Use of AXMI277 for the control of rotylenchulus reniformis nematode pests

Abstract

Compositions and methods for conferring reniform nematicidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Methods for killing or controlling a reniform nematode pest population, including a Rotylenchulus spp., e.g., Rotylenchulus reniformis are provided. The methods include contacting the reniform nematode pest with a pesticidally-effective amount of a polypeptide comprising a reniform nematicidal toxin. Further included are methods for increasing yield in plants by expressing the genes of the embodiments in plants.

Description

USE OF AXMI277 FOR THE CONTROL OF ROTYLENCHULUS RENIFORMIS NEMATODE PESTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 668,133 filed on July 5, 2024, the entire contents of which is hereby incorporated in their entirety by reference.SUBMISSION OF SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is PF240563_SEQLISTING_St26.xml. The size of the text file is 22 KB, and the text file was created on June 5, 2024.BACKGROUND

[0003] Plant pests are a major factor in the loss of the world’s important agricultural crops. Some estimates claim that 40% of global crop production is lost due to infestations of invertebrate pests including nematodes. In addition to losses in field crops, nematode pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and to home gardeners. Plant-infesting nematodes, a majority of which are root feeders, are found in association with most plants. Some are endoparasitic, living and feeding within the tissue of the roots, tubers, buds, seeds, etc. Others are ectop arasitic, feeding externally through plant walls. A single endoparasitic nematode can kill a plant or reduce its productivity. Endoparasitic root feeders include such economically important pests as the root-knot nematodes (Meloidogyne species), the reniform nematodes (Rotylenchulus species), the cyst nematodes (Heterodera species), and the root-lesion nematodes (Pratylenchus species).

[0004] Damage caused by nematodes can drastically decrease a plant's uptake of nutrients and water. Nematodes have the greatest impact on crop productivity when they attack the roots of seedlings immediately after seed germination. Nematode feeding also creates open wounds that provide entry to a wide variety of plant-pathogenic fungi and bacteria. These microbial infections can be more economically damaging than the direct effects of nematode feeding.

[0005] Reniform nematodes are responsible for direct loss mainly in cotton but also in soybean yield and indirect loss due to cost of pesticides and non-optimal use of land for crop rotation. It has been described that reniform nematode caused up to 8% annual loss in cotton production in the US Mid-South over the last decade (Singh, B., et al., Agronomy Journal. 115(5): September / October 2023. Pages 2140-2158). Among the 11 known species ofreniform nematodes, Rotylenchulus reniformis is geographically the most widely distributed and economically important species on the genus Rotylenchulus proposed by Linford and Oliveira (See Linford M.B. and Oliveira J.M. 1940. Proceeding of the Helminthological Society of Washington. 7:5-42). Economically significant densities of reniform nematodes usually cause stunting of crop plants and significant reduction in canopy area.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 depicts the plant transformation vector for the expression of SEQ ID NO:2 in planta.

[0007] FIG. 2 depicts a bar showing the results from a greenhouse trial demonstrating nematicidal effect of Axmi277 against reniform nematodes (Rotylenchulus reniformis).BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

[0008] SEQ ID NO: 1 discloses the codon-optimized Axmi277 nucleotide sequence.

[0009] SEQ ID NO:2 discloses the amino acid sequence SEQ ID NO: 1.

[0010] SEQ ID NO:3 discloses the C-terminal truncated Axmi277 amino acid sequence.

[0011] SEQ ID NO:4 discloses the delta-endotoxin nucleotide sequence of an Axmi277 homolog.

[0012] SEQ ID NO:5 discloses the delta-endotoxin amino acid sequence of SEQ ID NO:4.

[0013] SEQ ID NO:6 discloses the C-terminal truncated nucleotide sequence of an Axmi277 homolog.

[0014] SEQ ID NO:7 discloses the C-terminal truncated amino acid sequence of SEQ ID NO:6.SUMMARY

[0015] Traditional practices for managing nematode infestations include maintaining proper fertility and soil pH levels in nematode-infested land; controlling plant diseases that aid nematode invasion, as well as controlling insect and weed pests; using sanitation practices such as plowing, planting, and cultivating of nematode-infested fields only after working non-infested fields; cleaning equipment thoroughly after working in infested fields; not using seed from plants grown on infested land for planting non-infested fields unless the seed has been properly cleaned; rotating infested fields and alternating host crops with non-host crops, such as, corn, oat and alfalfa and planting resistant or tolerant plant varieties. While many of these can be effective, they are time consuming and costly to implement. Reniform nematodes are difficult pests to control without the use of chemical pesticides or fumigants (e.g., nematicides) and these approaches come with many challenges such as nematode resistance, regulatory restrictions, etc.

[0016] Biological pest control agents, such as Bacillus thuringiensis strains expressing pesticidal toxins like delta-endotoxins (also called Cry proteins), have been applied to crop plants with satisfactory results, offering an alternative or compliment to chemical pesticides. Some Cry proteins, for example, Cry 5, Cry6, Cryl l, Cryl2, Cryl3, Cryl4, Cry21, and Cry 55 have been shown to provide some activity against certain nematode species in laboratory bioassays but so far data on which cry genes are effective against which nematode species are very limited and no systematic relationships are obvious so far.

[0017] W02014 / 003769 discloses Axmi277 nematode toxin identified from strain ATX- 47290. Therein Axmi277 is described as having 73.4% identity to Axmi031, whereas a truncated version of Axmi277 (trun) shows 74.4% identity to truncated Cryl4Aa.

[0018] W02014 / 003769.provides data showing activity of Axmi277 against C. elegans but is silent about reniform nematodes. Because of the devastation that nematodes can confer and constant threat of pest developing resistance against current control measures, there is a continual need to discover new methods for controlling reniform nematode plant pests that provide an economic benefit to farmers and that are environmentally acceptable and safe.

[0019] Various embodiments herein provide for new methods of controlling economically important reniform nematode pests. Transgenic plants and / or plant parts expressing the Axmi277 polypeptides, were found to be capable of inhibiting the ability of reniform nematode pests to survive, grow and reproduce, or of limiting reniform nematode-related damage or loss to crop plants. Further embodiments are drawn to methods of using the transgenic Axmi277 expressing plants alone or in combination with other nematode control strategies to confer maximal reniform nematode control efficiency with minimal environmental impact. Plants and plant parts expressing the proteins described herein are highly tolerant or resistant to reniform nematode infestation.

[0020] One embodiment provides a method of controlling a reniform nematode pest comprising contacting the reniform nematode pest with a protein comprising SEQ ID NOs:2 or 3 and any functional fragments thereof. In another embodiment, the protein can be an Axmi277 protein homolog with at least 95% identity with SEQ ID NOs:2 or 3 and any functional fragments thereof.

[0021] Another embodiment provides for a method of controlling a reniform nematode pest, comprising contacting the reniform nematode pest with a transgenic plant or plant part comprising a heterologous nucleic acid molecule that directs expression of an Axmi277 protein or a homolog with at least 95% identity in the transgenic plant or plant part, wherein the transgenic plant or plant part controls the reniform nematode pest compared to a plant orplant part of the same type that does not express the Axmi277 protein or its homolog with 95% identity.

[0022] Another embodiment provides for a transgenic plant or plant part selected from the group consisting of alfalfa, apple, apricot, Arabidopsis, artichoke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, a cereal, celery, cherry, citrus, Clementine, coffee, com, cotton, cucumber, eggplant, endive, eucalyptus, figs, grape, grapefruit, groundnuts, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, nut oat, okra, onion, orange, an ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soy, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, a vine, watermelon, wheat, yams and zucchini.

[0023] In another embodiment, the transgenic plant or plant part is a cotton or soybean plant or plant part. Another embodiment provides for where the plant part is a root. In another embodiment, the root is a cotton or soybean root.

[0024] In another embodiment, the Axmi277 protein comprises an amino acid sequence that is the translation product of a nucleotide sequence whose complement hybridizes to SEQ ID NO: under high-stringency conditions.

[0025] In another embodiment, an Axmi277 protein of the embodiments comprises SEQ ID NOs:2 or 3 and any functional fragments thereof or a reniform nematode-active homolog thereof having at 95%, 96%, 97%, 98% or 99 sequence identity with SEQ ID NOs:2 or 3 and any functional fragments thereof.

[0026] Another embodiment provides for a transgenic plant further comprising or expressing at least one additional pesticidal agent, for example without limitation, a patatin, a Bacillus thuringiensis insecticidal protein, a Bacillus thuringiensis nematicidal protein, a a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphearicus insecticidal protein, a vegetative insecticidal protein, VIP3 and / or an RNAi molecule that targets a nematode pest. In another aspect, a Bacillus thuringiensis nematicidal protein is selected from the group consisting of a Cry5, a Cry6, a Cryl3, a Cryl4, a Cry21, and a Cry55.

[0027] Another embodiment provides for a method of conferring reniform nematode resistance to a plant and / or a plant part comprising inserting in the plant and / or a plant part a heterologous nucleic acid molecule Axmi277 protein, wherein the plant and / or plant partexpresses the Axmi277 protein at a reniform nematode-inhibiting level so as to confer reniform nematode resistance to the plant and / or plant part compared to the same type of plant and / or plant part not expressing the Axmi277 protein. Such insertion may occur via transformation, gene editing or through breeding.

[0028] Another embodiment provides for a method of reducing reniform nematode infectivity to a plant and / or plant part comprising contacting the nematode with an Axmi277 protein, wherein reniform nematode infectivity is reduced compared to infectivity of a plant and / or plant part by a nematode not contacted with an Axmi277 protein.

[0029] A further embodiment provides for a transgenic cotton plant or plant part thereof comprising a heterologous nucleic acid molecule encoding a Axmi277 protein, wherein the transgenic cotton plant or plant part is resistant to reniform nematode infestation.

[0030] Another embodiment provides for a transgenic soybean plant or plant part thereof comprising a heterologous nucleic acid molecule encoding a Axmi277 protein, wherein the transgenic soybean plant or plant part is resistant to reniform nematode infestation.

[0031] A further embodiment provides a method of producing a cotton plant protected against reniform nematode infestation, comprising transforming a cotton plant cell with a nucleic acid molecule encoding a Axmi277 protein; and regenerating a transformed cotton plant from the cotton plant cell, wherein the transformed plant is protected against reniform nematode infestation.

[0032] Another embodiment provides for a method of producing a soybean plant protected against reniform nematode infestation, comprising transforming a soybean plant cell with a nucleic acid molecule encoding an Axmi277 protein; and regenerating a transformed soybean plant from the soybean plant cell, wherein the transformed plant is protected against reniform nematode infestation.

[0033] A further embodiment provides for a method of producing a cotton plant protected against reniform nematode infestation, comprising crossing a first parent cotton plant with a second parent cotton plant, wherein the first or second parent cotton plant comprises a heterologous nucleic acid molecule encoding a Axmi277 protein of the embodiments, thereby producing a plurality of progeny plants; and selecting from the plurality of progeny plants, a transgenic cotton plant that is protected against reniform nematode infestation.

[0034] Another embodiment provides for a method of producing a soybean plant protected against reniform nematode infestation, comprising crossing a first parent soybean plant with a second parent soybean plant, wherein the first or second parent soybean plant comprises a heterologous nucleic acid molecule encoding an Axmi277 protein of the embodiments,thereby producing a plurality of progeny plants; and selecting from the plurality of progeny plants, a transgenic soybean plant that is protected against reniform nematode infestation.

[0035] Another embodiment provides for a method of reducing reniform nematode development on roots of a plant infectable by a reniform nematode, comprising introducing into cells of the root, a nucleic acid molecule capable of directing the expression of a Axmi277 protein, thereby reducing reniform nematode development on roots of the plant.

[0036] Another embodiment provides for a method of controlling or preventing reniform nematode growth comprising providing a reniform nematode pest with plant material comprising a heterologous DNA capable of directing expression of an Axmi277 protein, wherein said plant inhibits a reniform nematode biological activity.

[0037] Another embodiment provides for a method of providing a grower with a means of controlling reniform nematode pests is provided, the method comprising supplying seed to a grower, wherein the seed comprises a heterologous nucleic acid molecule that encodes a Axmi277 protein and wherein the seed is capable of producing a plant that is resistant to reniform nematode infestation.

[0038] Another embodiment provides for a method of suppressing growth of a plant- pathogenic reniform nematode population in a location capable of supporting growth of the reniform nematode population comprising growing in the location a population of transgenic cotton plants comprising a heterologous nucleic acid molecule capable of directing expression of a Axmi277 protein, wherein growth of the plant-pathogenic reniform nematode population is suppressed.

[0039] A further embodiment provides for a method of suppressing growth of a plant- pathogenic reniform nematode population in a location capable of supporting growth of the reniform nematode population is provided comprising growing in the location a population of transgenic soybean plants comprising a heterologous nucleic acid molecule capable of directing expression of an Axmi277 protein, wherein growth of the plant-pathogenic reniform nematode population is suppressed.

[0040] Another embodiment provides for a method of controlling reniform nematode pest comprising providing a transgenic cotton plant comprising an expression cassette comprising a nucleic acids comprising SEQ ID NO: 1 or a functional fragment thereof operably linked to a promoter capable of driving expression of an encoded Axmi277 protein to levels sufficient to inhibit reniform nematodes, wherein the proliferation of reniform pest feeding on the cotton plant is reduced compared to reniform pest feeding on a non- transgenic cotton plant not comprising the expression cassette.

[0041] Another embodiment also provides for a method of controlling reniform nematode pest comprising providing a transgenic soybean plant comprising an expression cassette comprising a nucleic acids comprising SEQ ID NO: 1 or a functional fragment thereof operably linked to a promoter capable of driving expression of an encoded Axmi277 protein to levels sufficient to inhibit reniform nematodes, wherein the proliferation of reniform pest feeding on the soybean plant is reduced compared to reniform pest feeding on a non- transgenic soybean plant not comprising the expression cassette.

[0042] A further embodiment also provides for a method of controlling reniform nematode pest comprising providing a transgenic cotton plant comprising an expression cassette comprising a nucleic acid encoding a protein comprising SEQ ID NOs:2 or 3 or a functional fragment thereof operably linked to a promoter capable of driving expression of an encoded Axmi277 protein to levels sufficient to inhibit reniform nematodes, wherein the proliferation of reniform pest feeding on the cotton plant is reduced compared to reniform pest feeding on a non-transgenic cotton plant not comprising the expression cassette.

[0043] Another embodiment also provides for a method of controlling reniform nematode pest comprising providing a transgenic soybean plant comprising an expression cassette comprising a nucleic acid encoding SEQ ID NOs:2 or 3 or a functional fragment thereof operably linked to a promoter capable of driving expression of an encoded Axmi277 protein to levels sufficient to inhibit reniform nematodes, wherein the proliferation of reniform pest feeding on the soybean plant is reduced compared to reniform pest feeding on a non- transgenic soybean plant not comprising the expression cassette.

[0044] Another embodiment provides for a method of improving plant yield in reniform nematode infested fields, comprising expressing in the plant a Axmi277 protein, wherein plant yield is improved compared to yield of a plant of the same type not expressing a Axmi277 protein.

[0045] Another embodiment provides for a method of improving cotton yield in reniform nematode infested fields, comprising expressing in a plant an Axmi277 protein, wherein cotton yield is improved compared to yield of a plant of the same type not expressing an Axmi277 protein.

[0046] Another embodiment provides for a method of improving cotton seed yield in reniform nematode infested fields, comprising expressing in a plant an Axmi277 protein, wherein cotton seed yield is improved compared to seed yield of a plant of the same type not expressing an Axmi277 protein.

[0047] Another embodiment provides for a method of improving cotton boll yield in reniform nematode infested fields, comprising expressing in a plant an Axmi277 protein, wherein cotton boll yield is improved compared to boll yield of a plant of the same type not expressing an Axmi 77 protein.

[0048] Another embodiment provides for a method of improving soybean yield in reniform nematode infested fields, comprising expressing in a plant an Axmi277 protein, wherein soybean yield is improved compared to yield of a plant of the same type not expressing an Axmi277 protein.

[0049] Another embodiment provides for a method of improving soybean seed yield in reniform nematode infested fields, comprising expressing in a plant an Axmi277 protein, wherein soybean seed yield is improved compared to seed yield of a plant of the same type not expressing an Axmi277 protein.

[0050] A further embodiment provides for a method of increasing the vigor or yield in a transgenic cotton plant exposed to a population of reniform nematodes comprising: introgressing a transgenic cotton event into a cotton plant resulting in a transgenic cotton plant, wherein the transgenic cotton event comprises a heterologous DNA sequence encoding an Axmi277 protein that confers upon the transgenic cotton event resistance to reniform nematodes; and growing the transgenic cotton plant or progeny thereof at a location where reniform nematode infestation is yield limiting to a cotton plant not comprising the heterologous nucleic acid molecule encoding the Axmi277 protein, whereby the transgenic cotton plant has increased vigor or yield compared to the control plant.

[0051] Another embodiment provides for a method of increasing the vigor or yield in a transgenic soybean plant exposed to a population of reniform nematodes comprising: introgressing a transgenic soybean event into a cotton plant resulting in a transgenic soybean plant, wherein the transgenic soybean event comprises a heterologous DNA sequence encoding an Axmi277 protein that confers upon the transgenic soybean event resistance to reniform nematodes; and growing the transgenic soybean plant or progeny thereof at a location where reniform nematode infestation is yield limiting to a soybean plant not comprising the heterologous nucleic acid molecule encoding the Axmi277 protein, whereby the transgenic soybean plant has increased vigor or yield compared to the control plant.

[0052] Another embodiment provides for a method of improving yield of a cotton field, comprising: introducing into a cotton plant a nucleic acid molecule capable of directing expression of an Axmi277 protein, thus producing a transgenic plant; and cultivating a plurality of transgenic seeds from the transgenic plant in a field producing or resulting in acotton field comprising a plurality of transgenic cotton plants having enhanced resistance to reniform nematode infestation, thereby improving yield of the cotton field.

[0053] Another embodiment provides for a method of improving yield of a soybean field, comprising: introducing into a soybean plant a nucleic acid molecule capable of directing expression of an Axmi277 protein, thus producing a transgenic plant; and cultivating a plurality of transgenic seeds from the transgenic plant in a field producing or resulting in a soybean field comprising a plurality of transgenic soybean plants having enhanced resistance to reniform nematode infestation, thereby improving yield of the soybean field.

[0054] Another embodiment provides for a reniform nematicidal composition comprising an effective reniform nematode-controlling amount of Axmi277 protein and an acceptable agricultural carrier. In a further embodiment, the agricultural carrier is a transgenic plant. In yet a further embodiment, the transgenic plant is a transgenic cotton or soybean plant and the Axmi277 protein is an Axmi277 protein having at least 95%, 97%, or 99% sequence identity with SEQ ID NOs:2 or 3 and any functional fragments thereof. In another embodiment, the Axmi277 protein comprises either SEQ ID NOs:2 or 3 and any functional fragments thereof.

[0055] Another embodiment provides for a method of producing a reniform nematoderesistant transgenic plant, comprising introducing a nucleic acid molecule encoding an Axmi277 protein into a plant cell thereby making a transgenic plant cell; regenerating a transgenic plant from the transgenic plant cell, wherein the Axmi277 protein is expressible in the transgenic plant in an effective amount to control reniform nematodes. According to another embodiment, the plant is a cotton or soybean plant. In another embodiment, the Axmi277 protein is a protein having at least 95%, 97%, or 99% sequence identity with SEQ ID NOs:2 or 3 and any functional fragments thereof. In yet another embodiment, the Axmi277 protein comprises SEQ ID NOs:2 or 3 and any functional fragments thereof.

[0056] Other aspects and advantages of the embodiments will become apparent to those skilled in the art from a study of the following description of the embodiments and nonlimiting examples.DETAILED DESCRIPTION

[0057] Before explaining the various embodiments of the disclosure, it is to be understood that the embodiments are not limited in its application to the details of construction and the arrangement of the components set forth in the following description. Other embodiments can be practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0058] Throughout this disclosure, various publications, patents and published patent specifications are referenced. Where permissible, the disclosures of these publications, patents and published patent specifications are hereby incorporated by reference in their entirety into the present disclosure to more fully describe the state of the art. Unless otherwise indicated, the disclosure encompasses conventional techniques of plant breeding, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols in Molecular Biology [(F. M. Ausubel, et al. eds., (1987)]; Plant Breeding: Principles and Prospects (Plant Breeding, Vol 1) M. D. Hayward, N, O. Bosemark, I. Romagosa; Chapman & Hall, (1993.); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) CURRENT Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Flames and G. R. Taylor eds. (1995)], Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture [R. I. Freshney, ed. (1987)].

[0059] Unless otherwise noted, technical terms are used according to conventional usage in the art. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Wiley-Interscience, 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by 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.

[0060] In order to facilitate understanding of the disclosure, the following definitions are provided:

[0061] “Activity” of the Axmi277 proteins of the embodiments means that the Axmi277 proteins have a toxic effect on reniform nematodes by disrupting or deterring feeding, inhibiting the ability of reniform nematode pests to survive, grow and reproduce which may or may not cause death of the reniform nematode, or of limiting reniform nematode-related damage or loss to crop plants.

[0062] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as lack of combinations when interpreted in the alternative (or).

[0063] “Associated with / operatively linked” refer to two nucleic acid sequences that are related physically or functionally. For example, a promoter or regulatory DNA sequence is said to be “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.

[0064] As used herein, the term “contacting” refers to a process by which an Axmi277 protein of the embodiments or transgenic plant or plant part expressing the Axmi277 protein of the embodiments are delivered or administered to target reniform nematode pests or reniform nematode pest populations. Contacting describes physical proximity of Axmi277 proteins or transgenic plants or plant parts expressing an Axmi277 protein and the target reniform nematode so that they interact. The transgenic plants or plant parts may be contacted with a target reniform nematode or reniform nematode population by planting transgenic seed, transgenic seedlings, cuttings, plant runners, tubers, and the like in a location capable of supporting growth of a reniform nematode pest or reniform nematode pest population.

[0065] 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 that codes for an mRNA or which is expressed as a protein, such that the regulator nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid sequence. The regulator nucleic acid sequence of the chimeric gene is not normally operatively linked to the associated nucleic acid sequence as found in nature.

[0066] A “coding sequence” is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.

[0067] As used herein the terms “to control” or “controlling” reniform nematodes means to inhibit, through a toxic effect, the ability of reniform nematode pests to survive, grow, feed, and / or reproduce, or to limit reniform nematode-related damage or loss in crop plants. To “control” reniform nematodes may or may not mean killing the reniform nematodes.

[0068] To “deliver” a toxin means that the toxin comes in contact with a reniform nematode or reniform nematode population, resulting in a toxic effect and control of the reniform nematode or reniform nematode population. The toxin can be delivered in many recognized ways, e.g., orally by ingestion by the reniform nematode or by contact with the reniform nematode via transgenic plant expression, formulated protein composition(s), sprayable protein composition(s), a bait matrix, or any other art-recognized toxin delivery system.

[0069] The term “economic threshold” is defined as the pest reniform nematode population that produces incremental damage equal to the cost of controlling or preventing that damage. It is the level of reniform nematode population where the benefit of reniform nematode control is equal to its cost. In this regard, economic threshold may be defined as the reniform nematode pest damage level where the value of incremental reduction in crop yield is equal to the cost of preventing its occurrence. In other words, economic threshold attempts to determine the point at which it becomes economically feasible to control a reniform nematode pest population. Economic damage to the host crop normally is inflicted by the first generation progeny of reniform nematodes and is prevented by transgenic plants expressing an Axmi277 protein through lowering the concentration of progeny reniform nematodes in the plant root zone.

[0070] A “reniform nematode-controlling effective amount” or interchangeably a “pesticidal amount” as used herein refers to the concentration of an Axmi277 protein or functional fragment thereof capable of inhibiting, through a toxic effect, the ability of reniform nematodes to survive, grow, feed and / or reproduce, or of reducing or preventing reniform nematode-related damage or loss in crop plants. “Reniform Nematode-controlling effective amount” may or may not mean killing the reniform nematodes.

[0071] “Expression cassette” as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.

[0072] A “gene” is a defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

[0073] “Reniform Nematicidal” is defined as a toxic biological activity capable of controlling reniform nematodes, preferably by killing them.

[0074] A nucleic acid sequence is “isocoding with” a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence. For example, a native coding sequence from Bacillus spp. that encodes an Axmi277 protein is isocoding with a coding sequence codon optimized for expression in a plant that encodes the same Axmi277 protein.

[0075] An “isolated” nucleic acid molecule or an isolated protein or toxin is a nucleic acid molecule or protein or toxin that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or protein or toxin may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell or a transgenic plant.

[0076] The term “native” refers to a coding sequence or gene that is naturally present in the genome of a cell or plant.

[0077] The term “naturally occurring” is used herein to describe an object that can be found in nature as distinct from being artificially produced by man. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.

[0078] A “plant” is any plant at any stage of development, particularly a seed plant.

[0079] A “plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

[0080] “Plant material” refers to 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.

[0081] A “plant organ” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.

[0082] A “plant part” may be any part of a plant and include a plant cell, plant material, plant organ or plant tissue.

[0083] “Plant tissue” as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0084] A “promoter” is an untranslated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.

[0085] “Regulatory elements” refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.

[0086] As used herein, “resistant” or resistance means a transgenic plant variety preferably a cotton or soybean variety that prevents a majority of reniform nematodes from surviving and / or reproducing upon their attempted infestation.

[0087] The term “substantially identical,” in the context of two nucleic acid or protein sequences, refers to two or more sequences or subsequences that have at least 60%, 80%, 90, 95%, or at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The substantial identity may exist over a region of the sequences that is at least about 50 residues in length, over a region of at least about 100 residues, and the sequences may be substantially identical over at least about 150 residues. In one embodiment, the sequences are substantially identical over the entire length of the coding regions. Furthermore, substantially identical nucleic acid or protein sequences perform substantially the same function,

[0088] To determine the percent-identity (“Percent Identity”) between two sequences, in a first step, a pairwise sequence alignment is generated between those two sequences. Pairwise alignments in this first step can be generated by various tools known to a person skilled in theart, like e.g. programs “Blast” (Altschul et al. J. Mol. Biol. 215:403-410), “Blast2” (“gapped Blast”) (Altschul et al., Nucleic Acids Res. 25:3389-3402.), programs from The European Molecular Biology Open Software Suite (EMBOSS, Trends in Genetics 16 (6), 276 (2000)) like “Water”, “Matcher” or “Needle”, or by visual inspection.

[0089] After aligning the two sequences, in a second step, a percent-identity value can be determined from the alignment produced. Percent-identity between the two sequences can be calculated from the complete alignment produced, or from a region out of the alignment, e.g. the region of the alignment showing the sequences of the embodiments over its complete length, or the region showing the other sequence over its complete length, or from a region showing only parts of the sequences. The alignment region from which a percent-identity value is calculated may have a length of at least 100 positions, may have a length of at least 150 positions, and may have a length of more than 200 positions. For determination of percent-identity, first the sum over all positions is calculated, in which both sequences are showing identical residues in the alignment region, and this sum is then divided by the length of the alignment region, whereby positions in which a sequence has an introduced gap are either component of said length (length of alignment region), or are subtracted from said length (length of alignment region - total number of gaps in alignment region). The obtained value is then multiplied with 100 to result in percent-identity (% identity).

[0090] In an embodiment, the two sequences are first aligned over their complete length according the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453) as implemented in program “Needle” from EMBOSS (Trends in Genetics 16 (6), 276 (2000), preferably version 6.3.1.2 or later with using the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EBLOSUM62 (EMBOSS version of the BLOSUM62 substitution)) for protein sequences and default parameters (gapopen=10.0, gapextend=0.5 and matrix=EDNAFULL) for nucleotide sequences. Percent-identity (% identity) is then determined from the complete alignment produced and is calculated as follows: percent- identity = (sum of positions showing identical residues in alignment x 100) / ( length of alignment - total number of gaps in alignment ). This value can also be obtained directly from EMBOSS program “Needle” as program labeled "longest identity" when the parameter option “-nobrief’ is applied.

[0091] For nucleotide sequences encoding for a protein the pairwise alignment shall preferably be made over the complete length of the coding region of the sequence of the embodiments from start to stop codon excluding introns. Introns present in the other sequencemay also be removed for the pairwise alignment to allow comparison with the sequence of the embodiments.

[0092] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

[0093] “Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but to no other sequences.

[0094] A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity. As used herein, molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low-stringency” conditions. Similarly, the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions. Conventional stringency conditions are described by Sambrook, et al., In: Molecular Cloning A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and by Haymes, et al. In: Nucleic Acid Hybridization, A Practical Approach, IR.L Press, Washington, D.C. (1985), herein incorporated by reference in its entirety.

[0095] The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tmfor a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42° C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2* SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1 *SSC at 45° C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6*SSC at 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 ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2* (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0096] The following are examples of sets of hybridization / wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference Cryl4- like nucleotide sequences of the embodiments: a reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPC , 1 mM EDTA at 50° C. with washing in 2* SSC, 0.1% SDS at 50° C., in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPC , 1 mM EDTA at 50° C. with washing in l x SSC, 0.1% SDS at 50° C., in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPC , 1 mM EDTA at 50° C. with washing in 0.5xSSC, 0.1% SDS at 50° C., in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPC , 1 mM EDTA at 50° C. with washing in 0. l x SSC, 0.1% SDS at 50° C., or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1 xSSC, 0.1% SDS at 65° C.

[0097] A further indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.

[0098] “Synthetic” refers to a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an Axmi277 coding sequence, not naturally found in Bacillus, that resembles more closely the G+C content and the normal codon distribution of dicot and / or monocot genes is said to be synthetic.

[0099] “ Transformation” is a process for introducing heterologous nucleic acid into a host cell or organism. In particular, “transformation” means the stable integration of a DNA molecule into the genome of an organism of interest.

[0100] “Transformed / transgenic / recombinanf ’ refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A “non-transformed”, “non-transgenic”, or “non-recombinant” host refers to a wildtype organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

[0101] In general an Axmi277” protein in the context of the embodiments refers to a Bacillus Cry insecticidal protein which comprises SEQ ID NOs:2 or 3 or amino acid sequences which show at least 95% identity to SEQ ID NOs:2 or 3. A reniform nematodeactive “homologue” as used herein may include but is not limited to, 1) proteins which are at least 95%, or at least 96%, or at least 97%, or at least 98% or at least 99% identical at the sequence level to Axmi277 while also retaining reniform nematicidal activity.

[0102] As used herein, nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G) Amino acids are likewise indicated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (He; 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 (Vai; V).

[0103] The materials and methods of the subject embodiments are useful for killing or controlling reniform nematodes; retarding growth or reproduction of reniform nematodes; reducing reniform nematode populations; and / or reducing or retarding damage to plants caused by infestation of reniform nematode pests. In particular, the embodiments provide for methods of controlling reniform nematode pests of crop plants such as cotton or soybean by using transgenic crop plants such as cotton or soybean expressing an Axmi277 protein.

[0104] The expression in transgenic plants or plant parts of the Axmi277 proteins of the embodiments results in compositions that can be used to control reniform nematode pests, for example, without limitation, Rotylenchulus spp, In one embodiment, an embodiment encompasses a method of controlling a reniform nematode pest, comprising contacting the nematode pest with an Axmi277 protein comprising SEQ ID NOs:2 or 3 or any functional fragments thereof.

[0105] In another embodiment, the contacting step is carried out with a plant or plant part transformed with at least one nucleic acid molecule encoding the Axmi277 protein. In yet another embodiment, the plant or plant part is a cotton plant or plant part. In still another embodiment, the cotton plant part is a cotton root.

[0106] In yet another embodiment, the plant or plant part is a soybean plant or plant part. In still another embodiment, the soybean plant part is a soybean root.

[0107] In another embodiment, the transgenic plant or plant part is selected from the group consisting of alfalfa, apple, apricot, Arabidopsis, artichoke, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, Brassica, broccoli, Brussels sprouts, cabbage, canola, carrot, cassava, cauliflower, a cereal, celery, cherry, citrus, Clementine, coffee, com, cotton, cucumber, eggplant, endive, eucalyptus, figs, grape, grapefruit, groundnuts, ground cherry, kiwifruit, lettuce, leek, lemon, lime, pine, maize, mango, melon, millet, mushroom, nut oat, okra, onion, orange, an ornamental plant or flower or tree, papaya, parsley, pea, peach, peanut, peat, pepper, persimmon, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, soy, soybean, spinach, strawberry, sugar beet, sugarcane, sunflower, sweet potato, tangerine, tea, tobacco, tomato, a vine, watermelon, wheat, yams and zucchini. In yet another embodiment, the transgenic plant or plant part is a cotton or soybean plant or plant part.

[0108] In further embodiments, the Axmi277 sequences, e.g. the nucleic acids sequences encoding the Axmi277 protein for the methods of the present embodiments can be modified by incorporation of random mutations in a technique known as in vitro recombination or DNA shuffling to increase nematode activity for example. This technique is described inStemmer et al., Nature 370:389-391 (1994) and U.S. Pat. No. 5,605,793, which are incorporated herein by reference. Millions of mutant copies of a nucleotide sequence are produced based on an original nucleotide sequence of the embodiments and variants with improved properties, such as increased reniform nematicidal activity, enhanced stability, or different specificity or range of target reniform nematode pests are recovered. The method encompasses forming a mutagenized double-stranded polynucleotide from a template doublestranded polynucleotide comprising a nucleotide sequence of the embodiments, wherein the template double-stranded polynucleotide has been cleaved into double-stranded-random fragments of a desired size, and comprises the steps of adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded template polynucleotide; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide. In a preferred embodiment, the concentration of a single species of double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the total DNA. In a further embodiment, the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides. In another preferred embodiment, the size of the double-stranded random fragments is from about 5 by to 5 kb. In another embodiment, the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles.

[0109] In further embodiments the nucleotide sequences coding for the Axmi277 protein of can be modified through N- or C-terminal deletions to encode functional fragments. The term functional fragments relate to consecutive amino acids for Axmi277 proteins that are at least 10, 20, 30, 40, 50, or 60 consecutive amino acids of SEQ ID NOs: 2 or 3 Preferred deletions would remove the C-terminal crystallization domain downstream of a conserved amino acid motif called “DRIEF” or “DRIE” which is also has previously been described as “Block 5” inSchnepf et al 1998. Functional fragments with C-terminal deletions of the crystallization domain are expected to result in toxic core proteins, which are independent from proteolytic activation. An exemplary functional fragment through deletion of the c-terminal crystallization domain downstream of the conserved “DRIEF” domain comprise amino acid sequence SEQ ID NO: 3.

[0110] In one embodiment, the Axmi277 protein is expressed in a higher organism, e.g., a plant. In this case, transgenic plants expressing effective amounts of the toxins protect themselves from reniform nematode pests. When the reniform nematode starts feeding on such a transgenic plant, it also ingests the expressed Axmi277 protein toxin. This may deter the reniform nematode from further feeding in the plant tissue, may harm or kill the reniform nematode or may reduce the reniform nematodes ability to reproduce. A nucleotide sequence of the embodiments is inserted into an expression cassette, which is then preferably stably integrated in the genome of the plant. Plants transformed in accordance with the embodiments may be monocots or dicots and include, but are not limited to, maize, wheat, barley, rye, sweet potato, bean, pea, 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, and woody plants such as coniferous and deciduous trees.[oni] Once a desired nucleotide sequence has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques. A nucleotide sequence of the embodiments is expressed in transgenic plants, thus causing the biosynthesis of the corresponding toxin in the transgenic plants. In this way, transgenic plants with enhanced resistance to reniform nematodes are generated. For their expression in transgenic plants, the nucleotide sequences of the embodiments may require modification and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in the embodiments can be changed to conform with plant preferences, while maintaining the amino acids encoded thereby. Furthermore, highexpression in plants is best achieved from coding sequences that have at least about 35% GC content, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60%. Although preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. NucL Acids Res. 17:477-498 (1989)). In addition, the nucleotide sequences are screened for the existence of illegitimate splice sites that may cause message truncation. All changes required to be made within the nucleotide sequences such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods known in the art.

[0112] In various embodiments, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.

[0113] For efficient initiation of translation, sequences adjacent to the initiating methionine encoding start codon may require modification. For example, they can be modified by the inclusion of sequences known to be effective in plants. Joshi has suggested an appropriate consensus for plants (NAR 15:6643-6653 (1987)) and Clonetech suggests a further consensus translation initiator (1993 / 1994 catalog, page 210). Further patent publication WO2022 / 261348 suggest methods and compositions for altering protein accumulation (All preceding references herein incorporated in their entirety by reference). These consensuses are suitable for use with the nucleotide sequences of the embodiments. The sequences are incorporated into constructions comprising the nucleotide sequences, up to and including the ATG (whilst leaving the second amino acid unmodified).

[0114] The genes encoding the Axmi 277 protein of the embodiments, either as their native sequence or as optimized synthetic sequences as described above, can be operably fused to a variety of promoters for expression in plants including constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissuespecific promoters to prepare recombinant DNA molecules, i.e., chimeric genes. The choice of promoter will vary depending on the temporal and spatial requirements for expression. Thus, expression of the nucleotide sequences encoding Axmi277 proteins of the embodiments in leaves, in stalks or stems, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and / or seedlings can be achieved, but particularly preferred for control of reniform nematodes is expression in roots. In many cases, however, protection against more 1than one type of nematode pest is sought, and thus expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences of the embodiments in the desired cell.

[0115] Vectors

[0116] A pesticidal sequence of the embodiments may be provided in an expression cassette for expression in a host cell of interest, e.g. a plant cell or a microbe. By “plant expression cassette” is intended a DNA construct that is capable of resulting in the expression of a protein from an open reading frame in a plant cell. Typically, these contain a promoter and a coding sequence. Often, such constructs will also contain a 3' untranslated region. Such constructs may contain a signal sequence or leader sequence to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus.

[0117] By “signal sequence” is intended a sequence that is known or suspected to result in co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Insecticidal toxins of bacteria are often synthesized as protoxins, which are protolytically activated in the gut of the target pest (Chang (1987) Methods EnzymoL 153:507-516). In some embodiments, the signal sequence is located in the native sequence, or may be derived from a sequence of the embodiments.

[0118] By “leader sequence” is intended any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeting transport and / or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Thus, further provided herein is a polypeptide comprising an amino acid sequence of the present embodiments that is operably linked to a heterologous leader or signal sequence.

[0119] By “plant transformation vector” is intended a DNA molecule that is necessary for efficient transformation of a plant cell. Such a molecule may consist of one or more plant expression cassettes, and may be organized into more than one “vector” DNA molecule. For example, binary vectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).

[0120] “Vector” refers to a nucleic acid construct designed for transfer between different host cells.

[0121] “Expression vector” refers to a vector that has the ability to incorporate, integrate and express heterologous DNA sequences or fragments in a foreign cell. The cassette will include 5' and / or 3' regulatory sequences operably linked to a sequence of the embodiments. By “operably linked” is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. In some embodiments, the nucleotide sequence is operably linked to a heterologous promoter capable of directing expression of said nucleotide sequence in a host cell, such as a microbial host cell or a plant host cell. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.

[0122] In various embodiments, the nucleotide sequence is operably linked to a heterologous promoter, e.g., a plant promoter.

[0123] Such an expression cassette is provided with a plurality of restriction sites for insertion of the pesticidal sequence to be under the transcriptional regulation of the regulatory regions.

[0124] The expression cassette will include in the 5 '-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a translational and transcriptional termination region (i.e., termination region) functional in plants. The promoter may be native or analogous, or foreign or heterologous, to the plant host and / or to the DNA sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is “native” or “homologous” to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is foreign or heterologous to the DNA sequence of the embodiments, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the embodiments. The promoter may be inducible or constitutive. It may be naturally occurring, may be composed of portions of various naturally occurringpromoters, or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley and Reynolds (1987) Nucleic Acids Res. 15:2343-2361. Also, the location of the promoter relative to the transcription start may be optimized. See, e.g., Roberts et al. (1979) Proc. Natl. Acad. Sci. USA, 76:760-764. Many suitable promoters for use in plants are well known in the art.

[0125] For instance, suitable constitutive promoters for use in plants include: the promoters from plant viruses, such as the peanut chlorotic streak caulimovirus (PC1SV) promoter (U.S. Pat. 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; promoters of Chlorella virus methyltransferase genes (U.S. Pat. No. 5,563,328) and the full-length transcript promoter from figwort mosaic virus (FMV) (U.S. Pat. No. 5,378,619); the promoters from such genes as rice actin (McElroy et al. (1990) Plant Cell 2: 163-171 and U.S. Patent 5,641,876); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689) and Grefen et a / .(2010) Plant J, 64:355-365; pEMU (Last et al. (1991) Theor. Appl. Genet. 81 :581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730 and U.S. Patent 5,510,474); maize H3 histone (Lepetit et al. (1992) Mol. Gen. Genet. 231 :276-285 and Atanassova et al. (1992) Plant J. 2(3):291-300); Brassica napus ALS3 (PCT application WO97 / 41228); a plant ribulose-biscarboxylase / oxygenase (RuBisCO) small subunit gene; the circovirus (AU 689 311) or the Cassava vein mosaic virus (CsVMV, US 7,053,205); promoters from soybean (Pbdc6 or Pbdc7, described in WO / 2014 / 150449 or ubiquitin 3 promoter described in US Patent No. 7393948 and US Patent No. 8395021); and promoters of various Agrobacterium genes (see U.S. Pat. Nos. 4,771,002; 5,102,796; 5,182,200; and 5,428,147).

[0126] Suitable inducible promoters for use in plants include: the promoter from the ACE1 system which responds to copper (Mett et al. (1993) PNAS 90:4567-4571); the promoter of the maize In2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al. (1991) Mol. Gen. Genetics 227:229-237 and Gatz et al. (1994)AfoZ. Gen. Genetics 243: 32- 38); and the promoter of the Tet repressor from TnlO (Gatz et al. (1991) Afo / . Gen. Genet. 227:229-237). Another inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter of this type is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421) or the recent application of a chimeric transcription activator, XVE, for use in an estrogen receptor-based inducible plant expression system activated by estradiol(Zuo et al. (2000) Plant J., 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 herein incorporated by reference in their entirety. Promoters composed of portions of other promoters and partially or totally synthetic promoters can also be used. See, e.g., Ni et al. (1995) Plant J. 7:661-676 and PCT WO 95 / 14098 describing such promoters for use in plants.

[0127] One embodiment provides for a promoter sequence specific for particular regions or tissues of plants can be used to express the reniform nematicidal proteins of the embodiments, such as promoters specific for seeds (Datla, R. et al., 1997, Biotechnology Ann. Rev. 3, 269-296), especially the napin promoter (EP 255 378 Al), the phaseolin promoter, the glutenin promoter, the helianthinin promoter (WO92 / 17580), the albumin promoter (WO98 / 45460), the oleosin promoter (WO98 / 45461), the SAT1 promoter or the SAT3 promoter (PCT / US98 / 06978).Use may also be made of an inducible promoter advantageously chosen from the phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase, proteinase inhibitor (PI), PR1 family gene, nopaline synthase (nos) and vspB promoters (US 5 670 349, Table 3), the HMG2 promoter (US 5 670 349), the apple beta-galactosidase (ABG1) promoter and the apple aminocyclopropane carboxylate synthase (ACC synthase) promoter (WO98 / 45445). Multiple promoters can be used in the constructs of the embodiments, including in succession.

[0128] The promoter may include, or be modified to include, one or more enhancer elements. In some embodiments, the promoter may include a plurality of enhancer elements. Promoters containing enhancer elements provide for higher levels of transcription as compared to promoters that do not include them. Suitable enhancer elements for use in plants include the PC1SV enhancer element (U.S. Pat. No. 5,850,019), the CaMV 35S enhancer element (U.S. Pat. Nos. 5,106,739 and 5,164,316) and the FMV enhancer element (Maiti et al. (1997) Transgenic Res. 6: 143-156); the translation activator of the tobacco mosaic virus (TMV) described in Application WO87 / 07644, or of the tobacco etch virus (TEV) described by Carrington & Freed 1990, J. Virol. 64: 1590-1597, for example, or introns such as the adhl intron of maize or intron 1 of rice actin. See also PCT WO96 / 23898, WO2012 / 021794, WO20 12 / 021797, WO2011 / 084370, and WO2011 / 028914.

[0129] Often, such constructs can contain 5' and 3' untranslated regions. Such constructs may contain a signal sequence or leader sequence to facilitate co-translational or post- translational transport of the peptide of interest to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus, or to be secreted.For example, the construct can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum.

[0130] By “3' untranslated region” is intended a polynucleotide located downstream of a coding sequence. Polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor are 3' untranslated regions. By “5' untranslated region” is intended a polynucleotide located upstream of a coding sequence.

[0131] Other upstream or downstream untranslated elements include enhancers. Enhancers are polynucleotides that act to increase the expression of a promoter region. Enhancers are well known in the art and include, but are not limited to, the SV40 enhancer region and the 35S enhancer element.

[0132] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the DNA sequence of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. ( 99V)Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91 : 151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0133] Where appropriate, the gene(s) may be optimized for increased expression in the transformed host cell (synthetic DNA sequence). That is, the genes can be synthesized using host cell-preferred codons for improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. Expression of the open reading frame of the synthetic DNA sequence in a cell results in production of the polypeptide of the embodiments.Synthetic DNA sequences can be useful to simply remove unwanted restriction endonuclease sites, to facilitate DNA cloning strategies, to alter or remove any potential codon bias, to alter or improve GC content, to remove or alter alternate reading frames, and / or to alter or remove intron / exon splice recognition sites, polyadenylation sites, Shine-Delgarno sequences, unwanted promoter elements and the like that may be present in a native DNA sequence. Generally, the GC content of the gene will be increased. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage.Methods are available in the art for synthesizing plant-preferred genes. 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, herein incorporated by reference.

[0134] It is also possible that synthetic DNA sequences may be utilized to introduce other improvements to a DNA sequence, such as introduction of an intron sequence, creation of a DNA sequence that in expressed as a protein fusion to organelle targeting sequences, such as chloroplast transit peptides, apoplast / vacuolar targeting peptides, or peptide sequences that result in retention of the resulting peptide in the endoplasmic reticulum. Thus, in one embodiment, the reniform nematicidal protein is targeted to the chloroplast for expression. In this manner, where the reniform nematicidal protein coding expression cassette is not directly inserted into the chloroplast genome, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the reniform nematicidal protein to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark etal. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233:478-481.

[0135] The pesticidal gene to be targeted to the chloroplast; see, for example, U.S. Patent No. 5,380,831, herein incorporated by reference.

[0136] Plant Transformation

[0137] Methods of the embodiments involve introducing a nucleotide construct into a plant. By “introducing” is intended to present to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the embodiments do not require that a particular method for introducing a nucleotide construct to a plant is used, only that the nucleotide construct gains access to 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.

[0138] “Transgenic plants” or “transformed plants” or “stably transformed” plants or cells or tissues refers to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not present in the untransformed plant cell, as well as those that may be endogenous, or present in the untransformed plant cell.

[0139] “Heterologous” generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.

[0140] The transgenic plants of the embodiments express one or more of the novel toxin sequences disclosed herein. In some embodiments, the protein or nucleotide sequence of the embodiments is advantageously combined in plants with other genes which encode proteins or RNAs that confer useful agronomic properties to such plants. Among the genes which encode proteins or RNAs that confer useful agronomic properties on the transformed plants, mention can be made of the DNA sequences encoding proteins which confer tolerance to one or more herbicides, and others which confer tolerance to certain insects, those which confer tolerance to certain diseases, DNAs that encodes RNAs that provide nematode or insect control, and the like. Such genes are in particular described in published PCT Patent Applications W091 / 02071 and WO95 / 06128 and in U.S. Patents 7,923,602 and US Patent Application Publication No. 20100166723, each of which is herein incorporated by reference in its entirety. In various embodiments, the transgenic plant further comprises one or more additional genes for nematode or insect resistance (e.g., Cryl, such as members of the Cryl A, Cry IB, CrylC, Cry ID, Cry IE, and Cry IF families; Cry2, such as members of the Cry2A family; Cry9, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; etc.). It will be understood by one of skill in the art that the transgenic plant may comprise any gene imparting an agronomic trait of interest.

[0141] Among the DNA sequences encoding proteins which confer tolerance to certain herbicides on the transformed plant cells and plants, mention can be made of a bar or PAT gene or the Streptomyces coelicolor gene described in WO2009 / 152359 which confers tolerance to glufosinate herbicides, a gene encoding a suitable EPSPS which confers tolerance to herbicides having EPSPS as a target, 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 5,633,435), a gene encoding glyphosate-n-acetyltransferase (for example, 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), a gene encoding glyphosate oxydoreductase (for example, US 5,463,175), or a gene encoding an HPPD inhibitor-tolerant protein (for example, the HPPD inhibitor tolerance genes described in WO 2004 / 055191, WO 199638567, US 6791014, WO2011 / 068567, WO201 1 / 076345, WO2011 / 085221, WO2011 / 094205, WO2011 / 068567, WO2011 / 094199, WO20 11 / 094205, WO2011 / 145015, W02012 / 056401, and WO2014 / 043435).

[0142] Among the DNA sequences encoding a suitable EPSPS which confer tolerance to the herbicides which have EPSPS as a target, mention will more particularly be made of the genewhich encodes a plant EPSPS, in particular maize EPSPS, particularly a maize EPSPS which comprises two mutations, particularly a mutation at amino acid position 102 and a mutation at amino acid position 106 (W02004 / 074443), and which is described in Patent Application US 6566587, hereinafter named double mutant maize EPSPS or 2mEPSPS, or the gene which encodes an EPSPS isolated from Agrobacterium and which is described by sequence ID No. 2 and sequence ID No. 3 of US Patent 5,633,435, also named CP4.

[0143] Among the DNA sequences encoding a suitable EPSPS which confer tolerance to the herbicides which have EPSPS as a target, mention will more particularly be made of the gene which encodes an EPSPS GRG23 from Arthrobacter globiformis, but also the mutants GRG23 ACE1, GRG23 ACE2, or GRG23 ACE3, particularly the mutants or variants of GRG23 as described in W02008 / 100353, such as GRG23(ace3)R173K of SEQ ID No. 29 in W02008 / 100353.

[0144] In the case of the DNA sequences encoding EPSPS, and more particularly encoding the above genes, the sequence encoding these enzymes is advantageously preceded by a sequence encoding a transit peptide, in particular the “optimized transit peptide” described in US Patent 5,510,471 or 5,633,448.

[0145] Exemplary herbicide tolerance traits that can be combined with the nucleic acid sequence of the embodiments further include at least one ALS (acetolactate synthase) inhibitor (W02007 / 024782); a mutated Arabidopsis ALS / AHAS gene (U.S. Patent 6,855,533); genes encoding 2,4-D-monooxygenases conferring tolerance to 2,4-D (2,4- dichlorophenoxyacetic acid) by metabolization (U.S. Patent 6,153,401); and, genes encoding Dicamba monooxygenases conferring tolerance to dicamba (3, 6-di chi oro-2 -methoxybenzoic acid) by metabolization (US 2008 / 0119361 and US 2008 / 0120739).

[0146] In various embodiments, the nucleic acid is stacked with one or more herbicide tolerant genes, including one or more HPPD inhibitor herbicide tolerant genes, and / or one or more genes tolerant to glyphosate and / or glufosinate.

[0147] Among the DNA sequences encoding proteins concerning properties of tolerance to insects, mention will more particularly be made of the Bt proteins widely described in the literature and well known to those skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorhabdus (WO97 / 17432 & WO98 / 08932).

[0148] Among such DNA sequences encoding proteins of interest which confer novel properties of tolerance to insects, mention will more particularly be made of the Bt Cry or VIP proteins widely described in the literature and well known to those skilled in the art. These include the CrylF protein or hybrids derived from a CrylF protein (e.g., the hybridCrylA-CrylF proteins described in US 6,326,169; US 6,281,016; US 6,218,188, or toxic fragments thereof), the Cryl A-type proteins or toxic fragments thereof, preferably the Cry 1 Ac protein or hybrids derived from the Cryl Ac protein (e.g., the hybrid Cryl Ab-Cryl Ac protein described in US 5,880,275) or the Cryl Ab or Bt2 protein or insecticidal fragments thereof as described in EP451878, the Cry2Ae, Cry2Af or Cry2Ag proteins as described in W02002 / 057664 or toxic fragments thereof, the Cryl A.105 protein described in WO 2007 / 140256 (SEQ ID No. 7) or a toxic fragment thereof, the VIP3Aal9 protein of NCBI accession ABG20428, the VIP3Aa20 protein of NCBI accession ABG20429 (SEQ ID No. 2 in WO 2007 / 142840), the VIP3A proteins produced in the COT202 or COT203 cotton events (W02005 / 054479 and W02005 / 054480, respectively), the Cry proteins as described in WO2001 / 47952, the VIP3Aa protein or a toxic fragment thereof as described in Estruch et al. (1996), Proc Natl Acad Sci U S A. 28;93(l l):5389-94 and US 6,291,156, the insecticidal proteins from Xenorhabdus (as described in WO98 / 50427), Serratia (particularly from S. entomophila) or Photorhabdus species strains, such as Tc-proteins from Photorhabdus as described in WO98 / 08932 (e.g., Waterfield et al., 2001, Appl Environ Microbiol. 67(l l):5017-24; Ffrench-Constant and Bowen, 2000, Cell Mol Life Sci.; 57(5):828-33). Also any variants or mutants of any one of these proteins differing in some (1-10, preferably 1-5) amino acids from any of the above sequences, particularly the sequence of their toxic fragment, or which are fused to a transit peptide, such as a plastid transit peptide, or another protein or peptide, is included herein.

[0149] In various embodiments, the nucleic acid can be combined in plants with one or more genes conferring a desirable trait, 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 reproduction, and the like.

[0150] Particularly useful transgenic cotton events which may be combined with the genes of the current embodiments in plants of the same species (e.g., by crossing or by re-transforming a plant containing another transgenic event with a chimeric gene of the embodiments), include Event EE-GH7 (WO2017 / 182420), Event 531 / PV-GHBK04 (cotton, insect control, described in WO 2002 / 040677), Event 1143-14A (cotton, insect control, not deposited, described in WO 06 / 128569); Event 1143-5 IB (cotton, insect control, not deposited, described in WO 06 / 128570); Event 1445 (cotton, herbicide tolerance, not deposited, described in US-A 2002-120964 or WO 02 / 034946), Event 281-24-236 (cotton, insect control — herbicide tolerance, deposited as PTA-6233, described in WO 05 / 103266 or US-A2005-216969); Event 3006-210-23 (cotton, insect control — herbicide tolerance, deposited as PTA-6233, described in US-A 2007-143876 or WO 05 / 103266); Event CE43-67B (cotton, insect control, deposited as DSM ACC2724, described in US-A 2009-217423 or WO 06 / 128573); Event CE44-69D (cotton, insect control, not deposited, described in US-A 2010- 0024077); Event CE44-69D (cotton, insect control, not deposited, described in WO 06 / 128571); Event CE46-02A (cotton, insect control, not deposited, described in WO 06 / 128572); Event COT102 (cotton, insect control, not deposited, described in US-A 2006- 130175 or WO 04 / 039986); Event COT202 (cotton, insect control, not deposited, described in US-A 2007-067868 or WO 05 / 054479); Event COT203 (cotton, insect control, not deposited, described in WO 05 / 054480); Event GHB 119 (cotton, insect control — herbicide tolerance, deposited as ATCC PTA-8398, described in WO 08 / 151780); Event GHB614 (cotton, herbicide tolerance, deposited as ATCC PTA-6878, described in US-A 2010-050282 or WO 07 / 017186); Event LLcotton25 (cotton, herbicide tolerance, deposited as ATCC PTA- 3343, described in WO 03 / 013224 or US-A 2003-097687); Event MON15985 (cotton, insect control, deposited as ATCC PTA-2516, described in US-A 2004-250317 or WO 02 / 100163); Event MON88913 (cotton, herbicide tolerance, deposited as ATCC PTA-4854, described in WO 04 / 072235 or US-A 2006-059590); Event MON88701 (cotton, herbicide tolerance, deposited as PTA-11754, described in WO 2012 / 134808), Event T304-40 (cotton, insect control — herbicide tolerance, deposited as ATCC PTA-8171, described in US-A 2010- 077501 or WO 08 / 122406); Event T342-142 (cotton, insect control, not deposited, described in WO 06 / 128568); event MON88701 (cotton, ATCC Accession N° PTA-11754, WO 2012 / 134808A1), event MON88702 (WO2017 / 182420) event pDAB4468.18.07.1 (cotton, herbicide tolerance, ATCC Accession N° PTA-12456), WO2013112525A2, event pDAB4468.19.10.3 (cotton, herbicide tolerance, ATCC Accession N° PTA-12457), WO2013112527A1, event A26-5 (Cotton, insect control) WO2013170398, event A2-6 (Cotton, insect control) WO2013170399, event A26-5 (Cotton, as described in WO2013170398A1), event A2-6 (Cotton, as described in WO2013170399A1), cotton event TAM66274 (W02019 / 014126 ) and event kjcOO3 (CN112011565)

[0151] Particularly useful soybean transgenic events which may be combined with the genes of the current embodiments in plants of the same species (e.g., by crossing or by retransforming a plant containing another transgenic event with a chimeric gene of the embodiments), include Event BPS-CV127-9 (soybean, herbicide tolerance, deposited as NCIMB No. 41603, described in WO2010 / 080829); Event DAS21606-3 / 1606 (soybean, herbicide tolerance, deposited as PTA-11028, described in WO2012 / 033794), Event DAS-44406-6 / pDAB8264.44.06.1 (soybean, herbicide tolerance, deposited as PTA-11336, described in WO2012 / 075426), Event DAS-14536-7 / pDAB8291.45.36.2 (soybean, herbicide tolerance, deposited as PTA-11335, described in WO2012 / 075429), Event DAS68416 (soybean, herbicide tolerance, deposited as ATCC PTA-10442, described in WO201 1 / 066384 or WO2011 / 066360); Event DP-305423-1 (soybean, quality trait, not deposited, described in US-A 2008-312082 or W02008 / 054747); Event DP-356043-5 (soybean, herbicide tolerance, deposited as ATCC PTA-8287, described in US-A 2010- 0184079 or W02008 / 002872); Event FG72 (soybean, herbicide tolerance, deposited as PTA- 11041, described in WO2011 / 063413), Event LL27 (soybean, herbicide tolerance, deposited as NCIMB41658, described in W02006 / 108674 or US-A 2008-320616); Event LL55 (soybean, herbicide tolerance, 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 W02009 / 064652); Event MON87705 (soybean, quality trait - herbicide tolerance, deposited as ATCC PTA-9241, described in US-A 2010- 0080887 or W02010 / 037016); Event MON87708 (soybean, herbicide tolerance, deposited as ATCC PTA-9670, described in WO2011 / 034704); Event MON87712 (soybean, yield, deposited as PTA-10296, described in W02012 / 051199), Event MON87754 (soybean, quality trait, deposited as ATCC PTA-9385, described in WO2010 / 024976); Event MON87769 (soybean, quality trait, deposited as ATCC PTA-8911, described in US-A 2011- 0067141 or W02009 / 102873); Event MON89788 (soybean, herbicide tolerance, deposited as ATCC PTA-6708, described in US-A 2006-282915 or W02006 / 130436); Event SYHT0H2 / SYN-000H2-5 (soybean, herbicide tolerance, deposited as PTA-11226, described in WO2012 / 082548), event EE-GM3 / FG72 (soybean, herbicide tolerance, ATCC Accession N° PTA-11041) optionally stacked with event EE-GM1 / LL27 or event EE-GM2 / LL55 (WO2011 / 063413A2); Event DAS-68416-4 (soybean, herbicide tolerance, ATCC Accession N° PTA-10442, WO2011 / 066360A1); Event DAS-68416-4 (soybean, herbicide tolerance, ATCC Accession N° PTA-10442, WO2011 / 066384A1); Event DAS-21606-3 (soybean, herbicide tolerance, ATCC Accession No. PTA-11028, WO2012 / 033794A2); Event MON- 87712-4 (soybean, quality trait, ATCC Accession N°. PTA-10296, WO2012 / 051199 A2); Event DAS-44406-6 (soybean, stacked herbicide tolerance, ATCC Accession N°. PTA- 11336, WO2012 / 075426A1); Event DAS-14536-7 (soybean, stacked herbicide tolerance, ATCC Accession N°. PTA-11335, WO2012 / 075429A1); Event SYN-000H2-5 (soybean, herbicide tolerance, ATCC Accession N°. PTA-11226, WO2012 / 082548A2); Event 8264.44.06.1 (soybean, stacked herbicide tolerance, Accession N° PTA-11336,WO2012075426A2); Event 8291.45.36.2 (soybean, stacked herbicide tolerance, Accession N°. PTA-11335, WO2012075429A2); Event SYHT0H2 (soybean, ATCC Accession N°. PTA-11226, WO2012 / 082548A2); Event pDAB8264.42.32.1 (soybean, stacked herbicide tolerance, ATCC Accession N° PTA-11993, W02013 / 010094A1).

[0152] Further, provided herein is a method for producing a cotton or soybean plant or seed comprising a nucleotide sequence encoding SEQ ID NOs: 2 or 3 or any functional fragments thereof combined with another reniform resistance locus / gene, such as by combining a cotton plant or seed comprising a nucleotide sequence encoding SEQ ID NOs: 2 or 3 or any functional fragments thereof with another reniform resistance locus / gene occurring in the same cotton plant / seed, and planting seed comprising a nucleotide sequence encoding SEQ ID NOs: 2 or 3 and any functional fragments thereof and said other reniform nematode resistance locus / gene. In one embodiment, the plants, cells or seeds of the embodiments contain one or more other reniform nematode resistance loci / genes that occur in cotton, to get a combination of different reniform nematode resistance sources in the cotton plants, cells or seeds of the embodiments. Several cotton reniform nematode resistance loci or genes are known and one or more of those can be combined with a plant comprising SEQ ID NOs: 2 or 3, or any functional fragments thereof in the same plant, cell or seed, such as any one of the reniform nematode genes / loci from the resistance sources from Gossypium barbardense accessions TAM RKRNR-9 (PI662039), TAM RKRNR-9 (PI662040) (Starr et al 2011), FB- 713 (PI608139) (Robinson et al, 2004). Further cotton reniform nematode resistance loci or genes which can be combined with a plant comprising SEQ ID NOs: 2 or 3 or any functional fragment therefore are provided by G. barbadense introgressions, 08SS110-NE06.0P and 08SS100, 08SS110-NE06.0P which showed significantly greater agronomic and physiological performance than commercial susceptible cultivars PHY 490 W3FE in reniform nematode-infested fields (Singh et al., 2021).

[0153] Several soybean reniform nematode resistance loci or genes are known and one or more of those can be combined with a plant comprising SEQ ID NOs: 2 or 3, or any functional fragments thereof in the same plant, cell or seed, such as any one of the reniform nematode genes / loci from the resistance soybean cultivars ‘Peking’ (MG IV), ‘Dyer’ (MG V), ‘Custer’ (MG IV), ‘Pickett’ (MG VI), ‘Forrest’ (MG V), ‘Hartwig’ (MG V), and ‘Anand’ (MG V) (Davis, Koenning, Burton, & Barker, 1996; Robbins et al., 1994, Rebois, Johnson, & Cairns, 1968). In some cases, soybean reniform nematode resistance loci have been shown to overlap with loci for soybean cyst nematode resistance (SCN). Usovsky et all (2020) showed that GmSNAP18 gene could control resistance to both SCN and reniform nematodesat the rhgl-a and rhgl-b and rhgl-bl allele. The paralog of this gene, mSNAPl l(Lakhssassi et al. ,2017b), also plays a role in controlling resistance to both SCN and RN at qSCNl 1 locus.

[0154] Transformation of plant cells can be accomplished by one of several techniques known in the art. The pesticidal gene of the embodiments may be modified to obtain or enhance expression in plant cells. Typically, a construct that expresses such a protein would contain a promoter to drive transcription of the gene, as well as a 3' untranslated region to allow transcription termination and polyadenylation. The organization of such constructs is well known in the art. In some instances, it may be useful to engineer the gene such that the resulting peptide is secreted, or otherwise targeted within the plant cell. For example, the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. It may also be preferable to engineer the plant expression cassette to contain an intron, such that mRNA processing of the intron is required for expression.

[0155] Typically, a plant expression cassette will be inserted into a plant transformation vector. This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as “binary vectors.” Binary vectors as well as vectors with helper plasmids are most often used for ^grotoctenwm-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA1O5, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

[0156] In general, 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 a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park (1994) Critical Reviews in Plant Science 13 :219-239 and Bommineni and Jauhar (1997) Maydica 42: 107-120. Since the transformed material contains many cells; both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove nontransformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants.

[0157] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Generation of transgenic plants may be performed by one of several methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mQd\&iQ transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, ballistic particle acceleration, aerosol beam transformation (U.S. Published Application No. 20010026941; U.S. Patent No. 4,945,050; International Publication No. WO 91 / 00915; U.S. Published Application No. 2002015066), Led transformation, and various other non-particle direct-mediated methods to transfer DNA.

[0158] Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91 :7301- 7305.

[0159] Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with appropriate selection, one identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.

[0160] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the embodiments provide for transformed seed (also referred to as “transgenic seed”) having a nucleotide construct of the embodiments, for example, an expression cassette of the embodiments, stably incorporated into their genome.

[0161] Evaluation of Plant Transformation

[0162] Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

[0163] PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrookand Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). PCR is carried out using oligonucleotide primers specific to the gene of interest o Agrobacterium vector background, etc.

[0164] Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, 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, radiolabeled32P target DNA fragment to confirm the integration of introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).

[0165] In Northern blot analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by the pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a pesticidal gene, by methods known in the art (Sambrook and Russell, 2001, supra).

[0166] Western blot, biochemical assays and mass spectrometric methods and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the reniform nematicidal protein.

[0167] Pesticidal Activity in Plants

[0168] Another aspect provides for generating transgenic plants expressing a reniform nematicidal protein that has pesticidal activity against a reniform nematode pest. Methods described above by way of example may be utilized to generate transgenic plants, but the manner in which the transgenic plant cells are generated is not critical to the embodiments. Methods known or described in the art such as Agrobacterium-moC\a.io transformation, biolistic transformation, and non-particle-mediated methods may be used at the discretion of the experimenter. Plants expressing a reniform nematicidal protein may be isolated by common methods described in the art, for example by transformation of callus, selection of transformed callus, and regeneration of fertile plants from such transgenic callus. In such process, one may use any gene as a selectable marker so long as its expression in plant cells confers ability to identify or select for transformed cells.

[0169] A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like. Other genes thatencode a product involved in chloroplast metabolism may also be used as selectable markers. For example, genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone may find particular use. Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314 (bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990) NucL Acids Res. 18:2188 (AHAS imidazolinone resistance gene). Additionally, the genes disclosed herein are useful as markers to assess transformation of bacterial or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell, propagule, embryo or progeny of the same are well known in the art. In one embodiment, the presence of the transgene is detected by testing for pesticidal activity against a reniform nematode pest.

[0170] Fertile plants expressing a reniform nematicidal protein may be tested for pesticidal activity against a nematode pest, and the plants showing optimal activity selected for further breeding. Methods are available in the art to assay for pest activity. Generally, the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology 78:290-293. Methods for testing nematicidial efficiency of plants expressing such nematicidial proteins are known in the art and have been described for example in Kahn et al. (2021) Nature Communications, 12(1), 3380 (herein incorporated by reference)).

[0171] One embodiment may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, com (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.

[0172] Use in Pesticidal Control

[0173] General methods for employing strains comprising a nucleotide sequence of the embodiments, or a variant thereof, in pest control or in engineering other organisms as pesticidal agents are known in the art. See, for example U.S. Patent No. 5,039,523 and EP 0480762A2.

[0174] Microorganisms can be genetically altered to contain a nucleotide sequence encoding SEQ ID NOs: 2 or 3 or nematicidally-active variants or fragments thereof, and protein may be used for protecting agricultural crops and products from pests. One aspect provides for whole, i.e., unlysed, cells of a toxin (pesticide)-producing organism are treated with reagentsthat prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s).

[0175] Alternatively, the pesticide is produced by introducing a pesticidal gene into a cellular host. Expression of the pesticidal gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. One aspect provides for these cells which are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of the target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein. Alternatively, one may formulate the cells expressing a gene of the embodiments such as to allow application of the resulting material as a pesticide.

[0176] The active ingredients of the embodiments are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be fertilizers, weed killers, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and / or timerelease or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or applicationpromoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise the formulations may be prepared into edible “baits” or fashioned into pest “traps” to permit feeding or ingestion by a target pest of the pesticidal formulation.

[0177] Methods of applying an active ingredient of the embodiments or an agrochemical composition of the embodiments that contains at least one of the reniform nematicidal proteins disclosed herein as SEQ ID NOs:2 or 3 or reniform nematicidally-effective variants or fragments thereof, include leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.

[0178] The composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenation, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present in a concentration of from about 1% to about 99% by weight.

[0179] Reniform nematode pests may be killed or reduced in numbers in a given area by the methods of the embodiments, or may be prophylactically applied to an environmental area to prevent infestation by a susceptible pest. Preferably the pest ingests, or is contacted with, a pesticidally-effective amount of the polypeptide.

[0180] The pesticide compositions described may be made by formulating either the bacterial cell, the crystal and / or the spore suspension, or the isolated protein component with the desired agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil / water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term “agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and / or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in U.S. Patent No. 6,468,523, herein incorporated by reference.

[0181] Methods for Increasing Plant Yield

[0182] Methods for increasing plant yield are provided. The methods comprise providing a plant or plant cell expressing a polynucleotide encoding the reniform nematicidal polypeptide sequence disclosed herein and growing the plant or a seed thereof in a field infested with (or susceptible to infestation by) a reniform nematode pest against which said polypeptide has nematicidal activity. In a preferred embodiment, the Axmi277 polypeptide described herein has nematicidal activity against a Rotylenchulus spp., and said field is infested with saidRotylenchulus spp. In various embodiments, the Rotylenchulus spp. is Rotylenchulus reniformis.

[0183] As defined herein, the “yield” of the plant refers to the quality and / or quantity of biomass produced by the plant. By “biomass” is intended any measured plant product. An increase in biomass production is any improvement in the yield of the measured plant product. In a preferred embodiment biomass refers to cotton boll weight and / or Cotton seed weight. In another preferred embodiment biomass refers to soybean seed weight. Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase in yield compared to a plant not expressing the reniform nematicidial protein described herein. In specific methods, plant yield is increased as a result of improved reniform nematode resistance of a plant expressing the reniform nematicidal protein disclosed herein. Expression of the reniform nematicidal protein results in a reduced ability of a reniform nematicidial pest to infest or feed. In various embodiments, expression of the reniform nematicidal protein results in 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 recovery from stress, increased mechanical strength, improved drought resistance, reduced fungal disease infection, preferably reduced Fusarium infection, and improved plant health compared to a plant not expressing the reniform nematicidal protein of the embodiments.

[0184] The plants, preferably the cotton plants can also be treated with one or more chemical compositions, including one or more herbicide, insecticides, or fungicides. Exemplary chemical compositions include:

[0185] Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, and Thidiazuron.

[0186] Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda- Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron,Rynaxypyr,Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2- difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, and Sulfoxaflor.

[0187] Cotton Fungicides: Azoxystrobin. Bixafen, Boscalid, Carbendazim, Chlorothalonil, Copper, Cy proconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fenamidone, Fluazinam, Fluopyram, Fluoxastrobin, Fluxapyroxad, Iprodione, Isopyrazam, Isotianil, Mancozeb, Maneb, Metominostrobin, Penthiopyrad, Picoxystrobin, Propineb, Prothioconazole, Pyraclostrobin, Quintozene, Tebuconazole, Tetraconazole, Thiophanate- methyl, and Trifloxystrobin.

[0188] The plants, preferable the soybean plants can also be treated with one or more chemical compositions, including one or more herbicide, insecticides, or fungicides. Exemplary chemical compositions include:

[0189] Soybean Herbicides: Alachlor. Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, and Glufosinate

[0190] Soybean Insecticides: Lambda-cyhalothrim Methomyl, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, 13- Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2- difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, and beta-Cyfluthrin.

[0191] Soybean Fungicides: Azoxystrobin. Bixafen, Boscalid, Carbendazim, Chlorothalonil, Copper, Cy proconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam, Fluopyram, Fluoxastrobin, Flutriafol, Fluxapyroxad, Isopyrazam, Iprodione, Isotianil, Mancozeb, Maneb, Metconazole, Metominostrobin, Myclobutanil, Penthiopyrad, Picoxystrobin, Propi conazole, Propineb, Prothioconazole, Pyraclostrobin, Tebuconazole, Tetraconazole, Thiophanate-methyl, and Trifloxystrobin.

[0192] Furthermore, the plants, preferably the cotton or soybean plants may be treated with biological control agents like NemOut, VOTiVO® or Nordi ca.

[0193] The following examples are offered by way of illustration and not by way of limitation.EXPERIMENTAL EXAMPLESExample 1. Expression of reniform nematicidial Axmi277 in soybean

[0194] Soybean events expressing Axmi277 (SEQ ID NO:2) were developed through Agrobacterium-mediated transformation of Thorne soybean plants using a construct containing a gene encoding a 4-hydroxyphenylpyruvate dioxygenase protein (HPPD) inhibitor tolerant herbicide gene (described in WO2014043435) and Axmi277 coding sequence. FIG. 1 depicts the plant transformation vector for the expression of SEQ ID NO:2 in planta. Table 1 shows a description of the genetic elements of pBAS01306 and Table 2 shows the references for the genetic elements described in Table 1. The Wild-type Thorne soybean served as the non-nematode resistant control. Axmi277, when expressed in soybean plants, reduces the number of Rotylenchulus reniformis that reproduce in the roots compared with wild-type plants. (FIG.2).Tablet : Description of the genetic elements of pBAS01306Table 2: References for genetic elements of pBAS01306Example 2: Soybean Transformation

[0195] Soybean transformation is achieved using methods well known in the art, such as the one described using the Agrobacterium tumefaciens mediated transformation soybean halfseed explants using essentially the method described by Paz et al. (2006), Plant cell Rep. 25:206. Transformants are identified using tembotrione as selection marker. The appearance of green shoots was observed, and documented as an indicator of tolerance to the herbicide isoxaflutole or tembotrione. The tolerant transgenic shoots will show normal greening comparable to wild-type soybean shoots not treated with isoxaflutole or tembotrione, whereas wild-type soybean shoots treated with the same amount of isoxaflutole or tembotrione will be entirely bleached. This indicates that the presence of the HPPD protein enables the tolerance to HPPD inhibitor herbicides, like isoxaflutole or tembotrione.

[0196] Tolerant green shoots are transferred to rooting media or grafted. Rooted plantlets are transferred to the greenhouse after an acclimation period. Plants containing the transgene are then sprayed with HPPD inhibitor herbicides, as for example with tembotrione at a rate of 100g Al / ha or with mesotrione at a rate of 300g Al / ha supplemented with ammonium sulfate methyl ester rapeseed oil. Ten days after the application the symptoms due to the application of the herbicide are evaluated and compared to the symptoms observed on wild type plants under the same conditions.Example 3: Greenhouse Trials

[0197] Soybean events for SEQ ID NO:2 were tested in the greenhouse. Data were log- transformed for analyses. Twenty total replications were performed across two plantings (10 reps per planting). Axmi277 was represented by six events providing a total of 120 experimental units. The wild-type comparator was included in two separate entries with 10 replications for each planting for a total of 40 experimental units. FIG. 2 shows a greenhouse trial demonstrating nematicidal effect of Axmi277 against reniform nematodes (Rotylenchulus reniformis). Expression of Axmi277 (SEQ ID NO:2 ) reduces the number of nematodes and eggs per plant by more than 80% compared with wild-type Thorne.

[0198] Parallel greenhouse trials were conducted for Melodogyne incognita w\ Melodogyne javonica (Root Knot Nematode) and results indicated that the Axmi277 was not effective against these nematode species, supporting the results obtained for Rotylenchulus reniformis as being nonobvious and unexpected.Example 4: Cotton Transformation

[0199] Cotton transformation is achieved using methods well known in the art, and see for example, WO 00 / 71733 and WO 2018 / 119336. Regenerated plants are transferred to the greenhouse. Following an acclimation period, sufficiently grown plants are sprayed with HPPD inhibitor herbicides as for example tembotrione equivalent to 100 or 200 gAI / ha supplemented with ammonium sulfate and methyl ester rapeseed oil. Seven days after the spray application, the symptoms due to the treatment with the herbicide are evaluated and compared to the symptoms observed on wild type cotton plants subjected to the same treatment under the same conditions. Additional methods of cotton tranformation include Trolinder, N. L., et al. (1987). Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum If. Plant Cell Rep. 6, 231-234; Vijaya, N. J., et al. (2015). Current status of tissue culture and genetic transformation research in cotton (Gossypium spp.). Plant Cell Piss Org. 120, 841; and Zhou L, et al. (2022) Highly efficient Agrobacterium rhizogenes-m Cxai hairy root transformation for gene editing analysis in cotton. Front. Plant Sci. 13: 1059404.

[0200] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the embodiments pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0201] Although the foregoing embodiments been described in some detail by way of illustration and example for purposes of clarity of understandng, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

CLAIMSWhat is claimed is:Listing of Claims:

1. A method for controlling a Rotylenchulus reniformis pest population comprising contacting said population with a pesticidally-effective amount of an Axmi277 protein.

2. A method for controlling a Rotylenchulus reniformis pest population comprising contacting said population with a pesticidally-effective amount of the polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of any of SEQ ID NO:2 or 3; and b) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:2 or 3.

3. A method for killing a Rotylenchulus reniformis pest, comprising contacting said pest with, or feeding to said pest, a pesticidally effective amount of the polypeptide of claim 2.

4. A method for protecting a plant from a Rotylenchulus reniformis pest, comprising expressing in a plant or cell thereof a nucleotide sequence that encodes a pesticidal polypeptide of claim 2.

5. A method for increasing yield in a plant comprising growing in a field a plant having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes the polypeptide of claim 2.

6. A method for increasing boll yield in a cotton plant comprising growing in a field a cotton having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes the polypeptide of claim 2.

7. The method of claim 2, wherein the Rotylenchulus reniformis pest population is six times less after 60 days after contacting said population with a pesticidally-effective amount of the polypeptide when compared with a Rotylenchulus reniformis pest population that has not been contacted with said population.

8. The methods of claims 5 and 6, wherein said plants exhibits an increase in root biomass, main stem length, leaf thickness, or shoot biomass.

9. The method of claim 2, wherein the nematode population is suppressed or maintained at or below the limit of detection.

10. A method of increasing yield of a nematode-susceptible plant comprising growing a plant containing the nematode-resistant polypeptide of claim 2 for at least one growing season and planting a nematode-susceptible plant in a subsequent growing season at the same location.

11. The method of claim 10, wherein the yield of the nematode-susceptible plant is increased compared to the yield of the nematode-susceptible plant not grown in the same location in which the nematode-resistant crop has not been grown during the most recent crop rotation cycle.

12. The method of claim 2, wherein controlling of said pest is combined with native trait resistance of a plant.

13. The method of claim 4, wherein said method comprises further applying a pesticide, a fungicide, a bactericide, a nematicide, an insecticide or an herbicide to the pest and / or plant.

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