allowing the use of the homologous ribosome binding site of the heterologous gene. Techniques for overexpression in Gram-positive species (such as *Bacillus*) are also known in the art and can be used in the context of this invention (Quax et al., in *Industrial Microorganisms: Basic and Applied Molecular Genetics*, edited by Baltz et al., American Society for Microbiology, Washington (1993)). Alternative systems for overexpression include those that rely on yeast vectors and include those using Pichia, Saccharomyces, and Kluyveromyces (Sreekrishna, in: Industrial Microorganisms: Basic and Applied Molecular Genetics, edited by Baltz, Hegeman, and Skautrud, American Society for Microbiology, Washington (1993); Dequin and Bane, Biotechnology L2:173-177 (1994); van den Berg et al., Biotechnology 8:135-139 (1990)).

[0159] In one embodiment, at least one Cry21 protein of the present invention is expressed in a higher organism (e.g., a plant). In this case, the transgenic plant expressing an effective amount of the toxin protects itself from nematode pests. When nematodes begin to feed on such a transgenic plant, they also ingest the expressed Cry21 toxin. This may prevent nematodes from further feeding in plant tissues, may harm or kill nematodes, or may reduce nematode reproductive capacity. The nucleotide sequence of the present invention is inserted into an expression cassette, which is then stably integrated into the plant genome. The plants transformed according to the present invention can be monocotyledonous or dicotyledonous plants, and include, but are not limited to, corn, wheat, barley, rye, sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis thaliana, and woody plants (such as conifers and deciduous trees).

[0160] Once the desired nucleotide sequence has been transferred into a specific plant species, it can be propagated in that species or transferred to other varieties of the same species, especially commercial varieties, using conventional breeding techniques.

[0161] 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 generated. For expression in transgenic plants, the nucleotide sequences of the present invention may require modification and optimization. Although in many cases genes from microorganisms can be expressed at high levels in plants without modification, low expression in transgenic plants may be due to the presence of codons in the microbial nucleotide sequences that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in the present invention can be altered to conform to plant preferences while maintaining the amino acids thereby encoded. Furthermore, high expression in plants is best achieved by coding sequences having a GC content of at least about 35% (preferably greater than about 45%, more preferably greater than about 50%, and most preferably greater than about 60%). Although preferred gene sequences can be adequately expressed in both monocot and dicot species, sequences can be modified to account for specific codon and GC content preferences in monocots or dicots, as these preferences have been shown to be distinct (Murray et al., Nucl. Acids Res. 17:477-498 (1989)). Furthermore, nucleotide sequences are screened for inappropriate splicing sites that could lead to information truncation. All necessary modifications to the nucleotide sequence, such as those described above, are performed using methods known in the art, employing well-known techniques such as site-directed mutagenesis, PCR, and synthetic gene construction.

[0162] In various embodiments, the nucleotide sequences of the present invention can be optimized for expression in any plant. It should be recognized that all or any part of the gene sequence can be optimized or synthetic. That is, synthetic or partially optimized sequences can also be used.

[0163] For translation to be effectively initiated, the sequence adjacent to the start methionine encoding the start codon may need to be modified. For example, this can be done by including sequences known to be effective in plants. Joshi proposed consensus sequences suitable for plants (NAR 15:6643-6653 (1987)), and Clonetech proposed further consensus translation initiators (1993 / 1994 table of contents, page 210). These consensus sequences are applicable to the nucleotide sequences of this invention. The sequence is incorporated into constructs containing the nucleotide sequence up to and including ATG (while keeping the second amino acid unmodified), or alternatively up to and including GTC after ATG (with the possibility of modifying the second amino acid of the transgene).

[0164] The Cry21 toxin gene of the embodiments, whether as its natural sequence or as the optimized synthetic sequence described above, can be operatively fused to a variety of promoters for expression in plants, including constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters, to prepare recombinant DNA molecules, i.e., chimeric genes. The choice of promoter will vary depending on the temporal and spatial requirements of expression. Therefore, expression of the nucleotide sequence encoding the Cry21 protein of the embodiments can be achieved in leaves, stems or stalks, spikes, inflorescences (e.g., spike tips, panicles, rachis, etc.), roots, and / or seedlings, but for nematode control, expression in roots is particularly preferred. However, in many cases, protection against more than one type of nematode pest is sought, thus requiring expression in multiple tissues. Although many promoters from dicotyledons have proven to be operable in monocotyledons and vice versa, ideally, dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters are selected for expression in monocotyledons. However, there are no restrictions on the source of the selected promoter; it is acceptable as long as it can drive the expression of the nucleotide sequence of the present invention in the desired cells.

[0165] carrier

[0166] The pest-killing sequences of the present invention can be provided in an expression cassette for expression in target host cells (e.g., plant cells or microorganisms). A "plant expression cassette" refers to a DNA construct capable of causing protein expression from an open reading frame in plant cells. Typically, these contain a promoter and a coding sequence. Typically, such constructs will also contain a 3' untranslated region. Such constructs may contain a signal sequence or leader sequence to facilitate co-translation or post-translational transport of the peptide to certain intracellular structures such as chloroplasts (or other plastids), endoplasmic reticulum, or Golgi apparatus.

[0167] A “signal sequence” is a sequence known or suspected of causing co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, resulting in some form of glycosylation. Bacterial insecticidal toxins are often synthesized as prototoxins, which are activated by proteolysis in the gut of the target pest (Chang (1987) Methods Enzymol. [Enzymological Methods] 153:507-516). In some embodiments, the signal sequence is located in a natural sequence or may be derived from the sequence of this invention.

[0168] "Lead sequence" refers to any amino acid sequence that, during translation, generates a sequence sufficient to trigger co-translational transport of the peptide chain to subcellular organelles. Therefore, this includes leader sequences for targeted transport and / or glycosylation via the endoplasmic reticulum, vacuoles, plastids (including chloroplasts), mitochondria, etc. Accordingly, this document further provides a polypeptide comprising the amino acid sequence of the present invention operatively linked to a heterologous leader sequence or signal sequence.

[0169] “Plant transformation vectors” refer to DNA molecules necessary for the efficient transformation of plant cells. Such molecules can consist of one or more plant expression cassettes and can be organized into more than one “vector” DNA molecule. For example, a binary vector is a plant transformation vector that utilizes two discontinuous DNA vectors to encode all the necessary cis and trans-acting functions for transforming plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).

[0170] A "vector" is a nucleic acid construct designed for transfer between different host cells.

[0171] "Expression vector" refers to a vector capable of incorporating, integrating, and expressing a heterologous DNA sequence or fragment in a foreign cell. The cassette will include 5' and / or 3' regulatory sequences operatively linked to the sequences of the present invention. "Operably linked" refers to a functional link between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Typically, operatively linked means that the linked nucleic acid sequences are adjacent and located within the same reading frame when two protein-coding regions need to be joined. In some embodiments, the nucleotide sequence is operatively linked to a heterologous promoter capable of directing the expression of the nucleotide sequence in a host cell (such as a microbial host cell or a plant host cell). Additionally, the cassette may contain at least one additional gene to be co-transformed into an organism. Alternatively, one or more additional genes may be provided on multiple expression cassettes.

[0172] In various embodiments, the nucleotide sequence of the present invention is operatively linked to a heterologous promoter, such as a plant promoter.

[0173] Such expression cassettes provide multiple restriction sites for inserting harmful organism-killing sequences that will be regulated by transcription in the regulatory region.

[0174] The expression cassette includes, in a 5'-3' transcriptional orientation, a transcription and translation initiation region (i.e., the promoter), the DNA sequence of the present invention, and a transcription and translation termination region (i.e., the termination region). The promoter may be natural or similar, foreign or heterologous, for the plant host and / or the DNA sequence of the present invention. Furthermore, the promoter may be a natural sequence or an alternatively synthesized sequence. When a promoter is "natural" or "homologous" for the plant host, this means that the promoter is present in the natural plant in which it is introduced. When a promoter is foreign or heterologous for the DNA sequence of the present invention, this means that the promoter is not natural or naturally occurring for the operatively linked DNA sequence of the present invention. The promoter may be inducible or constitutive. It may be naturally occurring, may consist of portions of various naturally occurring promoters, or may be partially or completely synthetic. Promoter design guidelines are provided through studies of promoter structure, such as those by Harley and Reynolds (1987) Nucleic Acids Res. [Nucleic Acid Research] 15:2343-2361. Additionally, the position of the promoter relative to the transcription start point can be optimized. See, for example, Roberts et al. (1979) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 76:760-764. Many suitable promoters for plants are well known in the art.

[0175] For example, suitable constitutive promoters for use in plants include: promoters from plant viruses, such as the peanut chlorosis virus (PClSV) promoter (US Patent No. 5,850,019); the 35S promoter from cauliflower mosaic virus (CaMV) (Odell et al. (1985) Nature 313:810-812); the 35S promoter described in Kay et al. (1987) Science 236:1299-1302; the promoter of the Chlorella virus methyltransferase gene (US Patent No. 5,563,328) and the full-length transcript promoter from Scrophularia mosaic virus (FMV) (US Patent No. 5,378,619); and promoters from genes such as rice actin (McElroy et al. (1990) Plant Cell). 2:163-171 and US Patent 5,641,876); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. [Plant Molecular Biology] 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. [Plant Molecular Biology] 18:675-689) and Grefen et al. (2010) Plant J [Plant Journal], 64:355-365; pEMU (Last et al. (1991) Theor. Appl. Genet. [Theoretical and Applied Genetics] 81:581-588); MAS (Velten et al. (1984) EMBO J. [Journal of the European Society for Molecular Biology] 3:2723-2730 and US Patent 5,510,474); maize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. [Molecular Genetics and Genomics]) 231:276-285 and Atanassova et al. (1992) Plant J. 2(3):291-300; European rapeseed (Brassica napus) ALS3 (PCT application WO97 / 41228); plant ribulose-dicarboxylase / oxygenase (RuBisCO) small subunit gene; porcine circovirus (AU 689 311) or cassava vein mosaic virus (CsVMV, US 7,053,205; promoters from soybean (Pbdc6 or Pbdc7 as described in WO / 2014 / 150449, or ubiquitin 3 promoters as described in U.S. Patent Nos. 7,393,948 and 8,395,021); and promoters of various Agrobacterium genes (see U.S. Patent Nos. 4,771,002, 5,102,796, 5,182,200 and 5,428,147).

[0176] Suitable inducible promoters for use in plants include: copper-responsive promoters from the ACE1 system (Mett et al. (1993) PNAS 90:4567-4571); promoters of the corn In2 gene responsive to the benzyl sulfonamide herbicide safener (Hershey et al. (1991) Mol. Gen. Genetics 227:229-237 and Gatz et al. (1994) Mol. Gen. Genetics 243:32-38); and promoters of the Tet repressor protein from Tn10 (Gatz et al. (1991) Mol. Gen. Genet. 227:229-237). Another type of inducible promoter for use in plants is an inducible promoter responsive to inducers that plants do not normally respond to. Exemplary inducible promoters of this type are inducible promoters derived from steroid hormone genes whose transcriptional activity is induced by glucocorticoids (Schena et al. (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 88:10421) or the recent application of the chimeric transcription activator XVE (for use in estrogen receptor-based inducible plant expression systems activated by estradiol) (Zuo et al. (2000) Plant J [Plant Journal], 24:265-273). Other inducible promoters for use in plants are described in EP 332104, PCT WO 93 / 21334 and PCT WO 97 / 06269, which are incorporated herein by reference in their entirety. Promoters composed of partially and partially or fully synthesized promoters from other promoters may also be used. See, for example, Ni et al. (1995) Plant J. 7:661-676 and PCT WO95 / 14098 describing such promoters for use in plants.

[0177] In one embodiment of the invention, promoter sequences specific to a specific region or tissue of a plant can be used to express the nematicidal protein of the invention, such as seed-specific promoters (Datla, R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), especially napin promoter (EP255 378 A1), phaseolin promoter, glutenin promoter, helianthinin promoter (WO92 / 17580), albumin promoter (WO98 / 45460), oleosin promoter (WO98 / 45461), SAT1 promoter, or SAT3 promoter (PCT / US98 / 06978).

[0178] Inducible promoters advantageously selected from the following can also be used: phenylalanine ammonia-lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, protease inhibitor (PI), PR1 family genes, carmine synthase (nos) and vspB promoters (US 5 670 349, Table 3), HMG2 promoter (US 5 670 349), apple β-galactosidase (ABG1) promoter and apple aminocyclopropane carboxylic acid synthase (ACC synthase) promoter (WO98 / 45445). Multiple promoters can be used in the constructs of the present invention, including sequentially.

[0179] Promoters may contain or be modified to contain one or more enhancer elements. In some embodiments, a promoter may contain multiple enhancer elements. Promoters containing enhancer elements provide higher levels of transcription compared to promoters that do not contain enhancer elements. Suitable enhancer elements for use in plants include the PC1SV enhancer element (US Patent No. 5,850,019), the CaMV 35S enhancer element (US Patent Nos. 5,106,739 and 5,164,316), and the FMV enhancer element (Maiti et al. (1997) Transgenic Res. 6:143-156); transcriptional activators of tobacco mosaic virus (TMV) as described in application WO87 / 07644 or, for example, tobacco etch virus (TEV) as described by Carrington and Freed 1990, J. Virol. 64:1590-1597; or introns, such as the adh1 intron of corn or the intron 1 of rice actin. See also PCT WO96 / 23898, WO 2012 / 021794, WO 2012 / 021797, WO 2011 / 084370 and WO 2011 / 028914.

[0180] Typically, such constructs may contain 5' and 3' untranslated regions. These constructs may also contain signal or leader sequences to facilitate co-translation or post-translational transport of the target peptide to certain intracellular structures, such as chloroplasts (or other plastids), the endoplasmic reticulum, or the Golgi apparatus, or to promote the secretion of the target peptide. For example, constructs may be engineered to contain a signal peptide to facilitate peptide translocation to the endoplasmic reticulum.

[0181] The "3' untranslated region" refers to a polynucleotide located downstream of the coding sequence. The polyadenylation signal sequence and other sequences encoding regulatory signals that can affect the addition of polyadenylation nucleotides to the 3' end of the mRNA precursor constitute the 3' untranslated region. The "5' untranslated region" refers to a polynucleotide located upstream of the coding sequence.

[0182] Other upstream or downstream non-translational elements include enhancers. Enhancers are polynucleotides that increase the expression of promoter regions. Enhancers are well known in the art and include, but are not limited to, SV40 enhancer regions and 35S enhancer elements.

[0183] Termination regions can be natural with respect to transcription initiation regions, natural with respect to the target DNA sequence to which operative linking is possible, natural with respect to the plant host, or derived from another source (i.e., foreign or heterologous with respect to the promoter, target DNA sequence, plant host, or any combination thereof). Convenient termination regions can be obtained from Ti-plasmids of Agrobacterium tumefaciens, such as the termination regions of octopaline synthase and carmine synthase. See also Guerineau et al. (1991) Mol. Gen. Genet. [Molecular Genetics and Genomics] 262:141-144; Proudfoot (1991) Cell [Cell] 64:671-674; Sanfacon et al. (1991) Genes Dev. [Genes and Development] 5:141-149; Mogen et al. (1990) Plant Cell [Plant Cell] 2:1261-1272; Munroe et al. (1990) Gene [Genes] 91:151-158; Ballas et al. (1989) Nucleic Acids Res. [Nucleic Acid Research] 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. [Nucleic Acid Research] 15:9627-9639.

[0184] Where appropriate, one or more genes can be optimized to increase expression in transformed host cells (synthetic DNA sequences). That is, genes can be synthesized using host-preferred codons to improve expression, or codons can be used at the frequency of host-preferred codon usage. Expression of the open reading frames of the synthetic DNA sequence in cells results in the production of the polypeptides of the present invention. Synthetic DNA sequences can be used to simply remove unwanted restriction endonuclease sites to favor DNA cloning strategies, alter or remove any potential codon bias, alter or improve GC content, remove or alter altered reading frames, and / or alter or remove intron / exon splicing recognition sites, polyadenylation sites, Shine-Delgarno sequences, unwanted promoter elements, etc., that may be present in native DNA sequences. Typically, the GC content of the gene will be increased. For a discussion of host-preferred codon usage, see, for example, Campbell and Gowri (1990) Plant Physiol. [Plant Physiology] 92:1-11. Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, U.S. Patent Publication No. 20090137409, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, which are incorporated herein by reference.

[0185] It is also possible that the synthetic DNA sequence can be used to introduce other modifications into the DNA sequence, such as introducing intron sequences, producing DNA sequences that express as protein fusions with organelle-targeting sequences, such as chloroplast transport peptides, apoplast / vacuole-targeting peptides, or peptide sequences that cause the resulting peptides to be retained in the endoplasmic reticulum. Thus, in one embodiment, the nematicide protein is targeted to the chloroplast for expression. In this way, without directly inserting the expression cassette encoding the nematicide protein into the chloroplast genome, the expression cassette will additionally contain nucleic acids encoding a transport peptide to guide the nematicide protein to the chloroplast. Such transport peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. [Journal of Plant Molecular Biology] 9:104-126; Clark et al. (1989) J. Biol. Chem. [Journal of Biochemistry] 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. [Plant Physiology] 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. [Biochemical and Biophysical Research Communications] 196:1414-1421; and Shah et al. (1986) Science [Science] 233:478-481.

[0186] The gene that kills harmful organisms will be targeted to the chloroplast; see, for example, U.S. Patent No. 5,380,831, which is incorporated herein by reference.

[0187] Plant transformation

[0188] The methods of the embodiments involve introducing nucleotide constructs into plants. "Introduction" is defined as presenting the nucleotide construct to a plant in a manner that allows the construct to enter the interior of plant cells. The methods of the present invention do not require the use of specific methods for introducing nucleotide constructs into plants; they only require that the nucleotide construct enter the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art, including but not limited to stable transformation methods, transient transformation methods, gene editing, and virus-mediated methods.

[0189] "Transgenic plant," "transformed plant," or "stable transformed" plant, cell, or tissue refers to a plant in which exogenous nucleic acid sequences or DNA fragments have been incorporated or integrated into the plant cell. These nucleic acid sequences include those that are exogenous or not present in untransformed plant cells, as well as those that may be endogenous or present in untransformed plant cells.

[0190] "Heterogeneous" usually refers to nucleic acid sequences that are not endogenous to the cell or part of the natural genome in which they reside and have been added to the cell through infection, transfection, microinjection, electroporation, microprojection, etc.

[0191] The transgenic plants of this invention express one or more of the novel toxin sequences disclosed herein. In some embodiments, the protein or nucleotide sequences of this invention are advantageously combined in the plant with other genes encoding proteins or RNA that confer useful agronomical characteristics to such plants. Among the genes encoding proteins or RNA that confer useful agronomical characteristics to the transformed plants, reference may be made to DNA sequences encoding proteins that confer tolerance to one or more herbicides, other proteins that confer tolerance to certain insects, those proteins that confer tolerance to certain diseases, DNA sequences encoding RNA that provides nematode or insect control, etc. Such genes are particularly described in published PCT patent applications WO91 / 02071 and WO95 / 06128, and U.S. Patent 7,923,602 and U.S. Patent Application Publication No. 20100166723, each of which is incorporated herein by reference in its entirety. In various embodiments, the transgenic plant further comprises one or more additional insect resistance genes (e.g., Cry1, such as members of the Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, and Cry1F families; Cry2, such as members of the Cry2A family; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc.). Those skilled in the art will understand that transgenic plants can contain any gene that confers the desired agronomic trait.

[0192] Among the DNA sequences encoding proteins that confer tolerance to certain herbicides in transformed plant cells and plants, references may be made to the bar or PAT genes or Streptomyces coelicolor genes that confer tolerance to glufosinate herbicides as described in WO 2009 / 152359, and genes encoding appropriate EPSPS that confer tolerance to herbicides targeting EPSPS (such as glyphosate and its salts) (US 4,535,060, US 4,769,061, US 5,094,945, US 4,940,835, US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5,312,910, US 5,627,061, US 4,535,060, US 5,312,910, US 5,627,061, US 5,310,667, US 5,312,910, US 5,627,061 ...312,910, US 5,627,061, US 5,312,910, US 5,312,910, US 5,312,91 Genes encoding glyphosate-n-acetyltransferase (e.g., US 8,222,489, US 8,088,972, US 8,044,261, US 8,021,857, US 8,008,547, US 7,999,152, US 7,998,703, US 7,863,503, US 7,714,188, US 7,709,702, US 7,666,644, US 7,666,643, US 7,531,339, US 7,527,955, and US 7,405,074), genes encoding glyphosate oxidoreductase (e.g., US 5,463,175), or genes encoding HPPD inhibitor tolerance proteins (e.g., WO HPPD inhibitor tolerance genes described in WO 2004 / 055191, WO199638567, US 6791014, WO 2011 / 068567, WO 2011 / 076345, WO 2011 / 085221, WO 2011 / 094205, WO 2011 / 068567, WO 2011 / 094199, WO 2011 / 094205, WO 2011 / 145015, WO 2012 / 056401 and WO 2014 / 043435).

[0193] Among the DNA sequences encoding suitable EPSPS that confer tolerance to herbicides targeting EPSPS, more particular reference will be made to the gene encoding plant EPSPS, especially corn EPSPS, particularly the corn EPSPS containing two mutations (specifically a mutation at amino acid position 102 and a mutation at amino acid position 106) described in patent application US 6566587 (hereinafter referred to as double mutant corn EPSPS or 2mEPSPS) (WO 2004 / 074443), or the gene encoding EPSPS isolated from Agrobacterium and described by sequence ID No. 2 and sequence ID No. 3 of US Patent 5,633,435 (also known as CP4).

[0194] In the DNA sequence encoding suitable EPSPS that confers tolerance to herbicides targeting EPSPS, more specifically will be mentioned the EPSPS, GRG23, or ACE5 encoded from Arthrobacter globiformis, and also the mutants GRG23 ACE1, GRG23 ACE2, or GRG23 ACE3, particularly the GRG23 mutants or variants as described in WO 2008 / 100353, such as the gene for GRG23(ace3)R173K in SEQ ID No. 29 of WO 2008 / 100353.

[0195] In the case of DNA sequences encoding EPSPS and more specifically encoding the aforementioned genes, the sequences encoding these enzymes are advantageously preceded by sequences encoding transport peptides, particularly the sequences of “optimized transport peptides” described in U.S. Patents 5,510,471 or 5,633,448.

[0196] Exemplary herbicide tolerance traits that can be combined with the nucleic acid sequences of the present invention further include at least one ALS (acetolactate synthase) inhibitor (WO 2007 / 024782); a mutant Arabidopsis ALS / AHAS gene (US Patent 6,855,533); a gene encoding a 2,4-D-monooxygenase that confers tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid) through metabolism (US Patent 6,153,401); and a gene encoding a dicamba monooxygenase that confers tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid) through metabolism (US 2008 / 0119361 and US 2008 / 0120739).

[0197] In various embodiments, the nucleic acid of the present invention is superimposed with one or more herbicide tolerance genes, including one or more HPPD inhibitor herbicide tolerance genes, and / or one or more genes resistant to glyphosate and / or glufosinate.

[0198] Among the DNA sequences encoding proteins involved in insect resistance, Bt proteins, which are widely described in the literature and well known to those skilled in the art, will be mentioned in particular. Proteins extracted from bacteria such as *Bacillus luminifera* (WO97 / 17432 and WO98 / 08932) will also be mentioned.

[0199] Among such DNA sequences encoding target proteins that confer novel insect resistance properties, the Bt Cry or VIP proteins, which are widely described in the literature and well known to those skilled in the art, will be mentioned in particular. These include Cry1F proteins or hybrids derived from Cry1F proteins (e.g., hybrid Cry1A-Cry1F proteins or their toxic fragments as described in US 6,326,169, US 6,281,016, US 6,218,188); Cry1A type proteins or their toxic fragments, preferably Cry1Ac proteins or hybrids derived from them (e.g., hybrid Cry1Ab-Cry1Ac proteins as described in US 5,880,275); or Cry1Ab or Bt2 proteins or their insecticidal fragments as described in EP451878; Cry2Ae, Cry2Af, or Cry2Ag proteins or their toxic fragments as described in WO 2002 / 057664; and the Cry1A.105 protein (SEQ ID) as described in WO 2007 / 140256. No. 7) or its toxic fragments; VIP3Aa19 protein of NCBI accession number ABG20428; VIP3Aa20 protein of NCBI accession number ABG20429 (SEQ ID No. 2 in WO 2007 / 142840); VIP3A protein produced in the COT202 or COT203 cotton events (WO 2005 / 054479 and WO 2005 / 054480, respectively); Cry protein as described in WO 2001 / 47952; VIP3Aa protein or its toxic fragments as described in Estruch et al. (1996), Proc Natl Acad Sci US A. [Proceedings of the National Academy of Sciences] 28;93(11):5389-94 and US 6,291,156; VIP3Aa protein or its toxic fragments derived from pathogenic bacilli (as described in WO98 / 50427), Serratia (particularly from Serratia tinctoria (S. tumefaciens)). Insecticidal proteins from *Epiluminobacterium* species or strains; Tc proteins from *Epiluminobacterium* as described in WO98 / 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). This document also includes any variants or mutants of any of these proteins with some (1-10, preferably 1-5) amino acids different from any of the sequences above, particularly sequences of their toxic fragments, or sequences fused to transport peptides such as plasmid transport peptides, or sequences of another protein or peptide.

[0200] In various embodiments, the nucleic acids of the present invention can be combined in plants with one or more genes that confer desired traits, such as herbicide tolerance, insect tolerance, drought tolerance, nematode control, water use efficiency, nitrogen use efficiency, improved nutritional value, disease resistance, improved photosynthesis, improved fiber quality, stress tolerance, improved regeneration, etc.

[0201] Particularly useful transgenic events that can be combined with the genes of the present invention in the same species of plant (e.g., by hybridization or by re-transforming a plant containing another transgenic event with the chimeric gene of the present invention) include event BPS-CV127-9 (soybean, herbicide tolerance, deposited as NCIMB 41603, described in WO 2010 / 080829); event DAS21606-3 / 1606 (soybean, herbicide tolerance, deposited as PTA-11028, described in WO 2012 / 033794); event DAS-44406-6 / pDAB8264.44.06.1 (soybean, herbicide tolerance, deposited as PTA-11336, described in WO2012 / 075426); and event DAS-14536-7. / pDAB8291.45.36.2 (soybean, herbicide tolerance, deposited as PTA-11335, described in WO 2012 / 075429), event DAS68416 (soybean, herbicide tolerance, deposited as ATCC PTA-10442, described in WO 2011 / 066384 or WO 2011 / 066360); event DP-305423-1 (soybean, quality traits, not deposited, described in US-A 2008-312082 or WO 2008 / 054747); event DP-356043-5 (soybean, herbicide tolerance, deposited as ATCC PTA-8287, described in US-A 2010-0184079 or WO Event FG72 (soybean, herbicide tolerant, deposited as PTA-11041, described in WO 2011 / 063413); Event LL27 (soybean, herbicide tolerant, deposited as NCIMB41658, described in WO 2006 / 108674 or US-A 2008-320616); Event LL55 (soybean, herbicide tolerant, deposited as NCIMB 41660, described in WO 2006 / 108675 or US-A 2008-196127); Event MON87701 (soybean, insect control, deposited as ATCC PTA-8194, described in US-A 2009-130071 or WO Event MON87705 (Soybean, quality trait – herbicide tolerance, deposited as ATCC PTA-9241, described in US-A 2010-0080887 or WO 2010 / 037016); Event MON87708 (Soybean, herbicide tolerance, deposited as ATCCPTA-9670, described in WO 2011 / 034704).Event MON87712 (soybean, yield, deposited as PTA-10296, described in WO 2012 / 051199), Event MON87754 (soybean, quality traits, deposited as ATCC PTA-9385, described in WO2010 / 024976); Event MON87769 (soybean, quality traits, deposited as ATCC PTA-8911, described in US-A2011-0067141 or WO 2009 / 102873); Event MON89788 (soybean, herbicide tolerance, deposited as ATCCPTA-6708, described in US-A 2006-282915 or WO 2006 / 130436); Event SYHT0H2 / Event SYN-000H2-5 (soybean, herbicide tolerance, deposited as PTA-11226, described in WO 2012 / 082548), event EE-GM3 / FG72 (soybean, herbicide tolerance, ATCC accession number PTA-11041), which optionally overlaps with event EE-GM1 / LL27 or event EE-GM2 / LL55 (WO 2011 / 063413A2); event DAS-68416-4 (soybean, herbicide tolerance, ATCC accession number PTA-10442, WO 2011 / 066360A1); event DAS-68416-4 (soybean, herbicide tolerance, ATCC accession number PTA-10442, WO 2012 / 082548). Event 2011 / 066384A1); Event DAS-21606-3 (Soybean, herbicide tolerance, ATCC Registry No. PTA-11028, WO 2012 / 033794A2); Event MON-87712-4 (Soybean, quality traits, ATCC Registry No. PTA-10296, WO2012 / 051199A2); Event DAS-44406-6 (Soybean, superimposed herbicide tolerance, ATCC Registry No. PTA-11336, WO2012 / 075426A1); Event DAS-14536-7 (Soybean, superimposed herbicide tolerance, ATCC Registry No. PTA-11335, WO2012 / 075429A1); Event SYN-000H2-5 (Soybean, herbicide tolerance, ATCC Registry No. PTA-11226, WO Event 2012 / 082548A2); Event 8264.44.06.1 (Soybean, herbicide tolerance, ATCC accession number PTA-11336, WO2012075426A2); Event 8291.45.36.2 (Soybean, herbicide tolerance, ATCC accession number PTA-11335, WO2012075429A2); Event SYHT0H2 (Soybean, ATCC accession number PTA-11226, WO 2012 / 082548A2);Event pDAB8264.42.32.1 (Soybean, superimposed herbicide tolerance, ATCC accession number PTA-11993, WO 2013 / 010094A1).

[0202] Furthermore, this document provides a method for producing soybean plants or seeds comprising a combination of a nucleotide sequence encoding SEQ ID NO: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54 or any functional fragment thereof with another SCN resistance locus / gene, such as by combining soybean plants or seeds comprising a nucleotide sequence encoding SEQ ID NO: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54 or any functional fragment thereof with another SCN resistance locus / gene appearing in the same soybean plant / seed, and planting seeds comprising a nucleotide sequence encoding SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 and any functional fragment thereof and said 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 present in soybeans 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 these genes can be combined with plants containing SEQ ID NO: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54 or any functional fragment thereof in the same plant, cell, or seed, such as those from resistance sources PI 88788, PI 548402 (Peking), PI Any SCN resistance gene / locus of 437654 (Hartwig or CystX®) or any combination thereof, or one or more of the natural SCN resistance loci / genes rhg1, rhg1-b, rhg2, rhg3, Rhg4, Rhg5, qSCN11, cqSCN-003, cqSCN-005, cqSCN-006, cqSCN-007, or any SCN resistance locus identified on soybean chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof (Kim et al. 2016, Theor. Appl. Genet. [Theoretical and Applied Genetics] 129(12):2295-2311; Kim and Diers 2013, CropScience 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 combined with one or more SCN resistance loci in soybean, wherein the one or more SCN resistance loci are obtained from SCN resistance sources PI 548316, PI 567305, PI 437654, PI 90763, PI 404198B, PI 88788, PI 468916, PI 567516C, PI 209332, PI 438489B, PI 89772, Peking, PI548402, PI 404198A, PI 561389B, PI 629013, PI 507471, PI 633736, PI 507354, PI404166, PI 437655, PI 467312, PI 567328, PI Either 22897 or PI 494182.

[0203] The transformation of plant cells can be achieved using one of several techniques known in the art. The pest-killing gene of the present invention can be modified to obtain or enhance expression in plant cells. Typically, constructs expressing such proteins will contain a promoter driving gene transcription, as well as a 3' untranslated region allowing transcription termination and polyadenylation. The organization of such constructs is well known in the art. In some cases, it may be useful to engineer the gene so that the resulting peptide is secreted or otherwise targeted within plant cells. For example, the gene can be engineered to contain a signal peptide to promote peptide translocation to the endoplasmic reticulum. It may also be preferred to engineer the plant expression cassette to contain introns such that the mRNA processing of the introns is required for expression.

[0204] Typically, a plant expression cassette is inserted into a plant transformation vector. This transformation vector can consist of one or more DNA vectors required to achieve plant transformation. For example, plant transformation vectors consisting of more than one contiguous DNA segment are commonly used in the art. These vectors are commonly referred to in the art as “binary vectors.” Binary vectors, as well as vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation, where the size and complexity of the DNA segments required for efficient transformation are considerable, and it is advantageous to segregate functions onto different DNA molecules. Binary vectors typically contain a plasmid vector containing cis-acting sequences (such as left and right borders) required for T-DNA transfer, an engineered selection marker that can be expressed in plant cells, and a “target gene” (a gene engineered for expression in plant cells, with the intention of generating transgenic plants). The plasmid vector also contains sequences required for bacterial replication. The arrangement of the cis-acting sequences allows for efficient transfer to and expression in plant cells. For example, the selection marker gene and the pest-killing gene are located between the left and right borders. Typically, the second plasmid vector contains a trans-acting factor that mediates the transfer of T-DNA from Agrobacterium to plant cells. This plasmid typically contains virulence functions (Vir genes) that allow Agrobacterium to infect plant cells and transfer DNA via cleavage at the boundary sequence and vir-mediated DNA transfer, as understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. A second plasmid vector is not essential for transforming plants via other methods such as microspraying, microinjection, electroporation, polyethylene glycol, etc.

[0205] Typically, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying an appropriately selected maximum threshold level (depending on the selective marker gene) to recover the transformed plant cells from a cluster of untransformed cell masses. Typically, explants are transferred to a fresh supply of the same medium and cultured routinely. Subsequently, after being placed on a regeneration medium supplemented with a maximum threshold level of selectant, the transformed cells differentiate into shoots. The shoots are then transferred to a selective rooting medium to recover the rooted shoots or plantlets. The transgenic plantlets then grow into mature plants and produce fertile seeds (e.g., Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Typically, explants are transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods used to generate transgenic plants can be found in Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Because transformed material contains numerous cells; both transformed and untransformed cells are present in any patch of target callus or tissue or cell population. The ability to kill untransformed cells and allow transformed cells to proliferate produces transformed plant cultures. Typically, the ability to remove untransformed cells limits the rapid recovery of transformed plant cells and the successful generation of transgenic plants.

[0206] Transformation protocols, and protocols for introducing nucleotide sequences into plants, can vary depending on the type of plant or plant cell targeted for transformation (i.e., monocot or dicot). Transgenic plants can be generated by one of several methods, including but not limited to microinjection, electroporation, direct gene transfer, introduction of heterologous DNA into plant cells via Agrobacterium (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous DNA adhering to particles, ballistic particle acceleration, aerosol micelle transformation (US Publication No. 20010026941; US ​​Patent No. 4,945,050; International Publication No. WO 91 / 00915; US Publication No. 2002015066), Lec1 transformation, and various other non-particle-directly mediated methods for DNA transfer.

[0207] Methods for transforming chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:913-917; Svab and Maliga (1993) EMBO J. [Journal of the European Society for Molecular Biology] 12:601-606. These methods rely on particle gun delivery of selectively labeled DNA and targeting the DNA to the plastid genome via homologous recombination. Alternatively, plastid transformation can be accomplished by transactivating silent plastid-carrying transgenes through tissue-biased expression of a nuclear-encoded, plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 91:7301-7305.

[0208] After the heterologous DNA is integrated into plant cells, an appropriate maximum selection threshold level is applied to the culture medium to kill untransformed cells, and the presumed transformed cells that survived this selection treatment are isolated and proliferated by periodically transferring them to fresh culture medium. Cells transformed with plasmid vectors are identified and proliferated through continuous passage and stimulation with appropriate selection. The presence of the target heterologous gene integrated into the genome of the transgenic plant can then be confirmed using molecular and biochemical methods.

[0209] The transformed cells can be grown into plants using conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same or different transformed lines, and the resulting hybrids with constitutive expression of the desired phenotypic trait can be identified. Two or more generations can be grown to ensure that the expression of the desired phenotypic trait is stably maintained and inherited, and the seeds are then harvested to ensure that the expression of the desired phenotypic trait has been achieved. In this way, the present invention provides transformed seeds (also known as “transgenic seeds”) having nucleotide constructs of the present invention stably incorporated into their genome, such as the expression cassette of the present invention.

[0210] Evaluation of plant transformation

[0211] After introducing heterologous DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods, such as analyzing nucleic acids, proteins and metabolites associated with the integrated gene.

[0212] PCR analysis is a rapid method for screening the presence of genes incorporated into transformed cells, tissues, or shoots at an early stage before transplantation into soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Spring Harbor Laboratory Press, Cold Spring Harbor, New York). PCR is performed using oligonucleotide primers specific to the target gene or the Agrobacterium vector background.

[0213] Plant transformation can be confirmed by DNA blot analysis of genomic DNA (Sambrook and Russell, 2001, ibid.). Typically, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel, and transferred to a nitrocellulose or nylon membrane. The membrane, or “blot,” is then probed with, for example, a radiolabeled 32P target DNA fragment according to standard techniques (Sambrook and Russell, 2001, ibid.) to confirm the integration of the introduced gene into the plant genome.

[0214] In RNA blot analysis, RNA was isolated from specific tissues of the transformant according to standard procedures routinely used in the art (Sambrook and Russell, 2001, ibid.), fractionated in formaldehyde agarose gel, and blotted onto a nylon filter membrane. The expression of RNA encoded by the nemesis gene was then tested by hybridizing the filter membrane with a radioactive probe derived from the nemesis gene, using methods known in the art (Sambrook and Russell, 2001, ibid.).

[0215] Western blotting and biochemical assays can be performed on transgenic plants. Using antibodies that bind to one or more epitopes present on nematicide proteins, the presence of proteins encoded by pest-killing genes can be confirmed through standard procedures (Sambrook and Russell, 2001, ibid.). In many biological studies, mass spectrometry is currently the most widely used and accepted method for quantifying low-abundance proteins and peptides. Mass spectrometry assays were developed for each target protein and used to quantify gene expression in transformed tissues. Reference: (J Proteome Res. Feb. 2009; 8(2): 787–797. Mass spectrometry based targeted protein quantification: methods and applications [Mass spectrometry based targeted protein quantification: 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.

[0216] Pesticide activity in plants

[0217] On the other hand, transgenic plants expressing nematicidal proteins with pest-killing activity against nematode pests can be generated. The methods described above by way of example can be used to generate transgenic plants, but the manner in which transgenic plant cells are generated is not decisive for this invention. Methods known or described in the art, such as Agrobacterium-mediated transformation, gene gun transformation, and non-particle-mediated methods, can be used according to the experimenter's judgment. Plants expressing nematicidal proteins can be isolated by common methods described in the art, such as by transforming callus, selecting transformed callus, and regenerating fertile plants from such transgenic callus. In such methods, any gene can be used as a selective marker, provided that its expression in plant cells confers the ability to identify or select transformed cells.

[0218] Many markers for plant cells have been developed, such as those for resistance to chloramphenicol, aminoglycoside G418, and hygromycin. Other genes encoding products involved in chloroplast metabolism can also be used as selective markers. For example, genes providing resistance to plant herbicides such as glyphosate, bromobenzonitrile, or imidazolinone can have specific uses. Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314 (bromobenzonitrile resistant nitrile hydrolase gene); and Sathasivan et al. (1990) Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene). Furthermore, the genes disclosed herein can be used as markers for assessing bacterial or plant cell transformation. Methods for detecting the presence of transgenes in plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos, or their progeny are well known in the art. In one embodiment, the presence of transgenes is detected by testing for pest-killing activity against nematode pests.

[0219] The biocidal activity of fertile plants expressing nematicides against nematode pests can be tested, and plants exhibiting the best activity can be selected for further breeding. A standard greenhouse method has been developed for evaluating soybean cyst 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), which is incorporated herein by reference. Modifications to this standard protocol can be used to demonstrate nematicidal activity in fertile plants expressing nematicidal proteins, see Kahn, TW, Duck, NB, McCarville, MT, et al. A Bacillus thuringiensis Cryprotein controls soybean cyst nematode in transgenic soybean plants. Nat Commun. [Nature Communications] 12,3380 (2021). https: / / doi.org / 10.1038 / s41467-021-23743-3 herein, which is incorporated herein by reference.

[0220] This 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, corn, sorghum, wheat, sunflower, tomato, cruciferous plants, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and rapeseed, Brassica species, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, nectarine, fig, guava, mango, olive, papaya, cashew, macadamia nut, apricot, oats, vegetables, ornamental plants, and conifers.

[0221] Applications in pest control

[0222] General methods for employing strains containing the nucleotide sequence of the present invention or variants thereof in pest control or in the engineering of other organisms as pest control agents are known in the art. See, for example, U.S. Patent Nos. 5,039,523 and EP 0480762A2.

[0223] Microorganisms can be genetically modified to contain nucleotide sequences encoding SEQ ID NO: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54 or their nematicidal active variants or fragments, and proteins can be used to protect crops and products from pests. In one aspect of the invention, when cells are applied to an environment containing one or more target pests, the intact (i.e., unlyzed) cells of an organism producing a toxin (pesticide) are treated with an agent that prolongs the activity of the toxin produced in the cells.

[0224] Alternatively, pest control agents are produced by introducing a pest-killing gene into a cell host. Expression of the pest-killing gene directly or indirectly leads to the intracellular production and maintenance of the pest control agent. In one aspect of the invention, when cells are applied to an environment containing one or more target pests, these cells are then treated under conditions that prolong the activity of the toxins produced in the cells. The resulting product retains the toxicity of the toxin. These naturally encapsulated pest control agents can then be formulated according to conventional techniques for use in environments containing target pests, such as soil, water, and plant leaves. See, for example, EPA 0192319 and the references cited therein. Alternatively, cells expressing the genes of the invention can be formulated to allow the resulting material to be used as a pest control agent.

[0225] The active ingredients of this invention are typically applied in the form of a composition and can be applied simultaneously or sequentially with other compounds to the crop area or plant to be treated. These compounds can be fertilizers, herbicides, cryoprotectants, surfactants, detergents, biocidal soaps, dormant oils, polymers, and / or timed release or biodegradable carrier formulations that allow for long-term administration to the target area after a single application of the formulation. They can also be selective herbicides, chemical insecticides, viricides, microbial agents, amoebics, pest control agents, fungicides, bactericides, nematicides, molluscicides, or mixtures of several of these formulations, if desired, along with other agriculturally acceptable carriers, surfactants, or application-promoting adjuvants commonly used in the field of formulations. Suitable carriers and adjuvants can be solids or liquids and correspond to substances commonly used in formulation technology, such as natural or recycled minerals, solvents, dispersants, wetting agents, thickeners, binders, or fertilizers. Similarly, preparations can be made into edible "bait" or pest "traps" to allow target pests to ingest or consume pest-killing preparations.

[0226] Methods of applying the active ingredient of the present invention or the agrochemical composition of the present invention containing at least one nematicidal protein disclosed herein as SEQ ID NO: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, 54 or a nematicidal effective variant or fragment thereof include foliar application, seed coating, and soil application. The amount and proportion of application depend on the intensity of the corresponding pest infestation.

[0227] The composition can be formulated as a powder, dust, pill, granule, spray, emulsion, colloid, solution, or the like, and can be prepared by conventional methods such as dehydration, freeze-drying, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of cell cultures containing the polypeptide. In all such compositions containing at least one such biocidal polypeptide, the polypeptide may be present at a concentration of about 1% to about 99% by weight.

[0228] The method of this invention can kill or reduce the number of nematode pests in a given area, or it can be applied preventively to environmental areas to prevent the infestation of susceptible pests. Preferably, the pests ingest or come into contact with a biocidally effective amount of the polypeptide.

[0229] The described biocidal composition can be prepared by formulating suspensions of bacterial cells, crystals, and / or spores, or isolated protein components having a desired agriculturally acceptable carrier. The composition can be formulated prior to application by suitable methods, such as lyophilization, freeze-drying, dehydration, or in an aqueous carrier, medium, or suitable diluent such as saline or other buffer. The formulated composition can be in the form of powder or granular material, or in the form of a suspension in oil (vegetable or mineral oil), or in the form of an oil or oil / water emulsion, or as a wettable powder, or in combination with any other carrier material suitable for agricultural applications. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally acceptable carrier" encompasses all adjuvants, inert components, dispersions, surfactants, thickeners, binders, etc., commonly used in biocidal formulation techniques; these are well known to those skilled in the art in biocidal formulation. The formulation can be mixed with one or more solid or liquid adjuvants and prepared by various means, such as homogenizing, blending, and / or grinding the biocidal composition with a suitable adjuvant using conventional formulation techniques. Suitable formulations and methods of application are described in U.S. Patent No. 6,468,523, which is incorporated herein by reference.

[0230] Methods for increasing plant yield

[0231] Methods for increasing plant yields are provided. These methods include providing a plant or plant cell expressing a polynucleotide encoding a nematicidal polypeptide sequence disclosed herein, and growing the plant or its seeds in a field infested (or susceptible to) a nematode pest, the polypeptide having nematicidal activity against that nematode pest. In some embodiments, the Cry21 polypeptide described herein has nematicidal activity against a species of the genus *Pratylenchus*, and the field is infested with said *Pratylenchus* species. In various embodiments, the *Pratylenchus* species are *Pratylenchus zeae* or *Pratylenchus zeae*. In other embodiments, the nematode is a root-knot nematode, a disease nematode, a soybean cyst nematode, or a spear nematode, or a target pest.

[0232] As used herein, the plant term “yield” refers to the quality and / or quantity of biomass produced by a plant. “Biomass” means any measured plant product. An increase in biomass production means any improvement in the yield of a measured plant product. Increased plant yield has several commercial applications. For example, increasing the biomass in plant leaves can increase the yield of leafy green vegetables for human or animal consumption. Additionally, increased leaf biomass can be used to increase the production of plant-derived pharmaceuticals or industrial products. Increased yield can include any statistically significant increase, including but not limited to increases of at least 1%, at least 3%, at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, or more, compared to plants that do not express the pest-killing proteins described herein. In certain methods, plant yield is increased due to improved nematode resistance in plants expressing the nematicidal proteins disclosed herein. Expression of nematicidal proteins leads to a reduction in the ability of pests to infect or consume them. In various embodiments, compared with plants that do not express the nematicidal proteins of the present invention, expression of the nematicidal proteins leads to improved root development (e.g., improved root or root hair growth), improved yield, faster emergence, improved plant stress management (including increased stress tolerance and / or improved stress recovery), increased mechanical strength, improved drought resistance, reduced fungal disease infection, and improved plant health.

[0233] Plants can also be treated with one or more chemical compositions, including one or more herbicides, insecticides, or fungicides. Exemplary chemical compositions include: fruit / vegetable herbicides: atrazine, chlorpyrifos, diuron, glyphosate, linuron, cyprodinil, simazine, trifluralin, quizalofop-P-ethyl, glufosinate, chlorpyrifos, paraquat, pendimethalin, haloxyfop-R-methyl, flupropargyl, chlorpyrifos, and triazine-indomethacin.

[0234] Fruit / vegetable pesticides: Aldicarb, Bacillus thuringiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Delmethrin, Abamectin, Cypermethrin / β-cypermethrin, Centrifugerin, Lambda-cyhalothrin, Acaricide, Bifenazate, Methoxyfenozide, Flufenoxuron, Cyclofenozide, Thiamethoxam, Dinotefuran, Pyrimethanil, Spirodiclofen, Gamma-cyhalothrin, Spirodiclofen, Spinosad, Chlorfenapyr, Brofenoxuron, Cyclofenozide, Spirodiclofen, Imidacloprid, Flufenoxuron, Thiamethoxam, Cyclofenozide, Flufenoxuron, Flufenoxuron, Difenoconazole, Cyanopyr, Thiamethoxam, Thiamethoxam, Spinotoraram, Thiamethoxam, Flufenoxuron, Cypermethrin, Emamectin benzoate, Indoxacarb, Benzophos, Pyriproxyfen Ethers, benzoyl tin oxide; Fruit / vegetable fungicides: azoxystrobin, pyraclostrobin, benomyl, boscalid, captan, carbendazim, chlorothalonil, copper, cyazofamid, cycloflufenoxam, cymoxanil, cyclopyridaben, cyclopyridaben, difenoconazole, dimethomorph, dioxin, anthraquinone, imidacloprid, cyclopyridaben, fluazinam, fluopyram, fluopyram, flupyraclostrobin, fluopyram. The following are listed: 1. Captan, 2. Triethylphosphonic acid, 2. Iprodione, 2. Propiconazole, 2. Pyraclostrobin, 2. Azoxystrobin, 2. Mancozeb, 2. Dimethomorph, 2. Metalaxyl / Metazan, 2. Mancozeb, 2. Benomyl, 2. Cyproconazole, 2. Tebuconazole, 2. Thiophanate-methyl, 2. Azoxystrobin, 2. Pyrimethanil, 2. Quinoxystrobin, 2. Spirocyclohexane, 3. Thiophanate-methyl, 2. Azoxystrobin.

[0235] Cereal herbicides: 2,4-D, pyrimisulfuron, bromosulfuron, chlorpyrifos-E, chlorpyrifos, clodinafop-P, dichloropyridine, dicamba, quizalofop-M, pyrfluthrin, quizalofop-P, flusulfuron-methyl, fluoxetine, fluoxetine-NA, fluthiamethoxam, flupyrimisulfuron-M, fluroxypyr, furazolidone, glyphosate, iodosulfuron-methyl, iodosulfuron-methyl, isoproturon, MCPA, mesosulfuron-methyl, mesosulfuron-methyl, pendimethalin, clodinafop-methyl, bensulfuron-methyl, sulfadiazine, sulfonylsulfuron-methyl, thifensulfuron-methyl, oxadiazon, fensulfuron-methyl, bensulfuron-methyl, trifluralin, trifluralin.

[0236] Grain fungicides: azoxystrobin, bifenthrin, boscalid, carbendazim, chlorothalonil, cycloflufenoxam, cyclopyrrolidone, azoxystrobin, pyrimethanil, fenpropathrin, flutriafol, benzyl benzoate, butylmorph, flupyroxypyr, fluquinazole, fluazinam, fluazinam, fluazinam, pyraclostrobin, fenpropathrin, tebuconazole, pyraclostrobin, azoxystrobin, prochloraz, propiconazole, propoxyquin, prothioconazole, azoxystrobin ester, quinoxalic acid, spirocycline, tebuconazole, thiophanate-methyl, azoxystrobin.

[0237] Cereal insecticides: dimethoate, lambda-cyhalothrin, deltamethrin, α-cyhalothrin, β-cyhalothrin, bifenthrin, imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, dinotefuran, chlorpyrifos, pirimicarb, thiamethoxam, flupyridine; Corn herbicides: atrazine, metolachlor, bromobenzonitrile, acetochlor, dicamba, dichloropyridine acid, (S-)-methylphenidate, glufosinate, glyphosate, isoxaflutole, (S-)-metolachlor, mesosulfuron, nicosulfuron, flusulfuron, sulfadiazine, sulfadiazine, formamide sulfadiazine, benzoxazine, cyclosulfuron, pyrimisulfuron, thiamethoxam, flusulfiram, pyrazosulfuron-methyl, pyrazosulfuron-methyl.

[0238] Insecticides for corn: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-cyhalothrin, Heptamethrin, Terbufos, Thiamethoxam, Thiamethoxam, Spirodiclofen, Fipronil, Lufenuron, Cypermethrin, Chlorantraniliprole, Deltamethrin, Thiamethoxam, Bifenthrin, Lufenuron, Butylpyridinium, Ethylbutazone, Brofenac, Thiamethoxam, Acetamiprid, Dinotefuran, Abamectin; Fungicides for corn: Azoxystrobin, Bifenopram, Cyclopyralid, Cyclopyralid, Azoxystrobin, Fluopyram, Seed coat ester, Fluopyram, Flupyraclostrobin, Fluopyram, Pyraclostrobin, Pyraclostrobin, Pyraclostrobin, Azoxystrobin, Propiconazole, Prothioconazole, Azoxystrobin, Tebuconazole, Azoxystrobin.

[0239] Rice herbicides: butachlor, propargite, tetrazole sulfidon, bensulfuron-methyl, cyhalofop-butyl, chlorpyrifos, tetrazole sulfidon, imazalil, benzylthiamethoxam, oxadiazon, pyrimisulfuron, barnyardgrass, quinclorac, quizalofop-p-ethyl, indicarb, fluthiamethoxam, tetrazole sulfidon, chlorpyrifos, benzo[a]cyclohexane, cyclohexyl-methyl, penoxsulam, bispyribac-sodium, propyzoxystrobin, ethoxysulfuron, pretilachlor, mesotrione, tebufenozide, oxadiazon, quizalofop-p-ethyl, pyrimisulfan.

[0240] Rice insecticides: diazinon, tebufenozide, carbofuran, thiamethoxam, fipronil, fipronil, imidacloprid, isoprocarb, thiamethoxam, cyclonepropalin, thiamethoxam, acetamiprid, flufenoxuron, chlorantraniliprole, deltamethrin, acetamiprid, thiamethoxam, brofenoxuron, spinosad, snotomum, emamectin benzoate, cypermethrin, chlorpyrifos, permethrin, carbofuran, carbofuran, and flupyradifurone.

[0241] Rice fungicides: azoxystrobin, carbendazim, cyproconazole, diclofenac, difenoconazole, cymoxanil, azoxystrobin, gentamicin, hexaconazole, oxamyl, isoprothiolane (IBP), isoprothiolane, isothiazinam, kasugamycin, mancozeb, fenoxystrobin, oxadiazon, pendimethalin, thiamethoxam, propiconazole, propineb, quinclorac, tebuconazole, thiophanate-methyl, thiamethoxam, tricyclazole, oxadiazon, jinggangmycin.

[0242] Cotton herbicides: diuron, fenflur, MSMA, ethoxysulfuron, prochloraz, trifluralin, cyclohexane, clethodim, butylpyrrolidone, glyphosate, pyrazosulfuron, pendimethalin, pyrimethanil, trifluralin, pyrazosulfuron, glufosinate, propyzoxystrobin, thidium-methyl; Cotton insecticides: acephate, aldicarb, chlorpyrifos, cypermethrin, deltamethrin, abamectin, acetamiprid, emamectin, imidacloprid, indoxacarb, lambda-cyhalothrin, spinosad, thiamethoxam, γ-cyhalothrin Spirodiclofen, Pyridalyl, Flupyridine, Flufenoxuron, Chlorantraniliprole, β-cypermethrin, Spirodiclofen, Thiamethoxam, Thiamethoxam, Thiamethoxam, Dinotefuran, Flufenoxuron, Bromhizometamide, Sponsauce, Ethyl Sponsauce, γ-Cypermethrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one), Thiamethoxam, Abamectin, Flupyridine, Pyridalyl, Spirodiclofen, Flupyridine,

[0243] Cotton fungicides: azoxystrobin, bifenthrin, cyazofamid, carbendazim, chlorothalonil, copper, cyclohexane, difenoconazole, fenpropathrin, flutriafol, imidacloprid, fluazinam, fluopyram, flupyraclostrobin, fluazinam, iprodione, pyraclostrobin, isothiazin, mancozeb, mancozeb, fenoxystrobin, pyraclostrobin, cyazofamid, propineb, prothioconazole, azoxystrobin, pentachloronitrobenzene, tebuconazole, flufenoxuron, thiophanate-methyl, oxadiazon; Soybean herbicides: metolachlor, bentazon, trifluralin, chlorpyrifos, chlorpyrifos-methyl, quizalofop-p-ethyl, flusulfanilamide, pyraclostrobin, glyphosate, methoxyfenozide, metolachlor, imazalofop-p-ethyl, (S-)-metolachlor, cyhalofop-p-ethyl, pendimethalin, pyranofop-p-ethyl, glufosinate.

[0244] Soybean insecticides: λ-cyhalothrin, methomyl, imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, fipronil, flufenoxuron, chlorantraniliprole, spinosad, snutomom, emamectin benzoate, fipronil, acetamiprid, deltamethrin, β-cyhalothrin, γ and λ-cyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, spirotetramat, spirodiclofen, chlorfenapyr, flufenoxuron, thiamethoxam, β-cyhalothrin.

[0245] Soybean fungicides: azoxystrobin, bifenthrin, cyazofamid, carbendazim, chlorothalonil, copper, cyclohexane, difenoconazole, fenpropathrin, flutriafol, fluazinam, fluopyram, flupyraclostrobin, fenpyroxacin, fluopyram, pyraclostrobin, iprodione, isothiazine, mancozeb, mancozeb, tebuconazole, fenoxystrobin, pyrimethanil, cyazofamid, cyazofamid, cyazofamid, propiconazole, propineb, prothioconazole, thiophanate-methyl, cycloheximetris.

[0246] Beet herbicides: chlorpyrifos, betaine, betaine furazolidone, betaine nitrate, chlorpyrifos, dichloropyridine, pyrifluquinazon, cyclopyridamole, benzoxazon, quinacrine, thiamethoxam, flusulfanilamide, pyranoxam, quizalofop-p-ethyl; Beet insecticides: imidacloprid, thiamethoxam, thiamethoxam, acetamiprid, dinotefuran, deltamethrin, β-cyhalothrin, γ / λ cyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, heptaflupyr, chlorantraniliprole, cyyaxypyr, fipronil, carbofuran.

[0247] Canola herbicides: dichloropyridine, quizalofop-P-ethyl, quizalofop-P-ethyl, glyphosate, glufosinate, pyraclostrobin, trifluralin, sulfanilamide, quinaloxam, quizalofop-P-ethyl, clethodim, pyranofop-P-ethyl; Canola fungicides: azoxystrobin, bifenthrin, cyazofamid, carbendazim, cyclopyrrolidone, difenoconazole, fenpropathrin, flutriafol, fluazinam, fluopyram, flupyraclostrobin, flusilazole, fluazinam, iprodione, pyraclostrobin, mepiquat chloride, tebuconazole, fenoxystrobin, paclobutrazol, pyraclostrobin, cyazofamid, prochloraz, prothioconazole, azoxystrobin, tebuconazole, thiophanate-methyl, oxadiazon, vinclozolin.

[0248] Canola insecticides: Carbofuran, Thiamethoxam, Deltamethrin, Imidacloprid, Thiamethoxam, Thiamethoxam, Acetamiprid, Dinotefuran, β-Cypermethrin, γ and λ-Cypermethrin, tau-Fluvaleriate, Ethylbutazone, Spondimethalin, Snomenom, Flufenoxuron, Chlorantraniliprole, Bromhexanil, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one.

[0249] The following examples are provided in an illustrative rather than restrictive manner. Experimental Examples

[0250] Example 1. Expression of the nematicide Cry21 gene in soybean.

[0251] Using constructs containing a gene encoding a 4-hydroxyphenylpyruvate dioxygenase protein (HPPD) inhibitor-resistant herbicide gene (described in WO 2014043435) and a Cry21 coding sequence, soybean events expressing novel Cry21 homologs (SEQ ID NO:2 (Axmi296) and SEQ ID NO:54 (Cry21Aa2), respectively) were developed via Agrobacterium-mediated transformation of Thorne soybean plants. Wild-type Thorne soybean served as a non-nematode resistance control. When Cry21 was expressed in soybean plants, it reduced the number of the shortest-tailed, short-bodied nematodes that reproduce in the roots compared to wild-type plants. This particular Cry21 (SEQ ID NO:2) gene shares approximately 65% ​​sequence homology with Cry21Aa. Alignment of the Cry21s indicated in this application with known Cry genes, including Cry21s, suggests a potential new or different mode of action that can be combined with other nematode resistance genes to control nematodes.

[0252] Figures and tables depicting plant transformation vectors used for Axmi296 (SEQ ID NO:2) plant expression.

[0253] Figure 1 Plant transformation vectors for expressing Axmi296 in plants are described.

[0254] Table 1 shows a description of the genetic elements of the construct.

[0255] Table 2 shows the references for the genetic elements of the construct.

[0256] Table 1: Description of genetic elements of the construct

[0257]

[0258]

[0259] Table 2: References for Constructs

[0260]

[0261]

[0262]

[0263] Figure 2 The NEEDLE sequence alignment of Axmi296 and Cry21Aa2 was depicted. The "DRIE" motif, separating the N-terminal and C-terminal crystal domains, was described using... “” indicates the sequence. Identical amino acids are indicated by black shading, and the consensus sequence (Cons) is given below the aligned sequence. Overall, there is 66% sequence identity between Axmi296 and Cry21Aa2 (74% sequence identity when vacancies are ignored). Internal data suggest that both genes are active against nematodes and are not limited by theory; the alignment below suggests that these two proteins may utilize the same mode of action to combat plant nematodes.

[0264] Example 2. Soybean Conversion

[0265] Soybean transformation was achieved using methods well-known in the art, such as the described method of transformation of soybean hemi-seed explants mediated by Agrobacterium tumefaciens, which essentially follows the method described by Paz et al. (2006), Plant Cell Reports 25:206. Cyclosulfonyl was used as a selection marker to identify transformants. The appearance of green shoots was observed and recorded as an indicator of tolerance to the herbicides isoxaflutole or cyclosulfonyl. Tolerant transgenic shoots would show normal greening comparable to wild-type soybean shoots not treated with isoxaflutole or cyclosulfonyl, while wild-type soybean shoots treated with the same amount of isoxaflutole or cyclosulfonyl would be completely bleached. This indicates that the presence of HPPD proteins enables tolerance to HPPD inhibitor herbicides, such as isoxaflutole or cyclosulfonyl.

[0266] Tolerant green shoots were transferred to rooting medium or transplanted. After the acclimatization period, the rooted plantlets were transferred to a greenhouse. Transgenic plants were selected for the transformation medium.

[0267] Example 3. Greenhouse and field trials

[0268] Overall, soybean events testing novel Cry21 homologs and Cry21Aa2 against RKN, SCN, and the shortest-tailed nematode were conducted in greenhouses and eight field trials in the US Midwest and Brazil. Exemplary field trial results are shown below.

[0269] Figure 3a and 3b The efficacy of Axmi296 against the shortest-tailed nematode was demonstrated. Greenhouse data showed increased mortality from the shortest-tailed nematode compared to ThorneWT (wild-type, non-resistant control), and a slight advantage compared to soybean Cry14 (a known resistant control).

[0270] Figure 4aand 4b This indicates that Axmi296 provides increased yield benefits compared to the non-resistant SCN control in SCN field trials (3 independent sites).

[0271] Figure 4c The results of a field trial show that the novel Cry21 homolog (Axmi296) reduced SCN sporangia on roots by 20% compared to wild-type Thorne.

[0272] Figure 4d The results show that in SCN-infected fields, the novel Cry21 homolog (Axmi296) produced an average yield increase of approximately 2% compared to wild-type Thorne.

[0273] Figure 5 The image depicts an Axmi296 plant (left) next to a non-resistant control soybean plant in a field infected with SCN (right).

[0274] Table 3 depicts novel Cry21 homologs and other Cry21 homologous sequences. Protein sequence identity with the closest Cry21 holotype was determined by pairwise comparison, according to Crickmore et al., 2021.

[0275] Table 3: Identification of other Cry21 homologous sequences

[0276]

[0277] Table 4 presents the sequence listing of the novel Cry21 homologous sequences disclosed in this paper.

[0278] Table 4: List of novel Cry21 homolog sequences

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349] All publications and patent applications mentioned in this specification indicate the level of skill of a person skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

[0350] Although the invention has been described in detail by way of illustration and examples for purposes of clarity, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

Claims

1. A method for controlling nematode pests, the method comprising contacting the nematode pest with a Cry21 protein, the Cry21 protein comprising an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 or an effective nematicidal variant or fragment thereof.

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

3. The method as described in claims 1-2, wherein the nematode pest is a target pest and / or the shortest-tailed nematode.

4. The method of claims 1-3, wherein the nematicidal effective variant or fragment thereof comprises at least 10, 20, 30, 40, 50 or 60 consecutive amino acids of SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51 or 53.

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

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

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

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

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

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

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

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

13. An expression cassette comprising nucleic acid encoding a Cry21 protein, the Cry21 protein comprising an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 or their nematicidal effective variants or fragments thereof.

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

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

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

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

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

19. A nematicide-effective fusion protein comprising any one of SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51 or 53.

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

21. A method for controlling the shortest-tailed nematode, the method comprising expressing a protein Cry21, Cry21Aa, Cry21Aa2 or Cry21Aa1 in a plant, wherein the protein comes into contact with the shortest-tailed nematode pest, thereby controlling the shortest-tailed nematode pest.

22. The method of 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 of SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53, or an effective nematicidal variant or fragment thereof.

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

24. A nucleic acid encoding a protein comprising an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any of SEQ ID NO: 1, 7, 9, 13, 15, 21, 23, 33, 35, 39, 41, 51, 53 or their nematicidal effective variants or fragments thereof.

25. The nucleic acid of claim 24, wherein the nucleic acid comprises a nucleic acid sequence having a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 2, 8, 10, 14, 16, 22, 24, 34, 36, 40, 42, 52, or 54.

26. The nucleic acid of claims 24 and 25, wherein the nucleic acid sequence comprises any one of SEQ ID NO: 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 as described in claims 24-26 in its cells.

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

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

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

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

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

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

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

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

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

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

38. A plant comprising an expression cassette as described in claims 29-32, wherein the promoter is capable of driving the expression of the protein to a level sufficient to inhibit target pests and / or the shortest-tailed nematode, wherein the proliferation of target pests and / or the shortest-tailed nematode fed on the plant is reduced compared to that fed on a control plant not containing the expression cassette.

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

40. A method for controlling nematode pests, the method comprising contacting the nematode pest with a Cry21 protein, the Cry21 protein comprising 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 NO: 1, 3, 7, 9, 11, 13, 15, 17, 21, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 or an effective nematode-killing variant or fragment thereof.

41. The method of claim 40, wherein the nematode pest is selected from any one of the following species: small ring nematode, stem nematode, golden nematode, spiral nematode, cyst nematode, long needle nematode, root-knot nematode, pseudo-spinous nematode, short-bodied nematode, perforating nematode, coiled nematode, kidney-shaped nematode, pad-shaped nematode, and dagger nematode.

42. An expression cassette comprising nucleic acid encoding a Cry21 protein, the Cry21 protein comprising 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 NO: 1, 3, 7, 9, 11, 13, 15, 17, 21, 23, 25, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 or their nematicidal effective variants or fragments thereof.

43. The expression cassette of claim 42, wherein the nucleic acid is operatively ligated to the nucleic acid.

44. The expression cassette as claimed in claims 42-43, wherein the expression cassette is in a plant expression vector.

45. The nucleic acid of claim 42, wherein the nucleic acid comprises a nucleic acid sequence having a sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO: 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.

Images