Compositions and methods to increase resistance to root knot nematode

Abstract

The present disclosure provides compositions and methods for producing plants having resistance to diseases caused by Meloidogyne such as, for example, root-knot nematode. The methods involve expressing in plants a heterologous nucleic acid associated with increased resistance to Meloidogyne. Additionally, compositions and methods useful in producing, identifying and / or selecting plants and plant materials comprising the heterologous nucleic acid associated with increased resistance to Meloidogyne are provided.

Description

Docket # 211912-WO-SEC-lCOMPOSITIONS AND METHODS TO INCREASE RESISTANCE TO ROOT KNOT NEMATODEREFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The official copy of the sequence listing is submitted electronically via Patent Center as an XML formatted sequence listing with a file named 211912_SequenceListing created on October 30, 2024 and having a size of 51,223 bytes and is filed concurrently with the specification. The sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND

[0002] Soybean diseases are major threat for soybean production, resulting in yield losses and decrease in grain quality. One important soybean pathogen is Meloidogyne which causes rootknot nematode disease, that can severely impact yield. Meloidogyne species are a particular genera of plant-parasitic nematodes. They are distributed worldwide and are obligate parasites of the roots of thousands of plant species, including soybean. Meloidogyne infestation results in poor growth, reduced crop yield, and reduced resistance to secondary stressors, such as other diseases.

[0003] Accordingly, there is a need to develop new compositions and methods for conferring resistance to Meloidogyne . This disclosure provides such compositions and methods.SUMMARY

[0004] Provided are genome edited or transgenic plants comprising a heterologous polynucleotide encoding a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 3, the heterologous polynucleotide associated with increased resistance to Meloidogyne.

[0005] Also provided are nucleic acid constructs comprising a polynucleotide operably linked to a regulatory element, the polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%Docket # 211912-WO-SEC-l sequence identity to SEQ ID NO: 3, wherein said polypeptide when expressed in the cells of a plant confers resistance to Meloidogyn .

[0006] Further provided are methods of modifying plant material comprising introducing into the genome of the plant material a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid is associated with increased resistance to Meloidogyne and (i) encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ ID NO: 1, or (iii) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ ID NO: 2.

[0007] Also provided are methods for conferring resistance io Meloidogyne in a legume crop species comprising introducing into a regenerable plant cell of a legume crop species a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3; and generating a plant from the plant cell, wherein the plant comprises the polynucleotide and has increased resistance io Meloidogyne as compared to a control plant not comprising the polynucleotide.

[0008] Provided are methods of introducing a gene associated with increased resistance to Meloidogyne into a plant comprising crossing a first plant having Meloidogyne resistance with a second plant to obtain progeny plants, obtaining a sample containing nucleic acid from each of one or more of the progeny plants, screening the sample for a nucleic acid that (i) encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ IDDocket # 211912-WO-SEC-lNO: 1, or (iii) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ ID NO: 2, and selecting one or more progeny plants that have the screened-for nucleic acid.

[0009] Also provided are methods of preventing Meloidogyne associated damage to a legume crop species comprising growing the genome edited or transgenic plants described herein.

[0010] Further provided are methods of introgressing Meloidogyne resistance gene into a soybean plant, the Meloidogyne resistance gene comprising a polynucleotide encoding a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 comprising i) crossing an Meloidogyne resistant soybean plant comprising a polynucleotide encoding a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3 with a second soybean plant to produce progeny; ii) screening the progeny with a nucleic acid marker to detect progeny comprising a polymorphism genetically linked to the Meloidogyne resistance gene; and iii) selecting progeny that comprise the polymorphism to obtain soybean plants that contain the Meloidogyne resistance gene.

[0011] Provided are methods for producing a soybean plant having increased resistance to Meloidogyne comprising genotyping a soybean population comprising a plurality of soybean plants or soybean germplasm for the presence of at least one maker genetically linked to a locus comprising or corresponding to ^Meloidogyne resistance gene encoding a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3, selecting from the soybean population one or more soybean plants or soybean germplasm comprising the at least one marker, and crossing the selected soybean plant or soybean germplasm with a second soybean plant or soybean germplasm to produce a progeny population, wherein at least one soybean plant or soybean germplasm of the progeny population comprises the at least one marker and has increased resistance o Meloidogyne as compared to a control plant.Docket # 211912-WO-SEC-lBRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0012] The disclosure can be more fully understood from the following detailed description and the accompanying Sequence Listing, which form a part of this application. The sequence descriptions (Table 1) and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§1.831-1.835.Table 1: Sequence Listing DescriptionDETAILED DESCRIPTION

[0013] The present disclosure provides compositions and methods for producing plants having resistance to diseases caused by Meloidogyne species (e.g., Meloidogyne incognita (RKI)) such as, for example, the root-knot nematode.

[0014] The methods involve expressing in plants a heterologous nucleic acid associated with increased resistance o Meloidogyne, such as for example Meloidogyne incognita (RKI). Additionally, compositions and methods useful in producing, identifying and / or selecting plants and plant materials comprising the heterologous nucleic acid associated with increased resistance to Meloidogyne , such as for example Meloidogyne incognita (RKI) are provided.Docket # 211912-WO-SEC-l

[0015] Accordingly, one aspect of the disclosure provides polynucleotides (e.g., isolated polynucleotides and recombinant polynucleotides) associated with increased resistance to Meloidogyne. The polynucleotides encoding polypeptides (e.g., isolated polypeptides and recombinant polypeptides) comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 and variants thereof. In certain embodiments, the polynucleotides confer resistance or increased resistance to at least one species of Meloidogyne, such as for example Meloidogyne incognita (RKI) when expressed in plants.

[0016] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0017] In the present disclosure, "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

[0018] An "isolated" polynucleotide (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a heterologous recombinant bacterial or plant host cell. An isolated polynucleotide, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated polynucleotide is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. A “recombinant” polynucleotide (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is in a recombinant bacterial or plant host cell. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used toDocket # 211912-WO-SEC-l refer to nucleic acid molecules excludes isolated chromosomes. For example, in certain embodiments, the recombinant nucleic acid molecules encoding the polypeptides of the disclosure can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

[0019] Nucleic acid molecules that are fragments of the polynucleotide sequences described herein are also encompassed by the disclosure. A fragment of a nucleic acid sequence may encode a biologically active portion of the encoded polypeptide or it may be a fragment that can be used as a hybridization probe or PCR primer. Nucleic acid molecules that are fragments can comprise at least about 150, 180, 210, 240, 270, 300, 330, 360, 400, 450, or 500 contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence encoding SEQ ID NO: 3. “Contiguous nucleotides” is used herein to refer to nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences will encode protein fragments that retain the biological activity of the full-length polypeptide and, hence, retain resistance to at least one species of Meloidogyne . “Retains Meloidogyne resistance” is used herein to refer to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of the Meloidogyne resistance of the full-length polypeptide.

[0020] As used herein "percent (%) sequence identity" with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence *100).Docket # 211912-WO-SEC-l

[0021] Unless otherwise stated, sequence identity / similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402).

[0022] As used herein "variant" when used with a protein or polypeptide refers to a protein or polypeptide derived from a native protein or polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein or polypeptide and / or a substitution of one or more amino acids at one or more sites in a native protein or polypeptide. Variants encompassed by the present disclosure exhibit a biological activity of the native protein or polypeptide sequence (e.g., increase resistance oMeloidogyne). For polynucleotides, a variant comprises a polynucleotide having a deletion (i.e., truncations) at the 5' and / or 3' end and / or a deletion and / or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and / or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. One of skill in the art can recognize that variants of the nucleic acids of the embodiments will be constructed such that the open reading frame is maintained. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, such as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a protein of the embodiments. Generally, variants of a particular polynucleotide of the present disclosure can have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide. Variants of a particular polynucleotide of the embodiments (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.Docket # 211912-WO-SEC-l

[0023] For polypeptides, a variant comprises a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and / or a substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, that is they continue to possess the desired biological activity of the native protein, which is, the ability to confer or enhance plant resistance (i.e., Meloidogyne resistance) as described herein. Such variants can result, for example, from genetic polymorphism or from human manipulation. Biologically active variants of a native protein of the embodiments can have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the amino acid sequence for the native protein. A biologically active variant of a protein of the present disclosure can differ from that protein by as few as about 1-15 amino acid residues, as few as about 1-10, such as about 6-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0024] The proteins disclosed herein can be altered, for example, by including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are known in the art. For example, amino acid sequence variants and fragments of the resistance proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are known in the art.

[0025] Variant polynucleotides and polypeptides also encompass sequences and proteins derived from mutagenic or recombinogenic procedures, including, and not limited to, procedures such as DNA shuffling. Libraries of recombinant polynucleotides can be generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest can be shuffled between the protein gene of the present disclosure and other known protein genes to obtain a new gene coding for a protein with an improved property of interest, such as increased ability to confer or enhance plant resistance to a plant pathogen. Strategies for such DNA shuffling are known in the art.

[0026] Variants may be made by making random mutations or the variants may be designed. In the case of designed mutants, there is a high probability of generating variants with similar activity to the native polypeptide when amino acid identity is maintained in critical regions of theDocket # 211912-WO-SEC-l polypeptide which account for biological activity or are involved in the determination of three- dimensional configuration which ultimately is responsible for the biological activity. A high probability of retaining activity will also occur if substitutions are conservative. Amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type are least likely to materially alter the biological activity of the variant.

[0027] The polynucleotides described herein can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods, such as PCR or hybridization, can be used to identify such sequences based on their sequence identity to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire sequences set forth herein or to variants and fragments thereof are encompassed by the present disclosure. Such sequences include sequences that are orthologs of the disclosed sequences. The term "orthologs" refers to genes derived from a common ancestral gene, and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and / or their encoded protein sequences share at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode for a protein that confers or enhances Meloidogyne resistance and that hybridize to the sequences disclosed herein, or to variants or fragments thereof, are encompassed by the present disclosure.

[0028] As used herein an “amino acid deletion,” “deletion mutation,” or the like, refers to a mutation in which the indicated amino acid residue is removed from the polypeptide sequence, so that, when aligned to the reference sequence (e.g., SEQ ID NO: 3) the mutated sequence does not have an amino acid corresponding to the indicated position of the reference sequence. An “amino acid addition,” “addition mutation,” “amino acid insertion,” “insertion,” or the like, refers to a mutation in which at least one amino acid residue is added to the polypeptide sequence, so that, when aligned to the reference sequence (e.g., SEQ ID NO: 3) the mutated sequence contains an additional amino acid corresponding to the indicated position of the reference sequence.Docket # 211912-WO-SEC-l

[0029] An “amino acid substitution,” “substitution mutation,” or the like, refers to a mutation in which the indicated amino acid residue is replaced with a different amino acid residue, so that, when aligned to the reference sequence (e.g., SEQ ID NO: 3) the mutated sequence does not have the same amino acid at the indicated position. When the amino acid residue is substituted for a residue that has similar properties (e.g., size, charge, and / or hydrophobicity) the substitution is referred to as a conservative amino substitution. Conservative amino acid substitutions are well known in the art. Alternatively, when the amino acid residue is substituted for an amino acid that has dissimilar properties the mutation is referred to as a radical amino acid substitution.

[0030] As used herein “encoding,” “encoded,” or the like, with respect to a specified nucleic acid, means comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as is present in some plant, animal and fungal mitochondria, the bacterium Mycoplasma capricolum (Yamao, et al., (1985) Proc. Natl. Acad. Sci. USA 82:2306-9) or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.

[0031] When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray, et al., (1989) Nucleic Acids Res. 17:477-98 and herein incorporated by reference).

[0032] In certain embodiments, the polynucleotide encoding the polypeptide described herein comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1 and 2.Docket # 211912-WO-SEC-l

[0033] In certain embodiments, the polynucleotide encoding the polypeptide is operably linked to at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7 or more) regulatory element. In certain embodiments, the regulatory element is a promoter. In certain embodiments, the polynucleotide is operably linked to its native promoter. In certain embodiments, the regulatory element is a heterologous regulatory element (e g., heterologous promoter). In certain embodiments, the heterologous regulatory element is heterologous to the polynucleotide sequence encoding the polypeptide. In certain embodiments, in which the polynucleotide operably linked to the heterologous regulatory element is introduced in a cell the regulatory element is heterologous to the cell.

[0034] As used herein, the term “heterologous” in reference to a nucleic acid means a nucleic acid sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, as used herein, a “heterologous” gene can refer to a gene sequence (that is the same or only minimally altered) as the native gene, but which has been deliberately inserted at a genomic locus that differs from the native gene’s locus. Where the promoter is a native or natural sequence, the expression of the operably linked sequence can be altered from the wild-type expression, which results in an alteration in phenotype.

[0035] As used herein the term “native gene” refers to a gene as found in its natural endogenous location with its own regulatory sequences. In the context of this disclosure, a “transgenic”, “mutated”, “modified” gene is not a native gene.

[0036] As used herein "operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, operably linked is intended that the coding regions are in the same reading frame.

[0037] As used herein “regulatory element” generally refers to a transcriptional regulatory element involved in regulating the transcription of a nucleic acid molecule such as a gene or a target gene. The regulatory element is a nucleic acid and may include a promoter, an enhancer, an intron, expression modulating elements (EMEs), a 5 ’-untranslated region (5’-UTR, also known as a leader sequence), or a 3’-UTR or a combination thereof. A regulatory element mayDocket # 211912-WO-SEC-l act in "cis" or "trans", and generally a regulatory element acts in "cis", i.e., it activates expression of genes located on the same nucleic acid molecule, e.g., a chromosome, where the regulatory element is located.

[0038] An “enhancer” element is any nucleic acid molecule that increases transcription of a nucleic acid molecule when functionally linked to a promoter regardless of its relative position. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9: 1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments described herein.

[0039] A “repressor” (also sometimes called herein silencer) is defined as any nucleic acid molecule which inhibits the transcription when functionally linked to a promoter regardless of relative position. The term "cis-element" generally refers to transcriptional regulatory element that affects or modulates expression of an operably linked transcribable polynucleotide, where the transcribable polynucleotide is present in the same DNA sequence. A cis-element may function to bind transcription factors, which are trans-acting polypeptides that regulate transcription.

[0040] An “intron” is an intervening sequence in a gene that is transcribed into RNA but is then excised in the process of generating the mature mRNA. The term is also used for the excised RNA sequences. An “exon” is a portion of the sequence of a gene that is transcribed and is found in the mature mRNA derived from the gene but is not necessarily a part of the sequence that encodes the final gene product. The 5' untranslated region (5’UTR) (also known as a translational leader sequence or leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. This region is involved in the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. The “3' non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affectingDocket # 211912-WO-SEC-l mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0041] As used herein “promoter” refers to a region of DNA upstream from the start of transcription involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. In certain embodiments, the polynucleotides described herein are operably linked to a promoter that drives expression in a plant cell. Any promoter known in the art can be used in the methods of the present disclosure including, but not limited to, constitutive promoters, pathogeninducible promoters, wound-inducible promoters, tissue-preferred promoters, and chemical- regulated promoters. The choice of promoter may depend on the desired timing and location of expression in the transformed plant as well as other factors, which are known to those of skill in the art. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in U.S. Patent No. 6,072,050; the core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS; and the like. Other constitutive promoters which are known in the art can be contemplated for use in the present disclosure.

[0042] Generally, it can be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen, e.g., PR proteins, SAR proteins, beta-1,3 -glucanase, chitinase, etc.

[0043] Of interest are promoters that are expressed locally at or near the site of pathogen infection. Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter can be used in the constructions of the disclosure. Such woundinducible promoters include potato proteinase inhibitor (pin II) gene, wunl and wun2, winl and win2, systemin, WIP1, MPI gene, and the like.

[0044] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter can be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical -inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are usedDocket # 211912-WO-SEC-l as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (e.g., the glucocorticoid-inducible promoter, and tetracycline-inducible and tetracycline-repressible promoters).

[0045] Tissue-preferred promoters can be utilized to target enhanced expression of the target genes or proteins within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Examples of tissue-preferred promoters include those described in Yamamoto et al. (1997) Plant J. 12(2): 255 -265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol.112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified.

[0046] Leaf-specific promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2)255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0047] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seedgerminating" promoters (those promoters active during seed germination). Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message), cZ19Bl (maize 19 kDa zein), milps (myo-inositol-1 -phosphate synthase), and cel A (cellulose synthase) (see WO 00 / 11177). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean -phaseolin, napin, -conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDaDocket # 211912-WO-SEC-l zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00 / 12733, where seedpreferred promoters from endl and end2 genes are disclosed; herein incorporated by reference.

[0048] In certain embodiments, the polynucleotides of the present disclosure can involve the use of the intact, native genes, wherein the expression is driven by a cognate 5' upstream promoter sequence(s), also referred to herein as the native promoter.

[0049] Specific soybean promoters include but are not limited to soy ubiquitin (subi-1), elongation factor 1 A, and S-adenosyl methionine synthase for constitutive expression and Rpp4, RPG1-B, and promoters contained in gene models such as Glyma promoters.

[0050] Also provided are nucleic acid (e.g., DNA) constructs comprising any of the polynucleotides described herein (e.g., SEQ ID NOs: 1 and 2). The use of the term "nucleotide constructs" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.

[0051] In certain embodiments, the polynucleotides described herein are provided in expression cassettes (e.g., a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence) for expression in a plant of interest or any organism of interest. The cassette can include 5' and 3' regulatory sequences operably linked to a polynucleotide. 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. Such an expression cassette is provided with aDocket # 211912-WO-SEC-l plurality of restriction sites and / or recombination sites for insertion of the polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0052] The expression cassette can include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (e.g., a promoter), a polynucleotide (e.g., SEQ ID NO: 1 or 2), and a transcriptional and translational termination region (e.g., termination region) functional in plants. The regulatory regions (e.g., promoters, transcriptional regulatory regions, and translational termination regions) and / or the polynucleotide may be native / analogous to the host cell or to each other. Alternatively, the regulatory regions and / or the polynucleotide may be heterologous to the host cell or to each other.

[0053] The termination region may be native with the transcriptional initiation region, with the plant host, or may be derived from another source (i.e., foreign or heterologous) than the promoter, the polynucleotide, the plant host, or any combination thereof.

[0054] The expression cassette may additionally contain a 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include viral translational leader sequences.

[0055] Generally, the expression cassette can comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present disclosure.

[0056] In preparing the expression cassette, the various DNA fragments may be manipulated, to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.Docket # 211912-WO-SEC-l

[0057] In certain embodiments, the nucleic acid constructs or expression cassettes, described herein, are expressed in a host cell, plant or seed. The nucleic acid constructs or expression cassettes disclosed herein may be used for transformation of any plant species.

[0058] Also provided are host cells that are engineered (e.g., transduced, transformed, or transfected) to express one or more of any of the polynucleotides encoding a polypeptide described herein. The polypeptides, polynucleotides or nucleic acid constructs can be expressed in any organism, including in non-animal cells such as yeast, fungi, bacteria and the like. Details regarding non-animal cell culture can be found in Payne el al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture,' Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin, Heidelberg, New York); and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

[0059] Host cells of interest can include, for example, a eukaryotic cell, an animal cell, a protoplast, a tissue culture cell, a prokaryotic cell, a bacterial cell, such as E. coli, B. subtilis, Streptomyces, Salmonella typhimurium, a gram positive bacteria, a purple bacteria, a green sulfur bacteria, a green non-sulfur bacteria, a cyanobacteria, a spirochetes, a thermatogale, a flavobacteria, bacteroides; a fungal cell, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; an insect cell such as Drosophila and Spodoptera frugiperda,' a mammalian cell such as CHO, COS, BHK, HEK 293 or Bowes melanoma; an archaebacteria such as Korarchaeota, Thermoproteus, Pyrodictium, Thermococcales, Methanogens, Archaeoglobus, and extreme Halophiles.

[0060] Further provided are genome edited or transgenic plants, plant cells, plant parts, seeds, and grain comprising at least one of the polynucleotide sequences, nucleic acid constructs, or expression cassettes described herein, so that the plants, plant cells, plant parts, seeds, and / or grain express any of the polypeptides described herein. In certain embodiments, the plants, plant cells, plant parts, seeds, and / or grain have stably incorporated at least one polynucleotide into its genome.

[0061] For example, in certain embodiments provided is a genome edited or transgenic plant, plant cell, plant part, seed and / or grain comprising a heterologous polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,Docket # 211912-WO-SEC-l96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, the heterologous polynucleotide associated with increased resistance to at least one species of Meloidogyne, such as, for example, Meloidogyne incognita (RKI). In certain embodiments, the heterologous polynucleotide is operably linked to a regulatory element. In certain embodiments, the regulatory element is the native gene promoter. In certain embodiments, the regulatory element is a heterologous regulatory element such as, for example, a heterologous promoter.

[0062] In certain embodiments, the plant is a soybean plant and the heterologous nucleic acid is inserted into a location on soybean chromosome (Chr) Chr 1, Chr 2, Chr 3, Chr 4, Chr 5, Chr 6, Chr 7, Chr 8, Chr 9, Chr 11, Chr 12, Chr 13, Chr 14, Chr 15, Chr 16, Chr 17, Chr 18, Chr 19, or Chr 20. In certain embodiments, the plant is a soybean plant and the heterologous nucleic acid is inserted into a location other than its native locus corresponding to physical positions 1419209 to 1652513 on soybean chromosome 10 in soybean genome Williams 82 (w82.a2.vl) public genome (Schmutz, J. et al. et al. 2010, Nature, 463(7278): 178-183). In certain embodiments, the plant is a soybean plant and the heterologous nucleic acid is inserted into a location other than between the positions corresponding to physical positions 1504127 and 1504874 on soybean chromosome 10 in soybean genome Williams 82 (w82.a2.vl) public genome.

[0063] In certain embodiments, the plant or a plant generated from the plant cell, plant part, or seed, has resistance to at least one species of Meloidogyne, such as, for example, Meloidogyne incognita (RKI). In certain embodiments, the plant or a plant generated from the plant cell, plant part, or seed, has increased resistance to at least one species of Meloidogyne , such as, for example, Meloidogyne incognita (RKI) as compared to a control plant (e.g., a plant of the same genetic background not comprising the polynucleotide). For example, in certain embodiments, the genome edited or transgenic plant has increased resistance to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 species of Meloidogyne.

[0064] Each of the genome edited or transgenic plants or genome edited or transgenic plants generated from the plant cells, plant parts, or seeds disclosed herein can be used to control rootknot nematode inasmuch as the modified plant expresses an effective amount a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3. Thus, disclosed herein is the use of the modified plants disclosed herein to control root-knot nematode by expressing a heterologous nucleic acid sequence the encodes a polypeptide having at least 70%, 75%, 80%, 85%, 90%,Docket # 211912-WO-SEC-l91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3.

[0065] As used herein, the term "resistance" refers to an absence or reduction of one or more disease symptoms in a plant caused by a plant pathogen. Resistance can mean that disease symptoms, such as, for example, number of lesions, defoliation, and associated yield loss, are reduced, minimized or lessened, when compared to a plant that is susceptible to the disease or a plant that does not contain an effective resistance gene. Resistance can also include the prevention or delay of proliferation of a pathogen (e.g., nematode pathogen).

[0066] In certain embodiments, the polynucleotides described herein are transiently expressed in the plant, plant cell, plant part, seed or grain using a transient transformation technique. In certain embodiments, the polynucleotides described herein are stably expressed in the plant, plant cell, plant part, seed or grain using a stable transformation technique. In certain embodiments, the polynucleotides are introduced into the plant, plant cell, plant part, seed or grain using a nucleic acid construct or an expression cassette described herein. In certain embodiments, the polynucleotides described herein are introduced into the plant, plant cell, plant part, seed or grain using a genome editing technique.

[0067] In certain embodiments, the plant cells or plant parts are grown into plants. The method for generating the plants from the plant cells or plant parts is not particularly limited. These plants can then be grown, and either pollinated with the same strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations can 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 certain embodiments of the present disclosure, the transformed seed or transgenic seed have a nucleotide construct described herein or an expression cassette described herein stably incorporated into their genome.

[0068] In certain embodiments, the present disclosure encompasses seeds comprising a polynucleotide sequence disclosed herein that can develop into or can be used to develop a plant or plants with increased or enhanced resistance to a pathogen (e.g., Meloidogyne) or infection caused by a pathogen as compared to, for example, a wild-type variety of the plant seed. InDocket # 211912-WO-SEC-l certain embodiments, the present disclosure features seeds from genome edited or transgenic legume crop plants wherein the seed comprises a polynucleotide disclosed herein.

[0069] In certain embodiments, the plants described herein are elite plant lines (e.g., elite soybean line). In certain embodiments, the plant cells, plant parts, seeds, and grain are isolated from or produced by an elite plant line. As used herein, “elite line” refers to any line that has resulted from breeding and selection for superior agronomic performance that allows a producer to harvest a product of commercial significance. Numerous elite lines are available and known to those of skill in the art of plant breeding (e.g., soybean breeding). An “elite population” is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as soybean.

[0070] As used herein, the term “plant” includes plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the introduced polynucleotides.

[0071] The plant species of the compositions and methods of the present disclosure can be any plant species for which expression of a polynucleotide or polypeptide described herein is desired, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, com (Zea mays), Brassica spp. (e.g., Brassica napus, Brassica rapa, Brassica juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Per sea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (OleaDocket # 211912-WO-SEC-l europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[0072] In certain embodiments, the plant of the compositions and methods described herein is a legume crop species, including, but not limited to, alfalfa (Medicago sativa); clover or trefoil (Trifolium spp.); pea, including (Pisum satinum), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata) and Lathyrus spp.; bean (Fabaceae or Leguminosae); lentil (Lens culinaris); lupin (Lupinus spp.); mesquite (Prosopis spp.); carob (Ceratonia siliqua), soybean (Glycine max), peanut (Arachis hypogaea) or tamarind (Tamarindus indica). The terms "legume species" and "legume crop species" are used herein to refer to plants, and can be for example, a plant of interest. In certain embodiments, the plant, plant part or plant cell is a legume species or legume crop species.

[0073] In certain embodiments, the plants, plant cells, plant parts, seeds, and grain further comprise a polynucleotide encoding at least one additional Meloidogyne resistance gene.

[0074] In certain embodiments of the present disclosure, the polynucleotide sequences can be stacked with any combination of polynucleotide sequences of interest to create plants with a desired phenotype. This stacking can be accomplished by a combination of genes within a DNA construct, or by crossing one or more plants having transgenes with another plant line that comprises a desired combination. For example, the polynucleotides of the present disclosure or variants thereof can be stacked with any other polynucleotides of the disclosure or with other genes. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present disclosure can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including and not limited to traits desirable for animal feed such as high oil genes, balanced amino acids, increased digestibility, insect, disease or herbicide resistance, avirulence and disease resistance genes, agronomic traits (e.g, male sterility, flowering time) and / or transformation technology traits (e.g., cell cycle regulation or gene targeting).

[0075] These stacked combinations can be created by any method including, but not limited to, cross breeding plants by any conventional or known methodology, genetic transformation, or genome editing. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example,Docket # 211912-WO-SEC-l a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a cotransformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that can suppress the expression of the polynucleotide of interest. This can be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant.

[0076] In one embodiment, the stacked combination includes one or more genes encoding pesticidal proteins including, but not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13); from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58: 12343-12349) and from Pseudomonas pseudoalcaligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89: 159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal, 3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069); US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide of US 9,688,730; an AfIP-lA and / or AfTP-lB polypeptide of US9,475,847; a PIP-47 polypeptide of US Publication Number US20160186204; an IPD045 polypeptide, an IPD064 polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077 polypeptide of PCT Publication Number WO 2016 / 114973; an IPD080 polypeptide of International Patent Application Publication Number W02018 / 075350; an IPD078 polypeptide, an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, an IPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide of International Patent Application Publication Number WO2018 / 084936; PIP-72 polypeptide of US Patent Publication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide of US Publication Number US20170166921; an IPD098 polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109 polypeptide of International Patent Application Publication Number WO2018 / 232072; aDocket # 211912-WO-SEC-lPtIP-83 polypeptide of US Publication Number US20160347799; a PtIP-96 polypeptide of US Publication Number US20170233440; an IPD079 polypeptide of PCT Publication Number WO20 17 / 23486; an IPD082 polypeptide of PCT Publication Number WO 2017 / 105987, an IPD090 polypeptide of International Patent Application Publication Number WO2017 / 192560, an IPD093 polypeptide of International Patent Application Publication Number WO2018 / 111551; an IPD103 polypeptide of International Patent Application Publication Number W02018 / 005411; an IPD101 polypeptide of International Patent Application Publication Number WO2018 / 118811; an IPD121 polypeptide of International Patent Application Publication Number WO2018 / 208882; and 5-endotoxins including, but not limited to a Cryl, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, CrylO, Cryl 1, Cryl2, Cryl3, Cryl4,Cry 15, Cry 16, Cry 17, Cry 18, Cry 19, Cry20, Cry21, Cry22, Cry23, Cry 24, Cry25, Cry26, Cry27,Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54,Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67,Cry68, Cry69, Cry70, Cry71, and Cry 72 classes of 8-endotoxin polypeptides and the B. thuringiensis cytolytic cytl and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins can be found in Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), at lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / which can be accessed on the world-wide web using the "www" prefix).

[0077] In another embodiment, the stacked combination includes a polynucleotide encoding resistance to an herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J. 7: 1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449, respectively. See also, US Patent Numbers 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824; US Patent Application Serial Number 11 / 683,737 and International Publication WO 1996 / 33270.

[0078] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to Glyphosate (resistance imparted by mutant 5-enolpyruvl-3- phosphikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids andDocket # 211912-WO-SEC-l cyclohexones (ACCase inhibitor-encoding genes). See, for example, US Patent Number 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS which can confer glyphosate resistance. US Patent Number 5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes. See also, US Patent Numbers 6,566,587; 6,338,961; 6,248,876; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 5,094,945, 4,940,835; 5,866,775; 6,225,114; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 and 5,491,288 and International Publications EP 1173580; WO 2001 / 66704; EP 1173581 and EP 1173582, which are incorporated herein by reference for this purpose. Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido-reductase enzyme as described more fully in US Patent Numbers 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose. In addition, glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyl transferase. See, for example, US Patent Numbers 7,462,481; 7,405,074 and US Patent Application Publication Number US 2008 / 0234130. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC® Accession Number 39256, and the nucleotide sequence of the mutant gene is disclosed in US Patent Number 4,769,061 to Comai. EP Application Number 0 333 033 to Kumada, et al., and US Patent Number 4,975,374 to Goodman, et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0 242 246 and 0 242 236 to Leemans, et al.; De Greef, et al., (1989) Bio / Technology 7:61, describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. See also, US Patent Numbers 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 and 5,879,903, which are incorporated herein by reference for this purpose. Exemplary genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop, are the Accl- Sl, Accl-S2 and Accl-S3 genes described by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

[0079] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell 3: 169, describeDocket # 211912-WO-SEC-l the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in US Patent Number 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC® Accession Numbers 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes, et al., (1992) Biochem. J. 285: 173.

[0080] In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants (see, e.g., Hattori, et al., (1995) Mol Gen Genet. 246:419). Other genes that confer resistance to herbicides include: a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant Physiol 106: 17), genes for glutathione reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36: 1687) and genes for various phosphotransferases (Datta, et al., (1992) Plant Mol Biol 20:619).

[0081] In another embodiment, the stacked combination includes a polynucleotide encoding resistance to an herbicide targeting Protoporphyrinogen oxidase (protox) which is necessary for the production of chlorophyll. The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in US Patent Numbers 6,288,306; 6,282,83 and 5,767,373 and International Publication WO 2001 / 12825.

[0082] In another embodiment, the stacked combination includes an aad-1 gene (originally from Sphingobhim herbicidovorans) encoding the aryloxyalkanoate dioxygenase (AAD-1) protein. The trait confers tolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly referred to as “fop” herbicides such as quizalofop) herbicides. The aad-1 gene, itself, for herbicide tolerance in plants was first disclosed in WO 2005 / 107437 (see also, US 2009 / 0093366). The aad-12 gene, derived from Delftia acidovorans, which encodes the aryl oxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to 2,4- dichlorophenoxyacetic acid and pyridyloxyacetate herbicides by deactivating several herbicides with an aryloxyalkanoate moiety, including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxy auxins (e.g., fluroxypyr, triclopyr).Docket # 211912-WO-SEC-l

[0083] In another embodiment, the stacked combination includes a polynucleotide encoding an herbicide resistant dicamba monooxygenase, such as a polynucleotide disclosed in US Patent Application Publication 2003 / 0135879 for imparting dicamba tolerance.

[0084] In another embodiment, the stacked combination includes a polynucleotide encoding bromoxynil nitrilase (Bxn), such as a polynucleotide disclosed in US Patent Number 4,810,648 for imparting bromoxynil tolerance.

[0085] In another embodiment, the stacked combination includes a polynucleotide encoding CcRppl disclosed in U.S. 10,842,097 for imparting ASR resistance. In another embodiment, the stacked combination includes a polynucleotide encoding CcRpp2 disclosed in WO2022140257 for imparting ASR resistance.

[0086] In another embodiment, the stacked combination includes a polynucleotide encoding phytoene (crtl) described in Misawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994) Plant J. 6:481-489 for norflurazon tolerance.

[0087] In another embodiment, the stacked combination includes a polynucleotide encoding a protein that confers or contributes to an altered grain characteristic, such as altered fatty acids, for example, by:

[0088] (1) Down-regulation of stearoyl-ACP to increase stearic acid content of the plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999 / 64579 (Genes to Alter Lipid Profdes in Com).

[0089] (2) Elevating oleic acid via FAD-2 gene modification and / or decreasing linolenic acid via FAD-3 gene modification (see, US Patent Numbers 6,063,947; 6,323,392; 6,372,965 and WO 1993 / 11245).

[0090] (3) Altering conjugated linolenic or linoleic acid content, such as in WO 2001 / 12800.

[0091] (4) Altering LEC1, AGP, Dekl, Superall, mil ps, and various Ipa genes such as Ipal, Ipa3, hpt or hggt. For example, see, WO 2002 / 42424, WO 1998 / 22604, WO 2003 / 011015, WO 2002 / 057439, WO 2003 / 011015, US Patent Numbers 6,423,886, 6,197,561, 6,825,397 and US Patent Application Publication Numbers US 2003 / 0079247, US 2003 / 0204870 and Rivera- Madrid, et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.

[0092] (5) Genes encoding delta-8 desaturase for making long-chain polyunsaturated fatty acids (US Patent Numbers 8,058,571 and 8,338,152), delta-9 desaturase for lowering saturated fatsDocket # 211912-WO-SEC-l(US Patent Number 8,063,269), Primula delta 6-desaturase for improving omega-3 fatty acid profdes.

[0093] (6) Isolated nucleic acids and proteins associated with lipid and sugar metabolism regulation, in particular, lipid metabolism protein (LMP) used in methods of producing transgenic plants and modulating levels of seed storage compounds including lipids, fatty acids, starches or seed storage proteins and use in methods of modulating the seed size, seed number, seed weights, root length and leaf size of plants (EP 2404499).

[0094] (7) Altering expression of a High-Level Expression of Sugar-Inducible 2 (HSI2) protein in the plant to increase or decrease expression of HSI2 in the plant. Increasing expression of HSI2 increases oil content while decreasing expression of HSI2 decreases abscisic acid sensitivity and / or increases drought resistance (US Patent Application Publication Number 2012 / 0066794).

[0095] (8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oil content in plant seed, particularly to increase the levels of omega-3 fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (US Patent Application Publication Number 2011 / 0191904).

[0096] (9) Nucleic acid molecules encoding wrinkled 1 -like polypeptides for modulating sugar metabolism (US Patent Number 8,217,223).

[0097] Also provided are methods for providing, conferring, or enhancing resistance of a plant to Meloidogyne, such that, for example, the Meloidogyne (e.g., Meloidogyne incognita (RKI)) can no longer reproduce. As used herein the term "enhance" means to improve, increase, amplify, multiply, elevate and / or raise, thereby reducing one or more disease symptoms. Accordingly, plants (e.g., soybean) exhibit an increased resistance to a disease (e.g., root-knot nematode) when compared to plants that are susceptible or tolerant to the root-knot nematode. In certain embodiments, methods described herein can reduce one or more symptoms (i.e., disease symptoms) of a legume plant disease.

[0098] As used herein "plant pathogen" or "nematode pathogen" can be used herein to mean nematode pathogens of, for example, the genus Meloidogyne, including the species Meloidogyne incognita. These species are known to cause root-knot nematode disease in plants.

[0099] “ Meloidogyne'' , “RKI”, “RKN”, “Meloidogyne incognita", “Meloidogyne sp" and “ eloidogyne species" are used interchangeably herein and refer to a plant parasitic nematode pathogen that is the causative agent for root-knot nematode disease.Docket # 211912-WO-SEC-l

[0100] In certain embodiments, the method for providing, conferring, or enhancing resistance comprises introducing into a regenerable plant cell at least one of the polynucleotides, nucleic acid constructs, or expression cassettes described herein. In certain embodiments, the plant is a legume crop species (e.g., soybean).

[0101] In certain embodiments of the methods for providing, conferring, or enhancing resistance of a plant to a. Meloidogyne species described herein, the method comprises the introduction of a polynucleotide (e.g., heterologous polynucleotide) encoding a polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, optionally operably linked to a regulatory element (e.g., heterologous promoter or native promoter); and generating a plant from the plant cell wherein the plant comprises the polynucleotide and has enhanced resistance, increased resistance, or resistance to a Meloidogyne species.

[0102] In certain embodiments, the method for providing, conferring, or enhancing resistance of a plant io Meloidogyne (e.g., Meloidogyne incognita (RKI)) comprises expressing in a regenerable plant cell a nucleic acid construct or expression cassette comprising a polynucleotide described herein; and generating the plant from the plant cell. In certain embodiments, the polynucleotide is operably linked to at least one regulatory sequence. In certain embodiments, the at least one regulatory sequence is a heterologous promoter. The nucleic acid construct or expression cassette for use in the method may be any nucleic acid construct or expression cassette provided herein. In certain embodiments, the nucleic acid construct or expression cassette is expressed by introducing into a plant, plant cell, plant part, seed, and / or grain the nucleic acid construct or expression cassette, whereby the polypeptide is expressed in the plant, plant cell, plant part, seed, and / or grain. In certain embodiments the nucleic acid construct or expression cassette is incorporated into the genome of the plant.

[0103] Various methods can be used to introduce the sequences into a plant, plant part, plant cell, seed, and / or grain. "Introducing" is intended to mean presenting to the plant, plant cell, seed, and / or grain the polynucleotide or resulting polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the disclosure do not depend on a particular method for introducing a sequence into a plant, plant cell, seed, and / or grain, only that the polynucleotide or polypeptide gains access to the interior of at least one cell of the plant.Docket # 211912-WO-SEC-l

[0104] "Stable transformation" is intended to mean that the polynucleotide introduced into a plant integrates into the genome of the plant of interest and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant of interest and does not integrate into the genome of the plant or organism, or a polypeptide is introduced into a plant or organism.

[0105] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), 4<t,v / ?ac7c / 7z / / ??-mediated transformation (U.S. Patent No. 5,563,055 and U.S. Patent No. 5,981,840), Ochrobacterium-medGted transformation (U.S. Patent Application Publication 2018 / 0216123 and WO20 / 092494) direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Patent Nos. 4,945,050; U.S. Patent No. 5,879,918; U.S. Patent No. 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00 / 28058). D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tume fact ens ,' all of which are herein incorporated by reference.

[0106] In certain embodiments, the sequences can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the protein directly into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58,' Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:115-184, all of which are herein incorporated by reference.

[0107] In other embodiments, the polynucleotides disclosed herein may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the disclosure within a DNA or RNA molecule. It isDocket # 211912-WO-SEC-l recognized that the inventive polynucleotide sequence may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters disclosed herein also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta e / al. (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.

[0108] Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In certain embodiments, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99 / 25821, WO99 / 25854, WO99 / 25840, WO99 / 25855, and WO99 / 25853, all of which are herein incorporated by reference. Briefly, the polynucleotide disclosed herein can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided, and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome. Other methods to target polynucleotides are set forth in WO 2009 / 114321 (herein incorporated by reference), which describes “custom" meganucleases produced to modify plant genomes, in particular the genome of maize. See, also, Gao et al. (2010) Plant Journal 7: 176-187.

[0109] One of skill will recognize that after the nucleic acid constructs and / or expression cassettes containing the polynucleotides described herein are stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of several standard breeding techniques can be used, depending upon the species to be crossed.

[0110] Parts obtained from the regenerated plants described herein, such as flowers, seeds, leaves, branches, fruit, and the like are included, provided that these parts comprise cells comprising the inventive polynucleotide. Progeny and variants, and mutants of the regeneratedDocket # 211912-WO-SEC-l plants are also included, provided that these parts comprise the introduced nucleic acid sequences.

[0111] In certain embodiments, a homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced. Backcrossing to a parental plant and outcrossing with a non-transgenic plant are also contemplated.

[0112] In certain embodiments, the method for providing, conferring, or enhancing resistance of a plant to Meloidogyne comprises introducing a heterologous polynucleotide encoding a polypeptide described herein into the genome of the regenerable plant cell using genome editing technologies. In certain embodiments, the method comprises editing genes or previously introduced genes of a plant to produce the polynucleotides provided herein using genome editing technologies. In certain embodiments, the method comprises inserting genes or polynucleotides described herein (e.g., heterologous polynucleotides) in soybean into a location on (Chr) Chr 1, Chr 2, Chr 3, Chr 4, Chr 5, Chr 6, Chr 7, Chr 8, Chr 9, Chr 11, Chr 12, Chr 13, Chr 14, Chr 15, Chr 16, Chr 17, Chr 18, Chr 19, or Chr 20 using genome editing technologies. In certain embodiments, the method comprises inserting genes or polynucleotides described herein (e.g., heterologous polynucleotides) in soybean into a location other than its native locus corresponding to physical positions 1419209 to 1652513 on soybean chromosome 10 in soybean Williams 82 (w82.a2.vl) public genome (Schmutz, J. et al. et al. 2010, Nature, 463(7278): 178- 183) using genome editing technologies. In certain embodiments, the method comprises inserting genes or polynucleotides described herein (e.g., heterologous polynucleotides) in soybean into a location other than between the positions corresponding to physical positions 1504127 and 1504874 on soybean chromosome 10 in soybean genome Williams 82 (w82.a2.vl) public genome.

[0113] The genome editing technology for use in the methods and compositions described herein is not particularly limited and may be any genome editing technique that allows for the introduction and / or targeted introduction of the desired polynucleotide.

[0114] In certain embodiments the genome editing technique uses an enzyme selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, baseDocket # 211912-WO-SEC-l editing deaminases, zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) or engineered site-specific meganuclease.

[0115] The desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with an existing trait of interest. Existing traits of interest could be either an endogenous trait or a previously introduced trait. Thus, for example, a native gene (Meloidogyne susceptible allele) can be altered though genome editing to the Meloidogyne resistant allele disclosed herein. Alternatively or additionally, the disclosed Meloidogyne resistant allele (that (i) encodes a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 1, or (iii) comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 2 can be introduced by genome editing at a different genomic location (e.g., in soybean).

[0116] The use of double-stranded break technologies such as Cas endonuclease-gRNA complexes, has been described, for example in U.S. Patent Application Publications 2015 / 0082478, and 2015 / 0059010, International Application Publications WO2015 / 026886, W02016 / 007347, and WO2016 / 25131, and US Patent No. 10,934,536. As used herein, a Cas endonuclease refers to a polypeptide encoded by a Cas (CRISPR- associated) gene. A Cas protein includes but is not limited to: a Cas9 protein, a Cpfl (Casl2) protein, a C2cl protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, CaslO, or combinations or complexes of these. When complexed with a guide polynucleotide, the guide polynucleotide / Cas endonuclease complex”, (or “guide polynucleotide / Cas endonuclease system”, “ guide polynucleotide / Cas complex”, “guide polynucleotide / Cas system” and “guided Cas system” or “Polynucleotide-guided endonuclease”, “PGEN”” are capable of directing the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and nick or cleave (introduce a single or double-strand break) the DNA target site. A guided Cas system referred to herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any known CRISPR systems (Horvath and Barrangou, 2010, Science 327: 167-170; Makarova et al. 2015,Docket # 211912-WO-SEC-lNature Reviews Microbiology No\. 13: 1-15; Zetsche et al., 2015, Cell 163, 1 -13; Shmakov et al., 2015, Molecular Cell 60, 1-13).

[0117] In some examples, a. Meloidogyne susceptible gene allele can be modified by genome editing technology to include the Meloidogyne resistance allele. Site specific modifications can include those any method for introducing site specific modification, including, but not limited to, through the use of gene repair oligonucleotides (e.g. US Publication 2013 / 0019349), or through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Such technologies can be used to modify the previously introduced polynucleotide through the insertion, deletion or substitution of nucleotides within the introduced polynucleotide. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include, additional expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing technologies may be used to position additional disease or pest resistant genes in close proximity to the Meloidogyne resistant gene within the genome of a plant, in order to generate molecular stacks with multiple disease / pest resistant proteins.

[0118] An “altered target site,” “altered target sequence.” “modified target site,” and “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).

[0119] The present disclosure also provides a method for screening or assaying legume plants for resistance, immunity, or susceptibility to a plant disease. General methods for determination of resistance, immunity, or susceptibility of a plant to a particular pathogen are known to one skilled in the art. For example, a method for screening or assaying legume plants for resistance, immunity or susceptibility to a plant disease may comprise exposing a plant cell, tissue or organ (e g., root) to a pathogen (e.g., Meloidogyne incognita) and then determining and / or measuring in the exposed plant, the degree of resistance, immunity and / or susceptibility to a plant disease (e.g., root-knot nematode) caused by the pathogen. The method can further comprise measuring any observable plant disease symptoms on the plant exposed to the plant pathogen and thenDocket # 211912-WO-SEC-l comparing the plant disease symptoms to a reference standard to determine the degree or extent of disease resistance.

[0120] Methods of exposing a plant cell, tissue or organ to a pathogen are known in the art. Methods of measuring, comparing, and determining the level of resistance, immunity and / or susceptibility (e.g., plant disease symptoms) to a disease, such as, for example, root-knot nematode, caused by the pathogen are also known in the art. The exposed plants can be further assessed to isolate polynucleotides, amino acid sequences and / or genetic markers that are associated with, linked to, and / or confer resistance, immunity or susceptibility of a plant to a particular pathogen or disease. Further assessments include, but are not limited to, isolating polynucleotides, nucleic acids, or amino acids sequences from the exposed plant, carrying out an assay of the isolated polynucleotides or nucleic acids, for example, to detect one or more biological or molecular markers associated with one or more agronomic characteristics or traits, including but not limited to, resistance, immunity and / or susceptibility. The information gleaned from such methods can be used, for example, in a breeding program.

[0121] In certain embodiments, the plants expressing the polynucleotides and polypeptides disclosed herein may also have one or more nematicides applied to the plants as a method of further preventing Meloidogyne associated damage to a legume crop species. These nematicidal compounds may also be applied to supplement the protection of a legume crop species comprising the polynucleotides described herein to a wider variety of undesirable diseases. These nematicides may be formulated or tank-mixed with other fungi cide(s) disclosed herein or applied sequentially with the other fungicide(s).

[0122] Accordingly, provided is a method for preventing Meloidogyne associated damage to a legume crop species comprising planting a field with seed comprising at least one polynucleotide (e g., heterologous polynucleotide) encoding at least one polypeptide described herein. In certain embodiments, the method further comprises treating the field with a nematicide.

[0123] Also provided herein is a method of identifying germplasm or plants comprising a gene or polynucleotide of the disclosure. The method comprises obtaining a nucleic acid sample from one or more plants, and contacting said nucleic acid sample with a nucleic acid sequence that specifically binds to a polynucleotide of the disclosure and detecting the specific binding of the nucleic acid to its target sequence. For example, the method can detect the target sequence through the use of a labeled probe or by conducting a PCR reaction with suitable PCR primersDocket # 211912-WO-SEC-l that only produce an amplicon in the presence of the target sequence. In one embodiment the method comprises obtaining a nucleic acid sample from one or more plants, and contacting the nucleic acid sample with either:

[0124] i) a polynucleotide that comprises a sequence of at least 8 nucleotides that are identical or have at least 90-95% sequence identity to a contiguous sequence selected from the group consisting of SEQ ID NOs: 1 and 2, or complements thereof; wherein said method further comprises subjecting said sample and said polynucleotide to stringent hybridization conditions; and assaying said sample for hybridization of said polynucleotide to said DNA; or

[0125] ii) a pair of PCR primers, wherein a first and second PCR primer each specifically bind to a sequence selected from the group consisting of SEQ ID NOs: 1 and 2, wherein said first and second PCR primers are capable of producing an amplicon when bound to their target complementary sequences and subjected to standard PCR reaction conditions; subjecting said sample to polymerase chain reaction conditions; and assaying for an amplicon generated between said first and second primers.

[0126] As used herein, “stringent conditions” encompass conditions under which hybridization will only occur if there is less than 20% mismatch between the hybridization molecule and a sequence within the target nucleic acid molecule. “Stringent conditions” include further particular levels of stringency. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize; and conditions of “very high stringency” are those under which sequences with more than 5% mismatch will not hybridize. The following are representative, non-limiting hybridization conditions.

[0127] High Stringency condition (detects sequences that share at least 90% sequence identity): Hybridization in 5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 16 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65 °C for 20 minutes each.Docket # 211912-WO-SEC-l

[0128] Moderate Stringency condition (detects sequences that share at least 80% sequence identity): Hybridization in 5x-6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 65-70 °C for 16-20 hours; wash twice in 2x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature for 5-20 minutes each; and wash twice in lx SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at 55-70 °C for 30 minutes each.

[0129] Non-stringent control condition (sequences that share at least 50% sequence identity will hybridize): Hybridization in 6x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55 °C for 16-20 hours; wash at least twice in 2x-3x SSC buffer (wherein the SSC buffer contains a detergent such as SDS, and additional reagents like salmon sperm DNA, EDTA, etc.) at room temperature to 55 °C for 20-30 minutes each.

[0130] As used herein, the term "germplasm" refers to genetic material of, or from, an individual (e g., a plant), a group of individuals (e.g., a plant line, variety, or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell or can be separate from the organism or cell. The germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. Germplasm in the context of the present disclosure includes cells, seed or tissues from which new plants can be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.

[0131] Also provided is a method of modifying plant material, comprising introducing into the plant material’s genome a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid (i) encodes a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 1, or (iii) comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%Docket # 211912-WO-SEC-l nucleotide sequence identity to SEQ ID NO: 2. In particular examples, the heterologous nucleic acid is associated with increased resistance to Meloidogyn .

[0132] For example, provided is a method of modifying plant material, comprising introducing into the genome of the plant material a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid encodes a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3. Provided is a method of modifying plant material, the method comprising introducing into the plant material’s genome a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0133] As used herein, the term “plant material” refers to any processed or unprocessed material derived, in whole or in part, from a plant. For example, and without limitation, a plant material may be a plant part, a seed, a fruit, a leaf, a root, a plant tissue, a plant tissue culture, a plant explant, or a plant cell.

[0134] In certain embodiments of the methods of modifying plant material, the plant material is preferably soybean and the heterologous nucleic acid can be inserted into a genomic locus on soybean chromosome (Chr) Chr 1, Chr 2, Chr 3, Chr 4, Chr 5, Chr 6, Chr 7, Chr 8, Chr 9, Chr 11, Chr 12, Chr 13, Chr 14, Chr 15, Chr 16, Chr 17, Chr 18, Chr 19, or Chr 20. Alternatively, in each of the foregoing instances, the plant material is preferably soybean and the heterologous nucleic acid can be inserted into a genomic locus on soybean chromosome 10 at location other than its native locus, which corresponds to physical positions 1419209 to 1652513 on soybean chromosome 10 in soybean Williams 82 (w82.a2.vl) public genome (Schmutz, J. et al. et al. 2010, Nature, 463(7278): 178-183). Alternatively, in each of the foregoing instances, the plant material is preferably soybean and the heterologous nucleic acid can be inserted into a genomic locus on soybean chromosome 10 at location other than between the positions corresponding to physical positions 1504127 and 1504874 on soybean chromosome 10 in soybean genome Williams 82 (w82.a2.vl) public genome.

[0135] In certain embodiments of the methods of modifying plant material, the plant material is preferably soybean and the heterologous nucleic acid can be operably linked to its nativeDocket # 211912-WO-SEC-l promoter. Alternatively, the heterologous nucleic acid can be operably linked to a heterologous promoter.

[0136] In certain embodiments of the methods of modifying plant material, the plant material is preferably soybean and the method comprises introducing the heterologous nucleic acid by creating a double-stranded break in the plant’s genome. Thus, the heterologous nucleic acid can be introduced using TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas technology. In some examples, the method comprises introducing the heterologous nucleic acid using a Cas endonuclease.

[0137] In another aspect, provided herein is a method of introducing a gene associated with increased resistance to Meloidogyne into a plant by crossing two plants. The crossing method can comprise (a) crossing a plant having Meloidogyne resistance trait with a plant from a second plant line (“second plant”) to obtain progeny plants; (b) obtaining a sample containing nucleic acid from each of one or more of the progeny plants; (c) screening the sample for a nucleic acid that (i) encodes a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 1, or (iii) comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 2; and selecting one or more progeny plants that have the screened-for nucleic acid. Typically, the plant having the Meloidogyne resistance trait comprises the screened-for nucleic acid.

[0138] For example, the crossing method can comprise screening the sample for and selecting one or more progeny plants that comprise a nucleic acid that (i) encodes a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 1, or (iii) comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 2.

[0139] In certain embodiments, the screened-for nucleic acid is a heterologous nucleic on soybean chromosome (Chr) Chr 1, Chr 2, Chr 3, Chr 4, Chr 5, Chr 6, Chr 7, Chr 8, Chr 9, ChrDocket # 211912-WO-SEC-l11 , Chr 12, Chr 13, Chr 14, Chr 15, Chr 16, Chr 17, Chr 18, Chr 19, or Chr 20. In other examples of each instance of the foregoing crossing method, the screened-for nucleic acid is a heterologous nucleic acid located on soybean chromosome 10 at location other than its native locus, which corresponds to physical positions 1419209 to 1652513 on soybean chromosome 10 in soybean Williams 82 (w82.a2.vl) public genome (Schmutz, J. et al. et al. 2010, Nature, 463(7278): 178-183). Alternatively, the screened-for nucleic acid is a heterologous nucleic acid located on soybean chromosome 10 at location other than between the positions corresponding to physical positions 1504127 and 1504874 on soybean chromosome 10 in soybean genome Williams 82 (w82.a2.vl) public genome.

[0140] In certain embodiments, the screened for nucleic acid can be a heterologous nucleic acid that was introduced to the plant having Meloidogyne resistance or was introduced to a progenitor of the plant having Meloidogyne resistance by genome editing or by transgenic modification.

[0141] Each of the foregoing crossing methods disclosed herein can further comprise crossing the selected one or more progeny plants with the second plant to produce backcross progeny plants. Samples containing nucleic acid from one or more backcross progeny plant can each be screened for the presence of the disclosed screened-for nucleic acid, and one or more backcross progeny plants having the screened-for nucleic acid can be selected.

[0142] This further process can be repeated such that the crossing method includes crossing the selected one or more backcross progeny plants with the second plant to produce additional backcross progeny plants; obtaining a sample containing nucleic acid from one or more of the additional backcross progeny plants; screening each sample for the screened-for nucleic acid; and selecting one or more additional backcross progeny plants that have the screened for nucleic acid. This method can include additional rounds of backcrossing such that the method further includes (a) crossing the selected one or more additional backcross progeny plants with the second plant to produce further additional backcross progeny plants, (b) obtaining samples containing nucleic acid from one or more further additional backcross progeny plants; (c) screening each sample for the screened-for nucleic acid; and (d) selecting one or more further additional backcross progeny plants that have the screened-for nucleic acid. Optionally, steps (a) (d) can be repeated one or more times using the selected one or more further additional backcross progeny plants as the selected additional backcross progeny plants.Docket # 211912-WO-SEC-l

[0143] Further provided is method of introgressing a Meloidogyne resistance gene into a soybean plant, the Meloidogyne resistance gene comprising a polynucleotide encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 comprising crossing an Meloidogyne resistant soybean plant comprising a polynucleotide encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3 with a second soybean plant to produce progeny, screening the progeny with a nucleic acid marker to detect progeny comprising a polymorphism genetically linked to the Meloidogyne resistance gene, and selecting progeny that comprise the polymorphism to obtain soybean plants that contain the Meloidogyne resistance gene. In certain embodiments, the screened for nucleic acid (e.g., the Meloidogyne resistance gene) can be a heterologous nucleic acid that was introduced to the plant having Meloidogyne resistance or was introduced to a progenitor of the plant having Meloidogyne resistance by genome editing or by transgenic modification.

[0144] The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, a transgene, or the like. Offspring comprising the desired allele may be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.

[0145] The process of “introgressing” is often referred to as “backcrossing” when the process is repeated two or more times. Backcrossing methods may be used to introduce a nucleic acid sequence into plants. The backcrossing technique has been widely used for decades to introduce new traits into plants. N. Jensen, Ed., Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (non-recurrent parent) that carries a gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent, and the processDocket # 211912-WO-SEC-l is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent plant are recovered in the converted plant, in addition to the transferred gene from the non-recurrent parent.

[0146] In certain embodiments of the method of introgressing a Meloidogyne resistance gene, the polymorphism is within 20 centimorgan (cM), 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, or 1 cM of the nucleic acid encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. In certain embodiments of the method of introgressing a Meloidogyne resistance gene, the polymorphism is within about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85 kb, 90 kb, 95 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, or about 200 kb of the nucleic acid encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. In certain embodiments, the polymorphism is a single nucleotide polymorphism (SNP). In certain embodiments, the polymorphism comprises the resistance allele P at marker RKN-ChrlO-RES. In certain embodiments, the polymorphism comprises the presence of a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4.

[0147] Also provided are methods for producing a soybean plant having increased resistance to Meloidogyne, comprising genotyping a soybean population comprising a plurality of soybean plants or soybean germplasm for the presence of at least one maker genetically linked to a locus comprising or corresponding to an Meloidogyne resistance gene encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3, selecting from the soybean population one or more soybean plants or soybean germplasm comprising the at least one marker, and crossing the selected soybean plant or soybean germplasm with a second soybean plant or soybean germplasm to produce a progeny population, wherein at least one soybean plant or soybean germplasm of the progeny population comprises the at least one marker and has increased resistance to Meloidogyne as compared to a control plant. In certain embodiments, the screened for nucleic acid (e g., the Meloidogyne resistance gene) can be a heterologous nucleic acid that wasDocket # 211912-WO-SEC-l introduced to the plant having Meloidogyne resistance or was introduced to a progenitor of the plant having Meloidogyne resistance by genome editing or by transgenic modification.

[0148] As used herein, the term "linkage" or "linked" is used to describe the degree with which one marker locus is associated with another marker locus or some other locus (for example, a Meloidogyne locus). The linkage relationship between a molecular marker and a phenotype is given as a "probability" or "adjusted probability". Linkage can be expressed as a desired limit or range. For example, in some embodiments, any marker is linked (genetically and physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units for cM). In certain embodiments, it is advantageous to define a bracketed range of linkage, for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes. Thus, "closely linked loci" such as a marker locus and a second locus display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10 (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be "proximal to" each other. Since one cM is the distance between two markers that show a 1% recombination frequency, any marker is closely linked (genetically and physically) to any other marker that is in close proximity, e.g., at or less than 10 cM distant. Two closely linked markers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each other.

[0149] A "marker" is a nucleotide sequence or encoded product thereof (e.g., a protein) used as a point of reference. For markers to be useful at detecting recombinations, they need to detect differences, or polymorphisms, within the population being monitored. For molecular markers, this means differences at the DNA level due to polynucleotide sequence differences (e.g. SSRs, RFLPs, FLPs, SNPs). The genomic variability can be of any origin, for example, insertions,Docket # 211912-WO-SEC-l deletions, duplications, repetitive elements, point mutations, recombination events, or the presence and sequence of transposable elements. Molecular markers can be derived from genomic or expressed nucleic acids (e.g., ESTs) and can also refer to nucleic acids used as probes or primer pairs capable of amplifying sequence fragments via the use of PCR-based methods. A large number of soybean molecular markers are known in the art, and are published or available from various sources, such as the SoyBase internet resource.

[0150] Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well-established in the art. These include, e.g., DNA sequencing, PCR- based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Well established methods are also known for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).

[0151] A "marker allele", alternatively an "allele of a marker locus", can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population that is polymorphic for the marker locus.

[0152] A "marker locus" is a specific chromosome location in the genome of a species when a specific marker can be found. A marker locus can be used to track the presence of a second linked locus, e.g., a linked locus that encodes or contributes to expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a locus, such as a QTL or single gene, that are genetically or physically linked to the marker locus.

[0153] The term "molecular marker" may be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus. A marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A "molecular marker probe" is a nucleic acid sequence or molecule that can be used toDocket # 211912-WO-SEC-l identify the presence of a marker locus, e g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in certain embodiments, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Nucleic acids are "complementary" when they specifically hybridize in solution, e.g., according to Watson-Crick base pairing rules. Some of the markers described herein are also referred to as hybridization markers when located on an indel region, such as the noncollinear region described herein. This is because the insertion region is, by definition, a polymorphism vis a via a plant without the insertion. Thus, the marker need only indicate whether the indel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g., SNP technology is used in the examples provided herein.

[0154] In certain embodiments of the method for producing a soybean plant having increased resistance oMeloidogyne, the at least one marker genetically linked to the locus is within 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, or 1 cM of the locus comprising or corresponding to an Meloidogyne resistance gene encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. In certain embodiments of the for producing a soybean plant having increased resistance o Meloidogyne the at least one marker genetically linked to the locus is within about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85 kb, 90 kb, 95 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, or about 200 kb of the nucleic acid encoding a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. In certain embodiments, the marker is resistance allele P at RKN-ChrlO-RES. In certain embodiments, the marker comprises detecting the presence of a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4. In certain embodiments, the genotyping comprises amplifying a nucleic acid sequence comprising the at least one marker and detecting the resulting amplified nucleic acid comprising the marker. In certain embodiments, the amplification comprising amplification of at least a portion of one or more genomic regions of the soybeanDocket # 211912-WO-SEC-l genome comprising SEQ ID NO: 1 or 4. Tn certain embodiments, the amplification comprises providing one or more nucleic acid primers, wherein the nucleic acid primers comprise the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8. In certain embodiments of the detection methods described herein, the amplicon can be amplified using a (1) a first set of two primers: (i) a first primer comprising SEQ ID NO: 5 and a second primer comprising SEQ ID NO: 6; or (ii) a first primer comprising SEQ ID NO: 7 and a second primer comprising SEQ ID NO: 8. In certain embodiments, the detecting comprises hybridization with one or more nucleic acid probes, the one or more nucleic acid probes comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9 and 10.

[0155] Any suitable detection method known in the art can be used to detect the polynucleotides, markers, and / or polymorphisms described herein. In certain embodiments, the presence of the polynucleotide, marker, and / or polymorphism is directly detected in unamplified genomic DNA by performing a Southern blot on a sample of genomic DNA using probes to the marker loci. In other examples, amplification-based techniques are employed. PCR, RT-PCR, and LCR are in particularly broad use as amplification and amplification-detection methods for amplifying nucleic acids of interest, thus facilitating detection of the polynucleotide. Procedures for performing Southern blotting, amplification (PCR, LCR, or the like), and many other nucleic acid detection methods are well established and are taught, e.g., in Sambrook et al., Molecular Cloning — A Laboratory Manual (3d ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2000 (“Sambrook”); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2002) (“Ausubel”)) and PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990) (Innis). Additional details regarding detection of nucleic acids in plants can also be found, e.g., in Plant Molecular Biology (1993) Croy (ed.) BIOS Scientific Publishers, Inc.

[0156] “Primer” refers to an oligonucleotide (synthetic or occurring naturally), which is capable of acting as a point of initiation of nucleic acid synthesis or replication along a complementary strand when placed under conditions in which synthesis of a complementary strand is catalyzed by a polymerase. Typically, primers are oligonucleotides from 10 to 30 nucleic acids in length, but longer or shorter sequences can be employed. Primers may be provided in double-strandedDocket # 211912-WO-SEC-l form, though the single-stranded form is preferred. A primer can further contain a detectable label, for example a 5' end label.

[0157] “Probe” refers to an oligonucleotide (synthetic or occurring naturally) that is complementary (though not necessarily fully complementary) to a polynucleotide of interest and forms a duplexed structure by hybridization with at least one strand of the polynucleotide of interest. Typically, probes are oligonucleotides from 10 to 50 nucleic acids in length, but longer or shorter sequences can be employed. A probe can further contain a detectable label. The terms “label” and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, semiconductor nanocrystals, ligands (e.g., biotin, avidin, streptavidin, or haptens), and the like. A detectable label can also include a combination of a reporter and a quencher, such as are employed in FRET probes or TaqMan™ probes. The term “reporter” refers to a substance or a portion thereof which is capable of exhibiting a detectable signal, which signal can be suppressed by a quencher. The detectable signal of the reporter is, e.g., fluorescence in the detectable range. The term “quencher” refers to a substance or portion thereof which is capable of suppressing, reducing, inhibiting, etc., the detectable signal produced by the reporter. As used herein, the terms “quenching” and “fluorescence energy transfer” refer to the process whereby, when a reporter and a quencher are in close proximity, and the reporter is excited by an energy source, a substantial portion of the energy of the excited state nonradiatively transfers to the quencher where it either dissipates nonradiatively or is emitted at a different emission wavelength than that of the reporter.

[0158] Further provided herein are methods of detecting the presence or absence of a Meloidogyne resistance allele comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 in a plant genome. The method can comprise amplifying genomic DNA containing the resistance allele to thereby generate an amplicon. The amplicon is then contacted with one or more probes comprising one or more of SEQ ID NOs: 9 or 10. The method includes detecting whether the one or more probes bind to the amplicon to thereby determine the presence or absence of the Meloidogyne resistance allele.Docket # 211912-WO-SEC-l

[0159] In certain embodiments, the detection method comprises detecting the zygosity of the Meloidogyne resistance allele in a plant genome. Zygosity can be detected by (a) amplifying genomic DNA containing the resistance allele to thereby generate an amplicon; (b) contacting the amplicon with one or both of a first probe comprising SEQ ID NO: 9 and a second probe comprising SEQ ID NO: 10; and (c) detecting whether one or both probes from each set of the contacted probes bind to the amplicon to thereby determine the zygosity of the Meloidogyne resistance allele.

[0160] The following are examples of specific embodiments of some aspects of the invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way.EXAMPLE 1

[0161] This example demonstrates the identification of a candidate gene on Chromosome 10 that provides soybean resistance to the root-knot nematode Meloidogyne incognita (RKI).

[0162] An RKI resistance QTL named Rmil from the donor cultivars Palmetto and PI 96354 has been previously reported within a 235-kb region flanked by BARCSOYSSR 10-0090 and BARCSOYSSR 10-0105 (Pham, A. T., (2013) Theoretical and applied genetics, 126, 1825- 1838).

[0163] To identify the gene or genes responsible for the RKI resistance from the Rmil QTL, a candidate gene discovery based on the genomic sequence of an RKI resistant line was utilized. Briefly, a whole genome reference was created for the elite breeding line 5055AJ05-01, which is resistant to RKI and contains the Rmil QTL. Predicted gene models within 5055AJ05-01 reference were then compared to analogous gene models from the susceptible line, Williams82 (w82.a2.vl). To generate the reference sequence for 5O55AJO5-O1, genomic DNA was sequenced on the PacBio Sequel2e platform (Pacific Biosciences of California, Inc, Menlo Park, CA USA) to generate HiFi long reads with greater than 99.9% predicted accuracy. HiFi reads were assembled with the de novo assembler hifiasm (github.com / chhylpl23 / hifiasm) with default parameters. The resulting contigs were filtered for a minimum length of 70kb and minimum coverage of lOx. Contigs were placed into hybrid scaffolds using BioNano optical maps (San Diego, CA USA). Hybrid scaffolds were ordered and orientated relative to the W82.a5 assembly available from Soybase. Brown et al. 2021, Nucl. Acids Res. 49(D1): D1496-Docket # 211912-WO-SEC-lD15012. doi: 10.1093 / nar / gkaal 107. To capture expression of genes and to support gene models within 5O55AJO5-O1, RNAseq and Isoseq experiments were performed on root and shoot tissue. Plants were removed from soil 7 days after planting and were briefly rinsed with tepid water; they were then severed at the stem / root junction. Roots and shoots from three plants were pooled separately into single 50 mL tubes, frozen with liquid nitrogen, and stored at -80°C. Three replicates of pooled root and shoot tissue were collected. Total RNA was collected from ground frozen tissue using a Qiagen RNeasy plant kit (Qiagen, Germantown, MD, USA) and then spiked with the Ambion ERCC Mix 1 (ThermoFisher Scientific, Inc.) as an internal control; mRNA libraries were prepared from the isolated RNA using the Illumina® Stranded mRNA Prep, Ligation kit (Illumina.com) and sequencing was performed on an Illumina Novaseq 6000 instrument. Additionally, the isolated RNA was used to generate long-read sequences using the PacBio Sequel2e platform (Pacific Biosciences of California, Inc, Menlo Park, CA USA) and the IsoSeq v3 chemistry. Paired-end RNA-seq and PacBio Iso-seq reads were aligned to the 5O55AJO5-O1 and Williams82 genomes using HISAT2 and MiniMap2, respectively.

[0164] Following development of the genomic resources for 5055AI05-01, re-analysis of gene content within the Rmil QTL interval was completed. Surprisingly, the QTL interval, delimited by markers BARCSOYSSR 10-0090 and BARCSOYSSR 10-0105, is ~56 Kbp longer in the resistance line when compared to the susceptible line Williams82. Additional genomic studies uncovered an approximately 50 Kbp insertion in 5O55AJO5-O1 relative to W82.a2.1 (Table 2).

[0165] The insertion contains a single ~40 Kbp gene, referred to herein as RKN STRG.19035 (SEQ ID NOs: 1 (genomic sequence), 2 (coding sequence), and 3 (amino acid sequence)), encoding an ankyrin repeat-containing membrane-anchored protein. This gene is unique to the resistant line and is absent from all susceptible genomes analyzed. The exact function of this gene is unknown but ankyrin repeats are thought to play a role in protein-protein interactions, and proteins containing them have been associated with resistance to plant pathogens.

[0166] The combination of genetic and genomic data described herein indicates that RKN STRG.19035 could confer enhanced RKI resistance in soybean.Table 2: Comparison Rmil QTL interval between a resistant (R) and susceptible (S) lineDocket # 211912-WO-SEC-lEXAMPLE 2

[0167] This example demonstrates the Rmil candidate RKN STRG.19035 conditions resistance to RKI.

[0168] To demonstrate that the Rmil candidate gene of Example 1 conditions resistance to RKI, RKN STRG.19035 (SEQ ID NO: 1) from 5055AJ05-01 is expressed in a variety that is susceptible to RKI, such as Williams82 or 93Y21.

[0169] In one example, the coding sequences of RKN STRG.19035 (SEQ ID NO: 2) was expressed under a constitutive promoter in 93Y21 using a hairy-root transformation system mediated by Agrobacterium-rhizogenes. Following transformation, transgenic hairy -roots were treated with root-knot nematodes in an assay adapted from Song, Jaehyo, et al. (Current protocols 1.7 (2021): e I95) and Coyne, D.L. et al. (Protocol for Nematode Resistance Screening: Root Knot Nematodes, Meloidogyne spp. International Institute of Tropical Agriculture (UTA), Ibadan, Nigeria. (2014)). A comparison was made between the final RKI gall numbers from the control plants and the transgenic plants expressing the Rmil candidate. As shown in Table 3, plants expressing RKN STRG.19035 had a decrease in the average root knot nematode gall score as compared to plants expressing an empty vector. Results demonstrate that the candidate gene RKN STRG.19035 coding sequence provided resistance to RKI in this assay.Table 3: Root knot nematode gall scoresEXAMPLE 3

[0170] This example demonstrates the Rmil candidate RKN STRG.19035 conditions resistance to RKI in soybean plants.Docket # 211912-WO-SEC-l

[0171] To demonstrate that the native genomic sequence comprising RKN STRG.19035 (SEQ ID NO: 1) confers RKI resistance, RKN STRG.19035 was expressed under its native promoter in 93Y21. Briefly, genomic sequence comprising RKN STRG.19035 and its native promoter was introduced into the RKI susceptible variety 93Y21 using Agrobact rium-meA aiQ<i transformation. Copy number of the inserted construct was determined using qPCR analysis, and single-copy TO plants were self-fertilized to produce segregating T1 seed. The T1 seed from two independent events were planted and seedlings were treated with root -knot nematodes in an assay adapted from Song, Jaehyo, et al. (Current protocols 1.7 (2021): el95) and Coyne, D.L. et al. (Protocol for Nematode Resistance Screening: Root Knot Nematodes, Meloidogyne spp. International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. (2014)). A comparison was made between the final RKI gall numbers from the control plants and the transgenic plants expressing the Rmil candidate. As shown in Table 3, the average root knot nematode gall score was lower in plants carrying one and two copies of RKN STRG.19035 as compared to plants carrying 0 copies and to the susceptible control. These results further demonstrate that RKI resistance is conditioned by the candidate gene.Table 3: Root knot nematode gall scoresEXAMPLE 4

[0172] This example demonstrates how molecular markers are used to track and select for the RKI resistance gene RKN STRG.19035 for developing soybean products with improved RKI resistance.

[0173] To enable efficient development of new material, plant breeders use genotypic data from molecular assays to select plants with favorable genetic alleles, including those alleles that condition disease resistance, improve agronomic performance, and impart higher yield. MarkerDocket # 211912-WO-SEC-l assisted selection of plants obviates the need for phenotypic observation, which saves costs and time during the breeding process.

[0174] Table 4 provides a molecular assay that can be used to genotype plant material for the purpose of identifying plants with an RKI resistant QTL on ChrlO and selecting for SEQ ID NO: 1. The “P” indicates the presence of the resistant allele while the “X” is a control that indicates the absence the resistant allele. Tables 5 and 6 provide primer and probe sequences that can be used to detect and select for one or more of the resistance alleles disclosed in Table 4.Table 4: Molecular Marker for Selecting and Developing Soybean Products Having the ChrlO QTL Associated with Improved RKI ResistanceTable 5: Primer Sequences for Detecting the Marker on ChrlOTable 6: Probe Sequences for Detecting the Marker on ChrlO

[0175] A population or germplasm with improved resistance to RKI is generated by genotyping with marker RKN-ChrlO-RES and breeding with plants that have the “P” allele, indicating the presence of the resistant allele. Additional molecular markers genetically linked to SEQ ID NO: 1 can be used either independently from or in addition to marker RKN-ChrlO-RES to select for resistant plants.EXAMPLE 5Docket # 211912-WO-SEC-l

[0176] This example demonstrates how inserting native resistance gene sequences of Rmil using a gene-editing approach will improve RKI resistance.

[0177] Introgression of native alleles from exotic material or older varieties, like Palmetto and PI 96354, into current elite cultivars through breeding is complicated by the unintended transfer of unfavorable linked DNA that decreases yield (e g., linkage drag). To eliminate the negative effects of linked loci, an SDN3 CRISPR / Cas9 approach that introduces Rmil resistance gene sequences (e.g., SEQ ID NO: 1 or 2) into elite breeding material is performed. In one example, a guide RNA is used with Cas9 to integrate the Rmil candidate genes into favorable positions of the genome of a RKI susceptible variety. The Rmil candidate gene is introduced with 2 kb of the native protomer to drive transcript levels and patterns that mimic its expression in native positions. Plants expressing the Rmil candidate resistance gene are then used to create cultivars with improved RKI resistance. A marker assisted selection strategy as demonstrated in Example 4 can be used to select for plants harboring newly generated Rmil alleles. The new alleles can be further crossed into additional elite material through breeding.

[0178] All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention 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 by reference.

[0179] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

[0180] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.Docket # 211912-WO-SEC-l

[0181] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

Claims

Docket # 211912-WO-SEC-lWe claim:

1. A genome edited or transgenic plant comprising a heterologous polynucleotide encoding a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 3, the heterologous polynucleotide associated with increased resistance to Meloidogyne.

2. The genome edited or transgenic plant of claim 1, wherein the plant is a soybean plant, and the heterologous nucleic acid is inserted into a location on soybean chromosome 1-9 or 11- 20.

3. The genome edited or transgenic plant of claim 1 or 2, wherein the plant is a soybean plant and the heterologous nucleic acid is inserted into a location other than its native locus corresponding to physical positions 1419209 to 1652513 on soybean chromosome 10 in soybean genome w82.a2.vl .

4. The genome edited or transgenic plant of any one of claims 1-3, wherein the heterologous nucleic acid comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 or 2.

5. The genome edited or transgenic plant of claim 4, wherein the heterologous nucleic acid comprises a regulatory element operably linked to the nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 or 2.

6. The genome edited or transgenic plant of claim 5, wherein the regulatory element is a heterologous promoter.

7. The genome edited or transgenic plant of claim 5, wherein the regulatory element is the native promoter.

8. The genome edited or transgenic plant of any one of claims 1-7, wherein the genome edited or transgenic plant has increased resistance to at least one species of Meloidogyne as compared to a control plant that does not comprise the heterologous polynucleotide.

9. The genome edited or transgenic plant of any one of claims 1 and 4-8, wherein the plant is a legume crop plant.Docket # 211912-WO-SEC-l10. The genome edited or transgenic plant of claim 9, wherein the legume crop plant is selected from the group consisting of alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

11. The genome edited or transgenic plant of claim 10, wherein the legume crop plant is soybean.

12. A nucleic acid construct comprising a polynucleotide operably linked to a regulatory element, the polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3, wherein said polypeptide when expressed in the cells of a plant confers resistance to Meloidogyne .

13. The nucleic acid construct of claim 12, wherein the regulatory element is a heterologous regulatory element.

14. The nucleic acid construct of claim 13, wherein the heterologous regulatory element is a heterologous promoter.

15. The nucleic acid construct of claim 12, wherein the regulatory element is a native promoter.

16. A host cell comprising the nucleic acid construct of any one of claims 12-15.

17. A genome edited or transgenic plant cell comprising the nucleic acid construct of any one of claims 12-15.

18. A plant or plant part comprising the genome edited or transgenic plant cell of claim 17.

19. The plant or plant part of claim 18, wherein the plant part is a seed.

20. The plant or plant part of claim 18 or 19, wherein the plant is a legume crop plant.

21. The plant or plant part of claim 20, wherein the legume crop plant is selected from alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

22. The plant or plant part of claim 21, wherein the legume crop plant is soybean.

23. A method of modifying plant material, the method comprising introducing into the genome of the plant material a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid is associated with increased resistance to Meloidogyne and (i) encodes a polypeptide having atDocket # 211912-WO-SEC-l least 90% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 1, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 2.

24. The method of claim 23, wherein the plant material is soybean plant material, and the heterologous nucleic acid is inserted into a location on soybean chromosome 1-9 or 11-20.

25. The method of any one of claims 23 or 24, wherein the plant material is soybean plant material, and the heterologous nucleic acid is inserted into a location other than its native locus corresponding to physical positions 1419209 to 1652513 on soybean chromosome 10 in soybean genome w82.a2.vl.

26. The method of any one of claims 23-25, wherein the heterologous nucleic acid further comprises a heterologous promoter.

27. The method of any one of claims 23-25, wherein the heterologous nucleic acid further comprises its native promoter.

28. The method of any one of claims 23-27, wherein the method comprises introducing the heterologous nucleic acid using a double-stranded break.

29. The method of claim 28, wherein the method comprises introducing the heterologous nucleic acid using TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas technology.

30. The method of claim 28, wherein the method comprises introducing the heterologous nucleic acid using Cas endonuclease.

31. A method for conferring resistance to Meloidogyne in a legume crop species, the method comprising introducing into a regenerable plant cell of a legume crop species a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3; and generating a plant from the plant cell, wherein the plant comprises the polynucleotide and has increased resistance o Meloidogyne as compared to a control plant not comprising the polynucleotide.Docket # 211912-WO-SEC-l32. The method of claim 31, wherein the polynucleotide is introduced into the regenerable plant cell using a nucleic acid construct comprising the polynucleotide operably linked to a regulatory element.

33. The method of claim 32, wherein the regulatory element is a heterologous regulatory element.

34. The method of claim 33, wherein the heterologous regulatory element is a promoter.

35. The method of claim 31, wherein the polynucleotide is introduced into the regenerable plant cell using genome editing technologies.

36. The method of claim 35, wherein the genome editing technology uses an enzyme selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, base editing deaminases, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and engineered site-specific endonucleases.

37. The method of claim 35 or 36, wherein the legume crop species is soybean and the polynucleotide is inserted into a location on soybean chromosome 1-9 or 11-20.

38. The method of claim 35 or 36, wherein the legume crop species is soybean and the polynucleotide is inserted into a location other than its native locus corresponding to physical positions 1419209 to 1652513 on soybean chromosome 10 in soybean genome w82.a2.vl.

39. The method of any one of claims 31-36, wherein the legume crop species is selected from the group consisting of alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, pigeon pea, peanut and tamarind.

40. The method of claim 39, wherein the legume crop species is soybean.

41. A method of introducing a gene associated with increased resistance to Meloidogyne into a plant, the method comprising: a. crossing a first plant having Meloidogyne resistance with a second plant to obtain progeny plants; b. obtaining a sample containing nucleic acid from each of one or more of the progeny plants;Docket # 211912-WO-SEC-l c. screening the sample for a nucleic acid that (i) encodes a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 1, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 2; and d. selecting one or more progeny plants that have the screened-for nucleic acid.

42. The method of claim 41, wherein the first plant having Meloidogyne resistance comprises the screened-for nucleic acid.

43. The method of claim 41 or 42, wherein the screened-for nucleic acid comprises a nucleic acid that (i) encodes a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 3, (ii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 1, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 2.

44. The method of any one of claims 41-43, wherein the screened-for nucleic acid is a heterologous nucleic acid on soybean chromosome 1-9 or 11-20.

45. The method of any one of claims 41-43, wherein the screened-for nucleic acid is a heterologous nucleic on soybean chromosome 10 at a locus other than its native locus corresponding to physical positions 1419209 to 1652513 on soybean chromosome 10 in soybean genome w82.a2.vl.

46. The method of any one of claims 41-43, wherein the screened-for nucleic acid is operably linked to a heterologous regulatory element.

47. The method of any one of claims 44-46, wherein the heterologous nucleic acid has been introduced to the first plant having Meloidogyne resistance or was introduced to a progenitor of the plant having Meloidogyne resistance by genome editing or by transgenic modification.

48. The method of any one of claims 41-47, further comprising crossing the selected one or more progeny plants with the second plant to produce backcross progeny plants.

49. The method of claim 48, further comprising:Docket # 211912-WO-SEC-l a. obtaining a sample containing nucleic acid from one or more of the backcross progeny plants; b. screening each sample for the screened for screened-for nucleic acid; and c. selecting one or more backcross progeny plants that have the screened for nucleic acid.

50. The method of claim 49, further comprising d. crossing the selected one or more backcross progeny plants with the second plant to produce additional backcross progeny plants; e. obtaining a sample containing nucleic acid from one or more of the additional backcross progeny plants; f. screening each sample for the screened-for nucleic acid; and g. selecting one or more additional backcross progeny plants that have the screened for nucleic acid.

51. The method of claim 50, further comprising one or more repeated steps of h. crossing the selected one or more additional backcross progeny plants with the second plant to produce further additional backcross progeny plants, i. obtaining samples containing nucleic acid from one or more further additional backcross progeny plants; j . screening each sample for the screened-for nucleic acid; k. selecting one or more further additional backcross progeny plants that have the screened for nucleic acid; and l. optionally repeating steps h.-k. one or more times using the selected one or more further additional backcross progeny plants as the selected additional backcross progeny plants.Docket # 211912-WO-SEC-l52. A method of preventing Meloidogyne associated damage to a legume crop species, the method comprising growing the genome edited or transgenic plants of any one of claims 1-12 in a field.

53. The method of claim 52, wherein the field is treated with a nematicide.

54. A method of introgressing a Meloidogyne resistance gene into a soybean plant, the Meloidogyne resistance gene comprising a polynucleotide encoding a polypeptide having at least 90% sequence identity to SEQ ID NO: 3, the method comprising: i) crossing an Meloidogyne resistant soybean plant comprising a polynucleotide encoding a polypeptide having at least 90% sequence identity to SEQ ID NO: 3 with a second soybean plant to produce progeny; ii) screening the progeny with a nucleic acid marker to detect progeny comprising a polymorphism genetically linked to the Meloidogyne resistance gene; and iii) selecting progeny that comprise the polymorphism to obtain soybean plants that contain the Meloidogyne resistance gene.

55. The method of claim 54, wherein the polymorphism is within 10 centimorgans of the nucleic acid encoding a polypeptide having at least 90% sequence identity to SEQ ID NO: 3.

56. The method of claim 54 or 55, wherein the polymorphism comprises a single nucleotide polymorphism (SNP).

57. The method of claim 54 or 55 wherein the polymorphism comprises an insertion.

58. The method of claim 54 or 55, wherein the polymorphism comprises the presence of a polynucleotide having at least 95% sequence identity to SEQ ID NO: 4.

59. A method for producing a soybean plant having increased resistance io Meloidogyne, the method comprising: i) genotyping a soybean population comprising a plurality of soybean plants or soybean germplasm for the presence of at least one maker genetically linked to a locus comprisingDocket # 211912-WO-SEC-l or corresponding to a Meloidogyne resistance gene encoding a polypeptide having at least 90% sequence identity to SEQ ID NO: 3; ii) selecting from the soybean population one or more soybean plants or soybean germplasm comprising the at least one marker; and iii) crossing the selected soybean plant or soybean germplasm with a second soybean plant or soybean germplasm to produce a progeny population, wherein at least one soybean plant or soybean germplasm of the progeny population comprises the at least one marker and has increased resistance to Meloidogyne as compared to a control plant.

60. The method of claim 59, wherein the at least one marker genetically linked to the locus is within 20 centimorgans of the locus comprising or corresponding to an Meloidogyne resistance gene encoding a polypeptide having at least 90% sequence identity to SEQ ID NO:3.

61. The method of claim 59 or 60, wherein the marker is the presence of a polynucleotide having at least 95% sequence identity to SEQ ID NO: 4.

62. The method of any one of claims 59-61, wherein genotyping comprises amplifying a nucleic acid sequence comprising the at least one marker and detecting the resulting amplified nucleic acid comprising the marker.

63. The method of claim 62, wherein the amplification comprises amplification of at least a portion of one or more genomic regions of the soybean genome comprising SEQ ID NO: 1 or4.

64. The method of claim 62 or 63, wherein the amplification comprises providing one or more nucleic acid primers, wherein the nucleic acid primers comprise the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8.

65. The method of any one of claims 59-64, wherein detecting the presence of the at least one maker comprises hybridization with one or more nucleic acid probes, the one or more nucleic acid probes comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9 and 10.

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