Novel resistance genes associated with disease resistance in soybean

Abstract

Methods and compositions for identifying, selecting and / or producing pathogen resistant plants, plant cells or seeds (e.g., legume or soybean plants, plant cells or seeds) are provided. Polynucleotides and polypeptides are provided that when expressed in a plant increase disease and / or pathogen resistance (e.g., resistance to Asian soybean rust) of the plant. Plants, plant cells, and seeds comprising such polypeptides and polynucleotides are provided, as well as methods of their use.

Description

Technical Field

[0001] The present invention relates to compositions and methods for identifying, selecting and producing plants with enhanced disease and / or pathogen resistance using novel resistance genes.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 481,627, filed on January 26, 2023, the entire contents of which are incorporated herein by reference.

[0004] Statement Regarding Electronic Submission of Sequence Listings

[0005] This application is accompanied by a sequence listing named 82653_PCT.xml, which was created on January 18, 2024 and is approximately 100 kb in size. This sequence listing is submitted with this application via EFS-Web and is incorporated herein by reference in its entirety in accordance with 37 CFR §1.831-1.835. Background Art

[0006] It is well known that plant pathogens can cause significant damage to important crops, leading to significant agricultural losses and widespread impacts on the food supply and other industries that rely on plant raw materials. Similarly, applicants desire to reduce the incidence and / or impact of agricultural pathogens on crops.

[0007] Several pathogens are associated with the damage suffered by soybean, and these pathogens are likely to cause significant yield losses to the U.S. and the world individually and collectively. Exemplary pathogens include, but are not limited to, fungi (e.g., Phytophthora and Asian soybean rust Phakopsora pahyrhizi), nematodes (e.g., Meloidogyne, particularly Meloidogyne javanica), soybean stem canker pathogens, and bacteria (e.g., Pseudomonas syringae). In view of the significant threat these pathogens pose to global food supply and the time and expense of processing soybean crops to prevent yield losses, a novel method for producing pathogen-resistant soybean cultivars is needed. What is needed is a novel resistance gene (herein, "R gene") that can be introduced into plants to control pathogens. Summary of the Invention

[0008] Compositions and methods are provided for increasing disease resistance and / or pathogen resistance in plants, particularly leguminous plants, and more particularly soybean plants. The disclosure provides RG31 and RG35 polypeptides, variants and active fragments of RG31 and RG35 polypeptides, and fusion proteins of RG31 and RG35 polypeptides that can enhance disease resistance. The disclosure further provides nucleic acids encoding the disease resistance-enhancing polypeptides and fusion proteins, as well as plants expressing the polypeptides and fusion proteins. Also provided are methods for enhancing disease resistance by providing a plant with a nucleic acid encoding the disclosed polypeptide.

[0009] The foregoing and other objects and aspects of the present invention will be explained in detail in the drawings and description set forth below. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A depicts the results of a rust bioassay experiment conducted on leaves collected from primary soybean events containing the novel R gene RG35 (constructs 25227, 26228, and 26229) or RG31 (constructs 25905, 25951, and 25900). Events containing the novel R gene showed increased ASR resistance to rust population 21BR08 compared to the control.

[0011] Figure 1 B shows a comparison of fungal biomass accumulation measured by β-tubulin mRNA in events containing either the R gene RG35 (constructs 25227, 26228, 26229) or the R gene RG31 (constructs 25900, 25905, 25951) when infected with rust colony 21BR08. Figure 1 The phenotypic results of A.

[0012] Figure 2 A depicts the results of a rust bioassay experiment conducted on leaves collected from primary soybean events generated from binary vectors containing either the R gene RG35 (constructs 25227, 26228, and 26229) or the R gene RG31 (constructs 25905, 25951, and 25900). Events containing the R gene showed increased ASR resistance to rust population 21 BRM compared to the control.

[0013] Figure 2 B shows a comparison of fungal biomass accumulation measured by β-tubulin mRNA in events containing either the R gene RG35 (constructs 25227, 26228, 26229) or the R gene RG31 (constructs 25900, 25905, 25951) when infected with rust colony 21BRM. Figure 2 The phenotypic results of A.

[0014] Figure 3 Depicted are the results of rust bioassay experiments conducted on single-copy TO and T1 events containing either the RG35 gene (construct 26228) or the RG31 gene (construct 25905), wherein expression of the R gene in the constructs was driven by an alfalfa promoter (prMt51186, SEQ ID NO: 12). Means and standard deviations of disease rates were calculated for each construct. T-test analysis of the data indicated that events containing the RG35 gene exhibited significantly higher resistance to soybean rust than events containing the RG31 gene. The same trend was observed for the TO and T1 events. The results indicate that RG35 driven by the alfalfa promoter exhibited higher disease resistance than RG31 driven by the alfalfa promoter.

[0015] Figure 4 Depicted are the results of rust bioassay experiments conducted on single-copy TO events and single-copy T1 events containing the RG35 gene (construct 25227) or the RG31 gene (construct 25951), wherein expression of the R gene in the constructs was driven by the corresponding native promoter (i.e., RG31 expression was driven by the native RG31 promoter of SEQ ID NO: 7, while RG35 expression was driven by the native RG35 promoter of SEQ ID NO: 10). The mean and standard deviation of disease rates were calculated for each construct. T-test analysis of the data indicated that events containing the RG35 gene exhibited significantly higher resistance to soybean rust than events containing the RG31 gene. The same trend was observed for the TO and T1 events. The results indicate that RG35 driven by the native promoter had higher disease resistance than RG31 driven by the native promoter.

[0016] Figure 5Results of an effector protein and R protein interaction assay in a heterologous assay system are shown. The presence of an interaction between the effector protein and the R protein is indicated by the presence of infiltration zones in the plant cells that display a hypersensitive response (HR). Soybean rust effectors (SPE-35 and SPE-01 in this example) were expressed in different halves of tobacco leaves using Agrobacterium-mediated transient assays of RG31, RG35, or RG1. SPE-35 was found to specifically elicit a hypersensitive localized cell death response at the inoculation site when co-infiltrated with RG31 (construct 25905; infiltration position 3) or RG35 (construct 26228; infiltration position 2). Co-infiltration of the effector with the different R proteins RG01 (construct 23968, infiltration position 1; or construct 24732, infiltration position 4) did not elicit a similar response, indicating that SPE-35 is specific for RG31 and RG35. The results also indicate that, despite their sequence similarity, the mode of action of the resistance proteins RG31 and RG35 differs from that of the resistance protein RG1. As expected, co-infiltration of the effectors with a control construct expressing GUS (R gene control, infiltration position 5) also did not induce a hypersensitive local cell death response at the inoculation site.

[0017] Figure 6 Results from an effector protein and R protein interaction assay in a heterologous assay system are shown. The presence of an interaction between the effector protein and the R protein is indicated by the presence of infiltration zones in plant cells that display a hypersensitive response (HR). Soybean rust effectors (SPE-35 and SPE-01 in this example) were expressed in different halves of tobacco leaves using Agrobacterium-mediated transient assays with RG31, RG35, or RG1. SPE-35, when co-infiltrated with RG31 (construct 25905; infiltration position 4) or RG35 (construct 26228; infiltration position 5), specifically triggered a hypersensitive localized cell death response at the inoculation site. Co-infiltration of the effector with the different R proteins RG01 (construct 23968, infiltration position 3; or construct 24732, infiltration position 2) did not trigger a similar response, demonstrating that SPE-35 is specific for RG31 and RG35. These results also indicate that, despite their sequence homology, the mode of action of the resistance proteins RG31 and RG35 differs from that of the resistance protein RG1. As expected, co-infiltration of the effectors with a control construct expressing GUS (R gene control, infiltration position 1) also did not induce a hypersensitive local cell death response at the inoculation site.

[0018] Brief Description of Sequence Listing

[0019] SEQ ID NO: 1 is the amino acid sequence of the RG31 resistance protein encoded by the RG31 resistance gene of Glycine tomentella.

[0020] SEQ ID NO: 2 is the amino acid sequence of the RG35 resistance protein encoded by the RG35 resistance gene of Glycine max.

[0021] SEQ ID NO: 3 is the genomic sequence of the RG31 gene. SEQ ID NO: 4 is the intronless cDNA sequence of the RG31 gene. Each of SEQ ID NOs: 3-4 encodes the protein of SEQ ID NO: 1.

[0022] SEQ ID NO: 5 is the genomic sequence of the RG35 gene. SEQ ID NO: 6 is the intronless cDNA sequence of the RG35 gene. Each of SEQ ID NO: 5-6 encodes the protein of SEQ ID NO: 2.

[0023] SEQ ID NO: 7 is the native promoter prGtoRG31-01 of the RG31 gene from Glycine max. SEQ ID NO: 8 is the modified native promoter prGtoRG31-02 of the RG31 gene from Glycine max. SEQ ID NO: 9 is the native terminator tGtoRG31 of the RG31 gene from Glycine max.

[0024] SEQ ID NO: 10 is the native promoter prGtoRG35 of the RG35 gene from Glycine max. SEQ ID NO: 11 is the native terminator tGtoRG35 of the RG35 gene from Glycine max.

[0025] SEQ ID NO: 12 is the plant-active constitutive promoter prMt51186 from Medicago truncatula SEQ ID NO: 13 is the plant-active terminator tMt51186 from Medicago truncatula.

[0026] SEQ ID NO: 14 is the rust-responsive / inducible promoter prGmACO3, which is derived from the first intron of the rust-responsive promoter of the ACO3 gene encoding an oxidoreductase (1-aminocyclopropane-1-carboxylic acid oxidase 3-related) protein on chromosome 2 of soybean (Glyma.02G268200).

[0027] SEQ ID NO: 15 is the rust-responsive / inducible promoter prGmMYB of the gene encoding a Myb-like DNA binding protein on chromosome 2 of soybean (Glyma.19G164600).

[0028] SEQ ID NO: 16 is the amino acid sequence of the effector protein SPE-35 from Phakopsora pachyrhizi. SEQ ID NO: 17 is the polynucleotide sequence encoding the protein of SEQ ID NO: 16.

[0029] SEQ ID NO: 18 is the amino acid sequence of the effector protein SPE-01 from Phakopsora pachyrhizi. SEQ ID NO: 19 is the polynucleotide sequence encoding the protein of SEQ ID NO: 18.

[0030] SEQ ID NO: 20 is the amino acid sequence of a longer splice variant of the RG35 polypeptide from the Rg35 gene of Glycine canescens. SEQ ID NOs: 21 and 22 are nucleotide sequences encoding the splice variant proteins of SEQ ID NO: 20.

[0031] SEQ ID NO: 23 is the amino acid sequence of the effector protein SPE-38 from Phakopsora pachyrhizi. SEQ ID NO: 24 is the polynucleotide sequence encoding the protein of SEQ ID NO: 22. DETAILED DESCRIPTION

[0032] Provided are compositions and methods for improving disease resistance and / or pathogen resistance in plants, particularly soybean plants, by expressing novel resistance proteins RG31 and / or RG35. RG31 and RG35 proteins are unexpectedly beneficial because, when expressed in plants, these proteins can enhance disease resistance through a novel mode of action. This novel mode of action distinguishes the protein from other R proteins with high sequence homology to RG31 and RG35, such as the RG1 resistance protein disclosed in WIPO Publication No. WO2019103918 A1 (as SEQ ID NO: 47).

[0033] 1. definition

[0034] 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 the presently disclosed subject matter belongs.

[0035] While it is believed that the following terms are well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate understanding of the subject matter of this disclosure.

[0036] All references listed below, as well as all references cited in this disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (e.g., database entries and all annotations available therein) are incorporated herein by reference in their entirety to the extent they supplement, explain, provide context for, or teach the methods, techniques, and / or compositions employed herein.

[0037] The nucleotide sequences provided herein are presented from left to right in a 5' to 3' orientation and are represented using the standard code representing nucleotide bases, as set forth in 37 CFR §§1.821-1.825 and §§1.831-1.835 and World Intellectual Property Organization (WIPO) standards ST.25 and ST.26, e.g., adenine (A), cytosine (C), thymine (T), and guanine (G).

[0038] Amino acids are also indicated using WIPO Standard ST.25, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0039] The singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0040] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0041] As used herein, the term "about" when referring to a measurable value such as a dose, administration rate, or time period, is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. As used herein, phrases such as "between about X and Y" mean "between about X and about Y," and phrases such as "from about X to Y" mean "from about X to about Y."

[0042] Unless the context indicates otherwise, as used herein, phrases such as “between about X and Y,” “between about X and about Y,” “from X to Y,” and “from about X to about Y” (and similar phrases) should be interpreted to include X and Y.

[0043] As used herein, a "coding sequence" or "CDS" is a nucleic acid sequence that is transcribed into RNA (e.g., mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA). In an embodiment, the RNA is subsequently translated to produce a protein. In example embodiments, the CDS is derived from a cDNA sequence and includes the spliced ​​exon sequence of the transcript in the DNA annotation and does not include any introns or 5' or 3'-untranslated regions (UTRs). In other example embodiments, the CDS is derived from a genomic DNA sequence and includes the spliced ​​exon sequence of the transcript in the DNA annotation and one or more introns, and 5' and / or 3'-untranslated regions (UTRs).

[0044] As used herein, " codon optimized " nucleotide sequence means the nucleotide sequence of recombinant, transgenic or synthetic polynucleotide, wherein these codons are selected to reflect the specific codon preference that host cell or organism may have. This is typically accomplished in the following manner, and the mode is to maintain the amino acid sequence of the polypeptide encoded by the codon optimized nucleotide sequence. In certain embodiments, the nucleotide sequence is codon optimized for the cell (for example, animal, plant, fungus or bacterial cell) in which the construct is to be expressed. For example, the construct to be expressed in plant cells can have its whole or part of its sequence codon optimized for expression in plants. See, for example, U.S. Patent number 6,121,014. In an embodiment, the polynucleotide provided herein is codon optimized for expression in plant cells (for example, dicotyledonous plant cells, monocotyledonous plant cells, soybean cells) or bacterial cells.

[0045] The terms “comprise”, “comprises”, and “comprising” when used in this specification indicate the presence of stated features, integers, steps, operations, elements, or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0046] As used herein, the transition phrase "consisting essentially of (and grammatical variations) means that the scope of the claim is to be construed to encompass the specified materials or steps recited in the claim and those that do not materially alter one or more of the basic and novel characteristics of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of is not intended to be interpreted as equivalent to "comprising."

[0047] As used herein, "expression cassette" means a nucleic acid molecule capable of directing the expression of at least one polynucleotide of interest (such as a polynucleotide encoding an RG31 polypeptide or an active variant or fragment thereof, or a polypeptide encoding an RG35 polypeptide or an active variant or fragment thereof) in an appropriate host cell, and comprises a promoter operably linked to the polynucleotide of interest (which is operably linked to a termination signal). An "expression cassette" may comprise additional polynucleotides to promote the correct translation of the polynucleotide of interest. The expression cassette may comprise other polynucleotides that are not related to the expression of the polynucleotide of interest but are present due to convenient restriction sites for removing the expression cassette from the expression vector. In an embodiment, at least one component in the expression cassette may be heterologous (i.e., foreign) relative to at least one other component (e.g., a heterologous promoter, terminator, intron, and / or any regulatory element operably associated with the polynucleotide of interest). In other embodiments, the expression cassette may be naturally occurring and comprise natural regulatory elements, one or more or all natural introns, and natural genomic DNA to allow expression of the RG31 or RG35 polypeptide, or its active variant or fragment or fusion. The expression cassette may be heterologous with respect to the host, ie the expression cassette (or even the polynucleotide of interest) is not naturally present in the host cell and has been introduced into the host cell by transformation methods or breeding methods.

[0048] In an embodiment of the present invention, a cell is provided that comprises an RG31 polypeptide (or an active variant or fragment thereof) or an RG35 polypeptide (or an active variant or fragment thereof). In an exemplary embodiment, a cell is provided that comprises a heterologous polynucleotide encoding an RG31 polypeptide, for example, a polynucleotide encoding a polypeptide having at least 90% identity or at least 95% identity to SEQ ID NO: 1 or 2, or a polypeptide comprising SEQ ID NO: 1 or 2, wherein increased expression of the polypeptide in a plant increases disease resistance in the plant. In other exemplary embodiments, the cell comprises a heterologous polynucleotide comprising a nucleotide sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 3, 4, 5, or 6; or a nucleotide sequence comprising SEQ ID NO: 3, 4, 5, or 6. In particular embodiments, the cell is a plant cell, and the polynucleotide is stably integrated into the genome of the cell. In specific embodiments, the plant cell has increased expression levels of the polypeptide compared to a control plant cell and the plant cell has increased disease resistance.

[0049] The term "introduced / introducing" defines the process of changing the content of a cell or plant by using traditional breeding or recombinant transformation techniques. Any method can be used to introduce a polynucleotide into a cell or plant cell, including methods that result in stable transformation, transient transformation, or gene editing. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, PEG, electroporation, ultrasonic methods (e.g., sonoporation), liposomes, microinjection, naked DNA, plasmid vectors, viral vectors (including episomal and integrated), gene introgression, transgenics, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALEN) (Feng et al. 2013, Joung & Sander [Journal of Sandwich Structures and Materials] 2013), mega-nucleases or zinc finger nucleases (ZFNs), and any other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell.

[0050] As used herein, the term "wild soybean" refers to perennial plants of the genus Glycine max, such as G. argyrea, G. clandestine, G. latrobeana, G. albicans, G. aphyonota, G. arenaria, G. curvata, G. cyrtoloba, G. dolichocarpa, G. falcate, G. gracei, G. hirticum, G. truncat ... aulis), milk green soybean (G.lactovirens), broad-leaved soybean (G.latifolia), small-leaved soybean (G.microphylla), Monty-Douglas soybean (G.montis-douglas), Western Australian tobacco bean (G.peratosa), Penghu tobacco bean (G.pescadrensis), G.pindanica, broad-leaved sand tobacco bean (G.pullenii), yellow-purple soybean (G.rubiginosa), white-haired tobacco bean (G.stenophita), rose-red wild soybean (G.syndetika), or short-haired wild soybean.

[0051] As used herein, the term "allele" refers to one of two or more different nucleotides or nucleotide sequences occurring at a particular locus.

[0052] A marker is "associated" with a trait when it is linked to the trait and when the presence of the marker indicates whether and / or to what extent the desired trait or trait form will occur in a plant / germplasm containing the marker. Similarly, a marker is "associated" with an allele when it is linked to the allele and when the presence of the marker indicates whether the allele is present in the plant / germplasm containing the marker. For example, a "marker associated with enhanced pathogen resistance" or a "marker associated with enhanced disease resistance" refers to a marker whose presence or absence can be used to predict whether and / or to what extent a plant will display a pathogen resistance phenotype.

[0053] The marker can be, but is not limited to, an allele, a gene, a haplotype, a restriction fragment length polymorphism (RFLP), a simple sequence repeat (SSR), a random amplified polymorphic DNA (RAPD), a cleaved amplified polymorphic sequence (CAPS) (Rafalski and Tingey, Trends in Genetics 9:275 (1993)), an amplified fragment length polymorphism (AFLP) (Vos et al., Nucleic Acids Res. [Nucleic Acids Research] 23:4407 (1995)), single nucleotide polymorphisms (SNPs) (Brookes, Gene [Gene] 234:177 (1993)), sequence signature amplified regions (SCARs) (Paran and Michelmore, Theor. Appl. Genet. [Theoretical and Applied Genetics] 85:985 (1993)), sequence tagged sites (STSs) (Onozaki et al., Euphytica [Netherlands Plant Breeding Journal] 138:255 (2004)), single strand conformation polymorphisms (SSCPs) (Orita et al., Pr USA [Proceedings of the National Academy of Sciences of the United States] 86:2766 (1989)), intersimple sequence repeats (ISSRs) (Blair et al., Theor. Appl. Genet. [theoretical and applied genetics] 98:780 (1999)), inter-retrotransposon amplified polymorphisms (IRAPs), retrotransposon microsatellite amplified polymorphisms (REMAPs) (Kalendar et al., Theor. Appl. Genet. [theoretical and applied genetics] 98:704 (1999)), chromosomal intervals or RNA cleavage products (e.g., Lynx tags). Markers can be present in genomic nucleic acids or expressed nucleic acids (e.g., ESTs). The term marker can also refer to a nucleic acid used as a probe or primer (e.g., primer pair) for amplification, hybridization, and / or detection of nucleic acid molecules according to methods well known in the art (e.g., using PCR).

[0054] As used herein, the terms "backcross" and "backcrossed" refer to a process whereby a progeny plant is repeatedly crossed back to one of its parents. In a backcross protocol, the "donor" parent refers to the parent plant that has the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) or the "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is introgressed. For example, see Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, Proceedings of the Symposium "Analysis of Molecular Marker Data," pp. 41-53 (1994). The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on.

[0055] Centimorgan ("cM") is a unit of measure for recombination frequency. One cM equals a 1% chance that a marker at one genetic locus will segregate with a marker at a second locus due to hybridization in a single generation.

[0056] As used herein, the term "cross" or "crossed" refers to the fusion of gametes through pollination to produce progeny (e.g., cells, seeds, or plants). The term encompasses both sexual crossing (one plant being pollinated by another) and selfing (self-pollination, e.g., when the pollen and ovules are from the same plant). The term "crossing" refers to the act of fusing gametes through pollination to produce progeny.

[0057] As used herein, the terms "cultivar" and "variety" refer to a group of similar plants that can be distinguished from other varieties within the same species by structural or genetic characteristics and / or performance.

[0058] As used herein, the terms "desirable allele," "favorable allele," and "allele of interest" are used interchangeably to refer to an allele that is associated with a desired trait (eg, ASR resistance).

[0059] As used herein, a "disease resistance gene" or "resistance gene" or "R gene" refers to a nucleic acid having a nucleotide sequence (e.g., a DNA sequence) encoding a polypeptide, R protein, or resistance protein that, when expressed in a plant cell, is capable of enhancing or improving or increasing a defense or immune response in a plant cell, thereby conferring increased resistance to one or more plant pathogens on the plant. In a specific embodiment, the Rg31 and Rg35 genes of the present invention are disease resistance genes or R genes that encode polypeptides that confer enhanced pathogen resistance when expressed in a plant cell. The encoded RG31 and RG35 polypeptides or active variants or fragments thereof can be expressed in plants to enhance pathogen resistance to plant pathogens, such as fungal pathogens. As non-limiting examples, the R genes of the present invention and the polypeptides encoded therefrom can be used to enhance resistance to fungal pathogens (Phytophthora) and the disease Asian soybean rust. In other examples, the R genes of the present invention and the polypeptides encoded therefrom can be used to enhance resistance to other fungal pathogens (e.g., fungal pathogens that cause powdery mildew) as well as bacterial pathogens and nematodes (e.g., soybean cyst nematode (SCN) and root knot nematode).

[0060] R gene embodiments may include one or more motifs associated with one or more domains of the corresponding R protein. Embodiments of R genes (such as the RG31 and RG35 genes disclosed herein) encode polypeptides having a TNLWK motif comprising a Toll / interleukin-1 receptor (TIR) ​​domain, a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) domain, a protein kinase domain, and a WRKY domain. In other embodiments, the R gene may encode a polypeptide having a CNL motif comprising a coiled-coil (CC) domain, a nucleotide binding site (NBS), and a leucine-rich repeat (LRR) domain. In still other embodiments, the R gene may encode a polypeptide having a TNL motif comprising a Toll / interleukin-1 receptor (TIR) ​​domain, a nucleotide binding site (NBS), and a leucine-rich repeat (LRR) domain. In some embodiments, the R gene may additionally or alternatively encode a polypeptide having an unknown functional domain, wherein the function of the domain is unknown at the time of gene discovery. In still further embodiments, the R gene may additionally or alternatively encode a polypeptide having a putative effector binding domain and / or one or more putative transmembrane helices. In further embodiments, the R gene may encode a polypeptide having fewer or additional domains and motifs.

[0061] In an embodiment, the nucleic acid sequence of the R gene is derived from a wild plant that exhibits increased resistance to pathogens, and includes at least one coding sequence that encodes a polypeptide that imparts resistance. The nucleic acid sequence of the R gene may further include nucleic acid sequences corresponding to one or more natural regulatory elements (e.g., natural introns, natural promoters, natural UTRs), one or more heterologous regulatory elements (e.g., heterologous promoters and one or more introns), and combinations thereof. Inserting the R gene into a plant with reduced pathogen resistance (e.g., no resistance or partially or completely susceptible) at a chromosomal location (e.g., stably integrated into the plant genome) or an extrachromosomal location (e.g., on a vector or plasmid) can impart pathogen resistance derived from wild plants to the recipient plant. For example, in representative embodiments, the R gene of the present invention is derived from short-haired wild soybean, gray-haired soybean, or Penghu soybean, and can be inserted into soybean (Glycine max) plants to impart or enhance resistance to pathogens (e.g., those that cause Asian soybean rust) in soybean plants.

[0062] As used herein, "variants" of polynucleotide sequences encoding disease-resistant R genes and / or "variants" of polypeptide sequences encoding corresponding R proteins that confer disease resistance include (as non-limiting examples) annotations and splice variants. As used herein, annotation variants refer to sequences that differ from each other due to differences in regulatory sequence annotations, including but not limited to the position of the transcription start site, the position of the start ATG codon, the position of the splice site, the position of introns and / or exons, etc. In an embodiment, due to the choice of the upstream start codon, the first annotation variant of the polypeptide may be longer than the second annotation variant (for example, the upstream methionine is annotated as a start codon in the first annotation variant, and the downstream methionine is annotated as a start codon in the second annotation variant). In a further embodiment, the first annotation variant comprises the second annotation variant. The RG31 and RG35 annotation variants have RG31 and RG35 activities, respectively, including the ability to confer increased disease resistance when expressed in plants.

[0063] As used herein, "splice variant" refers to a sequence that is different from a reference sequence due to a DNA sequence change during splicing at the splice site. As used herein, "splice" refers to the process by which a pre-mRNA transcript is converted into an mRNA molecule that can be translated into a protein. Typically, this is achieved by removing introns and splicing the exons of the transcript together. However, genetic changes in the sequence may occur on the boundary of exons and introns, i.e., on the splice site. During splicing, the addition of one or more introns or the removal of one or more exons can result in different mRNA molecules being produced from the same gene, thereby resulting in corresponding different protein sequences being expressed from a single gene. These different sequences at the DNA, RNA, and / or protein levels are referred to herein as "alternative splice variants." Splice variant polypeptides encoded by the alternative splicing of a gene sequence can include variants with more or less amino acids in the sequence and / or variants with alternative amino acids at specific positions within the sequence. In an embodiment, alternative splice variants of a gene can be predicted based on different annotations of the gene sequence region as exons or introns (e.g., via analysis of the sequence using sequence prediction software). In embodiments, the RG31 and RG35 splice variants have RG31 and RG35 activity, respectively, including the ability to confer increased disease resistance when expressed in plants.

[0064] As used herein, the terms "disease tolerance," "disease tolerant," or "disease resistant," "disease resistance," mean a statistically significant increase in a plant's tolerance to a disease caused by a plant pathogen and / or a statistically significant decrease or absence of one or more disease symptoms when compared to an appropriate control plant. Thus, these terms refer to the ability of a plant to tolerate and / or reproduce despite infection with the corresponding disease. In some embodiments, the increase in disease tolerance or resistance can be measured (1) by the plant's ability to tolerate and / or reproduce despite infection with the corresponding disease; (2) by infected disease-resistant legume or soybean plants that have the same (or nearly the same) yield as uninfected legume or soybean plants; or (3) by the delay or prevention of pathogen (e.g., fungus) proliferation (including the delay or prevention of disease-associated symptoms). In still other embodiments, a plant or germplasm can be labeled as "disease resistant" if it exhibits "enhanced or increased pathogen resistance" compared to a control plant.

[0065] As used herein, the terms "enhanced pathogen resistance," "enhanced disease resistance," "increased resistance to a pathogen," or "conferring pathogen resistance" refer to an improvement, enhancement, or increase in the ability of a plant to tolerate and / or reproduce despite infection with a pathogen or disease (e.g., Asian soybean rust) as compared to one or more control plants. Enhanced disease resistance includes a reduction in symptoms indicative of infection with a disease, such as Asian soybean rust ("ASR"). Enhanced plant pathogen resistance may include any statistically significant increase in resistance to a plant pathogen, including, for example, an increase of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. Conferring or enhancing or increasing resistance may include reducing (partially or completely) symptoms or phenotypic characteristics associated with susceptibility to a pathogen and / or increasing phenotypic characteristics associated with resistance to a pathogen. In example embodiments, conferring or increasing resistance to Asian soybean rust can include a statistically significant reduction in the number, size, and / or density of lesions, a change in lesion color (e.g., from tan to reddish-brown coloration), a reduction in the number and density of pustules, a reduction in sporulation, a reduction in leaf drop, a reduction in yield loss, or any combination thereof. Additionally, enhanced pathogen resistance can include preventing or delaying the proliferation of pathogens (e.g., fungi) in plants.

[0066] A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in the phenotype of a subject plant or plant cell. A control plant or plant cell can include, for example: (a) a wild-type plant or cell, i.e., having the same genotype as the starting material used to cause the genetic alteration of the subject plant or cell; (b) a plant or plant cell that has the same genotype as the starting material but has been transformed with a null construct (i.e., a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell that is a non-transformed isolate from a progeny of the subject plant or plant cell; (d) a plant or plant cell that is genetically identical to the subject plant or plant cell but has not been exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself under conditions in which the gene of interest is not expressed.

[0067] As used herein, "cisgenic" or "cisgenesis" refers to the insertion of one or more genes, optionally from the same or related species or from a hybridizable donor, into a genome (e.g., a plant genome). As used herein, a "cisgene construct" is a recombinant nucleic acid sequence present in a cell and optionally integrated into the genome of the cell, wherein the recombinant nucleic acid sequence comprises a regulatory element operably linked to the nucleic acid sequence of a gene of interest, wherein the regulatory element and the gene of interest are both native to the plant, or from a related species, or from a hybridizable donor, and are operably linked in the native cell at a genomic location different from the genomic location into which they are integrated as cisgene constructs. The introduction of specific alleles / genes existing in a gene pool via cisgenesis, without altering any DNA sequence, can accelerate the reproduction of species with long reproductive cycles and no linkage drag.

[0068] "Excellent lines" or "superior strains" are lines that are agronomically advantageous, and these lines are produced from many cycles of breeding for advantageous agronomic performance. Numerous superior lines are available and are known to those of ordinary skill in the field of soybean breeding. An "excellent population" is a class of superior individuals or lines that can be used to represent the prior art in terms of genotypes that are agronomically advantageous for a given crop species (such as soybean). Similarly, "superior germplasm" or superior strains of germplasm are germplasms that are agronomically advantageous, typically derived from and / or capable of producing plants with advantageous agronomic performance, such as existing or newly developed superior soybean lines.

[0069] A "superior" plant is any plant from a superior line, and thus a superior plant is a representative plant from a superior variety. Non-limiting examples of superior soybean varieties commercially available to farmers or soybean breeders include: AG00802, A0868, AG0902, A1923, AG2403, A2824, A3704, A4324, A5404, AG5903, AG6202, AG0934; AG1435; AG2031; AG2035; AG2433; AG2733; AG2933; AG3334; AG3832; AG4135; AG4632; AG4934; AG5831; AG6534; and AG7231 (Asgrow Seeds, Des Moines, Iowa, USA); BPR0144RR, BPR 4077NRR, and BPR 4390NRR (Bio-Plant Research Institute, Des Moines, Iowa, USA); Plant Research, Camp Point, IL, USA); DKB17-51 and DKB37-51 (DeKalb Genetics, DeKalb, IL, USA); DP 4546RR and DP 7870RR (Delta & Pine Land Company, Lubbock, TX, USA); JG03R501, JG 32R606CADD and JG 55R503C (JGL Inc.), Greencastle, Indiana, USA); NKS13-K2 (NK Division of Syngenta Seeds, Golden Valley, Minnesota, USA); 90M01, 91M30, 92M33, 93M11, 94M30, 95M30, 97B52, P008T22R2; P16T17R2; P22T69R; P25T51R; P34T07R2; P35T58R; P39T67R; P47T36R; P46T21R; and P56T03R2 (Pioneer Hi-Bred International, Inc. (Hi-Bred International, Johnston, IA, USA); SG4771NRR and SG5161NRR / STS (Soygenetics, LLC, Lafayette, IN, USA); S00-K5, S11-L2, S28-Y2, S43-B1, S53-A1, S76-L9, S78-G6, S0009-M2; S007 -Y4; S04-D3; S14-A6; S20-T6; S21-M7; S26-P3; S28-N6; S30-V6; S35-C3; S36-Y6; S39-C4; S47-K5; S48-D9; S52-Y2; S58-Z4; S67-R6; S73-S8; and S78-G6 (Syngenta Seeds, Henderson, Kentucky, USA); Richer (Northstar Seed Ltd., Alberta, Canada); 14RD62 (Stine Seed Co., Iowa, USA); or Armor 4744 (Armor Seed, LLC, Alaska, USA).

[0070] As used herein, the term "agronomically superior" means a genotype having a number of distinguishable traits (e.g., emergence, vigour, vegetative vigor, disease resistance, fruit set, standability, yield, and threshing) that allow the producer to harvest a commercially significant product.

[0071] "Native" or "wild-type" nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, "wild-type mRNA" is an mRNA that is naturally present in or endogenous to an organism.

[0072] The terms "nucleic acid," "nucleic acid molecule," "nucleotide sequence," "oligonucleotide," "polynucleic acid," and "polynucleotide" are used interchangeably herein, unless the context indicates otherwise, and refer to heteropolymers of nucleotides. These terms include, but are not limited to, DNA and RNA molecules, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and RNA, plasmid DNA, mRNA, antisense RNA, and RNA / DNA hybrids, any of which can be linear or branched, single-stranded or double-stranded, or combinations thereof. When dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications can also be made, such as modifications to the phosphodiester backbone or the 2'-hydroxyl group of the ribose group of the RNA.

[0073] As used herein, "operably linked" or "operably associated" means that the indicated elements are functionally related to each other, and are also typically physically related. Thus, as used herein, the term "operably linked" or "operably associated" refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Thus, a first nucleotide sequence that is operably linked to a second nucleotide sequence refers to a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For example, if a promoter affects the transcription or expression of a nucleotide sequence, the promoter is operably associated with the nucleotide sequence. It will be understood by those skilled in the art that control sequences (e.g., promoters, introns, terminators, enhancers) do not need to be adjacent to the nucleotide sequence to which they are operably associated, as long as the control sequence can function to direct its expression. Thus, for example, an intervening untranslated, transcribed sequence may be present between a promoter and a nucleotide sequence, and the promoter may still be considered to be "operably linked to" or "operably associated with" the nucleotide sequence.

[0074] As used herein, the term "endogenous" refers to a substance that originates within an organism or cell. "Exogenous" refers to a substance that originates outside an organism or cell. This typically applies to nucleic acid molecules used in the production of transformed or transgenic host cells and plants. For example, a nucleic acid molecule comprising an RG31 polypeptide or an active variant or fragment thereof is an exogenous nucleic acid for conferring or enhancing pathogen resistance in a plant cell transformed with the nucleic acid molecule. As another example, a nucleic acid molecule comprising an RG35 polypeptide or an active variant or fragment thereof is an exogenous nucleic acid for conferring or enhancing pathogen resistance in a plant cell transformed with the nucleic acid molecule.

[0075] As used herein, the terms "exotic," "exotic line," and "exotic germplasm" refer to any plant, line, or germplasm that is not superior. Typically, an exotic plant / germplasm is not derived from any known superior plant or germplasm, but is selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce novel alleles into a breeding program).

[0076] As used herein, the term "genome" as applied to plant cells includes not only the chromosomal DNA found within the nucleus, but also the organelle DNA found within subcellular components of the cell.

[0077] The term "gene" means a nucleic acid comprising chromosomal DNA, genomic DNA, plasmid DNA, cDNA, artificial DNA polynucleotides or other DNA encoding a polypeptide of interest. In particular embodiments, the nucleic acid sequence of a gene encodes a protein that, when expressed, is at least partially responsible for a particular characteristic or trait. In embodiments, a gene may be natural, modified (e.g., by directed recombination or site-specific mutation), or synthetic. In exemplary embodiments, a gene is transcribed into an RNA molecule (e.g., mRNA) in a cell, wherein the RNA may encode a peptide, polypeptide, or protein of interest, and in some instances, may also encode genetic elements flanking the coding sequence that participate in regulating the expression of the mRNA or polypeptide of the invention. Thus, a gene may comprise several operably linked sequences, such as a promoter sequence, a 5' leader sequence (including, for example, sequences involved in translation initiation), a (protein) coding region (comprising cDNA or genomic DNA), a 3' untranslated sequence (comprising, for example, a transcription termination sequence site, introns (e.g., one or more natural, foreign, or modified introns)). In example embodiments, the nucleic acid sequence of an isolated gene may include introns, exons, 5′ or 3′ untranslated regions (UTRs), and native regulatory elements (e.g., native promoters). In other example embodiments, the gene comprises a coding sequence for a polypeptide of interest but does not comprise any regulatory elements. Thus, a nucleic acid encoding an RG31 protein or an active variant or fragment thereof, or an RG35 protein or an active variant or fragment thereof, may comprise all native regulatory elements and / or all native introns. Alternatively, the nucleic acid encoding the RG31 protein or its active variant or fragment, or the RG35 protein or its active variant or fragment lacks all natural or exogenous / heterologous introns, can have only a subset of all natural introns (for example, only one or two or three of them, or all but one or two or three of them), can replace one, two, three or more or all natural introns with exogenous or modified introns, or replace one or more of the natural regulatory elements (promoter, 5'UTR, 3'UTR and / or terminator) with exogenous / heterologous or modified regulatory elements (promoter, 5'UTR, 3'UTR and / or terminator), or any combination thereof.

[0078] As used herein, "heterologous" with respect to a polypeptide or polynucleotide sequence refers to a 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. Thus, a heterologous sequence is in a configuration not found in nature and has been introduced through deliberate human intervention.

[0079] As used herein, the term "hybrid" refers to a seed and / or plant produced when at least two genetically non-identical parents are crossed.

[0080] As used herein, the term "inbred" refers to a substantially homozygous plant or variety. The term can refer to a plant or variety that is substantially homozygous throughout the genome, or a plant or plant variety that is substantially homozygous for a particular portion of the genome of interest.

[0081] As used herein, the terms "introgression," "introgressing," and "introgressed" refer to the natural and artificial transmission of a desired allele or combination of desired alleles of one or more genetic loci from one genetic background to another. For example, the desired allele at a given locus can be transmitted to at least one progeny by sexual hybridization between two parents of the same species, wherein at least one of the parents has the desired allele within its genome. Alternatively, for example, the transmission of the allele can occur by recombination between two donor genomes, such as in fused protoplasts, wherein at least one donor protoplast has the desired allele in its genome. The desired allele can be a selected allele of a marker, a QTL, a transgene, etc. The offspring comprising the desired allele can be repeatedly backcrossed with a strain having a desired genetic background and selected for the desired allele, with the result that the desired allele becomes fixed in a desired genetic background. For example, an RG31 polypeptide or an active variant or fragment or marker thereof associated with enhanced ASR tolerance or resistance can be infiltrated from a donor into a recurrent parent that does not have disease resistance. As another example, an RG35 polypeptide or an active variant or fragment or marker associated with enhanced ASR tolerance or resistance can be infiltrated from a donor into a recurrent parent that does not have disease resistance. The resulting offspring can then be repeatedly backcrossed and selected until the progeny have one or more ASR tolerance alleles in the recurrent parent background.

[0082] As used herein, an "isolated" nucleic acid molecule or gene is substantially separated from other nucleic acid or gene sequences with which the nucleic acid is normally associated, for example, from the chromosomal or extrachromosomal DNA of the cell in which the nucleic acid or gene is naturally present. A nucleic acid molecule is an isolated nucleic acid molecule when it comprises a transgene or a portion of a transgene present in the genome of another organism. The term also includes nucleic acids that have been biochemically purified to substantially remove contaminating nucleic acids and other cellular components.

[0083] A polypeptide is said to be "isolated" if it is separated from the cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) with which it naturally accompanies it, or is chemically synthesized or recombinant. A polypeptide molecule is an isolated polypeptide molecule when it is expressed transgenically in another organism. A monomeric polypeptide is isolated when at least 60%, preferably 90% or more, more preferably 95% or more, and most preferably more than 99% by weight of a sample is composed of monomeric polypeptide. Protein purity or homogeneity is indicated, for example, by polyacrylamide gel electrophoresis of a protein sample followed by visualization of individual polypeptide bands after staining the polyacrylamide gel; high pressure liquid chromatography; or other conventional methods. Proteins can be purified by any means known in the art, for example, as described in Guide to Protein Purification, Deutscher, ed., Meth. Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982.

[0084] A "locus" is a position on a chromosome where a gene, marker, or allele is located. In some embodiments, a locus can encompass one or more nucleotides.

[0085] A "non-naturally occurring legume or soybean variety" is any variety of legume or soybean that does not naturally occur in nature. A "non-naturally occurring legume or soybean variety" can be produced by any method known in the art, including but not limited to transforming legume or soybean plants or germplasm, transfecting legume or soybean plants or germplasm and crossing a naturally occurring legume or soybean variety with a non-naturally occurring soybean variety. In some embodiments, a "non-naturally occurring legume or soybean variety" can comprise one or more heterologous nucleotide sequences. In some embodiments, a "non-naturally occurring soybean variety" can comprise a non-natural combination of two or more naturally occurring nucleotide sequences (i.e., two or more naturally occurring genes that do not naturally occur in the same soybean, such as genes not found in soybean lines (e.g., polynucleotides from wild Glycine max species)).

[0086] As used herein, the terms "phenotype," "phenotypic trait," or "trait" refer to one or more traits and / or manifestations of an organism. A phenotype is a manifestation observable to the naked eye or by any other evaluation means known in the art (e.g., microscopy, biochemical analysis, or electromechanical determination). In some cases, a phenotype or trait is directly controlled by a single gene or genetic locus, i.e., a "monogenic trait." In other cases, a phenotype or trait is the result of multiple genes.

[0087] As used herein, the term "plant" can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, unless otherwise indicated, the term "plant" can refer to any one of the following: a whole plant, a plant component or organ (e.g., root, stem, leaf, bud, flower, pod, etc.), a plant tissue, a seed, and / or a plant cell. A plant cell is a plant cell obtained from a plant, or a plant cell derived from a cell taken from a plant by culturing. Thus, the term "soybean plant" can refer to a whole soybean plant, one or more parts of a soybean plant (e.g., root, root tip, stem, leaf, bud, flower, pod, seed, cotyledon, etc.), a soybean plant cell, a soybean plant protoplast, and / or a soybean plant callus.

[0088] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell can be in the form of an isolated single cell or a cultured cell, or as part of a higher-order organizational unit, such as, for example, a plant tissue, a plant organ, or a whole plant. In embodiments, a plant cell is sterile and / or incapable of regenerating a whole plant.

[0089] "Plant cell culture" means a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0090] "Plant material" or "plant part" refers to leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0091] A "plant organ" is a distinct and clearly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0092] As used herein, the term "plant part" includes, but is not limited to, individual cells and tissues from embryos, pollen, ovules, egg cells, seeds, leaves, flowers, flower parts, branches, fruits, stems, stalks, roots, root tips, anthers, cuttings and seeds, fertilized eggs, anthers, buds, scions, rhizomes and / or plant cells (including intact plant cells in plants and / or plant parts), plant protoplasts, plant tissues, plant cell tissue cultures, plant callus, plant clumps, etc. In some embodiments, the plant part or plant cell can be regenerated into a plant, while in other embodiments, the plant part or plant cell cannot be regenerated into a plant.

[0093] As used herein, "plant tissue" means a group of plant cells organized into a structural and functional unit. This includes any plant tissue in plants or in culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into a structural or functional unit. The use of this term in conjunction with or alone with any specific type of plant tissue, as listed above or otherwise encompassed by this definition, is not intended to exclude any other type of plant tissue.

[0094] "Plant pathogen" or "fungal pathogen" is used herein to refer to a fungal pathogen. In certain embodiments, the fungal pathogen is from the genus Phakopsora, including Phakopsora pachyrhizi and Phakopsora meibomiae species. These species are known to cause ASR in plants.

[0095] "Polyadenylation signal" or "poly A signal" refers to a nucleic acid sequence located 3' to a coding region that results in the addition of adenylate nucleotides to the 3' end of mRNA transcribed from the coding region.

[0096] "Polymerase chain reaction (PCR)" refers to a DNA amplification method that uses enzymatic technology to create multiple copies of a nucleic acid sequence (amplicon). Copies of DNA molecules are prepared by shuttling DNA polymerase between two amplicons. The basis of this amplification method is multiple cycles of temperature change to denature, then reanneal the amplicons (DNA primer molecules), followed by extension, and synthesizing new DNA chains in the region between the flanking amplicons. Nucleic acid amplification can be accomplished by any of the various nucleic acid amplification methods known in the art, including polymerase chain reaction (PCR). Various amplification methods are known in the art and are particularly described in: U.S. Patent Nos. 4,683,195 and 4,683,202 and PCR Protocols: A Guide to Methods and Applications [PCR Protocol: Methods and Application Guide], edited by Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed to amplify up to 22 kb of genomic DNA and up to 42 kb of phage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods, as well as other methods known in the art of DNA amplification, can be used in the practice of the present invention.

[0097] As used herein, the term "primer" refers to an oligonucleotide that can anneal to a nucleic acid target and serve as a starting point for DNA synthesis when placed under conditions that induce synthesis of primer extension products (e.g., in the presence of nucleotides and a reagent (such as a DNA polymerase) for polymerization and at a suitable temperature and pH). In order to obtain maximum efficiency in extension and / or amplification, in some embodiments, a primer (in some embodiments, an extension primer, and in some embodiments, an amplification primer) is single-stranded. In certain embodiments, a primer is an oligodeoxyribonucleotide. Primers are typically long enough to initiate the synthesis of extension and / or amplification products in the presence of a reagent for polymerization. The minimum length of a primer can depend on many factors, including but not limited to the temperature and composition (A / T vs. G / C content) of the primer. In the case of amplification primers, these amplification primers are typically provided as a pair of bidirectional primers consisting of a forward and a reverse primer, or as a pair of forward primers commonly used in the DNA amplification field (e.g., in PCR amplification). Thus, it should be understood that, as used herein, the term "primer" may refer to more than one primer, particularly in the case where there is some ambiguity in the information about one or more terminal sequences of the target region to be amplified. Therefore, a "primer" may include a set of primer oligonucleotides containing sequences representing possible variations in the sequence, or include nucleotides that allow typical base pairing. Primers can be prepared by any suitable method known in the art. The method for preparing the oligonucleotides of a specific sequence includes, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, phosphodiester or triester methods, diethylaminophosphoric acid ester methods, and solid phase support methods disclosed in U.S. Patent number 4,458,066. If necessary, primers can be labeled by incorporating detectable moieties, such as spectral moieties, fluorescent moieties, photochemical moieties, biochemical moieties, immunochemical moieties, or chemical moieties. Primers for diagnosing ASR resistance (i.e., being able to identify or select based on the presence of ASR resistance alleles) can be produced for any favorable SNP. The PCR method has been well described in the manual and is known to the technician. After PCR amplification, the target polynucleotide can be detected by hybridization with a probe polynucleotide that forms a stable hybrid with the target sequence under strict to moderately strict hybridization and washing conditions. If the probe is expected to be substantially completely complementary to the target sequence (i.e., about 99% or more), stringent conditions can be used. If it is expected that there are some mispairings, such as if the expected variant variety causes the probe to be incompletely complementary, the stringency of hybridization can be reduced. In certain embodiments, conditions are selected to exclude nonspecific / accidental binding. Conditions affecting hybridization and conditions selected for nonspecific binding are known in the art and are described in, for example, Sambrook and Russell (2001).Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, U.S.A. Generally, lower salt concentrations and higher temperatures for hybridization and / or washing increase the stringency of hybridization conditions.

[0098] As used herein, the terms "progeny" and "progeny plant" refer to plants produced by asexual or sexual reproduction from one or more parent plants. Progeny plants can be obtained by cloning or selfing a single parent plant (i.e., the same plant serves as the donor of both male and female gametes) or by crossing two parent plants. The one or more progeny can be, for example, F1, F2, or any subsequent generation.

[0099] The term "promoter" or "promoter region" refers to a polynucleic acid molecule that acts as a regulatory element, typically found upstream (5') of a coding sequence, which controls the expression of the coding sequence by controlling the production of messenger RNA (mRNA) by providing recognition sites for RNA polymerase and / or other factors necessary to initiate transcription at the correct site. As contemplated herein, promoters or promoter regions include variations of promoters derived by connection to various regulatory sequences, random or controlled mutagenesis, and the addition or duplication of enhancer sequences. The promoter regions disclosed herein and their biologically functional equivalents are responsible for driving transcription of the coding sequence under their control when introduced into a host as part of a suitable recombinant DNA construct, as demonstrated by their ability to produce mRNA. In some embodiments, the vector constructs or expression constructs or nucleic acid sequences disclosed herein comprise a promoter that is heterologous to the nucleic acid sequence encoding the RG31 and / or RG35 polypeptides or their active variants or fragments. In other examples, the vector constructs, expression constructs or nucleic acid sequences comprise a promoter that is native or endogenous to the nucleic acid sequence encoding the RG31 and / or RG35 polypeptides or their active variants or fragments. In particular embodiments, the vector construct, expression construct or nucleic acid sequence comprises a promoter native to the nucleic acid sequence of the genomic locus comprising the Rg31 or Rg35 gene.

[0100] As used herein, the term "recombinant" refers to non-naturally occurring DNA, proteins, cells, seeds or organisms that are the result of genetic engineering and are therefore not normally found in nature. A "recombinant DNA molecule" is a DNA molecule that comprises a DNA sequence that is not naturally occurring in nature and is therefore the result of human intervention, such as a DNA molecule composed of at least two DNA molecules that are heterologous to each other. An example of a recombinant DNA molecule is a DNA molecule encoding an RG31 and / or RG35 polypeptide or an active variant or fragment thereof as provided herein, which is operably linked to a heterologous regulatory element, such as a heterologous promoter, a heterologous terminator, or comprises one or more heterologous introns or lacks one or more native introns. A "recombinant protein" refers to a protein that comprises an amino acid sequence that is not naturally occurring and is therefore the result of human intervention, such as an engineered protein or a chimeric protein. A recombinant cell, seed or organism is a cell, seed or organism that comprises transgenic DNA, such as a transgenic cell, seed, plant or plant part that comprises a recombinant DNA molecule and is therefore produced as a result of plant transformation.

[0101] In the context of two nucleic acids or two amino acid sequences, the phrase "substantially identical" refers to two or more sequences or subsequences that have at least about 50% nucleotide or amino acid residue identity (as measured using a sequence comparison algorithm or by visual inspection) when comparing and aligning for maximum correspondence. In certain embodiments, substantially identical sequences have at least about 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 at the nucleotide or amino acid level. In certain embodiments, substantial identity exists over a region of at least about 50 amino acid residues, 100 amino acid residues, 150 amino acid residues, 200 amino acid residues, 250 amino acid residues, 300 amino acid residues, 350 amino acid residues, 400 amino acid residues, 450 amino acid residues, 500 amino acid residues, 525 amino acid residues, 526 amino acid residues, 527 amino acid residues, 528 amino acid residues, 529 amino acid residues, 530 amino acid residues, 531 amino acid residues, 532 amino acid residues, 533 amino acid residues, 534 amino acid residues, 535 amino acid residues, 536 amino acid residues of the sequence relative to the protein sequence or the nucleotide sequence encoding the same.

[0102] The terms "identity" or "identical" in the context of two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of a reference ("query") sequence (or its complementary strand) when the two sequences are globally aligned, as compared to a test ("subject") sequence. Unless otherwise indicated, sequence identity as used herein refers to the value obtained using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:443-453) as implemented in the EMBOSS Needle alignment tool, using the default matrix file EBLOSUM62 (for proteins) and default parameters (gap open=10, gap extension=0.5, terminal gap penalty=false, terminal gap open=10, terminal gap extension=0.5) or DNAfull (for nucleic acids) and default parameters (gap open=10, gap extension=0.5, terminal gap penalty=false, terminal gap open=10, terminal gap extension=0.5); or any equivalent program thereof. EMBOSS Needle is available, for example, from EMBL-EBI, for example at the following website: ebi.ac.uk / Tools / psa / emboss_needle / and as described in the following publication: "The EMBL-EBI search and sequence analysis tools APIs in 2019." Madeira et al. Nucleic Acids Research, 2019, June, 47(W1):W636-W641. As used herein, the term "equivalent program" refers to any sequence comparison program that, for any two sequences in question, generates an alignment with identical nucleotide or amino acid residue matches and an identical percentage of sequence identity when compared to the corresponding alignment generated by EMBOSS Needle. In some embodiments, substantially identical nucleic acid or amino acid sequences can perform substantially the same function.

[0103] When two nucleotide sequences hybridize to each other under stringent conditions, the two sequences can also be considered to be substantially identical. In representative embodiments, two nucleotide sequences that are considered to be substantially identical hybridize to each other under high stringency conditions.

[0104] The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a nucleic acid will selectively hybridize to a target sequence at a significantly higher degree than to other sequences (e.g., at least 2-fold greater than to non-target sequences), and optionally will substantially exclude binding to non-target sequences. Stringent conditions are sequence-dependent and will vary under different circumstances. By controlling the stringency of hybridization and / or washing conditions, target sequences that may be up to 100% complementary to a reference nucleotide sequence can be identified. Alternatively, conditions of moderate or even low stringency can be used to allow for some mismatches in the sequence, thereby detecting lower degrees of sequence similarity. For example, one skilled in the art will appreciate that in order to function as a primer or probe, a nucleic acid sequence only needs to be sufficiently complementary to the target sequence under the conditions employed to substantially bind thereto, thereby forming a stable double-stranded structure. Thus, primers or probes can be used under conditions of high, medium, or even low stringency. Similarly, conditions of low or medium stringency can be advantageous for detecting homologs, orthologs, and / or paralogs sequences that have a lower degree of sequence identity than can be identified under high stringency conditions.

[0105] As used herein, the term "complementary" or "complementarity" (and similar terms) refers to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "AGT" binds to the complementary sequence "TCA". The complementarity between two single-stranded molecules may be partial, where only some nucleotides bind, or complete when full complementarity exists between single-stranded molecules. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and intensity of hybridization between molecules. As used herein, the term "substantially complementary" (and similar terms) means that two nucleic acid sequences are at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more complementary. Alternatively, the term "substantially complementary" (and similar terms) can mean that two nucleic acid sequences can hybridize together under high stringency conditions (as described herein).

[0106] As used herein, "specifically" or "selectively" hybridizes (and similar terms) refers to the binding, duplexing, or hybridization of a molecule to a specific nucleic acid target sequence (when the sequence is present in a complex mixture (e.g., total cellular DNA or RNA)) under stringent conditions to the substantial exclusion of non-target nucleic acids, or even the absence of detectable binding, duplexing, or hybridization to non-target sequences. Specifically or selectively hybridizing sequences are typically at least about 40% complementary, and optionally are substantially complementary or even fully complementary (i.e., 100% identical).

[0107] For DNA-DNA hybrids, T mIt can be estimated from the equation of Meinkoth and Wahl, Anal. Biochem. 138:267-84 (1984): T m = 81.5°C + 16.6 (log M) + 0.41 (GC%) - 0.61 (formamide%) - 500 / L; where M is the molar concentration of monovalent cations, GC% is the percentage of guanosine and cytosine nucleotides in the DNA, formamide% is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. m is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. For every 1% mismatch, T m decrease by about 1°C; therefore, T m The , hybridization and / or washing conditions are such that hybridization occurs to sequences with the desired degree of identity. For example, if sequences with >90% identity are sought, T m Generally, stringent conditions are selected to be higher than the thermal melting point (T m ) is about 5°C lower. However, high stringency conditions can be used at the thermal melting point (T m ) or specific heat melting point (T m ) Hybridization and / or washing can be performed at 1°C, 2°C, 3°C or 4°C lower; moderate stringency conditions can be used at a temperature below the melting point (T m ) Hybridization and / or washing at 6°C, 7°C, 8°C, 9°C or 10°C lower; low stringency conditions can be used at a temperature below the melting point (T m ) Hybridization and / or washing is performed at 11°C, 12°C, 13°C, 14°C, 15°C or 20°C. If the desired degree of mismatch results in T mBelow 45°C (aqueous solution) or 32°C (formamide solution), the SSC concentration can optionally be increased to allow the use of higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York (1993); Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York (1995); and Green and Sambrook, In: Molecular Cloning, A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2012).

[0108] Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 M to 1.0 M Na ion concentration (or other salts) at about pH 7.0 to pH 8.3, and the temperature is at least about 30° C. (for short probes, e.g., 10 to 50 nucleotides) and at least about 60° C. (for long probes, e.g., greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide or Denhardt's (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water). Exemplary low stringency conditions include hybridization at 37° C. with a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) and washing in 1× to 2× SSC (20× SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary moderate stringency conditions include hybridization in 40% to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1× SSC at 55° C. to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1× SSC at 60° C. to 65° C. Another non-limiting example of high stringency conditions includes hybridization in 4× SSC, 5× Denhardt's, 0.1 mg / ml boiled salmon sperm DNA, and 25 mM sodium phosphate at 65° C., and a wash in 0.1× SSC, 0.1% SDS at 65° C. Another illustration of high stringency hybridization conditions includes hybridization in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50° C. and washing in 2X SSC, 0.1% SDS at 50° C., alternatively washing in 1X SSC, 0.1% SDS at 50° C., alternatively washing in 0.5X SSC, 0.1% SDS at 50° C., or alternatively washing in 0.1X SSC, 0.1% SDS at 50° C., or even washing in 0.1X SSC, 0.1% SDS at 65° C. Those skilled in the art will understand that specificity typically depends on post-hybridization washes, with related factors being the ionic strength and temperature of the final wash solution.

[0109] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical (eg, due to the degeneracy of the genetic code).

[0110] Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive with the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein when, for example, the two proteins differ only in conservative substitutions.

[0111] As used herein, the term "transgenic" refers to a DNA molecule that is artificially incorporated into the genome of an organism due to human intervention (e.g., plant transformation methods). As used herein, the term "transgenic" means comprising a transgene, for example, a "transgenic plant" refers to a plant that comprises a transgene in its genome, and a "transgenic trait" refers to a characteristic or phenotype conveyed or conferred by the presence of a transgene integrated into the plant genome. Due to such genomic alterations, transgenic plants are significantly different from related wild-type plants, and transgenic traits are traits that do not naturally occur in wild-type plants. Transgenic plants can comprise recombinant DNA molecules and engineered proteins as provided herein.

[0112] As used herein, the term "transgenic" and its grammatical variants refer to a plant in which a heterologous nucleic acid is integrated into the genome, including any part derived from the plant, such as a cell, tissue, or organ. In certain embodiments, the heterologous nucleic acid is a recombinant construct, vector, or expression cassette comprising one or more nucleic acids.

[0113] The term "vector" refers to a composition used to transfer, deliver or introduce one or more nucleic acids into a cell. A vector comprises a nucleic acid molecule containing one or more nucleotide sequences to be transferred, delivered or introduced.

[0114] 2. Polynucleotides and polypeptides that confer increased disease resistance

[0115] Compositions, polypeptides, polynucleotides, and active fragments and variants thereof that confer increased disease resistance are provided.

[0116] I. RG31 polypeptide and polynucleotide encoding RG31 polypeptide

[0117] Provided are RG31 polypeptides comprising SEQ ID NO: 1 or an active fragment or variant of SEQ ID NO: 1. Further provided are polynucleotides comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 or an active variant or fragment thereof; and polynucleotides comprising any one of SEQ ID NOs: 3 and 4 or an active fragment or variant thereof.

[0118] The RG31 polypeptide (SEQ ID NO: 1) encodes a resistance protein (also referred to herein as an R protein) of 1735 aa in length. The RG31 polypeptide (SEQ ID NO: 1) contains multiple conserved domains, including a TIR domain (Toll / interleukin-1 receptor domain; pfam Clan No. CL0173), a NB-ARC domain (a nucleotide binding adaptor shared by APAF-1, certain R gene products, and CED-4; pfam Clan No. CL0023), a LRR domain (leucine-rich repeat domain; pfam Clan No. CL0022); a WRKY domain (WRKY DNA binding domain; pfam Clan No. CL0274); a PK domain (protein kinase domain; pfam Clan No. CL0016), and a kinase-like domain (pfam Clan No. CL0016). The positions of the conserved domains in the RG31 polypeptide are listed in Table A.

[0119] Provided are polynucleotides comprising a coding sequence encoding an RG31 polypeptide (SEQ ID NO: 1) or an active variant or fragment thereof. In particular embodiments, the polynucleotide encoding an RG31 polypeptide (SEQ ID NO: 1) or an active variant or fragment thereof has a coding sequence comprising or derived from the genomic sequence of the Rg31 gene, for example, a polynucleotide comprising SEQ ID NO: 3 or an active fragment or variant thereof.

[0120] In exemplary embodiments, the polynucleotide encoding the RG31 polypeptide or an active variant thereof derived from the genomic sequence of the Rg31 gene comprises all native exons of the Rg31 gene and one or more native introns of the gene, such as only 1 native intron, only 2 native introns, only 3 native introns, all native introns except 1, all native introns except 2, all native introns except 3, etc. In exemplary embodiments, the polynucleotide encoding the RG31 polypeptide or an active variant thereof derived from the genomic sequence of the Rg31 gene comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 or all 14 native introns of the Rg31 genomic sequence (SEQ ID NO: 3).

[0121] In a particular embodiment, the polynucleotide encoding the RG31 polypeptide of SEQ ID NO: 1 or an active variant or fragment thereof comprises one or more of the following: (i) the first native intron of the Rg31 gene (RG31_intron 1, positions 996 to 1,171 of SEQ ID NO: 3), (ii) the second native intron of the Rg31 gene (RG31_intron 2, positions 4,173 to 4,357 of SEQ ID NO: 3), (iii) the third native intron of the Rg31 gene (RG31_intron 3, positions 4,532 to 8,649 of SEQ ID NO: 3), (iv) the fourth native intron of the Rg31 gene (RG31_intron 4, positions 8,805 to 9,496 of SEQ ID NO: 3), and (v) the fifth native intron of the Rg31 gene (RG31_intron 5, positions 8,906 to 9,113 of SEQ ID NO: 3). NO:3, positions 9,654 to 10,351); (vi) the sixth natural intron of the Rg31 gene (RG31_intron 6, SEQ ID NO:3, positions 10,515 to 12,562); (vii) the seventh natural intron of the Rg31 gene (RG31_intron 7, SEQ ID NO:3, positions 12,654 to 12,723); (viii) the eighth natural intron of the Rg31 gene (RG31_intron 8, SEQ ID NO:3, positions 12,764 to 13,781); (ix) the ninth natural intron of the Rg31 gene (RG31_intron 9, SEQ ID NO:3, positions 13,917 to 14,981); (x) the tenth natural intron of the Rg31 gene (RG31_intron 10, SEQ ID NO:3, positions 14,654 to 14,723); ID NO:3, positions 15,207 to 15,308); (xi) the eleventh natural intron of the Rg31 gene (RG31_intron 11, SEQ ID NO:3, positions 15,492 to 15,587); (xii) the twelfth natural intron of the Rg31 gene (RG31_intron 12, SEQ ID NO:3, positions 15,818 to 16,249); (xiii) the thirteenth natural intron of the Rg31 gene (RG31_intron 13, SEQ ID NO:3, positions 16,296 to 16,373); and (xiv) the fourteenth natural intron of the Rg31 gene (RG31_intron 14, SEQ ID NO:3, positions 16,441 to 16,803). In still further embodiments, one or more of the natural introns (RG31_intron1 to RG31_intron14) may be replaced by other introns.Furthermore, a polynucleotide derived from a genomic sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 of the native introns of the genomic sequence encoding the RG31 polypeptide may comprise one or more additional introns derived from other sources that enhance expression of the RG31 polypeptide.

[0122] In other specific embodiments, the polynucleotide encoding the RG31 polypeptide (SEQ ID NO: 1) or an active variant or fragment thereof has a coding sequence comprising or derived from an intronless cDNA sequence of the Rg31 gene, such as a polynucleotide comprising SEQ ID NO: 4 or an active fragment or variant thereof. In still further embodiments, the polynucleotide encoding the RG31 polypeptide or an active variant thereof derived from the cDNA sequence of the Rg31 gene comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 or all 14 natural introns of the Rg31 genomic sequence, as listed herein, and the cDNA sequence (SEQ ID NO: 4) to enhance expression of the RG31 polypeptide. In other embodiments, the polynucleotide encoding the RG31 polypeptide or an active variant thereof derived from the cDNA sequence of the Rg31 gene comprises one or more heterologous introns and the cDNA sequence (SEQ ID NO: 4) to enhance expression of the RG31 polypeptide. In still other embodiments, the coding sequence comprising or derived from the intronless cDNA sequence of the Rg31 gene (SEQ ID NO: 4) may include one or more of the native 3'UTR (RG31_3'UTR, positions 16,854 to 17,357 of SEQ ID NO: 3) and the native 5'UTR (RG31_5'UTR, positions 1 to 504 of SEQ ID NO: 3) to enhance expression of the RG31 polypeptide.

[0123] When expressed in a plant, plant part or seed, the RG31 polypeptide of SEQ ID NO: 1 or an active fragment or variant of SEQ ID NO: 1 confers disease resistance to the plant, plant part or seed (e.g., a legume plant, legume plant part or legume seed). In a specific embodiment, when expressed in a soybean plant, plant part or seed, expression of the RG31 polypeptide of SEQ ID NO: 1 or an active fragment or variant of SEQ ID NO: 1 confers ASR resistance to the soybean plant, plant part or seed.

[0124] Similarly, when expressed in a plant, plant part, or seed, a polynucleotide encoding the RG31 polypeptide of SEQ ID NO: 1 or an active fragment or variant of SEQ ID NO: 1, such as a polynucleotide comprising any one of SEQ ID NOs: 3-4 or an active fragment or variant thereof, confers disease resistance to a plant, plant part, or seed (e.g., a legume plant, legume plant part, or legume seed). In a specific embodiment, when expressed in a soybean plant, plant part, or seed, transcription and expression of the polynucleotide of any one of SEQ ID NOs: 3-4 or an active fragment or variant of any one of SEQ ID NOs: 3-4 confers ASR resistance to the soybean plant, plant part, or seed.

[0125] II. RG35 polypeptides and polynucleotides encoding RG35 polypeptides

[0126] Provided are RG35 polypeptides comprising SEQ ID NO: 2 or an active fragment or variant of SEQ ID NO: 2. Further provided are polynucleotides comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 or an active variant or fragment thereof; and polynucleotides comprising any one of SEQ ID NOs: 5 and 6 or an active fragment or variant thereof.

[0127] The RG35 polypeptide (SEQ ID NO: 2) encodes a resistance protein (also referred to herein as an R protein) of 1728 aa in length. The RG35 polypeptide (SEQ ID NO: 2) comprises multiple conserved domains, including a TIR domain (Toll / interleukin-1 receptor domain; pfam Clan No. CL0173), a NB-ARC domain (a nucleotide binding adaptor shared by APAF-1, certain R gene products, and CED-4; pfam Clan No. CL0023), a LRR domain (leucine-rich repeat domain; pfam Clan No. CL0022), a WRKY domain (WRKY DNA binding domain; pfam Clan No. CL0274), and a PK domain (protein kinase domain; pfam Clan No. CL0016). The positions of the conserved domains in the RG35 polypeptide are listed in Table A.

[0128] Provided are polynucleotides comprising a coding sequence encoding an RG35 polypeptide (SEQ ID NO: 2) or an active variant or fragment thereof. In particular embodiments, the polynucleotide encoding an RG35 polypeptide (SEQ ID NO: 2) or an active variant or fragment thereof has a coding sequence comprising or derived from the genomic sequence of the Rg35 gene, for example, a polynucleotide comprising SEQ ID NO: 5 or an active fragment or variant thereof.

[0129] In exemplary embodiments, the polynucleotide encoding the RG35 polypeptide or an active variant thereof derived from the genomic sequence of the Rg35 gene comprises all native exons of the Rg35 gene and one or more native introns of the gene, such as only 1 native intron, only 2 native introns, only 3 native introns, all native introns except 1, all native introns except 2, all native introns except 3, etc. In exemplary embodiments, the polynucleotide encoding the RG35 polypeptide or an active variant thereof derived from the genomic sequence of the Rg35 gene comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 or all 15 native introns of the Rg35 genomic sequence (SEQ ID NO: 5).

[0130] In a particular embodiment, the polynucleotide encoding the RG35 polypeptide of SEQ ID NO: 2 or an active variant or fragment thereof comprises one or more of the following: (i) the first native intron of the Rg35 gene (RG35_intron 1, positions 141 to 1,068 of SEQ ID NO: 5), (ii) the second native intron of the Rg35 gene (RG35_intron 2, positions 1,246 to 1,487 of SEQ ID NO: 5), (iii) the third native intron of the Rg35 gene (RG35_intron 3, positions 2,121 to 2,296 of SEQ ID NO: 5), (iv) the fourth native intron of the Rg35 gene (RG35_intron 4, positions 5,289 to 6,007 of SEQ ID NO: 5), (v) the fifth native intron of the Rg35 gene (RG35_intron 5, positions 1,246 to 1,487 of SEQ ID NO: 5), NO:5 positions 6,173 to 8,273); (vi) the sixth natural intron of the Rg35 gene (RG35_intron 6, SEQ ID NO:5 positions 8,426 to 9,122); (vii) the seventh natural intron of the Rg35 gene (RG35_intron 7, SEQ ID NO:5 positions 9,280 to 9,973); (viii) the eighth natural intron of the Rg35 gene (RG35_intron 8, SEQ ID NO:5 positions 10,139 to 12,192); (ix) the ninth natural intron of the Rg35 gene (RG35_intron 9, SEQ ID NO:5 positions 12,322 to 13,324); (x) the tenth natural intron of the Rg35 gene (RG35_intron 10, SEQ ID NO:5 positions 12,326 to 13,324); NO:5 positions 13,460 to 14,524); (xi) the eleventh native intron of the Rg35 gene (RG35_intron 11, SEQ ID NO:5 positions 14,750 to 14,851); (xii) the twelfth native intron of the Rg35 gene (RG35_intron 12, SEQ ID NO:5 positions 15,035 to 15,130); (xiii) the thirteenth native intron of the Rg35 gene (RG35_intron 13, SEQ ID NO:5 positions 15,361 to 15,780); (xiv) the fourteenth native intron of the Rg35 gene (RG35_intron 14, SEQ ID NO:5 positions 15,827 to 15,904); and (xv) the fifteenth native intron of the Rg35 gene (RG35_intron 15, SEQ ID NO:5 positions 15,035 to 15,130). In yet further embodiments, one or more of the natural introns (RG35_intron1 to RG35_intron15) may be replaced by other introns.Furthermore, a polynucleotide derived from a genomic sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or all 15 of the native introns of a genomic sequence encoding an RG35 polypeptide may comprise one or more additional introns derived from other sources that enhance expression of the RG35 polypeptide.

[0131] In other specific embodiments, the polynucleotide encoding the RG35 polypeptide (SEQ ID NO: 2) or an active variant or fragment thereof has a coding sequence comprising or derived from an intronless cDNA sequence of the Rg35 gene, such as a polynucleotide comprising SEQ ID NO: 6 or an active fragment or variant thereof. In still further embodiments, the polynucleotide encoding the RG31 polypeptide or an active variant thereof derived from the cDNA sequence of the Rg31 gene comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 or all 15 natural introns of the Rg35 genomic sequence, as listed herein, and the cDNA sequence (SEQ ID NO: 6) to enhance expression of the RG35 polypeptide. In other embodiments, the polynucleotide encoding the RG35 polypeptide or an active variant thereof derived from the cDNA sequence of the Rg35 gene comprises one or more heterologous introns and the cDNA sequence (SEQ ID NO: 6) to enhance expression of the RG35 polypeptide. In still other embodiments, the coding sequence comprising or derived from the intronless cDNA sequence of the Rg35 gene (SEQ ID NO: 6) may include one or more of the native 3'UTR (RG35_3'UTR, positions 16,381 to 16,798 of SEQ ID NO: 5) and the native 5'UTR (RG35_5'UTR, positions 1 to 1629 of SEQ ID NO: 5) to enhance expression of the RG35 polypeptide.

[0132] When expressed in a plant, plant part or seed, the RG35 polypeptide of SEQ ID NO: 2 or an active fragment or variant of SEQ ID NO: 2 confers disease resistance to the plant, plant part or seed (e.g., a legume plant, legume plant part or legume seed). In a specific embodiment, when expressed in a soybean plant, plant part or seed, expression of the RG35 polypeptide of SEQ ID NO: 2 or an active fragment or variant of SEQ ID NO: 2 confers ASR resistance to the soybean plant, plant part or seed.

[0133] Similarly, when expressed in a plant, plant part, or seed, a polynucleotide encoding the RG35 polypeptide of SEQ ID NO: 2 or an active fragment or variant of SEQ ID NO: 2, such as a polynucleotide comprising any one of SEQ ID NOs: 5-6 or an active fragment or variant thereof, confers disease resistance to a plant, plant part, or seed (e.g., a legume plant, legume plant part, or legume seed). In a specific embodiment, when expressed in a soybean plant, plant part, or seed, transcription and expression of the polynucleotide of any one of SEQ ID NOs: 5-6 or an active fragment or variant of any one of SEQ ID NOs: 5-6 confers ASR resistance to the soybean plant, plant part, or seed.

[0134] Table A: Functional annotation of RG31 and RG35 polypeptides

[0135]

[0136]

[0137] III. Fragments and variants of RG31 and RG35 polypeptides and polynucleotides

[0138] Also provided are active fragments and variants of the RG31 polypeptide (SEQ ID NO: 1) and / or active fragments or variants of the RG35 polypeptide (SEQ ID NO: 2). Further provided are polynucleotides comprising a nucleotide sequence encoding an active fragment or variant of the polypeptide of any one of SEQ ID NOs: 1 and 2; and polynucleotides comprising any active variant or fragment of any one of SEQ ID NOs: 3-6.

[0139] In certain embodiments, the polynucleotide sequences (SEQ ID NOs: 3-4) and polypeptide sequences (SEQ ID NO: 1) of RG31 and active variants and fragments thereof, when expressed in a plant, plant part or seed, increase disease resistance in a plant. In other certain embodiments, the polynucleotide sequences (SEQ ID NOs: 5-6) and polypeptide sequences (SEQ ID NO: 2) of RG35 and active variants and fragments thereof, when expressed in a plant, plant part or seed, increase disease resistance in a plant. In particular embodiments, the polynucleotide sequences (SEQ ID NOs: 3-4) and polypeptide sequences (SEQ ID NO: 1) of RG31 polypeptides and active variants or fragments thereof, and the polynucleotide sequences (SEQ ID NOs: 5-6) and polypeptide sequences (SEQ ID NO: 2) of RG35 polypeptides and active variants or fragments thereof, when expressed in a plant, plant part or seed, increase disease resistance in a plant when compared to an appropriate control plant. Various methods by which such increases in disease resistance can be measured are provided in the Examples and discussed elsewhere herein.

[0140] Fragments of the RG31 polypeptide that increase disease resistance when expressed in plants, plant parts or seeds include fragments of the RG31 polypeptide that are shorter than the full-length sequence and can contain truncations or internal deletions at the N- or C-termini. Active fragments of the RG31 polypeptide can be, for example, polypeptides of SEQ ID NO: 1 that are 10, 25, 50, 100, 150, 200, 250 or more amino acids in length when expressed in plants. Such biologically active portions can be prepared by recombinant techniques and evaluated for their ability to confer increased resistance when expressed in plants, plant parts or seeds. As used herein, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO: 1. Active fragments of the RG31 polypeptide include fragments that retain the ability to (i) elicit an immune response when expressed in a plant and / or (ii) increase disease resistance in a plant when expressed in a plant, plant part or seed.

[0141] Fragments of the RG35 polypeptide that increase disease resistance when expressed in plants, plant parts or seeds include fragments of the RG35 polypeptide that are shorter than the full-length sequence and can contain truncations or internal deletions at the N- or C-termini. Active fragments of the RG35 polypeptide can be, for example, polypeptides of SEQ ID NO:2 that are 10, 25, 50, 100, 150, 200, 250 or more amino acids in length when expressed in plants. Such biologically active portions can be prepared by recombinant techniques and evaluated for activity in conferring increased resistance when expressed in plants, plant parts or seeds. As used herein, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO:2. Examples of active fragments of the RG35 polypeptide include fragments that retain the ability to (i) elicit an immune response when expressed in a plant and / or (ii) increase disease resistance in a plant when expressed in a plant, plant part or seed.

[0142] Variant RG31 polypeptides comprise an amino acid sequence that is at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of SEQ ID NO: 1. Such active variants, when expressed in plants, plant parts, or seeds, will increase disease resistance in plants.

[0143] In some embodiments, the variant RG31 polypeptide comprises a deletion and / or addition of one or more amino acids at one or more internal sites within the native polypeptide of SEQ ID NO: 1 and / or a substitution (e.g., a conservative substitution) of one or more amino acids at one or more sites within the native polypeptide of SEQ ID NO: 1.

[0144] Variant RG35 polypeptides comprise an amino acid sequence that is at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of SEQ ID NO: 2. Such active variants, when expressed in plants, plant parts, or seeds, will increase disease resistance in the plant.

[0145] In some embodiments, the variant RG35 polypeptide comprises a deletion and / or addition of one or more amino acids at one or more internal sites within the native polypeptide of SEQ ID NO: 2 and / or a substitution (e.g., a conservative substitution) of one or more amino acids at one or more sites in the native polypeptide of SEQ ID NO: 2.

[0146] In some embodiments, variant RG31 polypeptides or variant RG35 polypeptides include annotated variants of polypeptides that contain a different (e.g., more or fewer) number of amino acids relative to the native polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2, respectively. These annotation variants may occur due to annotation of the Rg31 or Rg35 gene sequence or annotation of the translated gene sequence using an alternative start codon. In embodiments, the annotated variants of SEQ ID NO: 1 or 2 may comprise an amino acid sequence that is at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of SEQ ID NO: 1 or 2, respectively.

[0147] In still other embodiments, the variant RG31 polypeptide or variant RG35 polypeptide comprises an alternative splice variant (or simply "splice variant") of the native polypeptide comprising a different (e.g., more or less) number of amino acids relative to the polypeptide of SEQ ID NO: 1 or 2, respectively, and / or comprising one or more substitutions relative to the polypeptide of SEQ ID NO: 1 or 2, respectively. These splice variants may occur due to alternative splicing of exons and introns of the Rg31 or Rg35 gene sequence, thereby generating variant mRNA transcripts that, upon expression, form variant protein sequences. Splice variants may also occur due to alternate annotation of intron and exon positions in the gene sequence. In embodiments, a splice variant of SEQ ID NO: 1 or 2 may comprise an amino acid sequence that is at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of SEQ ID NO: 1 or 2, respectively.

[0148] In a particular embodiment, a splice variant of the RG35 polypeptide comprises the amino acid sequence of SEQ ID NO: 20 and encodes a resistance protein that is 1570 aa in length (i.e., shorter than the RG35 protein of SEQ ID NO: 2). The splice variant RG35 polypeptide of SEQ ID NO: 20 is encoded by the nucleotide sequence of SEQ ID NO: 21 (nucleotide sequence including UTRs) and / or SEQ ID NO: 22 (coding sequence without UTRs). In an embodiment, a DNA construct comprising a polynucleotide encoding the RG35 polypeptide of SEQ ID NO: 20 or an active variant or fragment thereof comprises the nucleotide sequence of SEQ ID NO: 5, 6, 21, or 22, and includes one or more alternatively spliced ​​native introns and exons. As non-limiting examples, the DNA construct may comprise one or more of the following: (i) a first natural intron at positions 141 to 1,068 of SEQ ID NO: 5, (ii) a second natural intron at positions 1,246 to 1,490 of SEQ ID NO: 5, (iii) a third natural intron at positions 2,121 to 2,296 of SEQ ID NO: 5, (iv) a fourth natural intron at positions 5,289 to 6,007 of SEQ ID NO: 5, (v) a fifth natural intron at positions 6,173 to 12192 of SEQ ID NO: 5; (vi) a sixth natural intron at positions 12,322 to 13,324 of SEQ ID NO: 5; (vii) a seventh natural intron at positions 13,460 to 14,524 of SEQ ID NO: 5; (xi) a fifth natural intron at positions 13,173 to 13,192 of SEQ ID NO: 5; NO:5 at positions 14,750 to 14,851; (xii) SEQ ID NO:5 at positions 15,035 to 15,130; (xiii) SEQ ID NO:5 at positions 15,361 to 15,780; (xiv) SEQ ID NO:5 at positions 15,827 to 15,904; and (xv) SEQ ID NO:5 at positions 15,972 to 16,334. In still further embodiments, one or more of the alternatively spliced ​​natural introns listed above can be replaced with other introns. Furthermore, a polynucleotide derived from a genomic sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or all 12 of the alternatively spliced ​​native introns of a genomic sequence encoding an RG35 splice variant polypeptide may comprise one or more additional introns derived from other sources that enhance expression of the RG35 splice variant.

[0149] In other cases, the RG31 polypeptide variant and / or the RG35 polypeptide variant comprises a tag, such as a His tag. In still other cases, the polypeptide variant comprises a detectable label, such as a detectable peptide label.

[0150] The fragment and variant of nucleotide sequence can encode the protein fragment that retains native protein biological activity and has increased disease resistance ability.Alternatively, the nucleotide sequence fragment or variant that can be used as hybridization probe or the recombinant DNA construct for gene editing does not necessarily encode the protein fragment that retains biological activity.Therefore, the fragment of nucleotide sequence can be at least about 15,50,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,750,900,950,1000 nucleotides, or less than the full-length nucleotide sequence (that is, SEQ ID NO:3-6 or 21-22) of the protein disclosed herein.

[0151] The variant of the nucleotide sequence has at least 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the nucleotide sequence of any one of SEQ ID NO:3-6 or 21-22. In a specific embodiment, the variant nucleotide sequence encodes an active polypeptide of the present invention. In other embodiments, the variant polynucleotide does not need to encode an active variant polypeptide and can be used as a component of a gene editing construct or as a probe or primer or other tools for producing plants and seeds provided herein.

[0152] In the context of nucleic acid sequences, the term "corresponding to" means that when the nucleic acid sequences of certain sequences are aligned with each other, the nucleic acids "corresponding to" certain enumerated positions in the present invention are those aligned with these positions in the reference sequence, but are not necessarily located in these precise numerical positions relative to the specific nucleic acid sequence of the present invention. Optimal alignment of sequences for comparison can be performed by computerized implementations of known algorithms or by visual inspection. Easily available sequence comparison and multiple sequence alignment algorithms are the Basic Local Alignment Search Tool (BLAST) and ClustalW / ClustalW2 / Clustal Omega programs available on the Internet (e.g., the website of EMBL-EBI), respectively. Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA, which are part of the Accelrys GCG software package available from Accelrys Corporation (San Diego, California, USA). See also Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; Ausubel et al., 1988; and Sambrook and Russell, 2001, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package at the Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection.

[0153] Unless otherwise indicated, the "corresponding" amino acid positions for a given SEQ ID NO were determined using Geneious as a global alignment tool / program with free end gaps and the following parameters: cost matrix Blossum 62, gap opening penalty 12, gap extension penalty 3, refinement iterations 2; or its equivalent programs. As used herein, the term "equivalent program" refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical corresponding nucleotide or amino acid residue matches when compared to the corresponding alignment generated by the program provided above.

[0154] An example of an algorithm suitable for determining percent sequence identity and sequence similarity, as well as for aligning sequences, is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (US National Library of Medicine, 8600 Rockville Pike, Bethesda, MD 20894). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with a word of the same length in a database sequence, match or satisfy some positive-valued threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., 1990).

[0155] These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. These word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score has decreased by the amount X from its maximum achieved value; when the cumulative score approaches 0 or below due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains as defaults. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad Sci. USA 89:10915 (1989)).

[0156] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0157] The variants and fragments 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 RG31 and / or RG35 polypeptides can be prepared by mutation of the corresponding polynucleotide sequences. Methods for mutagenesis and polynucleotide alteration are known in the art.

[0158] Variant polynucleotides and polypeptides also encompass sequences and polypeptides derived from mutagenesis or recombination procedures, including but not limited to procedures such as DNA shuffling. Strategies for such DNA shuffling are known in the art.

[0159] Variants can be generated by generating random mutations in the RG31 and / or RG35 polypeptide sequences. In other embodiments, variants can be specifically designed. In the case of designed mutants, when the amino acid identity is maintained in regions of the polypeptide that determine the biological activity of the polypeptide or are involved in determining the three-dimensional configuration of the polypeptide responsible for biological activity, variants with similar biological activity to the original polypeptide can be generated. Biological activity can also be retained if conservative substitutions are made, in which an amino acid of a given class is replaced by another amino acid of the same class. Thus, it is known that amino acids can be classified into one of the following categories: aliphatic or cyclic (glycine, alanine, valine, leucine, isoleucine, proline), aromatic (phenylalanine, tyrosine, tryptophan), acidic (aspartic acid, glutamic acid, asparagine, glutamine), basic (histidine, lysine, arginine), and sulfur or hydroxyl groups (serine, cysteine, methionine, threonine). Conservative substitutions refer to the replacement of one class of amino acids with another amino acid of the same class, which is the least likely to substantially alter the biological activity of the variant.

[0160] Variants of polypeptides and polynucleotides also include sequences from other organisms (particularly other plants) that are isolated based on their sequence identity to the polypeptide and polynucleotide sequences disclosed herein. Such sequences include sequences that are orthologs of the disclosed sequences. The term "ortholog" refers to genes that are derived from a common ancestral gene and are found in different species due to speciation. Genes found in different species are considered to be orthologs when their nucleotide sequences and / or their encoded polypeptide 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 more sequence identity. The functions of orthologs are often highly conserved between species. Therefore, the present disclosure encompasses isolated polynucleotides that encode RG31 or RG35 polypeptides that confer or enhance disease resistance and hybridize with sequences disclosed herein or variants or fragments thereof.

[0161] The variant of polypeptide and polynucleotide also comprises the sequence from the same organism, is present in different positions (for example different chromosomes) of genome, and these sequences are separated based on the sequence identity of polypeptide and polynucleotide sequence disclosed herein.Such sequence comprises the sequence of paralog as disclosed sequence.Term " paralog " refers to the gene copy produced by the duplication event on the same genome.When its nucleotide sequence and / or its encoded polypeptide sequence 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 higher sequence identity, the gene found on the same genome is considered to be paralog.The function of paralog can be retained, although they may only be expressed under different selection pressures.Due to the lack of selection pressure to gene duplicate copy, paralog also may develop different functions.

[0162] The variant of polypeptide and polynucleotide also comprises the sequence of the allelic variant of disclosed sequence.Term " allelic variant " refers to the different variants of the same gene on single locus, can cause identical or similar phenotypic expression (for example, increase disease resistance).These include the gene from the same locus of the same organism, and the gene from the same locus of the organism belonging to the same genus.Such allelic variant is based on its and the sequence identity of polypeptide and polynucleotide sequence disclosed herein and separates.When its nucleotide sequence and / or its encoded polypeptide sequence 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 higher sequence identity, the gene found on the same locus is considered to be allelic variant.The function of allelic variant can be retained.

[0163] Variants of polypeptides and polynucleotides also include sequences from other organisms (particularly other plants), which are identified based on their sequence identity to the polypeptides and polynucleotide sequences disclosed herein, and have functional identity to the polypeptides and polynucleotide sequences disclosed herein. Such variants with "functional identity" are referred to as "functional identity variants" in this article. As used herein, "functional identity" refers to the presence of a common functional activity, such as a common enzymatic activity or a common mode of action. Polypeptides with functional identity can have low sequence identity, for example, their nucleotide sequence and / or its encoded polypeptide sequence can share about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or higher sequence identity. However, they can share specificity or preference, share kinetic parameters, etc. to the same substrate. In one example, variant polypeptides and polynucleotides of RG31 and / or RG35 polypeptides, and polynucleotide sequences encoding such polypeptides, comprise proteins that interact with the same set of effector proteins to produce a hypersensitive response in plant cells.

[0164] As used herein, "effector" or "effector protein" or "plant pathogen effector" refers to a polypeptide molecule secreted by a pathogen to counteract the plant's endogenous defense system. Effector proteins interact (e.g., physically interact) with plant-based resistance proteins or polypeptides encoded by plant-based resistance genes to elicit a local immune response, including a hypersensitive programmed cell death response, in infected plant cells / tissues. Typically, each R protein interacts with a different set of effector proteins, thereby defining a unique mode of action or site of action that confers disease resistance. In an exemplary embodiment, the RG31 polypeptide and / or the RG35 polypeptide are R proteins, each of which interacts with a different set of effector proteins to elicit a hypersensitive response in plant cells. In particular embodiments, despite the presence of high sequence identity to the R protein RG1 (described as SEQ ID NO: 47 (protein) and SEQ ID NO: 6 (gene) in WO 2019103918 A1), RG31 and RG35 have a novel mode of action that is different from the mode of action of RG1, as indicated by the difference in specificity for the effector protein in an assay for detecting local hypersensitivity reactions (as disclosed in Example 3). As used herein, the term "site of action" or "mode of action" refers to the specific interaction between a pathogen-derived effector polypeptide and a plant-derived disease resistance protein (R protein).

[0165] In particular embodiments, variants of polypeptides and polynucleotides comprise sequences from other organisms (particularly other plants) that, when expressed in plants and assayed, interact with most or all of the effector proteins recognized by the RG31 and / or RG35 polypeptides to produce a localized hypersensitivity response. Example 3 herein discloses assays for identifying interactions of expressed R proteins, or variants or fragments thereof, with effector proteins and for identifying the presence of a localized hypersensitivity response.

[0166] 3. Expression cassettes and regulatory elements

[0167] The polynucleotides provided herein can be provided in expression cassettes (also referred to herein as "DNA constructs") for expression in a target organism. The expression cassette will include 5' and 3' regulatory sequences operably linked to a polynucleotide encoding an RG31 polypeptide or an RG35 polypeptide, or an active variant or fragment of an RG31 or RG35 polypeptide, which allow expression of the polynucleotide. The cassette may additionally contain at least one additional gene or genetic element to be co-transformed into the organism. Where additional genes or elements are included, these components are operably linked. Alternatively, the additional one or more genes or elements may be provided on multiple expression cassettes. Such expression cassettes are provided with multiple restriction sites and / or recombination sites to place the insertion of the polynucleotide under the transcriptional control of the regulatory element or region. The expression cassette may additionally contain a selective marker gene.

[0168] "DNA construct" refers to the operably linked genetic elements that constitute a recombinant DNA molecule and may include elements that provide for expression of the DNA polynucleotide molecule in a host cell as well as elements that provide for maintenance of the construct in the host cell. The various genetic elements within the DNA construct may be native to the polynucleotide encoding the polypeptide or may be heterologous to the native polynucleotide encoding the polypeptide.

[0169] DNA constructs, vectors, and expression cassettes incorporating nucleotide sequences encoding RG31 or RG35 polypeptides, or active variants or fragments thereof, can be prepared for directing expression of the sequences directly from host plant cells. Examples of such constructs and methods generally described for this purpose are described, for example, in Svab et al., Proc. Natl. Acad. Sci. USA 87:8526-8530 (1990) and Svab et al., Proc. Natl. Acad. Sci. USA 90:913-917 (1993) and U.S. Pat. No. 5,693,507.

[0170] A plant expression cassette comprises an operably linked set of genetic elements that, when introduced into plant cells, provide for expression of a desired gene product. A "plant expression cassette" refers to a DNA construct comprising regulatory elements operably linked to provide for expression of a desired nucleic acid in a plant. Promoters, leader sequences, introns, polynucleic acids encoding transit peptides, and 3' transcriptional termination regions are all genetic elements that can be operably linked by one skilled in the art of plant molecular biology to provide for the desired level of expression or function of an RG31 polypeptide or RG35 polypeptide, or any active variant or fragment thereof.

[0171] In embodiments, the DNA construct comprising a polynucleotide encoding the RG31 polypeptide of SEQ ID NO: 1 or an active variant or fragment thereof comprises one or more of the native introns of the genomic sequence of the Rg31 gene encoding the RG31 polypeptide (SEQ ID NO: 3). As non-limiting examples, the DNA construct may comprise one or more of the following: (i) the first native intron of the Rg31 gene (RG31_intron 1, positions 996 to 1,171 of SEQ ID NO: 3), (ii) the second native intron of the Rg31 gene (RG31_intron 2, positions 4,173 to 4,357 of SEQ ID NO: 3), (iii) the third native intron of the Rg31 gene (RG31_intron 3, positions 4,532 to 8,649 of SEQ ID NO: 3), (iv) the fourth native intron of the Rg31 gene (RG31_intron 4, positions 8,805 to 9,496 of SEQ ID NO: 3), (v) the fifth native intron of the Rg31 gene (RG31_intron 5, positions 8,906 to 9,113 of SEQ ID NO: 3), NO:3, positions 9,654 to 10,351); (vi) the sixth natural intron of the Rg31 gene (RG31_intron 6, SEQ ID NO:3, positions 10,515 to 12,562); (vii) the seventh natural intron of the Rg31 gene (RG31_intron 7, SEQ ID NO:3, positions 12,654 to 12,723); (viii) the eighth natural intron of the Rg31 gene (RG31_intron 8, SEQ ID NO:3, positions 12,764 to 13,781); (ix) the ninth natural intron of the Rg31 gene (RG31_intron 9, SEQ ID NO:3, positions 13,917 to 14,981); (x) the tenth natural intron of the Rg31 gene (RG31_intron 10, SEQ ID NO:3, positions 14,970 to 15,981); NO:3, positions 15,207 to 15,308); (xi) the eleventh natural intron of the Rg31 gene (RG31_intron 11, SEQ ID NO:3, positions 15,492 to 15,587); (xii) the twelfth natural intron of the Rg31 gene (RG31_intron 12, SEQ ID NO:3, positions 15,818 to 16,249); (xiii) the thirteenth natural intron of the Rg31 gene (RG31_intron 13, SEQ ID NO:3, positions 16,296 to 16,373); and (xiv) the fourteenth natural intron of the Rg31 gene (RG31_intron 14, SEQ ID NO:3, positions 16,441 to 16,803).In still further embodiments, one or more of the natural introns (RG31_intron 1 to RG31_intron 14) can be replaced by other introns. In addition, a polynucleotide derived from a genomic sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all 14 of the natural introns of a genomic sequence encoding an RG31 polypeptide may comprise one or more additional introns derived from other sources that enhance expression of the RG31 polypeptide.

[0172] In embodiments, the DNA construct comprising a polynucleotide encoding the RG35 polypeptide of SEQ ID NO: 2 or an active variant or fragment thereof comprises one or more of the native introns of the genomic sequence of the Rg35 gene encoding the RG35 polypeptide (SEQ ID NO: 5).As non-limiting examples, the DNA construct may comprise one or more of the following: (i) the first native intron of the Rg35 gene (RG35_intron 1, positions 141 to 1,068 of SEQ ID NO: 5), (ii) the second native intron of the Rg35 gene (RG35_intron 2, positions 1,246 to 1,487 of SEQ ID NO: 5), (iii) the third native intron of the Rg35 gene (RG35_intron 3, positions 2,121 to 2,296 of SEQ ID NO: 5), (iv) the fourth native intron of the Rg35 gene (RG35_intron 4, positions 5,289 to 6,007 of SEQ ID NO: 5), (v) the fifth native intron of the Rg35 gene (RG35_intron 5, positions 5,289 to 6,007 of SEQ ID NO: 5). NO:5 positions 6,173 to 8,273); (vi) the sixth natural intron of the Rg35 gene (RG35_intron 6, SEQ ID NO:5 positions 8,426 to 9,122); (vii) the seventh natural intron of the Rg35 gene (RG35_intron 7, SEQ ID NO:5 positions 9,280 to 9,973); (viii) the eighth natural intron of the Rg35 gene (RG35_intron 8, SEQ ID NO:5 positions 10,139 to 12,192); (ix) the ninth natural intron of the Rg35 gene (RG35_intron 9, SEQ ID NO:5 positions 12,322 to 13,324); (x) the tenth natural intron of the Rg35 gene (RG35_intron 10, SEQ ID NO:5 positions 12,326 to 13,324); NO:5 positions 13,460 to 14,524); (xi) the eleventh native intron of the Rg35 gene (RG35_intron 11, SEQ ID NO:5 positions 14,750 to 14,851); (xii) the twelfth native intron of the Rg35 gene (RG35_intron 12, SEQ ID NO:5 positions 15,035 to 15,130); (xiii) the thirteenth native intron of the Rg35 gene (RG35_intron 13, SEQ ID NO:5 positions 15,361 to 15,780); (xiv) the fourteenth native intron of the Rg35 gene (RG35_intron 14, SEQ ID NO:5 positions 15,827 to 15,904); and (xv) the fifteenth native intron of the Rg35 gene (RG35_intron 15, SEQ ID NO:5 positions 15,035 to 15,130). In yet further embodiments, one or more of the natural introns (RG35_intron1 to RG35_intron15) may be replaced by other introns.Furthermore, a polynucleotide derived from a genomic sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or all 15 of the native introns of a genomic sequence encoding an RG35 polypeptide may comprise one or more additional introns derived from other sources that enhance expression of the RG35 polypeptide.

[0173] In an embodiment, a DNA construct comprising a polynucleotide encoding the RG35 polypeptide of SEQ ID NO: 20 or an active variant or fragment thereof comprises a nucleotide sequence of SEQ ID NO: 5, 6, 21 or 22, and includes one or more alternatively spliced ​​natural introns and exons of the genomic sequence of the Rg35 gene encoding the RG35 polypeptide (SEQ ID NO: 5). As non-limiting examples, the DNA construct may comprise one or more of the following alternatively spliced ​​introns: (i) the first native intron at positions 141 to 1,068 of SEQ ID NO: 5, (ii) the second native intron at positions 1,246 to 1,490 of SEQ ID NO: 5, (iii) the third native intron at positions 2,121 to 2,296 of SEQ ID NO: 5, (iv) the fourth native intron at positions 5,289 to 6,007 of SEQ ID NO: 5, (v) the fifth native intron at positions 6,173 to 12192 of SEQ ID NO: 5; (vi) the sixth native intron at positions 12,322 to 13,324 of SEQ ID NO: 5; (vii) the seventh native intron at positions 13,460 to 14,524 of SEQ ID NO: 5; (xi) the seventh native intron at positions 13,460 to 14,524 of SEQ ID NO: 5. NO:5 at positions 14,750 to 14,851; (xii) SEQ ID NO:5 at positions 15,035 to 15,130; (xiii) SEQ ID NO:5 at positions 15,361 to 15,780; (xiv) SEQ ID NO:5 at positions 15,827 to 15,904; and (xv) SEQ ID NO:5 at positions 15,972 to 16,334. In still further embodiments, one or more of the alternatively spliced ​​natural introns listed above can be replaced with other introns. Furthermore, a polynucleotide derived from a genomic sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or all 12 of the alternatively spliced ​​native introns of a genomic sequence encoding an RG35 splice variant polypeptide may comprise one or more additional introns derived from other sources that enhance expression of the RG35 splice variant.

[0174] The DNA construct may comprise one or more plant expression cassettes for expressing a DNA molecule of the present invention or other DNA molecules used in crop plant genetic engineering. An example of a DNA construct useful for expressing an RG31 polypeptide or an active variant or fragment thereof is a vector comprising a nucleic acid sequence encoding an RG31 polypeptide or an active fragment or variant thereof. An example of a DNA construct useful for expressing an RG35 polypeptide or an active variant or fragment thereof is a vector comprising a nucleic acid sequence encoding an RG35 polypeptide or an active fragment or variant thereof.

[0175] In an embodiment, the nucleic acid sequence is operably linked to a heterologous regulatory element. In a particular example, the vector comprises an expression cassette comprising a polynucleotide encoding an RG31 polypeptide or an RG35 polypeptide, or an active fragment or variant thereof, operably coupled to a heterologous regulatory element, such as a plant-active promoter that drives expression of the RG31 polypeptide, RG35 polypeptide, or an active fragment or variant thereof in a plant, plant part, or seed.

[0176] The DNA constructs, expression cassettes, and vectors disclosed herein confer disease resistance to plants, plant parts, or seeds. In particular embodiments, expression of an RG31 polypeptide (or active fragments and variants thereof) or an RG35 polypeptide (or active variants and fragments thereof) confers disease resistance (e.g., fungal pathogen resistance and / or ASR resistance) to a leguminous plant, plant part, or seed (e.g., a soybean plant, plant part, or seed) via expression of a nucleic acid molecule comprising a nucleotide sequence comprising a polynucleotide encoding an RG31 polypeptide or a polynucleotide encoding an RG35 polypeptide.

[0177] The translation leader sequence refers to the DNA molecule located between the gene promoter and the coding sequence. The translation leader sequence is present in the translation initiation sequence upstream of the fully processed mRNA. The translation leader sequence can affect the processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences include maize and petunia heat shock protein leaders, plant virus coat protein leaders, plant ribulose diphosphate carboxylase (rubisco) gene leaders, etc. (Turner and Foster, Molecular Biotechnology [molecular biotechnology] 3:225,1995).

[0178] "3' non-translated sequence", or 3' untranslated sequence or 3'-UTR means a DNA sequence located downstream of the structural polynucleotide sequence and includes sequences encoding polyadenylation and other regulatory signals that can affect mRNA processing or gene expression. The function of the polyadenylation signal in plants is to cause multiple adenylate nucleotides to be added to the 3' end of the mRNA precursor. The polyadenylation sequence can be derived from a natural gene, from various plant genes, or from T-DNA. In a particular embodiment, the 3'-UTR of the RG31 gene can be included, wherein the 3'-UTR of the RG31 gene is derived from the genomic sequence of SEQ ID NO: 3 and can include at least 500bp, 1000bp or 2000bp region located immediately downstream of the stop codon. In a particular embodiment, the 3'-UTR of the RG31 gene (RG31_3'UTR) comprises positions 16,854 to 17,357 of SEQ ID NO: 3. In other specific embodiments, the 3'-UTR of the RG35 gene can be included, wherein the 3'-UTR of the RG35 gene is derived from the genomic sequence of SEQ ID NO: 5 and can include at least 500 bp, 100 bp, or 2000 bp immediately downstream of the stop codon. In a specific embodiment, the 3'-UTR of the RG35 gene comprises position 16,381 to position 16,798 of SEQ ID NO: 5.

[0179] An example of a polyadenylation sequence is the nopaline synthase 3' sequence (nos 3'; Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803-4807, 1983). The use of different 3' untranslated sequences is exemplified by Ingelbrecht et al., Plant Cell 1:671-680, 1989.

[0180] In particular embodiments, the various 3′-UTRs disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO 2022173659, WO 2021260673, WO 2021263249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609 may be used; each of which is incorporated herein by reference in its entirety, including, for example, those disclosed as SEQ ID NO: 20 in WO 2022173659.

[0181] A variety of transcription terminators can be used in expression cassettes. These transcription terminators are responsible for transcription termination beyond transgenic and correct mRNA polyadenylation. The terminator region can be naturally together with the transcription initiation region, can be naturally together with the operably connected target DNA sequence, can be naturally together with the plant host, or may be derived from another source (that is, external or heterologous to promoter, target DNA sequence, plant host, or any combination thereof). Suitable transcription terminators are those known to play a role in plants, and include CAMV 35S terminator, tml terminator, nopaline synthase terminator, and pea rbcs E9 terminator. These terminators can be used in both monocots and dicots. Other known plant terminators can also be used, for example, the terminator (for example, tMt51186; SEQ ID NO: 13) derived from alfalfa. In addition, the natural transcription terminator of the gene can be used. In particular embodiments, the natural terminator of the RG31 gene (tGtoRG31; SEQ ID NO: 9) and / or the natural terminator of the RG35 gene (tGtoRG35; SEQ ID NO: 11) can be used. The termination region used in the expression cassette can be obtained from, for example, the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0182] In certain embodiments, various terminators disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO 2022173659, WO 2021260673, WO 202163249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609 may be used, including, for example, those disclosed in WO 2019103918, including SEQ ID NO: 8 (RG1), SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, or SEQ ID NO: 32; those disclosed in WO 2022173659, including SEQ ID NO: 33; ID NO: 18 (RG30); those disclosed as SEQ ID NO: 10 (TirA terminator) or SEQ ID NO: 15 (TirB terminator) in WO 2021022022; or those disclosed as SEQ ID NO: 9 (RG31 terminator) or SEQ ID NO: 11 (RG35 terminator) in U.S. Provisional Application 63 / 481627; or those disclosed as SEQ ID NO: 28 (RG3a terminator) or SEQ ID NO: 29 (RG3b terminator) in U.S. Provisional Application 63 / 383609; each of which is incorporated herein by reference in its entirety.

[0183] "5' non-translated sequence", or 5' untranslated sequence or 5'-UTR means a DNA sequence located upstream of the start codon of a structural polynucleotide sequence, and includes sequences that can affect the translation of an mRNA sequence. The 5'-UTR sequence is also referred to as a leader sequence. In different organisms, the 5'-UTR can remain untranslated and form a complex secondary structure to regulate the translation of downstream sequences. The leader sequence can be derived from a natural gene or from various plant genes. In a particular embodiment, the 5'-UTR of the RG31 gene can be included, wherein the 5'-UTR of the RG31 gene is derived from the genomic sequence of SEQ ID NO: 3 and can include at least a 500bp, 1000bp or 2000bp region located immediately upstream of the start codon or a 500bp region located immediately downstream of the transcription start site. In a particular embodiment, the 5'-UTR of the RG31 gene (RG31_5'UTR) comprises positions 1 to 504 of SEQ ID NO: 3. In other specific embodiments, the 5'-UTR of the RG35 gene may be included, wherein the 5'-UTR of the RG35 gene is derived from the genomic sequence of SEQ ID NO: 5 and may include at least a 500 bp, 1000 bp, or 2000 bp region located immediately upstream of the start codon or a 500 bp region located immediately downstream of the transcription start site. In a specific embodiment, the 5'-UTR of the RG35 gene comprises positions 1 to 1,629 of SEQ ID NO: 5. In another embodiment, the 5'-UTR of the RG35 gene comprises the sequence of positions 1 to 1,629 of SEQ ID NO: 5, excluding the first natural intron of positions 141 to 1068 and the second natural intron of positions 1246 to 1487 of SEQ ID NO: 5. In a specific embodiment, the 5'-UTR of the RG35 gene comprises the sequence of positions 1 to 140 and 1488 to 1629 of SEQ ID NO: 5.

[0184] In particular embodiments, various 5′-UTRs disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO 2022173659, WO 2021260673, WO 2021263249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609 may be used; each of which is incorporated herein by reference in its entirety, including, for example, those disclosed as SEQ ID NO: 19 in WO 2022173659.

[0185] The present invention also can be used to express the non-translated leader sequence of the present invention.Also known multiple non-translated leader sequence enhanced expression that is derived from virus, and these sequences are particularly effective in dicotyledonous plant cells.Expression cassette can comprise one or more such leader sequences.Especially, it has been shown that the leader sequence from tobacco mosaic virus (TMV, " W sequence "), maize chlorotic mottle virus (MCMV) and alfalfa mosaic virus (AMV) is effective in enhancing expression (for example, Gallie et al. Nucl.Acids Res. [nucleic acids research] 15:8693-8711 (1987); Skuzeski et al. Plant Molec.Biol. [plant molecular biology] 15:65-79 (1990)). Other leader sequences known in the art include, but are not limited to, picornavirus leaders, e.g., the EMCV leader (encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, TR and Moss, B. PNAS USA 86:6126-6130 (1989)); potato virus Y leaders, e.g., the tobacco etch virus (TEV) leader (Allison et al., 1986); the maize dwarf mosaic virus (MDMV) leader; (Virology 154:9-20); the human immunoglobulin heavy chain binding protein (BiP) leader (Macejak, DG and Samow, P., Nature 353:90-94 (1991)); the untranslated leader sequence from the envelope protein mRNA of alfalfa mosaic virus (AMV RNA4) (Jobling, SA and Gehrke, L., Nature 325:622-625 (1987)); Tobacco mosaic virus leader (TMV) (Gallie, DR et al., Molecular Biology of RNA, 237-256 (1989)); and Maize chlorotic mottle virus leader (MCMV) (Lommel, SA et al., Virology 81:382-385 (1991)). See also Della-Cioppa et al., Plant Physiology 84:965-968 (1987).

[0186] In specific embodiments, the various 3'-UTRs disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO 2022173659, WO 2021260673, WO 2021263249, and WO 2022173659 are each incorporated by reference in their entirety. In further specific embodiments, the various 5'-UTRs disclosed in U.S. Provisional Application Nos. 63 / 426524, 63 / 509586, and 63 / 383609 are each incorporated by reference in their entirety.

[0187] Additional regulatory signals include, but are not limited to, transcription initiation start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, start codons, termination signals, etc. See, e.g., U.S. Patent Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, Maniatis et al., eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), hereinafter "Sambrook"; Davis et al., eds., (1980).

[0188] Expression cassette can also comprise selective marker gene for selecting transformed cells.Selective marker gene is utilized to select transformed cells or tissue.Marker gene comprises the gene of coding antibiotic resistance, such as coding neomycin phosphotransferase II (NEO) and hygromycin, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or acetolactate synthase (ALS). Selectable markers routinely used in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing and Vierra Gene 19:259-268 (1982); Bevan et al., Nature 304:184-187 (1983)); the pat and bar genes, which confer resistance to the herbicide glufosinate (also known as phosphinothricin; (see White et al., Nucl. Acids Res 18:1062 (1990), Spencer et al., Theor. Appl. Genet 79:625-631 (1990) and U.S. Pat. Nos. 5,561,236 and 5,276,268); the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger and Diggelmann, Mol. Cell. Res. 20:1061-1062 (1990)); Biol. Mol. Cell. Biol. 4: 2929-2931) and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. [European Journal of Molecular Biology] 2(7):1099-1104 (1983)); the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642); the glyphosate N-acetyltransferase (GAT) gene, which also confers resistance to glyphosate (Castle et al. (2004) Science, 304:1151-1154; U.S. Patent Application Publication Nos. 20070004912, 20050246798 and 20050060767); and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629).

[0189] A. Promoter

[0190] A variety of promoters can be used in the various methods and compositions disclosed herein. Promoters can be selected based on the desired results. Nucleic acids can be combined with constitutive, inducible, tissue-preferred, or other promoters for expression in the target organism. See, e.g., the promoters set forth in WO 99 / 43838 and U.S. Patent Nos. 8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939; 6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; which are incorporated herein by reference.

[0191] For expression in plants, constitutive promoters can be used. Non-limiting examples of constitutive promoters include the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730). Inducible promoters include those that drive expression of pathogenesis-related proteins (PR proteins), which are induced following infection by pathogens. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116; and WO 99 / 43819, which are incorporated herein by reference.Promoters that express locally at or near the site of pathogen infection can also be used (Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977; Chen et al. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 10:955-966. J. [Plant Journal] 3:191-201; Siebertz et al. (1989) Plant Cell [Plant Cell] 1:961-968; Cordero et al. (1992) Physiol. Mol. Plant Path. [Physiology and Molecular Plant Pathology] 41:189-200; U.S. Patent No. 5,750,386 (inducible nematodes); and references cited therein).

[0192] In particular embodiments, various constitutive promoters disclosed in WO 2019103918, WO 2021000878, WO 2021022022, WO2022173659, WO 2021260673, WO 2021263249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609 may be used; each of which is incorporated herein by reference in its entirety, including, for example, those disclosed in WO 2019103918 as SEQ ID NO: 16 (prGmUbil) and SEQ ID NO: 19 (prMt51186); disclosed in WO 2021022022 as SEQ ID NO: 19 (prAtEF1aA1), SEQ ID NO: 20 (prUBQ3), or SEQ ID NO: 21 (prMt51187); NO: 21 (prGmUbm); those disclosed as SEQ ID NOs: 6 and 8 in WO 2021260673 and WO 2021263249; those disclosed as SEQ ID NO: 13 (prMt12344) or SEQ ID NO: 14 (prMt51186) in WO 2022173659; those disclosed as SEQ ID NO: 13 (prUBQ3) and SEQ ID NO: 14 (prGmUbil) in U.S. Provisional Application No. 63 / 426524, each of which is incorporated herein by reference in its entirety. An exemplary embodiment of a constitutive promoter (prMt51186) is also disclosed herein as SEQ ID NO: 12.

[0193] Wound-inducible promoters can be used in the constructs of the present invention. Such wound-inducible promoters include the pin II promoter (Ryan (1990) Ann. Rev. Phytopath. [Annual Review of Plant Pathology] 28:425-449; Ouan et al. (1996) Nature Biotechnology [Nature Biotechnology] 14:494-498); wun1 and wun2 (U.S. Patent No. 5,428,148); win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 215:200-208); systemin (McGurl et al. (1992) Science [Science] 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Genetics 215:1571-1573); Mol. Biol. [Plant Molecular Biology] 22: 783-792; Eckelkamp et al. (1993) FEBS Letters [Federation of European Biochemical Societies Newsletter] 323: 73-76); MPI genes (Corderok et al. (1994) Plant J. [Plant Journal] 6(2): 141-150); etc., which are incorporated herein by reference).

[0194] Still other inducible promoters can be used for expressing the polypeptide of the present invention in the construct of the present invention. In an embodiment, the inducible promoter is a rust responsive or rust inducible promoter. As used herein, a "rust inducible promoter" is a plant promoter that is induced or activated in response to the rust exposure or rust infection of a plant. In a particular embodiment, a rust responsive or rust inducible promoter (such as prLuFIS1) from the Fis1 gene of flax (flax, Linum usitatissimum) can be used, for example, disclosed as SEQ ID NO: 22 in WO 2021022022 and disclosed as SEQ ID NO: 25 in U.S. Provisional Application No. 63 / 383609, the contents of which are incorporated herein by reference in their entirety. In other specific embodiments, a rust-responsive promoter derived from the ACO3 gene (Glyma.02G268200) of soybean (Glycine max) (e.g., prGmACO3; SEQ ID NO: 14) or a rust-responsive promoter from the MYB gene (Glyma.19G164600) of soybean (Glycine max) (e.g., prGmMYB; SEQ ID NO: 15) can be used.

[0195] Tissue-preferred promoters for use in the present invention include those shown in the following references: 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. [Plant Physiol.] 112(2):525-535; Canevascim et al. (1996) Plant Physiol. [Plant Physiol.] 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. [Plant Cell Physiol.] 35(5):773-778; Lam (1994) Results Probl. Cell Differ. [Results and Problems in Cell Differentiation] 20:181-196; Orozco et al. (1993) Plant Mol Biol. [Plant Molecular Biology] 23(6):1129-1138; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.[Plant Journal] 4(3):495-505.

[0196] Leaf-preferred promoters include those shown in 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.

[0197] Root-preferred promoters are known and include those shown in Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (mannopine synthase (MAS) gene from Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (cytosolic glutamine synthetase (GS)); Bogusz et al. (1990) Plant Cell 3(2):207-218 (soybean root-specific glutamine synthetase gene); Cell 2(7):633-641; Leach and Aoyagi (1991) Plant Science (Limerick) 79(1):69-76 (rolC and rolD); Teeri et al. (1989) EMBO J. 8(2):343-350; Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772 (VfENOD-GRP3 gene promoter); and Capana et al. (1994) Plant Mol. Biol. 25(4):681-691 (rolB promoter). See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

[0198] "Seed-preferred" promoters include "seed-specific" promoters (promoters active during seed development, such as promoters of seed storage proteins) and "seed germination" promoters (promoters active during seed germination). See Thompson et al. (1989) BioEssays 10:108. Seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase) (see WO 00 / 11177 and U.S. Patent No. 6,225,529). γ-zein is an endosperm-specific promoter. Globulin 1 (Gib-1) is a representative embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, rapeseed albumin (napin), β-conglycinin, soybean lectin, and cruciferin. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, cerein, corrugated protein 1, corrugated protein 2, globulin 1, etc. See also WO 00 / 12733, which discloses seed-preferred promoters from the end1 and end2 genes.

[0199] In some embodiments, promoters that control the expression of resistance genes can be used to express the polynucleotide of interest. Such promoters include, but are not limited to, the various native R gene promoters set forth in WO 2019103918, WO 2021000878, WO 2021022022, WO 2022173659, WO 2021260673, WO 2021263249, or U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, or 63 / 383609; each of which is incorporated herein by reference in its entirety, including, for example, those disclosed in WO 2019103918 as SEQ ID NO: 7 (RG1 promoter), SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, or SEQ ID NO: 31; 2021000878 as SEQ ID NO: 7 (Rpp6907 promoter); WO 2021022022 as SEQ ID NO: 9 (TirA promoter) and SEQ ID NO: 14 (TirB promoter); WO 2022173659 as SEQ ID NO: 15 (RG30 promoter); WO 2021260673 and WO202263249 as SEQ ID NO: 7 (RG21 promoter); and U.S. Provisional Application No. 63 / 383609 as SEQ ID NO: 26 (RG3a promoter) and SEQ ID NO: 27 (RG3b promoter), and U.S. Provisional Application Nos. 63 / 426524 and 63 / 509586 as SEQ ID NO: 8 (RG32 promoter), SEQ ID NOs: 10-11 (RG34 promoter) and SEQ ID NO: 12 (RG36 promoter). NO: 18 (bidirectional promoter); each of which is incorporated herein by reference in its entirety. Still other native promoters include the native Rg31 promoter (prGcaRG31) set forth herein in SEQ ID NO: 7 and variants thereof set forth herein in SEQ ID NO: 8, and the native Rg35 promoter (prGcaRG35) set forth herein in SEQ ID NO: 10.

[0200] In particular embodiments, the native promoter of an R gene is a promoter sequence derived from the genomic sequence or genomic locus of the corresponding R gene. In other specific embodiments, the native promoter sequence derived from the genomic sequence of a given R gene is at least 5 ′-UTR sequence or in the native promoter sequence one or more ORFs are modified, replaced or removed. In a particular example embodiment, 5 ′ The native promoter derived from the genomic sequence of the Rg31 gene in which one or more ORFs of the -UTR sequence are modified or deleted comprises the sequence set forth herein as SEQ ID NO: 8. Another specific example of a modified native promoter that can be used to control the expression of a gene of interest is provided as SEQ ID NO: 11 in U.S. Provisional Application No. 63 / 426,524, which is incorporated herein by reference in its entirety.

[0201] For expression in bacterial hosts, promoters that function in bacteria are known in the art. Such promoters include any known crystal protein gene promoter, including promoters for any of the proteins of the present invention, and promoters specific for the Bacillus thuringiensis sigma factor. Alternatively, mutagenized or recombinant crystal protein encoding gene promoters can be recombinantly engineered and used to promote expression of the novel gene segments disclosed herein.

[0202] B. Natural regulatory elements

[0203] Compositions comprising novel regulatory elements are provided. In one embodiment, polynucleotides comprising regulatory elements operably linked to a polynucleotide of interest are provided. Such regulatory elements include promoters and comprise the nucleotide sequences set forth in SEQ ID NOs: 7, 8, and 10, or active variants or fragments thereof. Active variants or fragments of the promoters will retain the ability to direct the expression of the operably linked polynucleotide sequence. Thus, active variants of the promoter sequence comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 7, 8, or 10 and retain the ability to direct the expression of the operably linked nucleotide sequence. Fragments of such promoter sequences are also provided, which may comprise at least 100, 200, 250, 300, 350, 400, or more nucleotides of the sequence set forth in SEQ ID NOs: 7, 8, or 10. Fragments of such promoters may be active fragments and retain the ability to direct the expression of the operably linked nucleotide sequence.

[0204] Provided are regulatory elements comprising a terminator sequence as set forth in SEQ ID NO: 9 or 11, or an active variant or fragment thereof. Active variants or fragments of the terminator sequence will retain the ability to regulate the expression of an operably linked polynucleotide sequence. Thus, an active variant of the terminator sequence comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 or 11 and retains the ability to direct the expression of an operably linked nucleotide sequence. Also provided are fragments of such terminator sequences that may comprise at least 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides of the sequence set forth in SEQ ID NO: 9 or 11. Fragments of such terminators can be active fragments and retain the ability to regulate the expression of an operably linked nucleotide sequence.

[0205] Provided are regulatory elements comprising the following intron sequences: (i) the first native intron of the Rg31 gene (RG31_intron 1, positions 996 to 1,171 of SEQ ID NO: 3), (ii) the second native intron of the Rg31 gene (RG31_intron 2, positions 4,173 to 4,357 of SEQ ID NO: 3), (iii) the third native intron of the Rg31 gene (RG31_intron 3, positions 4,532 to 8,649 of SEQ ID NO: 3), (iv) the fourth native intron of the Rg31 gene (RG31_intron 4, positions 8,805 to 9,496 of SEQ ID NO: 3), (v) the fifth native intron of the Rg31 gene (RG31_intron 5, positions 8,906 to 9,113 of SEQ ID NO: 3). NO:3, positions 9,654 to 10,351); (vi) the sixth natural intron of the Rg31 gene (RG31_intron 6, SEQ ID NO:3, positions 10,515 to 12,562); (vii) the seventh natural intron of the Rg31 gene (RG31_intron 7, SEQ ID NO:3, positions 12,654 to 12,723); (viii) the eighth natural intron of the Rg31 gene (RG31_intron 8, SEQ ID NO:3, positions 12,764 to 13,781); (ix) the ninth natural intron of the Rg31 gene (RG31_intron 9, SEQ ID NO:3, positions 13,917 to 14,981); (x) the tenth natural intron of the Rg31 gene (RG31_intron 10, SEQ ID NO:3, positions 14,970 to 15,981); NO:3, positions 15,207 to 15,308); (xi) the eleventh native intron of the Rg31 gene (RG31_intron 11, SEQ ID NO:3, positions 15,492 to 15,587); (xii) the twelfth native intron of the Rg31 gene (RG31_intron 12, SEQ ID NO:3, positions 15,818 to 16,249); (xiii) the thirteenth native intron of the Rg31 gene (RG31_intron 13, SEQ ID NO:3, positions 16,296 to 16,373); and (xiv) the fourteenth native intron of the Rg31 gene (RG31_intron 14, SEQ ID NO:3, positions 16,441 to 16,803), or active variants or fragments thereof. Active variants or fragments of intron sequences will retain the ability to regulate the expression of the operably linked polynucleotide sequence. Thus, active variants of intron sequences comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the intron sequences listed above and retain the ability to direct expression of the operably linked nucleotide sequence.Also provided are fragments of such intron sequences, which may comprise at least 100, 200, 250, 300, 350, 400 or more nucleotides of the sequence shown at any position below: SEQ ID NO:3 positions 996 to 1,171; positions 4,173 to 4,357; positions 4,532 to 8,649; positions 8,805 to 9,496; positions 9,654 to 10,351; positions 10,515 to 12,562; positions 12,654 to 12,723; positions 12,764 to 13,781; positions 13,917 to 14,981; positions 15,207 to 15,308; positions 15,492 to 15,587; positions 15,818 to 16,249; positions 16,296 to 16,373; and positions 16,441 to 16,803. Such intron fragments can be active fragments and retain the ability to regulate the expression of the nucleotide sequence to which they are operably linked.

[0206] Provided are regulatory elements comprising the following intron sequences: (i) the first native intron of the Rg35 gene (RG35_intron 1, positions 141 to 1,068 of SEQ ID NO: 5), (ii) the second native intron of the Rg35 gene (RG35_intron 2, positions 1,246 to 1,487 of SEQ ID NO: 5), (iii) the third native intron of the Rg35 gene (RG35_intron 3, positions 2,121 to 2,296 of SEQ ID NO: 5), (iv) the fourth native intron of the Rg35 gene (RG35_intron 4, positions 5,289 to 6,007 of SEQ ID NO: 5), (v) the fifth native intron of the Rg35 gene (RG35_intron 5, positions 6,173 to 8,273 of SEQ ID NO: 5); (vi) the sixth native intron of the Rg35 gene (RG35_intron 6, positions 2,121 to 2,296 of SEQ ID NO: 5). 5 positions 8,426 to 9,122 of SEQ ID NO: 5); (vii) the seventh natural intron of the Rg35 gene (RG35_intron 7, positions 9,280 to 9,973 of SEQ ID NO: 5); (viii) the eighth natural intron of the Rg35 gene (RG35_intron 8, positions 10,139 to 12,192 of SEQ ID NO: 5); (ix) the ninth natural intron of the Rg35 gene (RG35_intron 9, positions 12,322 to 13,324 of SEQ ID NO: 5); (x) the tenth natural intron of the Rg35 gene (RG35_intron 10, positions 13,460 to 14,524 of SEQ ID NO: 5); (xi) the eleventh natural intron of the Rg35 gene (RG35_intron 11, positions 13,470 to 14,524 of SEQ ID NO: 5). NO:5 positions 14,750 to 14,851); (xii) the twelfth native intron of the Rg35 gene (RG35_intron12, SEQ ID NO:5 positions 15,035 to 15,130); (xiii) the thirteenth native intron of the Rg35 gene (RG35_intron13, SEQ ID NO:5 positions 15,361 to 15,780); (xiv) the fourteenth native intron of the Rg35 gene (RG35_intron14, SEQ ID NO:5 positions 15,827 to 15,904); and (xv) the fifteenth native intron of the Rg35 gene (RG35_intron15, SEQ ID NO:5 positions 15,972 to 16,334), or active variants or fragments thereof. Active variants or fragments of intron sequences will retain the ability to regulate the expression of the operably linked polynucleotide sequence.Thus, active variants of intron sequences comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the intron sequences listed above and retain the ability to direct expression of an operably linked nucleotide sequence. Also provided are fragments of such intron sequences, which may comprise at least 100, 200, 250, 300, 350, 400 or more nucleotides of the sequence set forth at any position below: SEQ ID NO:5 position 141 to 1,068, position 1,246 to 1,487, position 2,121 to 2,296, position 5,289 to 6,007, position 6,173 to 8,273, position 8,426 to 9,122, position 9,280 to 9,973, position 10,139 to 12,192, position 12,322 to 13,324, position 13,460 to 14,524, position 14,750 to 14,851, position 15,035 to 15,130, position 15,361 to 15,780, position 15,827 to 15,904 and position 15,972 to 16,334. Such intronic fragment can be an active fragment and retains the ability to regulate and control the expression of the nucleotide sequence that can be operably connected.

[0207] Regulatory elements comprising intron sequences are also shown below: (i) the first natural intron at positions 141 to 1,068 of SEQ ID NO: 5, (ii) the second natural intron at positions 1,246 to 1,490 of SEQ ID NO: 5, (iii) the third natural intron at positions 2,121 to 2,296 of SEQ ID NO: 5, (iv) the fourth natural intron at positions 5,289 to 6,007 of SEQ ID NO: 5, (v) the fifth natural intron at positions 6,173 to 12192 of SEQ ID NO: 5; (vi) the sixth natural intron at positions 12,322 to 13,324 of SEQ ID NO: 5; (vii) the seventh natural intron at positions 13,460 to 14,524 of SEQ ID NO: 5; (xi) the eighth natural intron at positions 14,750 to 14,851 of SEQ ID NO: 5; (xii) the eighth natural intron at positions 14,750 to 14,851 of SEQ ID NO: 5. NO:5 at positions 15,035 to 15,130; (xiii) SEQ ID NO:5 at positions 15,361 to 15,780; (xiv) SEQ ID NO:5 at positions 15,827 to 15,904; and (xv) SEQ ID NO:5 at positions 15,972 to 16,334; or an active variant or fragment thereof. An active variant or fragment of an intron sequence will retain the ability to regulate the expression of an operably linked polynucleotide sequence. Thus, an active variant of an intron sequence comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the intron sequences listed above and retains the ability to direct the expression of an operably linked nucleotide sequence. Also provided are fragments of such intron sequences, which may comprise at least 100, 200, 250, 300, 350, 400 or more nucleotides of the sequence shown in any of the above-listed positions. Such intron fragments may be active fragments and retain the ability to regulate the expression of an operably linked nucleotide sequence.

[0208] In some aspects, the disclosure provides expression cassettes. In some embodiments, the expression cassette comprises a nucleotide sequence comprising any one of SEQ ID NOs: 3-4 or any one of SEQ ID NOs: 5-6 or 21-22, wherein the nucleotide sequence is operably linked to a heterologous nucleotide sequence. In some embodiments, the expression cassette further comprises a selective marker.

[0209] In some embodiments, the heterologous sequence of interest is a nucleic acid of interest encoding an RNA or protein of interest. In some embodiments, the RNA or protein of interest can confer desirable characteristics on plants, such as antibiotic resistance, virus resistance, insect resistance, disease resistance, resistance to other harmful organisms, herbicide tolerance, improved nutritional value, improved performance in industrial processes, or altered reproductive capacity. In some embodiments, the RNA or protein of interest comprises a genome editing agent, such as a CRISPR / Cas agent (such as a Cas protein and / or guide RNA), a TALEN, a DNA-guided nuclease, a mega-nuclease, a recombinase, or a zinc finger nuclease. In some embodiments, the heterologous nucleotide sequence encodes a selective marker.

[0210] The heterologous nucleotide sequence of interest may comprise a sequence encoding a polypeptide of interest, and in more specific embodiments, the heterologous nucleotide sequence of interest encodes a protein that increases disease resistance in a plant (eg, increases fungal pathogen resistance, such as ASR resistance). Such sequences include, but are not limited to, polynucleotides encoding proteins that confer increased ASR resistance, as described in U.S. Patent Application No. US20200354739, and PCT Application Nos. WO 2019103918, WO 2021000878, WO 2021154632A1, WO 2021022022, WO 2021022026, WO 2021022101, WO 2022173659, WO 2021260673, WO2021263249, and U.S. Provisional Applications 63 / 481627, 63 / 426524, 63 / 509586, and 63 / 383609, each of which is incorporated by reference in its entirety.

[0211] In some embodiments, the expression cassette is located in a vector, such as a plasmid, virus, or Agrobacterium. In some embodiments, the expression cassette is located in a plant cell, as discussed elsewhere herein.

[0212] 4. Plants, plant cells and plant parts

[0213] Provided are plants, plant parts, plant cells, and seeds comprising in their genome a nucleic acid sequence operably linked to a plant-active promoter, wherein the nucleic acid sequence comprises a polynucleotide encoding the RG31 polypeptide set forth in SEQ ID NO: 1, or an active variant or fragment thereof, or wherein the nucleic acid sequence comprises a polynucleotide encoding the RG35 polypeptide set forth in SEQ ID NO: 2 or 20, or an active variant or fragment thereof. In particular embodiments, the plants, plant parts, plant cells, and seeds express the RG31 or RG35 polypeptide, or active variants and fragments thereof, in their genome. In still further embodiments, provided are plants, plant parts, plant cells, and seeds comprising in their genome a heterologous nucleic acid sequence comprising a polynucleotide set forth in any one of SEQ ID NOs: 3-6 or 21-22, or active variants and fragments thereof. Such heterologous polynucleotides can be transiently expressed or stably integrated into the genome.

[0214] Although soybean plants are used to illustrate compositions and methods throughout the application, the polynucleotides provided herein can be introduced into any plant species, including but not limited to monocots and dicots. Examples of target plants include but are not limited to corn (maize), sorghum, wheat, sunflower, tomato, crucifers, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley and oilseed rape, Brassica, alfalfa, rye, millet, safflower, peanut, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, nectarine, fig, guava, mango, olive, papaya, cashew, macadamia, apricot, oat, vegetables, ornamental plants, and conifers.

[0215] In certain embodiments, the plant is of the family Leguminosae. Examples of the Leguminosae family include, but are not limited to, Phaseolus (e.g., French beans, green beans, climbing beans (Phaseolus vulgaris), lima beans (Phaseolus lunatus), tepary beans (Phaseolus acutifolius), runner beans (Phaseolus coccineus); Glycine max (L.); peas (Pisum); marrowfat peas (Pisum sativum), sugar peas (Pisum sativum), also known as snow peas, mangetouts (Pisum granda); peanuts (Arachis pecans). hypogaea), clover (Trifolium spp.), alfalfa (Medicago spp.), kudzu (Pueraria lobata), common alfalfa, alfalfa (Medicago sativa), chickpeas (Cicer), lentils (Lens culinaris), and lupins (Lupinus); vetch (Vicia), field bean, broad bean (Vicia faba), vetchling (Lathyrus) (e.g., chickling pea (Lathyrus sativus), heath pea (Lathyrus tuberosus);Vigna (e.g., moth bean (Vigna aconiti folia), adzuki bean (Vigna angularis), urad bean (Vignamungo), mung bean (Vigna radiata), bambara groundnut (Vigna subterrane), rice bean (Vigna umbellata), wild cowpea (Vigna vexillata), cowpea (Vigna unguiculata) (also known as asparagus bean, cowpea); pigeon pea (Cajanus cajari; Cajanus cajan), Macrotyloma (e.g., geocarpa groundnut (Macrotyloma geocarpum), horse bean (Macrotyloma uniflorum); goa bean (Psophocarpus tetragonolobus), African yam bean (Sphenostylis stenocarpa), Egyptian black bean, lablab bean (Lablab purpureus), yam bean (Pachyrhizus erosus), guar bean (Cyamopsis tetragonolobus); and / or Canavalia (e.g., jack bean (Canavalia ensiformis)), sword bean (Canavalia gladiata).

[0216] In one embodiment, the legume is soybean, and more particularly soybean (Glycine max).

[0217] Glycine (soybean or soya bean) is a genus of the soybean family, leguminous family. The Glycine plant provided herein can be Glycine arenaria, Glycine cyrtoloba ...

[0218] In certain embodiments, the plant (legume or soybean plant) provided herein is an elite plant, an elite germplasm, or is derived from an elite line or an elite germplasm. Numerous elite lines are available and are known to those of ordinary skill in the field of soybean breeding and are discussed in further detail elsewhere herein.

[0219] In some embodiments, the plants provided herein may comprise one or more additional polynucleotides encoding additional polypeptides that increase plant disease resistance. Such combinations are described in more detail elsewhere herein.

[0220] In certain embodiments, plants, plant parts or seeds having heterologous polynucleotides or polypeptides disclosed herein or active variants and fragments thereof can have increased expression of the polynucleotides or polypeptides. In other embodiments, plants, plant parts or seeds having heterologous polynucleotides or polypeptides disclosed herein or active variants and fragments thereof can have increased activity levels of the polypeptides. Methods for producing such increased expression or activity levels include, but are not limited to, breeding, gene editing and transgenic technologies.

[0221] In some embodiments, plant cells, seeds, or plant parts or harvested products can be obtained from the plants produced as described above, and the plant cells, seeds, or plant parts can be screened using the methods disclosed above to demonstrate stable incorporation of the polynucleotide. The term "stable incorporation" means that the nucleic acid sequence is integrated into the genome of the plant and that the nucleic acid sequence is capable of being inherited by its progeny.

[0222] In some embodiments, plant products can be harvested from the plants disclosed above and processed to produce processed products, such as flour, soybean meal, oil, starch, etc. These processed products are also within the scope of the present invention, provided that they comprise a polynucleotide or polypeptide disclosed herein or a variant thereof. Other soybean plant products include, but are not limited to, protein concentrates, protein isolates, soybean hulls, meal, flowers, oil, and whole soybeans themselves.

[0223] Provided are seed lots comprising a population of seeds comprising a heterologous nucleic acid sequence in their genome comprising a polynucleotide encoding the RG31 polypeptide set forth in SEQ ID NO: 1, or an active variant or fragment thereof, or a polynucleotide encoding the RG35 polypeptide set forth in SEQ ID NO: 2 or 20, or an active variant or fragment thereof; and having increased disease resistance. In other embodiments, the seed lot comprises a population of seeds comprising a heterologous nucleic acid sequence in their genome comprising a polynucleotide set forth in any one of SEQ ID NOs: 3-6 or 21-22, or variants and fragments thereof.

[0224] Such seeds can be from any plant, including but not limited to dicotyledonous crop plants, legumes or soybeans. The method of making a seed batch includes harvesting seeds from plants with increased resistance to plant pathogens. Such a seed batch can contain at least 50, 100, 1000, 100,000 or more seeds of the present invention.

[0225] Further provided are plant collections that produce seeds with increased resistance to plant pathogens as described herein. Such plant collections have stably integrated into their genomes a heterologous nucleic acid sequence comprising a polynucleotide encoding the RG31 polypeptide set forth in SEQ ID NO: 1 or an active variant or fragment thereof, or the heterologous nucleic acid sequence comprising a polynucleotide encoding the RG35 polypeptide set forth in SEQ ID NO: 2 or 20 or an active variant or fragment thereof; and wherein the plant collection has increased disease resistance. In other embodiments, the seed lot comprises a population of seeds comprising in their genomes a heterologous nucleic acid sequence comprising a polynucleotide set forth in any one of SEQ ID NOs: 3-6 or 21-22 or variants and fragments thereof. The term collection encompasses any collection of plants joined together by proximity, such as plants in a field, a greenhouse, or a tray. The plant collection comprises at least 50, 100, 1000, 10,000, 100,000, or more plants of the present invention.

[0226] 5. Method for producing plants with increased disease resistance

[0227] Provided herein are methods for producing plants, plant parts, or seeds with increased disease resistance by introducing into a plant, plant part, or plant cell a nucleic acid sequence comprising a polynucleotide encoding an RG31 polypeptide as set forth in SEQ ID NO: 1, or an active variant or fragment thereof, or by introducing into a plant, plant part, or plant cell a nucleic acid sequence comprising a polynucleotide encoding an RG35 polypeptide as set forth in SEQ ID NO: 2 or 20, or an active variant or fragment thereof, wherein expression of the RG31 polypeptide or RG35 polypeptide increases the disease resistance of the plant. In other embodiments, the method comprises introducing into a plant, plant cell, or plant part a nucleic acid sequence comprising a polynucleotide as set forth in any one of SEQ ID NOs: 3-4, 5-6, or 21-22, or variants and fragments thereof, wherein expression of the polynucleotide increases the disease resistance of the plant.

[0228] Nucleic acid sequences can be introduced into plant cells in a variety of ways, such as by transformation, by genome modification techniques (e.g., by genome editing or directed integration), or by breeding. In one aspect, plants can be produced by transforming a nucleic acid sequence encoding an RG31 or RG35 polypeptide, or an active variant or fragment thereof, into a recipient plant. In one aspect, the method can include editing the genome of the recipient plant so that the resulting plant comprises a polynucleotide encoding an RG31 or RG35 polypeptide, or an active variant or fragment thereof. In another aspect, the method can include breeding a donor plant comprising a polynucleotide encoding an RG31 or RG35 polypeptide, or an active variant or fragment thereof, as provided herein, with a recipient plant, and selecting for incorporation of the polynucleotide into the recipient plant genome.

[0229] A. Methods and compositions for increasing the expression and / or activity of a polypeptide of interest

[0230] Provided are methods and compositions for increasing plant disease resistance by increasing the expression and / or activity of an RG31 polypeptide or an active variant or fragment thereof, or increasing the expression and / or activity of an RG35 polypeptide or an active variant or fragment thereof.

[0231] In particular embodiments, the methods and compositions provided herein increase the expression of RG31 or RG35 polypeptides, or fragments and variants thereof. As used herein, "increased expression" or "increased expression" of an RG31 or RG35 polypeptide, or an active variant or fragment of an RG31 or RG35 polypeptide, means that the level of RG31 or RG35 polypeptide, or an active variant or fragment thereof, produced by a given plant, plant cell, plant part, or seed is statistically higher than the level of expression compared to an appropriate control plant, plant part, plant cell, or seed. In particular embodiments, the increase in expression can include any statistically significant increase in polypeptide concentration (e.g., an increase in RG31 polypeptide concentration or an increase in RG35 polypeptide concentration) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to an appropriate control. In other embodiments, the increase in expression can include an increase in the target protein concentration level by at least 1-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 175-fold, 200-fold or more compared to an appropriate control plant. Methods for determining the level or increase in expression of a target polypeptide are known and include, for example, detecting the protein via an antibody or detecting an increase in the expression level of an mRNA encoding the protein.

[0232] In other embodiments, methods and compositions are provided for increasing the activity of an RG31 polypeptide, or an active variant or fragment thereof, or increasing the activity of an RG35 polypeptide, or an active variant or fragment thereof, in a plant, plant cell, or plant part. As used herein, "increased activity" or "increased activity" of an RG31 or RG35 polypeptide, or an active variant or fragment thereof, means that the level of enzymatic activity or protein functionality of the RG31 or RG35 polypeptide is statistically higher compared to an appropriate control. In particular embodiments, increased activity of an RG31 polypeptide, or an active variant or fragment thereof, means one or more of the following: (i) an increase in the ability of the RG31 polypeptide (or its active variant or fragment) to elicit an immune response in a plant cell, and / or (ii) an increase in the ability of the RG31 polypeptide (or its active variant or fragment) to confer disease resistance to the plant when expressed in a plant, plant part, or seed. In other specific embodiments, increased activity of an RG35 polypeptide or an active variant or fragment thereof means one or more of the following: (i) an increased ability of the RG35 polypeptide (or an active variant or fragment thereof) to elicit an immune response in plant cells, and / or (ii) an enhanced ability of the RG35 polypeptide (or an active variant or fragment thereof) to confer disease resistance to the plant when expressed in a plant, plant part or seed.

[0233] In certain embodiments, the increase in activity can include an increase in the level of enzyme activity or protein functionality of the RG31 or RG35 polypeptide, or an active variant or fragment thereof, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to an appropriate control plant, plant part, plant cell, or seed. In other embodiments, the increase in activity of the RG31 or RG35 polypeptide, or an active variant or fragment thereof, can include an increase in the level of enzyme activity or protein functionality by at least 1-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 175-fold, 200-fold or more compared to an appropriate control plant, plant part, plant cell, or seed. Methods for determining an increase in enzyme activity or protein functionality include direct determination of the activity of a particular protein as well as indirect determination.

[0234] In some embodiments, increasing the activity and / or expression level of an RG31 or RG35 polypeptide comprises introducing a nucleic acid construct into a plant, thereby increasing the expression and / or activity of the RG31 or RG35 polypeptide or an active variant or fragment thereof. The nucleic acid construct can be stably integrated into the genome or provided temporarily. For example, the nucleic acid construct can comprise a nucleic acid sequence encoding the RG31 polypeptide of SEQ ID NO: 1 (e.g., a nucleic acid sequence of SEQ ID NO: 3 or 4) or encoding the RG35 polypeptide of SEQ ID NO: 2 or 20 (e.g., a nucleic acid sequence of SEQ ID NO: 5 or 6), or an active variant or fragment thereof.

[0235] Thus, provided are plants, plant parts, seeds and plant cells having stably incorporated into their genome a polynucleotide operably linked to a promoter active in a plant, wherein the polynucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, or an active variant or active fragment thereof, or wherein the polynucleotide encodes a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2 or 20, or an active variant or active fragment thereof, wherein increased expression or activity of the polypeptide of SEQ ID NO: 1 or 2 or 20, or an active variant or fragment thereof, results in increased disease resistance and / or pathogen resistance in the plant. After transformation, the target polypeptide is integrated into the genome and expressed in a manner that increases the activity or expression level of the RG31 or RG35 polypeptide in the plant.

[0236] Thus, provided are methods for increasing pathogen resistance in plants by introducing into the genome of a plant a nucleic acid construct resulting in increased expression and / or activity of a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 1, or an active variant or active fragment thereof, or resulting in increased expression and / or activity of a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2 or 20, or an active variant or active fragment thereof, wherein introduction of the nucleic acid construct into the genome increases the pathogen resistance and / or disease resistance of the plant. In one embodiment, the method comprises introducing into the genome of the plant a nucleic acid sequence encoding a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 1, or an active variant or active fragment thereof, or a polypeptide having an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 2 or 20, or an active variant or active fragment thereof, wherein increased activity or expression of the polypeptide results in increased pathogen resistance and / or disease resistance in the plant. In some embodiments, an expression cassette comprising a promoter active in a plant is introduced into the genome of the plant, the promoter being operably linked to a polynucleotide of interest encoding an RG31 or RG35 polypeptide or an active variant or fragment thereof. In other embodiments, the polynucleotide of interest can be introduced into the genome of the plant and integrated into a genomic location that allows for expression of the polypeptide (eg, by targeted integration).

[0237] In other embodiments, the method comprises introducing a nucleic acid construct that produces a modification in the genome of the plant, thereby resulting in increased expression or increased activity of an RG31 polypeptide having an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1, or an active variant or fragment thereof, or thereby resulting in increased expression or increased activity of an RG35 polypeptide having an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 2 or 20, or an active variant or fragment thereof, wherein the modification increases the pathogen resistance of the plant.

[0238] B. Transformation Methods

[0239] In some embodiments, the method of introducing a polynucleotide into a plant comprises transforming a polynucleotide disclosed herein or an active variant or fragment thereof into a recipient plant to obtain a transgenic plant, wherein the transgenic plant has increased pathogen resistance and / or disease resistance. An expression cassette comprising a polynucleotide encoding a polypeptide as described above can be used to transform the plant of interest.

[0240] Transformation produces the plant that introduces heterologous nucleic acid, including whole plant and plant organ (such as leaf, stem, root etc.), seed, plant cell, propagule and its embryo and progeny.Plant cell can be differentiated or undifferentiated (such as callus, suspension culture cell, protoplast, leaf cell, root cell, phloem cell, pollen).Conversion can make nucleic acid stable or transiently be incorporated into cell. " stable transformation " is intended to mean that the nucleotide construct introduced into the host cell is integrated into the genome of the host cell and can be inherited by its progeny.In certain embodiments, stable conversion occurs via random integration event.In other embodiments, stable transformation is realized via using genome modification mechanism (such as CRISPR or TALEN) that the target sequence is integrated into the genome." transient transformation " is intended to mean that polynucleotide is introduced into the host cell and is not integrated into the genome of this host cell.

[0241] The method for transformation typically involves introducing the nucleotide construct into the plant. In some embodiments, the transformation method is Agrobacterium-mediated transformation. In some embodiments, the transformation method is gene gun-mediated transformation. Transformation can also be carried out by infection, transfection, microinjection, electroporation, microprojection, gene gun or particle bombardment, electroporation, silica / carbon fiber, ultrasound mediation, PEG mediation, calcium phosphate coprecipitation, polycation DMSO technology, DEAE dextran procedure, Agrobacterium and virus mediation (e.g., cauliflower virus, geminivirus, RNA plant virus), liposome mediation, etc.

[0242] Transformation protocols and protocols for introducing polypeptide or polynucleotide sequences into plants may vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocot or dicot). Methods for transformation are known in the art and include those set forth in: U.S. Patent Nos. 8,575,425; 7,692,068; 8,802,934; and 7,541,517; each of which is incorporated herein by reference. See also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods, Vol. 1, No. 5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4:1-12; Bates, GW (1999) Methods in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:57 Sup' / Sup5-606; Christou, P. (1992) The Plant Plant Journal 2:275-281; ​​Christou, P. (1995) Euphytica 85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107:1041-1047.

[0243] Methods for transformation of plant cells or tissues include, but are not limited to, Agrobacterium-mediated transformation methods and gene gun or particle gun-mediated transformation methods. Suitable plant transformation vectors for Agrobacterium-mediated transformation purposes include those elements derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, such as the right border (RB) region and the left border (LB) region, as well as other elements disclosed by Herrera-Estrella et al., Nature 303:209 (1983); Bevan, Nucleic Acids Res. 12:8711-8721 (1984); Klee et al., Bio-Technology 3(7):637-642 (1985). In addition to plant transformation vectors derived from Agrobacterium Ti or root-inducing (Ri) plasmids, alternative methods can be used to insert the DNA constructs of the present invention into plant cells. These methods may involve, but are not limited to, for example, the use of liposomes, electroporation, chemicals that increase uptake of free DNA, free DNA delivery by microprojectile bombardment, and transformation using viruses or pollen.

[0244] Methods for transforming chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87(21):8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90(3):913-917; Staub and Maliga (1993) EMBO J. 12(2):601-606. This method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA into the plastid genome by homologous recombination. Alternatively, plastid transformation can be accomplished by transactivating a silent plastid-borne transgene through tissue-preferred expression of a nuclear-encoded, plastid-localized RNA polymerase. Such a system has been reported by McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91(15):7301-7305.

[0245] The transformed cells can be cultured into plants according to conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports [plant cell report] 5: 81-84. These plants can then be cultivated and pollinated with the same transformed strain or different strains, and the resulting hybrids with the constitutive expression of the desired phenotypic characteristics can be identified. Two or more generations can be cultivated to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and then seeds are harvested to ensure that the expression of the desired phenotypic characteristics has been achieved. In this way, the invention provides transformed seeds (also referred to as "transgenic seeds") having nucleotide constructs of the present invention that can be stably incorporated into their genome, such as expression cassettes of the present invention.

[0246] "Regeneration" refers to the process of growing a plant from a plant cell (e.g., a plant protoplast or explant). Such regeneration techniques rely on the manipulation of certain plant hormones in tissue culture growth media, typically relying on a biocide and / or herbicide marker that has been introduced along with the desired nucleotide sequence. The method chosen for the regeneration step is not critical; see, e.g., Ammirato et al., Handbook of Plant Cell Culture—Crop Species. Macmillan Publ. Co. (1984); Shimamoto et al., Nature 338:274-276 (1989); Fromm, UCLA Symposium on Molecular Strategies for Crop Improvement, April 16-22, 1990. Keystone, Colo. (1990); Vasil et al., Bio / Technology 8:429-434 (1990); Vasil et al., Bio / Technology 10:667-674 (1992); Hayashimoto, Plant Cell Culture 339:274-276 (1989); Fromm, UCLA Symposium on Molecular Strategies for Crop Improvement, April 16-22, 1990. Keystone, Colo. (1990); Vasil et al., Bio / Technology 8:429-434 (1990); Vasil et al., Bio / Technology 10:667-674 (1992); Hayashimoto, Plant Cell Culture 339:274-276 (1989); Physiol. [Plant Physiology] 93:857-863 (1990); and Datta et al., Bio-technology [Biotechnology] 8:736-740 (1990). Such regeneration techniques are generally described in Klee et al., Ann. Rev. Plant Phys. [Plant Physiology Annual Review] 38:467-486 (1987).

[0247] C. Hybridization

[0248] In some embodiments, the method comprises crossing a donor plant comprising a polynucleotide encoding an RG31 polypeptide or an active variant or fragment thereof, or an RG35 polypeptide or an active variant or fragment thereof, and the polypeptide is capable of conferring increased pathogen resistance on the recipient plant. As used herein, the terms "crossing" and "breeding" refer to the fusion of gametes to produce offspring (e.g., by fertilization, such as in plants by pollination to produce seeds). In some embodiments, "crossing," "breeding," or "cross-fertilization" is the fertilization of one individual by another individual (e.g., cross-pollination in plants). The plants disclosed herein can be whole plants, or can be plant cells, seeds, or tissues, or plant parts, such as leaves, stems, pollen, or cells that can be cultivated into whole plants.

[0249] In some embodiments, progeny plants produced by hybridization or breeding methods are repeatedly backcrossed to one of their parents by a process referred to herein as "backcrossing." In a backcrossing protocol, the "donor" parent refers to the parent plant that has the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is introgressed. For example, see Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, Techniqueset Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, Proceedings of the Symposium "Analysis of Molecular Marker Data," pp. 41-43 (1994). The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on.

[0250] In some embodiments, the donor soybean plant is a soybean plant. In some embodiments, the donor soybean plant is a wild soybean plant. In some embodiments, the recipient soybean plant is an elite soybean plant or an elite wild soybean plant.

[0251] D.Gene editing

[0252] Plants, plant cells, and seeds with genome modifications created by gene editing are further provided. Such methods include, but are not limited to, meganucleases designed for the target plant genome sequence CRISPR-Cas9, TALENs, and other technologies for precise genome editing (Feng et al. Cell Research 23:1229-1232, 2013, WO2013 / 026740); Cre-lox site-specific recombination; FLP-FRT recombination (Li et al. (2009) Plant Physiol 151:1087-1095); Bxbl-mediated integration (Yau et al. Plant J (2011) 701:147-166); zinc finger-mediated integration (Wright et al. (2005) Plant J 44:693-705; Cai et al. (2009) Plant Mol. Biol [Plant Mol Biol] 69:699-709); and homologous recombination (Lieberman-Lazarovich and Levy (2011) Methods Mol Biol [Molecular Biology Methods]:51-65).

[0253] Various embodiments of the methods described herein utilize gene editing. In some embodiments, gene editing is used to mutagenize the genome of a plant to produce a plant that possesses one or more of the polypeptides capable of increasing disease resistance in the plant. In other instances, gene editing is used to allow for the targeted insertion of a nucleotide sequence encoding an RG31 or RG35 polypeptide, or an active variant or fragment thereof, into the genome.

[0254] "Target site," "target sequence," "target DNA," "target locus," "genomic target site," "genomic target sequence," and "genomic target locus" are used interchangeably herein to refer to a polynucleotide sequence in the genome of a cell (including chloroplast and mitochondrial DNA) to which an endonuclease is recruited and optionally nicks or cuts the DNA at the target site. The target site can be an endogenous site in the plant genome, or alternatively, the target site can be heterologous to the plant and therefore not naturally occurring in the genome, or the target site can be located at a heterologous genomic location compared to where it occurs in nature.

[0255] In some embodiments, provided herein are plants transformed with and expressing the gene editing machinery as described above, which, when crossed with a target plant, result in gene editing in the target plant.

[0256] The term "polynucleotide modification template" includes a polynucleotide comprising at least one nucleotide modification compared to the nucleotide sequence to be edited. The nucleotide modification can be at least one nucleotide substitution, addition, or deletion. The polynucleotide modification template may further comprise a homologous nucleotide sequence flanked by at least one nucleotide modification, wherein the flanking homologous nucleotide sequence provides sufficient homology to the desired nucleotide sequence to be edited.

[0257] Gene editing generally refers to the use of site-directed nucleases (including but not limited to CRISPR / Cas, zinc fingers, meganucleases, etc.) to shorten a nucleotide sequence at a desired position. This may result in insertion / deletion ("indel") mutations (i.e., "SDN1"), base editing (i.e., "SDN2"), or allele insertion or replacement (i.e., "SDN3"). SDN2 or SDN3 gene editing can include providing one or more recombination templates (e.g., in a vector) comprising a target gene sequence (i.e., to be introduced into the plant genome) that can be used for homology-directed repair (HDR) in plants. In some embodiments, the target gene or allele is a gene or allele that can confer improved traits (e.g., increased protein content and / or altered seed composition) on the plant. The recombination template can be introduced into the plant by transforming or breeding a donor plant comprising a recombination template. The break in the plant genome can be introduced into the interior, upstream, and / or downstream of the target sequence. In some embodiments, a double-stranded DNA break is generated inside or near the target sequence locus. In some embodiments, a break is generated upstream and downstream of the target sequence locus, which can result in its excision from the genome. In some embodiments, one or more single-stranded DNA breaks (nicks) are generated within the target sequence, upstream and / or downstream (e.g., using a nickase Cas9 variant). Any of these DNA breaks, as well as those introduced via other methods known to those skilled in the art, can induce HDR. By HDR, the target sequence is replaced by the sequence of a provided recombination template, which comprises a polynucleotide of interest, such as any one of SEQ ID NOs: 3-4 or 5-6, or a variant or fragment thereof, provided on / as a template. By designing this system, one or more single-stranded or double-stranded breaks are introduced into the interior, upstream and / or downstream of the corresponding region in the plant genome that does not comprise the target gene sequence, and the region can be replaced by a template.

[0258] In certain embodiments, the mutation in the target gene described herein can be produced via targeted introduction of DNA double-strand breaks without using a recombinant template. Such a break can be repaired by non-homologous end joining (NHEJ) process, which may result in a small insertion or deletion (indel) at the repair site. Such indels may result in frameshift mutations, thereby causing premature termination codons or other types of loss-of-function mutations in the targeted gene.

[0259] In some embodiments, gene editing may involve transient, inducible, or constitutive expression of a gene editing component or system in a target plant. Gene editing may also involve genomic integration or episomal presence of a gene editing component or system in a target plant.

[0260] In certain embodiments, nucleic acid modification or mutation is achieved by a (modified) zinc finger nuclease (ZFN) system. The ZFN system uses an artificial restriction endonuclease generated by fusing a zinc finger DNA binding domain with a DNA cleavage domain that can be engineered to target a desired DNA sequence. Exemplary methods for genome editing using ZFNs can be found, for example, in the following: U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; and 6,979,539.

[0261] In certain embodiments, nucleic acid modification is achieved by (modified) meganucleases, which are endodeoxyribonucleases characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs). Exemplary methods using meganucleases can be found in: U.S. Patent Nos. 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134, which are specifically incorporated by reference.

[0262] In certain embodiments, nucleic acid modification is achieved by a (modified) CRISPR / Cas complex or system. In certain embodiments, the CRISPR / Cas system or complex is a Class 2 CRISPR / Cas system. In certain embodiments, the CRISPR / Cas system or complex is a Type II, Type V, or Type VI CRISPR / Cas system or complex. Instead of generating customized proteins to target specific sequences, the CRISPR / Cas system can program a single Cas protein to recognize a specific nucleic acid target via an RNA guide sequence (gRNA). In other words, the short RNA guide sequence can be used to recruit the Cas enzyme protein to a specific nucleic acid target locus of interest (which may contain or consist of RNA and / or DNA).

[0263] Typically, CRISPR / Cas or CRISPR systems, as used herein, collectively refer to transcripts and other elements involved in the expression of CRISPR-associated ("Cas") genes or directing their activity, including sequences encoding Cas genes and one or more of the following: tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-pairing sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or one or more "RNAs" as used herein (e.g., one or more RNAs for guiding Cas such as Cas9, such as CRISPR RNA and, where applicable, trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from the CRISPR locus. Typically, a CRISPR system is characterized by elements (also referred to as protospacers in the context of endogenous CRISPR systems) that promote CRISPR complex formation at the site of the target sequence. In the context of forming a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes formation of a CRISPR complex. The target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide.

[0264] In certain embodiments, gRNA is a chimeric guide RNA or a single guide RNA (sgRNA). In certain embodiments, gRNA comprises a guide sequence and a tracr pairing sequence (or a repeat sequence in the same direction). In certain embodiments, gRNA comprises a guide sequence, a tracr pairing sequence (or a repeat sequence in the same direction) and a tracr sequence. In certain embodiments, CRISPR / Cas systems or complexes as described herein do not include and / or do not rely on the presence of a tracr sequence (e.g., if the Cas protein is Cas12a).

[0265] Cas proteins as mentioned herein, such as, but not limited to, Cas9, Cas12a (formerly known as Cpf1), Cas12b (formerly known as C2c1), Cas13a (formerly known as C2c2), C2c3, Cas13b proteins, may be derived from any suitable source and may therefore include different orthologs from a variety of (prokaryotic) organisms, as are well documented in the art. In certain embodiments, the Cas protein is a (modified) Cas9, preferably a (modified) Staphylococcus aureus Cas9 (SaCas9) or a (modified) Streptococcus pyogenes Cas9 (SpCas9). In certain embodiments, the Cas protein is Cas12a, optionally from an Acidaminococcus species, such as an Acidaminococcus species BV3L6 Cpf1 (AsCas12a), or a Lachnospiraceae Cas12a, such as a Lachnospiraceae bacterium MA2020 or a Lachnospiraceae bacterium MD2006 (LBCas12a). See U.S. Patent No. 10,669,540, which is incorporated herein by reference in its entirety. Alternatively, the Cas12a protein can be from Moraxella bovoculi AAX08_00205 [Mb2Cas12a] or Moraxella bovoculi AAX11_00205 [Mb3Cas12a]. See, WO2017 / 189308, which is incorporated herein by reference in its entirety. In certain embodiments, the Cas protein is (modified) C2c2, preferably Leptotrichia wadei C2c2 (LwC2c2) or Listeria newyorkensis FSL M6-0635 C2c2 (LbFSLC2c2). In certain embodiments, the (modified) Cas protein is C2c1. In certain embodiments, the (modified) Cas protein is C2c3. In certain embodiments, the (modified) Cas protein is Cas13b. Other Cas enzymes are available to those skilled in the art.

[0266] Gene editing methods and compositions are also disclosed in U.S. Patent Nos. 10,519,456 and 10,285,348 82, the entire contents of which are incorporated herein by reference.

[0267] The gene editing machine (for example, DNA modifying enzyme) introduced into the plant can be controlled by any promoter that can drive the recombinant gene to be expressed in the plant. In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is a tissue-specific promoter, such as a pollen-specific promoter or a sperm cell-specific promoter, a zygote-specific promoter, or a promoter (for example, prOsActin1) highly expressed in sperm, ovum and zygote. Suitable promoters are disclosed in U.S. Patent No. 10,519,456, the entire contents of which are incorporated herein by reference.

[0268] In another aspect, provided herein are methods for editing plant genomic DNA. In some embodiments, the method comprises pollinating a target plant comprising genomic DNA to be edited using a first soybean plant expressing a DNA modifying enzyme and at least one optional guide nucleic acid as described above.

[0269] 6. Overlay

[0270] As provided herein, polynucleotides encoding RG31 polypeptides and active variants and fragments thereof, or encoding RG35 polypeptides and active variants and fragments thereof, can be stacked with one or more polynucleotides encoding desired traits, such as polynucleotides that confer insect, disease, or herbicide resistance or other desired agronomic traits of interest, including but not limited to traits associated with high oil content; traits associated with increased protein content, increased digestibility; balanced amino acid content; improved drought resistance, altered maturity and / or flowering time, and high energy content. Such traits can refer to properties of seeds and non-seed plant tissues, or properties of food or feed prepared from plants or seeds having such traits.

[0271] As used herein, gene or proterties " stacking " comprises combining desired gene or proterties into transgenic plant lines. Other polynucleotides can be introduced by a variety of methods (including by transgenic means, by breeding or by genome editing). As a method, plant breeders hybridize between parents (each parent has desired proterties) and then identify the offspring with these two desired proterties to stack transgenic traits (so-called " breeding stacking "). Another way of stacking genes is to transfer two or more genes to the nucleus of the plant while converting. In an embodiment, two or more genes can be transferred via different expression cassettes or via a common expression cassette. Another way of stacking genes is to re-transform the transgenic plant comprising the desired proterties with another target gene that gives another desired proterties, thereby providing the progeny transgenic plant comprising a combination of proterties. Such methods can include, for example, random integration technology or the targeted integration performed via a gene editing system (such as Crispr or meganuclease). For example, gene stacking can be used to combine two different insect resistance traits, two different herbicide resistance traits, two different agronomic performance traits, an insect resistance trait with a disease resistance trait, a herbicide resistance trait (e.g., Bt11), or an agronomic performance trait. In addition to the target gene, the use of a selective marker is also considered to be gene stacking. In an embodiment, offspring or progeny plants with the desired trait combination are identified using genetic markers or molecular markers, including but not limited to SNPs, QTLs, primers or probes for desired trait-related genes or transgenics, promoters, microRNAs, siRNAs, mRNAs, dsRNAs, transcriptional profiles, and methylation patterns.

[0272] In some embodiments, the nucleic acid molecules or vectors of the present disclosure may include additional coding sequences for one or more polypeptides of interest or double-stranded RNA molecules (dsRNA) for agronomic traits, the main beneficiaries of which are seed companies, growers, or grain processors. The polypeptide of interest may be any polypeptide encoded by a nucleotide sequence of interest. Non-limiting examples of polypeptides of interest suitable for production in plants include those that produce agronomically important traits, such as herbicide resistance (sometimes also referred to as "herbicide tolerance"), disease resistance, virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, or fungal resistance. See, for example, U.S. Patent Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. The polypeptide can also be a trait that increases plant vigor or yield (including traits that allow plants to grow under different temperatures, soil conditions, and sunlight and precipitation levels), or a trait that allows identification of plants that exhibit the trait of interest (e.g., selectable markers, seed coat color, relative maturity groups, etc.). Various polypeptides of interest and methods of introducing these polypeptides into plants are described, for example, in U.S. Patent Nos. 4,761,373; 4,769,061; 4,810,648; 4,940,835; 4,975,374; 5,013,659; 5,162,602; 5,276,268; 5,304,730; 5,495,071; 5,554,798; 5,561,236; 5,569,823; 5,767,366; 5,879,903; 5,928,937; 6,084,155; 6,329,504 and 6,337,431; and in U.S. Patent Publication No. 2001 / 0016956.

[0273] In particular embodiments, polynucleotides can be stacked (or, alternatively, multiple expression cassettes can be stacked on a single polynucleotide) to express more than one polypeptide that increases disease resistance in a plant. This is particularly advantageous when, for example, one polypeptide is particularly well-suited for providing resistance to one class of plant pathogens (e.g., a first rust isolate), while another provides resistance to a different class of plant pathogens (or different fruiting isolates). For example, a first polypeptide encoding an RG31 or RG35 protein is stacked with a second polypeptide encoding another disease resistance protein. In alternative embodiments, a first polypeptide is provided that provides resistance to a plant pathogen (e.g., an ASR) via a first mode of action, while another polypeptide provides resistance to the same plant pathogen via a second, different mode of action. Stacking polypeptides encoded by different polypeptides is also an advantage in situations where one polypeptide expresses inherent pathogen resistance but is somewhat unstable.

[0274] Such additional disease resistance proteins include, but are not limited to, one or more of the various resistance proteins and / or polypeptides encoded by the various resistance genes set forth in WO 2019103918 (for example, but not limited to, RG1 disclosed as SEQ ID NO: 47, and active variants or fragments thereof, or any one of SEQ ID NOs: 42, 43, 44, 45, 46, 49 or 50 of WO 2019103918); WO 2021000878 (for example, Rpp6907 shown in SEQ ID NO: 1, and active variants or fragments thereof); WO 2021022022 (for example, TIRA shown in SEQ ID NO: 11, or TIRB shown in SEQ ID NO: 16, or active variants or fragments thereof); WO 2021260673 and / or WO 2021263249 (for example, RG21 shown in SEQ ID NO: 1, or RG21 shown in SEQ ID NO: 1, or RG21 shown in SEQ ID NO: 2). NO: 2, or an active variant or fragment thereof); WO2022173659 (for example, but not limited to, RG30 or an active variant or fragment thereof as shown in SEQ ID NO: 5); WO2022159341 (for example, but not limited to, SEQ ID NOs: 1 and 148 of WO 2022159341, or an active variant or fragment thereof); US11492637 (for example, but not limited to, RG1 as shown in SEQ ID NO: 47); US11530419 (for example, but not limited to, FIT1 as shown in SEQ ID NO: 2, or an active variant or fragment thereof, or any FIT1 paralog or ortholog disclosed in SEQ ID NOs: 4, 6, 8, 10, 12, 15, 16, 18 or 20); US10842097 (for example, but not limited to, CcRPP1 as shown in SEQ ID NO: 2, or an active variant or fragment thereof, or any FIT1 paralog or ortholog disclosed in SEQ ID NOs: NO: 4, 6 or 8); and / or WO 2022140257 (e.g., CcRpp2-R1Aa shown in SEQ ID NO: 2 therein or CcRpp2-R3Aa shown in SEQ ID NO: 4 therein, or an active variant or fragment thereof, or any of the CcRpp2-R1 or CcRpp2-R3 homologs, orthologs or paralogs disclosed in SEQ ID NOs: 21-36 and 48-58 therein); and / or a gene encoding a resistance protein disclosed as RG32 (SEQ ID NO: 1) or RG34 (SEQ ID NOs: 2, 17) in U.S. Provisional Application Nos. 63 / 426524 and 63 / 509586; each of these documents is incorporated by reference in its entirety.

[0275] In other embodiments, a nucleic acid sequence encoding an RG31 polypeptide or an RG35 polypeptide, or an active variant or fragment of any of these polypeptides, is stacked with two or more polynucleotides encoding resistance polypeptides that are desired to be co-expressed to increase disease resistance in a plant. In particular embodiments, an RG31 polypeptide or an RG35 polypeptide is stacked with each of an RG32 polypeptide (as set forth in U.S. Provisional Application No. 63 / 426,524 as SEQ ID NO: 1), or an active variant or fragment thereof, and an RG34 polypeptide (as set forth in U.S. Provisional Application No. 63 / 426,524 as SEQ ID NO: 2), or an active variant or fragment thereof, wherein co-expression of the RG32 and RG34 polypeptides is desired to increase plant resistance to ASR via a first mode of action that is different from the mode of action of the RG31 or RG35 polypeptide. In another particular embodiment, the RG31 polypeptide or the RG35 polypeptide is stacked with each of the TIRA polypeptide (as shown in WO2021022022 as SEQ ID NO: 11) or an active variant or fragment thereof, and the TIRB polypeptide (as shown in WO2021022022 as SEQ ID NO: 16) or an active variant or fragment thereof, wherein co-expression of the TIRA and TIRB polypeptides is required to increase the plant's resistance to ASR via a first mode of action that is different from the mode of action of the RG31 or RG35 polypeptide. In yet another particular embodiment, the RG31 polypeptide or the RG35 polypeptide is stacked with each of the CcRpp2-R1 polypeptide (as shown in WO 2022140257 as SEQ ID NO: 2) or an active variant or fragment or homolog or ortholog thereof, and the CcRpp2-R3 polypeptide (as shown in WO 2022140257 as SEQ ID NO: 4) or an active variant or fragment or homolog or ortholog thereof, wherein co-expression of the CcRpp2-R1 and CcRpp2-R3 polypeptides is required to increase the plant's resistance to ASR via a first mode of action that is different from the mode of action of the RG31 or RG35 polypeptide. Each of these references is incorporated herein by reference in its entirety.

[0276] In still other embodiments, a nucleic acid sequence encoding an RG31 polypeptide or an RG35 polypeptide, or an active variant or fragment of any of these polypeptides, is stacked with a fusion protein comprising two or more resistance polypeptides, or an active variant or fragment of the fusion protein, wherein co-expression of two or more resistance polypeptides is desired to increase disease resistance in a plant. In particular embodiments, an RG31 polypeptide or an RG35 polypeptide is stacked with a fusion protein comprising the TIRA and TIRB polypeptides of WO 2021022022, or an active variant or fragment thereof, such as a fusion protein set forth in SEQ ID NO: 9, 10, 11, 12, 17, 18, or 21 of U.S. Provisional Application No. 63 / 383609, or an active variant or fragment thereof, wherein the fusion protein comprising the TIRA and TIRB polypeptides is desired to increase plant resistance to ASR via a first mode of action that is different from the mode of action of the RG31 or RG35 polypeptide. In yet another specific embodiment, the RG31 polypeptide or RG35 polypeptide is superimposed with a fusion protein comprising the RG32 polypeptide and the RG34 polypeptide of U.S. Provisional Application No. 63 / 426524, or an active variant or fragment thereof, wherein the fusion protein comprising the Rg32 and RG34 polypeptides is required to increase the plant's resistance to ASR via a first mode of action different from the mode of action of the RG31 or RG35 polypeptide. In yet another specific embodiment, the RG31 polypeptide or RG35 polypeptide is superimposed with a fusion protein comprising the CcRpp2-R1 polypeptide and the CcRpp2-R3 polypeptide of WO 2022140257, or an active variant or fragment thereof, wherein the fusion protein comprising the CcRpp2-R1 and CcRpp2-R3 polypeptides is required to increase the plant's resistance to ASR via a first mode of action different from the mode of action of the RG31 or RG35 polypeptide. Each of these references is incorporated herein by reference in its entirety.

[0277] In other embodiments, the nucleic acid sequence encoding the RG31 polypeptide or RG35 polypeptide, or an active variant or fragment of any of these polypeptides, is stacked with a natural trait that confers disease resistance. In specific embodiments, the natural trait that confers disease resistance is a protein that confers increased resistance to ASR or to pathogens from the genus Psoralea (including species of Psora pachyrhizi and Psora argenteus). For example, the various intervals, loci or resistance genes set forth in WO 2009079729, US9091681, WO 2010009404, WO2017222827, WO 2021000878, WO 2021022026, WO 2021022101, WO 2021154632, WO2022173659 and PCT Application No. PCT / US23 / 60373 (each of which is incorporated by reference in its entirety) can be bred into soybean plants comprising an RG31 polypeptide or an RG35 polypeptide, or any active variant or fragment thereof.

[0278] Thus, in some embodiments, the RG31 protein or the RG35 protein can be deployed as a "natural construct" or "natural overlay". As used herein, a natural construct comprises a natural gene (i.e., a natural regulatory region and a natural coding region) found in nature and not artificially modified. Such a natural construct is integrated into a heterologous position of the genome (i.e., a position different from the natural position in the genome). In such cases, the natural construct or natural overlay of RG31 (e.g., SEQ ID NO: 3) or RG35 (e.g., SEQ ID NO: 5) is stably integrated into the genome by any method (including, for example, TIN or random integration). In some embodiments, the natural construct employed comprises at least the natural gene of RG31 or the natural gene of RG35 or a natural variant or fragment thereof and at least one additional natural gene of interest. Additional natural genes of interest may include, for example, the natural gene of any R gene disclosed herein or a natural variant thereof or any other natural gene of interest, including natural genes associated with any other pathogen resistance or agronomic performance.

[0279] When integrated via targeted insertion, the natural construct or natural stack can be deployed adjacent to other natural traits of interest and / or other heterologous traits of interest, such as herbicide tolerance traits or insect control traits disclosed herein. See, for example, the stacks of WO 2022040134, US 20220056470, and WO 2023164453, the contents of which are incorporated herein by reference in their entirety.

[0280] The polynucleotides that impart resistance / tolerance to herbicides (such as imidazolinones or sulfonylureas) that inhibit growing points or meristems may also be applicable in some embodiments. For example, exemplary polynucleotides in this classification number for mutant ALS and AHAS enzymes are described in, for example, U.S. Patent numbers 5,767,366 and 5,928,937. U.S. Patent numbers 4,761,373 and 5,013,659 relate to plants that resist different imidazolinones or sulfonylurea herbicides. U.S. Patent number 4,975,374 relates to plant cells and plants containing following nucleic acid, the nucleic acid encoding mutant glutamine synthetase (GS), the mutant glutamine synthetase is resistant to the inhibitory effect of known herbicides that inhibit GS (for example, phosphinothricin and methionine sulfoximine). U.S. Patent number 5,162,602 discloses plants that resist the inhibitory effect of cyclohexanedione and aryloxyphenoxypropionic acid herbicides. This resistance is conferred by an altered acetyl-CoA carboxylase (ACCase) enzyme.

[0281] Polypeptides encoded by nucleotide sequences that confer resistance to glyphosate are also suitable for use in the present disclosure. See, for example, U.S. Patent No. 4,940,835 and U.S. Patent No. 4,769,061. U.S. Patent No. 5,554,798 discloses transgenic maize plants resistant to glyphosate, the resistance being conferred by an altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase gene.

[0282] Polynucleotides encoding resistance to phosphoryl compounds such as glufosinate or phosphinothricin, pyridinyloxypropionic acid or phenoxypropionic acid, and cyclohexanone are also suitable. See European Patent Application No. 0 242 246. See also U.S. Patent Nos. 5,879,903, 5,276,268, and 5,561,236.

[0283] Other suitable polynucleotides include those encoding resistance to herbicides such as triazines and benzocyanate (nitrile hydrolases) that inhibit photosynthesis, see U.S. Patent number 4,810,648. Other suitable polynucleotides encoding for herbicide resistance include those encoding resistance to 2,2-dichloropropionic acid, sethoxydim, haloxyfop-ethyl, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides and bromoxynil. Also suitable are polynucleotides that confer resistance to the original enzyme, or polynucleotides that provide enhanced resistance to plant diseases; Tolerance to the enhancement of adverse environmental conditions (abiotic stress), the adverse environment including but not limited to drought, supercooling, overheating or soil salinity is too high or extremely acidic or alkaline; and changes in plant structure or development, including changes in development time. See, for example, U.S. Patent Publication No. 2001 / 0016956 and U.S. Patent No. 6,084,155.

[0284] Additional herbicide tolerance traits include PPO tolerance traits, including, for example, one or more PPO traits set forth in US 20190062777, US10370677, US11124803, WO 2017217793, WO 2020251313, US10392630, US10378023, WO 2016099153, WO2019117579, WO 2019117578, and US10100329, each of which is herein incorporated by reference in its entirety. HPPD tolerance traits include: WO 2009144079, US 8642748, EP 2453012, WO2013026740, US 9078446, US10793872, US10508089, US10400249, US10597674, WO2018119364, WO 2018119361, US11180770, US20200157086, US20210147866, US11279944, US202000331866, WO 2019227036, WO 2019227028, WO 2022115296, and WO 2011068567, each of which is incorporated herein by reference in its entirety. ACCase tolerance traits include: US20120284812, US20120284853, US20160108423, US20160244780, US20160264990, US20170275645, US20210153448, US10696975B2, US10370678, CN 109082416, US10694694, US20170265469, US20170231225, each of which is incorporated herein by reference. Mediben tolerance traits include, for example, RE45048 or US7884262. Various traits that confer tolerance to AOPP herbicides, phenoxy acid herbicides, and / or pyridyloxy acid herbicides include, for example, US10174337, US8278505, WO 05107437, WO 11022469, US10023874, and US2019241903 (and other traits therein), each of which is incorporated herein by reference. Additional herbicide tolerance traits of interest for stacking include the glucosyltransferase polypeptides described in 2018213022 or the solanyl diphosphate synthase polypeptides described in WO 2020236790, each of which is incorporated herein by reference in its entirety.

[0285] Other suitable polynucleotides include those encoding insecticidal polypeptides. These polypeptides can be produced in an amount sufficient to control, for example, insect pests (i.e., an insect control amount). It should be recognized that the production of insecticidal polypeptides necessary for controlling insects or other harmful organisms in plants can vary, depending on the type of cultivated variety, harmful organism, environmental factors, etc. Polynucleotides that can be used for other insect or harmful organism resistance include, for example, those encoding toxins identified in Bacillus (Bacillus) organisms. Polynucleotides comprising nucleotide sequences encoding Bacillus thuringiensis (Bt) Cry proteins from several subspecies have been cloned, and it has been found that these recombinant clones are toxic to Lepidoptera, Diptera, and / or Coleopteran insect larvae. Examples of such Bt insecticidal proteins include Cry proteins such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9B, Cry9C, etc., and vegetative insecticidal proteins such as Vip1, Vip2, Vip3, etc. A complete list of Bt-derived proteins can be found on the World Wide Web in the Bacillus thuringiensis Toxin Nomenclature Database maintained by the University of Sussex (see also Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813).

[0286] In embodiments, the additional polypeptide is an insecticidal polypeptide derived from a non-Bt source, including but not limited to: alpha amylase, peroxidase, cholesterol oxidase, potato glycoprotein, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, Xenorhabdus species (such as X. nematophila or X. bovienii) insecticidal protein, Photorhabdus species (such as P. luminescens or P. asymobiotica) insecticidal protein, Brevibacillus species (such as B. laterosporous) insecticidal protein, Lysinibacillus species (such as B. spp.) (such as spherical lysine Bacillus (L. Sphearicus) insecticidal proteins, Chromobacterium species (such as C. subtsugae or C. piscinae) insecticidal proteins, Yersinia species (such as Yersinia entomophage (Y. entomophaga)) insecticidal proteins, Paenibacillus species (such as Gamma-type Paenibacillus species (P. propylaea)) insecticidal proteins, Clostridium species (such as C. bifermentans) insecticidal proteins, Pseudomonas species (Pseudomonas spp.) (such as Pseudomonas fluorescens (P. fluorescens) insecticidal proteins and lignin.

[0287] In certain embodiments, the additional polypeptide is a resistance protein that confers enhanced pathogen resistance, such as enhanced resistance to any of the following pathogens: soybean cyst nematode, bacterial pustule, root knot nematode, soybean gray spot, Phytophthora, brown stem rot, nematode, Asian soybean rust, powdery mildew, Golovinomyces cichoracearum, Erysiphe cichoracearum, Blumeria graminis, Podosphaera xanthii, Sphaerotheca fuliginea, Pythium ultimum, Uncinula necator, Mycosphaerella pinodes, Magnaporthe grisea, Bipolaris oryzae, Rice blast, Rhizoctonia solani, solani), Phytophthora sojae, Schizaphis graminum, Bemisia tabaci, Rhopalosiphum maidis, Deroceras reticulatum, Diatraea saccharalis, Schizaphis graminum, Myzus persicae, Sclerotinia sclerotiorum, Macrophomina phaseolina, Fusarium virguliforme, piercing-sucking pests (e.g., aphids, stink bugs, and whiteflies), or bacterial pathogens (including Pseudomonas aeruginosa). Exemplary polynucleotides encoding proteins that confer increased pathogen resistance that can be stacked with the polynucleotides of the present invention include polynucleotides encoding proteins that confer increased ASR resistance as described in: U.S. Patent Publication No. US20200354739 and PCT Publication Nos. WO 2019103918, WO 2021154632A1, WO 2021022022, WO 2021022026, WO 2021022101, WO2021260673, and WO 2021263249, each of which is incorporated by reference in its entirety.

[0288] Disease resistance proteins can enhance resistance to various plant diseases, including rust, and include, but are not limited to, one or more resistance genes listed in WO 2019103918; WO 202100878; WO 2021022022; WO 2021260673; WO 2022173659; WO 2022159341; WO 2021154632A1; WO 2021022026; WO 2021022101; US ​​20220135997; US 10842097; or WO 2022140257; each of which is incorporated by reference in its entirety. In other embodiments, nucleic acid sequences encoding RG32 and / or RG34 polypeptides, or active variants or fragments thereof, are stacked with natural traits that confer disease resistance. For example, various intervals, loci or resistance genes as shown in WO 2009079729, US9091681, WO 2010009404, WO2017222827, WO 2021000878, WO 2021022026, WO 2021022101, WO 2021154632, WO2022173659 (each of which is incorporated by reference in its entirety) can be used to introduce traits of interest into soybean. Disease resistance proteins and / or natural traits that increase resistance to various plant diseases, including Northern Corn Leaf Blight (NCLB), include, for example, US 8921646, US2021000059, US10858668, US20200199610, WO 2022 / 013268, WO 2022 / 013268, US 9040772, US10897862, EP 3839073, each of which is incorporated herein by reference.

[0289] Polypeptides suitable for production in plants further include those that improve or otherwise aid in the conversion of harvested plants or plant parts into commercially useful products, including, for example, increased or altered carbohydrate content or distribution, improved fermentation characteristics, increased oil content, increased protein content, improved digestibility, and increased nutrient content (e.g., increased phytosterol content, increased tocopherol content, increased stanol content, or increased vitamin content). Polypeptides of interest also include, for example, those that result in or contribute to a reduction in the level of undesirable components (e.g., phytic acid, or enzymes that degrade sugars) in the harvested crop. "Resulting in" or "contributing to" means that the polypeptide of interest can directly or indirectly contribute to the presence of the trait of interest (e.g., increased cellulose degradation through the use of heterologous cellulases).

[0290] In certain embodiments, the polypeptide contributes to the improved digestibility of food or feed. Xylanases are hemicellulolytic enzymes that improve the decomposition of plant cell walls, which results in animals making better use of these plant nutrients. This results in improved growth rates and feed conversion. Similarly, the viscosity of feeds containing xylanases can be reduced. Heterogeneous production of xylanases in plant cells can also promote the conversion of lignocellulose into fermentable sugars in industrial processing.

[0291] Many xylanases from fungal and bacterial microorganisms have been identified and characterized (see, e.g., U.S. Pat. No. 5,437,992; Coughlin et al. (1993) "Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases" Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial Fermentation Research 8:125-135; U.S. Pat. Pub. No. 2005 / 0208178; and PCT Pub. No. WO 03 / 16654). In particular, three specific xylanases (XYL-I, XYL-II, and XYL-III) have been identified in Trichoderma reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14:566; Torronen et al. (1992) Bio / Technology 10:1461; and Xu et al. (1998) Appl. Microbiol. Biotechnol. 49:718).

[0292] In other embodiments, the polypeptides useful for the present disclosure may be polysaccharide degrading enzymes. Plants of the present disclosure that produce such enzymes may be useful for producing fermentation feedstocks, for example, for bioprocessing. In some embodiments, enzymes useful in fermentation processes include alpha-amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycosyltransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolases, and other glucoamylases.

[0293] Polysaccharide degrading enzymes include: starch degrading enzymes such as α-amylase (EC 3.2.1.1), glucuronidase (EC 3.2.1.131); exo-1,4-α-D glucanases such as amyloglucosidase and glucoamylase (EC 3.2.1.3), β-amylase (EC 3.2.1.2), α-glucosidase (EC 3.2.1.20) and other exoamylases; starch debranching enzymes such as a) isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41), etc.; b) cellulase such as exo-1,4-3-cellobiohydrolase (EC 3.2.1.91), exo-1,3-β-D-glucanase (EC 3.2.1.39), β-glucosidase (EC 3.2.1.21); c) L-arabinase (arabinase), such as endo-1,5-α-L-arabinase (EC 3.2.1.99), α-arabinosidase (EC 3.2.1.55), etc.; d) galactanase, such as endo-1,4-β-D-galactanase (EC 3.2.1.89), endo-1,3-β-D-galactanase (EC 3.2.1.90), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), etc.; e) mannanase, such as endo-1,4-β-D-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), α-mannosidase (EC 3.2.1.24), etc.; f) xylanase, such as endo-1,4-β-xylanase (EC 3.2.1.8), β-D-xylosidase (EC 3.2.1.37), 1,3-β-D-xylanase, etc.; and g) other enzymes such as α-L-fucosidase (EC 3.2.1.51), α-L-rhamnosidase (EC 3.2.1.40), levanase (EC 3.2.1.65), inulinase (EC 3.2.1.7), etc. In one embodiment, the α-amylase is the synthetic α-amylase Amy797E described in U.S. Patent No. 8,093,453 (incorporated herein by reference in its entirety).

[0294] Other enzymes that can be used together with the present disclosure include proteases, such as fungi and bacterial proteases. Fungal proteases include but are not limited to those obtained from Aspergillus (Aspergillus), Trichoderma (Trichoderma), Mucor (Mucor) and Rhizopus (Rhizopus), such as Aspergillus niger (A.niger), Aspergillus awamori (A.awamori), Aspergillus oryzae (A.oryzae) and Mucor miehei (M.miehei). In certain embodiments, the polypeptide of the present disclosure can be a cellobiohydrolase (CBH) (EC3.2.1.91). In one embodiment, the cellobiohydrolase can be CBH1 or CBH2.

[0295] Other enzymes useful in the present disclosure include, but are not limited to, hemicellulases, such as mannanases and arabinofuranosidases (EC 3.2.1.55); ligninases; lipases (e.g., EC 3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, α1,6 glucosidases (e.g., EC 3.2.1.20); esterases, such as feruloyl esterases (EC 3.1.1.73) and acetylxylan esterases (EC 3.1.1.72); and cutinases (e.g., EC 3.1.1.74).

[0296] In other embodiments, the polynucleotides provided herein are stacked with polynucleotides that increase protein content and / or alter seed composition and / or fatty acid content. Such sequences include, but are not limited to, those disclosed in PCT Application Nos. PCT / CN2022 / 075977 and PCT Application Nos. PCT / CN2022 / 075982, both filed February 11, 2022, WO2021 / 044027, which discloses various amino acid permease (AAP) polypeptides from soybean and various other plants, including, for example, KJN37208 (soybean AAP8 polypeptide), XP XP_003526513 (soybean AAP8 polypeptide), NP_001242816LOC100777963 (soybean AAP polypeptide), and AA XP_028228300 (soybean AAP6-like polypeptide); US2020 / 0131524, which discloses various UPL3 polypeptides from various plants, including, for example, SEQ ID NO: 1131524. NO: 28) and the various UPL3 polypeptide homologs listed in Table 1; and US2021 / 0403933, which discloses various HECT E3 ligases (including HEL1 and HEL2) and other polypeptides of interest and gene editing, each of which is incorporated herein by reference in its entirety.

[0297] Double-stranded RNA molecules useful to this disclosure include but are not limited to those that inhibit target genes. As used herein, the term "gene inhibition" is intended to refer to any well-known method for reducing the level of protein produced as a result of gene transcription into mRNA and the subsequent translation of the mRNA when considered together. Gene inhibition is also intended to mean reducing the expression of protein from a gene or coding sequence, including post-transcriptional gene inhibition and transcriptional inhibition. Post-transcriptional gene inhibition is mediated by homology between all or a portion of the mRNA transcribed from the gene or coding sequence that is suppressed by targeting and the corresponding double-stranded RNA for inhibition, and refers to the substantial and measurable reduction of the amount of available mRNA available for ribosome binding in the cell. Transcribed RNA can be in the sense direction and play a role, referred to as co-suppression, in the antisense direction and play a role, referred to as antisense inhibition, or produce dsRNA in both directions and play a role, referred to as RNA interference (RNAi). Transcriptional inhibition is mediated by the presence in the cell of a dsRNA that exhibits substantial sequence identity to a promoter DNA sequence or its complement as a gene inhibitor, referred to as promoter trans inhibition. Gene suppression can be effective for the natural plant genes associated with traits, for example, to provide a plant with a reduced level of protein encoded by the natural gene or with an enhanced or reduced level of affected metabolites. Gene suppression can also be effective for target genes in plant pests, which can be taken in or contacted with plant materials containing gene inhibitors designed specifically to suppress or inhibit the expression of one or more homologous or complementary sequences in the cells of the pest. Such genes targeted for suppression can encode essential proteins whose predicted functions are selected from the group consisting of: muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, digestive enzyme synthesis, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antenna formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration and apoptosis.

[0298] As used herein, "selectable marker" means a nucleotide sequence that, when expressed, confers a different phenotype to a plant, plant part, and / or plant cell expressing the marker, and thus allows such transformed plants, plant parts, and / or plant cells to be distinguished from those that do not possess the marker. Such a nucleotide sequence can encode a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for chemically, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or whether the marker is simply a trait that one can identify by observation or testing, such as by screening. In some examples, traits can be identified by visual observation, such as by comparing plant height, plant vigor, or plant flowering time of plants having the desired combination of traits to control plants (e.g., plants not comprising the combination of traits or the parent plant from which the selected plants were derived).

[0299] 7. Assays, kits, and primers

[0300] Also provided are kits, probes, primers, and antibodies that can be used to introduce polynucleotide sequences as described herein into recipient plants or to detect polynucleotide or polypeptide sequences as described herein in plants. The polypeptides and polynucleotides provided herein, or variants and fragments thereof, can be packaged as components of kits along with instructions for performing the assays described herein. DNA detection kits are provided for detecting nucleotide sequences encoding RG31 or RG35 polypeptides, or variants and fragments thereof, or for detecting transgenic events or gene editing in plants that contain RG31 or RG35 polypeptides.

[0301] In some embodiments, the kit may include one or more probes having a sequence corresponding to a sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a specific region of the nucleotide sequence shown in any one of SEQ ID NOs: 3-6 or 21-22, or a sequence complementary thereto, thereby allowing detection of the sequence. In some embodiments, the kit may include any reagents and materials required for performing the assay or detection method. In particular embodiments, the probe can be used to specifically hybridize to a target polynucleotide, thereby detecting a nucleotide sequence shown in any one of SEQ ID NOs: 3-6 or 21-22, or a variant or fragment thereof.

[0302] Further provided are antibodies against the polypeptides of the present invention or variants or fragments thereof. Methods for generating antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; and U.S. Patent No. 4,196,265). These antibodies can be used in kits for detecting and isolating toxin polypeptides. Thus, the present disclosure provides kits comprising antibodies that specifically bind to polypeptides described herein, including, for example, polypeptides having a sequence of SEQ ID NO: 1 or 2, or variants or fragments thereof.

[0303] 8. How to use plants and seeds

[0304] Methods for preventing pathogen damage, disease damage, or ASR damage in plants are provided. Also provided are methods for controlling disease or ASR in a cultivation area. Such methods comprise planting a plant or seed having a nucleotide sequence encoding an RG31 or RG35 polypeptide, or an active variant or fragment thereof, stably integrated into its genome in the cultivation area, and cultivating the plant or seed, wherein expression of the RG31 or RG35 polypeptide, or an active variant or fragment thereof, increases the plant's resistance to plant diseases and / or plant pathogens.

[0305] The various compositions and methods disclosed herein can provide increased or enhanced resistance to various plant pathogens and / or plant diseases. Exemplary plant pathogens and / or plant diseases include, but are not limited to, soybean cyst nematode, bacterial pustule, root knot nematode, soybean gray spot, Phytophthora, brown stem rot, nematodes, Asian soybean rust, powdery mildew, Golovinomyces cichoracearum, Erysiphe cichoracearum, Blumeria graminis, Podosphaera xanthii, Sphaerotheca fuliginea, Pythium ultimum, Uncinula necator, Mycosphaerella pinodes, Magnaporthe grisea, Bipolaris oryzae, Rice blast, Rhizoctonia solani, Phytophthora sojae, Schizaphis graminum), Bemisia tabaci, Rhopalosiphum maidis, Deroceras reticulatum, Diatraea saccharalis, Schizaphis graminum, Myzus persicae, Sclerotinia sclerotiorum, Macrophomina phaseolina, or Fusarium virguliforme.

[0306] In certain embodiments, the plant disease is Asian soybean rust. In other embodiments, the plant pathogen is from the genus Phakopsora, including Psora pachyrhizi and Psora argenteus species, known to cause ASR in plants.

[0307] In other embodiments, plants expressing RG31 or RG35 polypeptides or active variants or fragments thereof are contacted with one or more fungicides to further prevent ASR-related damage or any other plant disease of interest in the plant (i.e., legume or soybean). Such fungicides can be applied to any part of the plant, including, for example, seeds or leaves or the area under cultivation. Such fungicide compounds can be formulated or mixed with other fungicides, or applied sequentially with other fungicides. Such fungicides can include fungicides from one or more of the following chemical classes: benzimidazoles, dicarboximides, azoles, pyrimidines, benzamides, morpholines, carboxamides, anilinopyrimidines, methoxyacrylates, carboxamides, inorganics, dithiocarbamates, or phthalimides. See Morton, V. and Staub, T. 2008 A Short History of Fungicides. Online, APSnet Features. doi: 10.1094 / APSnetFeature-2008-0308, incorporated herein by reference.

[0308] The invention provides a method for screening or measuring the resistance of plants (legumes or soybean plants) to determine the immunity or susceptibility of plants to plant diseases. Such methods include but are not limited to screening plants (legumes or soybean plants), measuring the immunity, resistance or susceptibility of plants to plant diseases (such as pachyrhizoma pachyrhizoma, pachyrhizoma spp.) by contacting plant cells, tissues or organs with pathogens and measuring the resistance, immunity or susceptibility of plants or plant parts to plant diseases (such as ASR) caused by pathogens. Any observable phenotype of plant pathogens and / or diseases can be measured. These include but are not limited to the number, size and / or density of disease-related lesions, the color (such as, from tan to reddish-brown color range) of lesions, the number and density of pustule formation, sporulation, cyst formation, yield loss or its any combination. Other embodiments include the change of any of the above-mentioned phenotypes. Still other embodiments include delaying or accelerating the proliferation of measurement pathogens (such as fungi).

[0309] 9. Methods and compositions for generating resistance and / or virulence traits in plant pathogens in areas of cultivation.

[0310] Currently, resistance management of fungi in crops involves applying a combination of fungicides with different modes of action and / or applying ASR genetic resistance traits throughout the crop, based on the availability of products for controlling specific pathogens. General knowledge and general advice on the presence of pathogen resistance in a region or country are currently provided, however, this only provides general information about the presence of resistance and advice on how to deal with resistance. The examples disclosed herein provide methods for how to improve and adjust resistance management for specific situations. This can be achieved by determining the resistance and / or virulence characteristics of plant pathogens through the qualitative and quantitative pesticide (e.g., fungicide) sensitivity of plant pathogens to specific pesticides and / or the qualitative and quantitative toxicity of plant pathogens to crops in a specific location. This can be determined quickly (e.g., near real time or within 48 hours), as explained in more detail below. By understanding the genetic variation of plant pathogen populations and their corresponding number or frequency in a specific location and at a given time, more targeted treatment programs or disease control measures can be provided within the season that also takes into account the development of resistance of plant pathogens. It can also be combined with recommendations for optimal or specific resistant crops. This will allow for more sustainable use of available pathogen management tools and avoid ineffective solutions tailored to that specific location. Furthermore, it could help improve agronomic practices to provide optimal control of pathogens and more rational resistance management strategies.

[0311] Methods and compositions for generating resistance and / or virulence traits of plant pathogens within a cultivated area are provided. The resistance and / or virulence traits of a cultivated area can be used to determine recommended pesticide application protocols to control the development, reproduction, and / or viability of ASR plant pathogen populations within the cultivated area and / or adjacent cultivated areas.

[0312] a. Resistance and / or virulence traits

[0313] Methods and compositions are provided for generating resistance and / or virulence traits against plant pathogens (eg, ASR plant pathogens) in areas of cultivation.

[0314] As used herein, "sensitivity" refers to the susceptibility of an ASR plant pathogen to an ASR pesticide (e.g., an ASR fungicide) or an ASR genetic resistance trait. Variation in sensitivity to ASR pesticides and / or ASR genetic resistance traits may be caused by a range of different mechanisms.

[0315] "Resistance" refers to the ability of an ASR plant pathogen to survive exposure to an ASR pesticide (including a fungicide) intended to control it or through exposure to an ASR genetic resistance trait. Several mechanisms can shape the level of resistance or adaptation. In contrast to sensitivity, resistance of an ASR plant pathogen means that the ASR plant pathogen or a population of ASR plant pathogens becomes less sensitive to the extent that the pesticide (e.g., fungicide) or ASR genetic resistance trait is no longer effective in controlling the development, reproduction, and viability of the ASR plant pathogen and / or the ASR plant pathogen population.

[0316] Resistance development refers to the development of reduced susceptibility of an ASR plant pathogen and / or ASR plant pathogen population to an ASR pesticide and / or ASR genetic resistance trait. Reduced susceptibility can lead to complete resistance or progressive resistance to a fungicide or ASR genetic resistance trait. A single mechanism or a combination of mechanisms can determine resistance development. Resistance development depends on the rate at which these mechanisms are selected across a population over time and space.

[0317] As used herein, resistance management programs are understood to be measures that can be taken to minimize the development of resistance in ASR pathogens and / or ASR pathogen populations to plant protection products (e.g. pesticides or ASR genetic resistance traits).

[0318] As used herein, a resistance trait refers to the ability of an ASR plant pathogen or a population of ASR plant pathogens to overcome and survive exposure to an ASR genetic resistance trait as defined by FRAC, a pesticide, a fungicide, a class of pesticides or a class of fungicides, or other chemical or biological agents used to control the development, viability and reproduction of ASR plant pathogens.

[0319] As used herein, virulence traits refer to the ability of an ASR plant pathogen or a population of ASR plant pathogens to infect and cause damage to a host, where the host may have some intrinsic resistance to the pathogen (ie, an ASR genetic resistance trait).

[0320] b. Methods for generating resistance and / or virulence traits to ASR plant pathogens

[0321] Various methods can be used to characterize resistance and / or virulence against an ASR phytopathogen population within a cultivation area. In one non-limiting embodiment, the frequency of presence of one or more ASR phytopathogen effectors from an ASR phytopathogen sample is obtained from locations within the cultivation area. In other embodiments, the frequency of presence of one or more ASR phytopathogen effectors is obtained from multiple locations within the cultivation area, and a frequency map is generated for the entire cultivation area. In other cases, the average frequency of presence of one or more ASR phytopathogen effectors is determined for the entire cultivation area.

[0322] i. Obtain samples and information demonstrating the susceptibility of plants under cultivation to ASR plant pathogens

[0323] The frequencies of the ASR effectors of interest and the ASR plant pathogen population can be obtained from an ASR plant pathogen sample collected from a single location within the cultivation area, or the information can be obtained from multiple locations within the cultivation area, such that there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or more samples within the cultivation area. The geographical location, the size of the cultivation area, and the disease pressure in the area will all influence the exact number of samples collected and the exact locations within the cultivation area from which the samples are obtained. In addition, the choice of location within the cultivation area for collecting ASR plant pathogens may be influenced by previously obtained disease pressure data. In certain embodiments, the resistance traits and / or virulence traits are generated from an ASR plant pathogen sample obtained from a single location within the cultivation area. In other embodiments, the resistance traits and / or virulence traits are generated from information obtained from multiple ASR plant pathogen samples collected from more than one location within the cultivation area.

[0324] A cultivation area can be any location where plants grow, including farmland, cultivated land, and protected areas (e.g., greenhouses).

[0325] A "sample" can be obtained by collecting at least some plant material or at least one environmental sample (air or soil sample) from the cultivation area. Thus, the sample can be a field sample (e.g., a leaf, several leaves, and / or other parts of a crop) collected from a crop or field or an alternative host plant (e.g., a crop blight-type form or a volunteer crop). Additionally or alternatively, the sample can be an environmental sample comprising soil and / or above-ground material collected from where a single crop is grown, or collected from several points in the cultivation area. In one example, a "sample" is to be understood as a sample of multiple plants, spore traps, or soil. The sample comprises at least one ASR plant pathogen and is to be understood as being representative of a local population of at least one plant pathogen.

[0326] In some embodiments, the cultivation area may contain plants and / or the collected samples are from plants or any part of plants. Plants within the cultivation area include any plant type including leguminous plants. Examples are listed elsewhere herein. The frequency of the presence of ASR plant pathogen effectors in ASR plant pathogen samples can be obtained via a variety of methods, including by analyzing proteomic information (protein expression / amino acid sequence), transcriptional information or genomic DNA information. Such information can be obtained by various biological materials and methods. As used herein, "biological material" means any material from an ASR plant pathogen that allows the frequency of one or more target ASR plant pathogen effectors to be determined. For example, the biological material may comprise a polynucleotide. The polynucleotide may comprise DNA (genomic DNA, a regulatory region affecting the expression of the target ASR effector or a variant or fragment thereof, an intron or coding region of the target ASR effector or a variant or fragment thereof, or a SNP or haplotype associated with the target ASR effector) or RNA encoding the target ASR effector. The polynucleotides within the biological material can be amplified and detected using a variety of techniques employed in the art. In other embodiments, the biological material comprises a polypeptide encoding the target ASR effector or an active variant or fragment thereof.

[0327] For example, the amino acid sequence of a given ASR effector or an active variant or fragment thereof can be detected by an antibody assay that specifically detects the ASR effector polypeptide of interest. The use of proteomics or gene expression to generate specific proteins can be employed to determine the frequency of a given protein within a population of ASR pathogens by detecting a single protein of interest or multiple differentially expressed proteins.

[0328] Genotypic information can be obtained by isolating, amplifying, and / or sequencing genomic DNA from a population of ASR plant pathogens in a sample and then detecting genomic DNA corresponding to the ASR effector sequence of interest (e.g., including haplotypes or SNPs). Alternatively, the transcriptome can be assayed to determine the frequency of ASR effectors in an ASR plant pathogen sample.

[0329] The test kit provided can detect a specific group of target ASR effector proteins via a variety of different diagnostic methods (including DNA detection, RNA detection and protein detection). Such a test kit may include a set of nucleic acid probes and / or primers, each probe and / or primer comprising a nucleotide sequence that specifically hybridizes with a nucleotide sequence encoding the target ASR effector protein. In other embodiments, the test kit includes antibodies that specifically recognize the target ASR effector polypeptide. In some embodiments, these test kits are used in a field environment. See, for example, Shin et al. (2015) Vector Ecology [Vector Ecology] 41: 1, 63-71, detecting specific resistance markers, and Tian et al. (2018) Scientific Rep 8 [Scientific Report 8], No. 12587, identifying pest resistance associated with increased detoxification gene expression.

[0330] Examples of nucleotide sequences and antibodies that can detect various ASR effector proteins or DNA or RNA sequences encoding ASR effector proteins can be found, for example, in Krasileva et al. (2010) Plant Cell 22:2444-2458, Activation of an Arabidopsis resistance protein is specified by the in planta association of its leucine-rich repeat domain with the cognateoomycete effector, and Gupta et al. (2023) Nature Communications 14, article 1835, Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi.

[0331] ii. ASR genetic resistance traits; ASR plant pathogen effectors; and detection methods

[0332] 1. ASR plant pathogen effectors

[0333] ASR plant pathogen effectors comprise polypeptides produced by ASR pathogens that can suppress the defense mechanisms of plant hosts and promote infection of plants by ASR pathogens. The frequency of any ASR plant pathogen effector can be detected by the methods provided herein. In some embodiments, the frequency of one or more of the following ASR effector proteins in an ASR plant pathogen population is determined: SEQ ID NO: 16-17 of effector protein SPE35, or active variants or fragments thereof; and SEQ ID NO: 23-24 of effector protein SPE38, or active variants or fragments thereof. The nucleotide (NT) sequence and protein (AA) sequence SEQ ID NO of each of these ASR effector proteins are given in Table B.

[0334] Table B. Effector sequences

[0335] effector SEQ ID SPE-35NT 17 SPE-35AA 16 SPE-38NT 24 SPE-38AA 23

[0336] 2. ASR genetic resistance traits

[0337] ASR genetic resistance traits include any trait (natural, gene-edited, or transgenic) that is stably integrated into the genome of a plant (particularly a legume or soybean plant) and whose expression enhances the plant's disease resistance to ASR plant pathogens. ASR genetic resistance traits include R genes or disease resistance polypeptides that encode proteins that recognize (directly or indirectly) ASR effector proteins. The specific protein encoded by the R gene or disease resistance polypeptide will recognize (directly or indirectly) a specific ASR effector protein or a specific combination of ASR effector proteins.

[0338] In a specific embodiment of the method provided herein, the frequency of the determined ASR pathogen effectors will vary depending on the plants planted in the cultivation area. For example, the frequency of the presence of a given ASR effector in an ASR pathogen population can be determined based on the ASR genetic resistance traits in legumes (e.g., soybean plants) within the cultivation area. Table C provides a summary of ASR genetic resistance traits and their corresponding ASR effectors. As used herein, a "corresponding" ASR effector protein or an active variant or fragment thereof comprises an ASR effector protein that interacts (directly or indirectly) with the ASR genetic resistance trait, whereby the interaction (directly or indirectly) increases the tolerance of the plant (soybean plant or legume) to the ASR pathogen.

[0339] Table C. Summary of ASR genetic resistance traits and corresponding ASR effectors

[0340]

[0341] A given legume or soybean plant or seed within a cultivation region may contain one or more ASR resistance proteins or active variants or fragments thereof as shown in Table C. Thus, the frequency of one or more ASR effector proteins or active variants or fragments thereof corresponding to a given ASR genetic resistance trait will be determined to generate resistance and / or virulence traits for the cultivation region.

[0342] In one embodiment, wherein the cultivated area comprises a plant comprising a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (RG31) and SEQ ID NO: 2 or 20 (RG35), or an active variant or active fragment thereof, wherein expression of the polypeptide or active variant or fragment thereof increases the plant's tolerance to an ASR plant pathogen; the frequency of one or more of the corresponding ASR effectors SPE-35 (SEQ ID NO: 16-17) and / or SPE-38 (SEQ ID NO: 23-24), or active variants or fragments thereof, in the sample and / or in the cultivated area can be determined. As discussed elsewhere herein, the frequency of the effector can be determined at the polynucleotide or polypeptide level.

[0343] In certain embodiments, the polynucleotide encoding the ASR genetic resistance trait is heterologous to the plant.

[0344] 3. Detection Methods

[0345] The frequency of an ASR effector polypeptide or active variant or fragment thereof in a sample from an area of ​​cultivation can be determined via detection of a polypeptide or polypeptides discussed elsewhere herein, including through the use of primers, probes, PCR, and the like.

[0346] In one embodiment, a PCR reaction is used to determine the frequency of an ASR effector polypeptide or its active variant or fragment within an ASR plant pathogen population in a cultivation area. In other embodiments, stringent hybridization conditions can be used when detecting a polynucleotide of interest (i.e., a polynucleotide of an ASR effective protein). A PCR reaction is used to determine the frequency of an ASR effector polypeptide or its active variant or fragment within an ASR plant pathogen population in a cultivation area. Such methods are described later herein. Such methods are described later herein.

[0347] In other embodiments, DNA sequencing is performed on the biological material to determine the presence of a given ASR effector. As used herein, genetic variation, genetic variation, or mutation of a plant pathogen is understood to be a mutation in a DNA sequence that differs from the wild-type sequence. For example, in a genetic variation, at least one nucleotide in the DNA sequence has been permanently changed, deleted, or inserted.

[0348] A genotype is a unique combination of multiple genetic variations, gene variants, or variations in a plant pathogen. If multiple independent genetic variations can be grouped into different unique combinations, each of these unique combinations can express different susceptibility or virulence traits.

[0349] In the embodiment of the nucleic acid sequence in the sample to which biological material is carried out DNA sequencing, the operation can include using a sequencer that can sequence at least 200, 300, 400 or 500 base pairs in a single read. For example, the PacBio Sequel system of the implementation single molecule real-time sequencing provided by Oxford Nanopore sequencing technology (such as MinION, GridION or PromethION) or provided by PACBIO company can provide required ability. These technologies can be referred to as third generation sequencers, and they provide the high throughput of the larger sequencing genetic region combination from hundreds of base pairs to 10,000 base pairs. The technology provided by Oxford Nanopore Technologies (Oxford Nanopore Technologies) includes flow cells, which contain micropore arrays called nanopores embedded in resistive membranes. Each nanopore corresponds to its own electrode connected to channel and sensor chip, and the electrode measures the current flowing through the nanopore. When a molecule passes through the nanopore, the electric current is disturbed to produce characteristic "curve" or current intensity value. The curve is then decoded using base recognition (base calling) algorithm to determine DNA. Base calling is the computational process of translating a curve into a DNA sequence. Specific bioinformatics pipelines can be combined to quantify one or more ASR effector peptides in a given ASR pathogen population.

[0350] Before the sample is subjected to DNA sequencing, DNA can be extracted from the received sample. The DNA sample can include multiple individuals from plant pathogenic species representing genetic variations of the disease. In one example, a specific set of multiplex PCR is designed to amplify multiple targets in a single PCR reaction. This optimization step requires identification of non-self-hybridizing primers in conserved regions to cope with the natural variability between the individuals making up the population. Through this optimization, genetic loci containing one or more target ASR effector polypeptides can be amplified from the largest proportion of strains in the natural population. Primers can also be checked to ensure that other plant pathogens or other ASR effector proteins that may occur in the same area will not be non-specifically amplified. Thereafter, if multiple samples are merged before sequencing the plant pathogen, DNA barcoding can be performed to identify the specific sample. Thereafter, sequencing preparation can be performed, and then the DNA sequencing operation can be performed using a third-generation sequencer as described above.

[0351] It should be understood that the present disclosure is not limited to the specific third generation sequencers as described herein, but may be applied to any sequencing technology that generates data within a timeframe that gives current insights into plant diseases.

[0352] Referring now again to the procedure of determining the presence of genetic polymorphisms based on DNA sequencing, this procedure may include comparing the DNA sequence of the sample with reference DNA sequences of plant pathogens and identifying genetic polymorphisms that are unique to a given ASR effector of interest.

[0353] Antibodies can be used to detect polypeptides. Techniques for using antibodies to detect polypeptides include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Antibodies can be polyclonal or monoclonal. Antibodies with specific binding affinity for the polypeptide can be produced using methods well known in the art. Antibodies can be attached to a solid support, such as a microtiter plate, using methods known in the art. In the presence of the polypeptide, an antibody-polypeptide complex is formed.

[0354] Detection (e.g., of an amplification product, hybridization complex, or polypeptide) is typically accomplished using a detectable label. The term "label" is intended to encompass the use of direct and indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances.

[0355] 4. Assays, Kits, and Primers

[0356] Also provided are kits, probes, primers, and antibodies useful for determining the frequency of ASR plant pathogen effectors in a cultivated area. ASR plant pathogen effector polypeptides and polynucleotides, or variants and fragments thereof, can be packaged as components of the kit along with instructions for performing the assays described herein. DNA or RNA detection kits are provided for use in detecting nucleotide sequences encoding ASR plant pathogen effector polypeptides, or variants and fragments thereof.

[0357] In some embodiments, the kit may comprise one or more probes having a sequence corresponding to a sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a specific region of the nucleotide sequence set forth in any one of SEQ ID NOs: 3-6 or 21-22, or a sequence complementary thereto, thereby allowing detection of the sequence of interest. In some embodiments, the kit may comprise any reagents and materials required to perform the assay or detection method. In particular embodiments, the probe can be used to specifically hybridize to a target polynucleotide, thereby detecting a nucleotide sequence set forth in any one of SEQ ID NOs: 3-6 or 21-22, or a variant or fragment thereof.

[0358] Further provided are antibodies against the polypeptides of the present invention or variants or fragments thereof. Methods for generating antibodies are well known in the art (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; and U.S. Patent No. 4,196,265). These antibodies can be used in kits for detecting and isolating toxin polypeptides. Thus, the present disclosure provides kits comprising antibodies that specifically bind to the polypeptides described herein, including, for example, polypeptides having the sequence of SEQ ID NO: 1, 2, or 20, or variants or fragments thereof.

[0359] c. Determine the frequency of ASR plant pathogen effectors in biological materials and generate recommendations for pesticide application

[0360] The quantification of one or more ASR effectors of interest (e.g., SPE-35, SPE-38, or active variants thereof) can be expressed as a frequency in the ASR plant pathogen population within a cultivation area. The frequency of occurrence can be determined as a percentage of the total population of ASR plant pathogens present in a sample. For example, one or more ASR effectors of interest can be determined to be present at a particular frequency in an ASR population. Such frequencies within an ASR population can be at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the ASR plant pathogen population from a sample and / or within a cultivation area; or alternatively, between about 1% to about 10%, about 10% to 20%, about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 80% to 90% or about 90% to 100% of the ASR effectors within a sample and / or ASR plant pathogen population within a cultivation area. The frequency of ASR-responsive polypeptides can also be expressed in alternative formats (e.g., heat maps, box plots, or pie charts with or without associated maps).

[0361] The legume or soybean plant in the cultivation area may comprise one or more ASR genetic resistance traits, such as RG31 or RG35 or its active variants or fragments. As explained elsewhere herein, the ASR effectors detected in the population will vary depending on the ASR genetic resistance traits of the crops planted in the cultivation area. For example, when the cultivation area comprises a legume or soybean plant expressing RG31 or RG35 or its active variants or fragments, any one of the corresponding ASR effectors of RG31 and / or RG35 can be detected in the ASR plant pathogen population. For example, SPE-35 or SPE-38 or its active variants or fragments can be detected. Resistance characteristics in such cultivation areas where the frequency of SPE-35 or SPE-38 or its active variants or fragments is present at a high frequency within the population indicate a need for a pesticide application scheme with greater ability to control ASR pathogens. The resistance characteristics in such cultivated areas where the frequency of SPE-35 or SPE-38 or its active variants or fragments is present at low frequencies within the population indicate the need for a pesticide application scheme that reduces the ability to control ASR pathogens, because the ASR genetic resistance in the plant will be able to control the ASR pathogen population. Such a method can improve the ASR pathogen resistance management in a given cultivated area. Although plants with RG31 and RG35 were used in the above examples, similar schemes can be used for any ASR genetic resistance trait disclosed herein and its corresponding ASR effector protein or any combination thereof. See, for example, Table C herein for information on the corresponding ASR effector protein for a given ASR genetic resistance trait.

[0362] As used herein, a "high frequency within a population" of an ASR effector protein corresponding to an ASR genetic resistance trait will result in a plant population being more susceptible to infection. A high effector protein frequency within a population of an ASR plant pathogen occurs when one or more ASR effectors, alone or in combination, are present at a frequency of at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%; or alternatively, between about 1% and about 10%, about 10% and 20%, about 20% and 30%, about 30% and 40%, about 40% and 50%, about 50% and 60%, about 60% and 70%, about 70% and 80%, about 80% and 90%, or about 90% and 100% of the ASR effectors within the ASR plant pathogen population.

[0363] As used herein, an ASR genetic resistance trait corresponds to a "low frequency within a population" of an ASR effector protein that will result in a plant population that is less susceptible to infection. A low ASR effector protein frequency within a population occurs when one or more ASR effectors, alone or in combination, are present within a population of an ASR plant pathogen at a frequency of at least about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%; or alternatively, between about 1% and about 10%, about 10% and 20%, about 20% and 30%, about 30% and 40%, about 40% and 50%, about 50% and 60%, about 60% and 70%, about 70% and 80%, about 80% and 90%, or about 90% and 100% of the ASR effectors within the ASR plant pathogen population.

[0364] The frequency of the ASR plant pathogen effector of interest at the sampling location can be used to generate the frequency of various ASR plant pathogen effectors within the cultivation area. By combining data from different sampling locations within the cultivation area, the predicted local frequency of a given ASR plant pathogen effector within the ASR plant pathogen population can be determined based on local conditions. A resistance map and / or virulence map can then be generated for that given cultivation area.

[0365] The resistance trait map and / or the virulence trait map can then be used to identify candidate pesticides for use in a pesticide application program to improve the resistance of legume or soybean plants to ASR plant pathogens in the cultivation area. A recommended pesticide application program can then be generated for the cultivation area, the program comprising at least one disease control measure that controls the development, reproduction, and / or viability of the ASR plant pathogen based on the resistance trait and / or the virulence trait.

[0366] Disease control measures or treatment programs are understood to be the management of ASR plant pathogens, for example by the application of plant protection products such as pesticides, the timing or intervals of application of plant protection products, or the selection of optimal or specifically resistant crops, in order to control the development, reproduction and / or viability of the pathogen while minimizing the development of resistance. As discussed elsewhere herein, the disease control measures developed will take into account the resistance characteristics and / or virulence characteristics of the ASR plant pathogens detected in the cultivation area.

[0367] Disease control measures may include the use of fungicides that can increase the growth, reproduction and / or viability of ASR plant pathogen populations within the cultivated area. Such fungicide applications include, but are not limited to, demethylation inhibitors (DMI-tebuconazole, cyproconazole, propiconazole (triazolintione), fluopicolide, flutriafol, etc.); quinone oxidase inhibitors (QoI-pyraclostrobin, trifloxystrobin, picoxystrobin and pyraclostrobin), succinate dehydrogenase inhibitors (SDHI-flupyraclostrobin, bixafen and benzovinflupyraclostrobin), copper (chloride oxide), nitriles (e3), strobilurins and mancozeb. Additional fungicide applications include fungicides belonging to the formamide class, which have a specific mode of action (inhibiting fungal respiration of complex II-succinate dehydrogenase (SDHI) to control ASR). Members of the formamide class include bixafungin, fluopicolide and benzovinflupyraclostrobin. The fungicides benzovinflumizone, bixafen, fluopyram, forabi, pyraclostrobin, fluopyram, penthiopyrad and silkxane; or multi-site fungicides such as mancozeb, chlorothalonil and / or metiram.

[0368] In other embodiments, the method further comprises determining at least one disease control measure for application to an adjacent location based on the resistance and / or virulence characteristics.

[0369] An exemplary method for determining the resistance characteristics and / or virulence characteristics of an ASR plant pathogen population present in a cultivated area is disclosed as follows. The method comprises collecting a sample from the cultivated area, wherein the sample can be a field sample or an environmental sample, such as aerial sampling by spore traps, plant material or soil material as described above. Biological material is obtained from the sample, including, for example, polynucleotides or polypeptides. In some embodiments, the polynucleotides from the biological material are sequenced and / or amplified and / or detected, or the polypeptides are detected to detect target ASR plant pathogen effectors that may confer higher virulence to the resistant plant or cultivated variety. The method further comprises determining the qualitative presence of ASR plant pathogen effectors in the plant pathogen or pathogen population, and quantifying the frequency of one or more target ASR plant pathogen effectors. Thereafter, the method further comprises generating resistance characteristics and / or virulence characteristics of the ASR plant pathogen or ASR pathogen population by combining the results with information or knowledge obtained from specialized studies to assess the impact of a given frequency of one or more ASR plant pathogen effectors on crop productivity.

[0370] Such methods can rapidly (e.g., in near real time, within 48 hours or days) quantitatively and qualitatively characterize the resistance and / or virulence characteristics of the ASR plant pathogen population present at a given location. This provides the opportunity to gain a clearer and more detailed understanding of the resistance characteristics that may exist at a given location and facilitate the identification of more targeted disease control measures and / or resistance management programs for the current and / or future seasons. This allows the selection of the most effective type of pesticide and / or optimal crop variety to control the pathogen, thereby avoiding the use of pesticides that confer high resistance.

[0371] Referring again to the process of collecting samples of ASR plant pathogens, this process can include receiving samples from farmers who have already collected samples from their fields, or receiving samples from another source, such as a wild population of a volunteer crop or flower, where the resistance characteristics of the plant pathogen population are to be determined. The samples can be specimens collected by farmers from various plants in their fields. Alternatively or additionally, the samples can be environmental samples (e.g., soil and / or above-ground samples).

[0372] Reference is now made again to the operation of generating resistance characteristics and / or virulence characteristics of plant pathogens. This operation may include interpreting data determined by polynucleotide sequencing, polynucleotide amplification and detection and / or polypeptide detection to detect ASR effectors within the biological material. By applying an understanding of the susceptibility of the legume and / or soybean plant in the cultivation area or to be planted in the cultivation area, combined with the frequency of different ASR effectors present in the cultivation area sample, it can be determined. More particularly, generating resistance characteristics may include associating the presence and corresponding frequency of one or more ASR effectors in the ASR plant pathogen population of the sample determined in the aforementioned operation 103 with the susceptibility level of the legume and / or soybean plant in the cultivation area or to be planted in the cultivation area. The resistance level can be considered as an index that condenses the complex information of the frequency and resistance factors of each ASR effector or combination of ASR effectors into a single value that is comparable between different samples. The resistance level of the plant pathogen population in the sample requires understanding the impact of each ASR effector detected on the resistance of the plant (legume and / or soybean) in the cultivation area.

[0373] Further provided is a method comprising determining at least one pesticide or other disease control measure for controlling the growth, reproduction and / or viability of crop pathogens based on resistance characteristics and / or virulence characteristics. For example, resistance characteristics and / or virulence characteristics can be determined before using any pesticide to control ASR pathogens. Resistance characteristics of plants to specific ASR effectors (e.g., SPE-35 or SPE-38 or their active variants) of pathogen colonies in a cultivated area can be generated or measured according to the above-mentioned example methods by monitoring the presence of one or more ASR effectors in a sample and quantifying their frequency. Similarly, virulence characteristics can be generated as described above. Based on resistance characteristics and / or virulence characteristics, pesticides or other disease control measures can be determined or recommended. The method can further comprise collecting or receiving subsequent or additional samples from the same location, crop or plant as the initial sample, wherein the subsequent samples have been processed with the pesticide or other disease control measures determined in the operation. The subsequent samples are subjected to the above-mentioned operation and then resistance characteristics and / or virulence characteristics similar to the operation are generated. Finally, the resistance characteristics and / or virulence characteristics generated are compared with the resistance characteristics and / or virulence characteristics of the initial sample. For example, if the resistance characteristics are generated for the initial sample, then it is compared with the resistance characteristics generated for the subsequent sample, and similarly, if the virulence characteristics are generated for the initial sample, then it is compared with the virulence characteristics of the subsequent sample. According to the comparison of these two resistance characteristics, it can be determined that the resistance of the colony to the fungicide class is due to the agronomy decision-making made for controlling the disease and improves or worsens. For example, the ratio between the sensitivity index generated before and after the pesticide treatment provides information about the sustainability of the disease control measures applied, and can be used as a tool for identifying and informing suboptimal disease control measures and resistance management. Sharing information about disease control measures (such as application date, the plant protection products used, application rate and other information) will be able to issue an alarm about risk decision-making, and for proposing alternatives for the ensuing season.

[0374] In one example, based on the comparison, the method can further include determining at least one pesticide or other disease control measure to use to control the plant pathogen. The pesticide or other disease control measure can be the same as or different from the pesticide or other disease control measure applied prior to collecting and receiving the subsequent sample.

[0375] The first or initial sample as described herein can be collected at the beginning of the season, and subsequent or additional samples can be collected during the season or at the end of the season (in the middle or at the end). The beginning and middle / end of the season can be a time period of one day, a few days, a week or weeks, a month or months, a year or years, depending on the crop, pathogen, or sampling frequency required for a particular situation. In the case where it is necessary to analyze resistance characteristics more frequently, additional sampling can be performed, for example, after each application or in a time series.

[0376] Although it has been described that samples are collected at different points in time relative to the season, it will be appreciated that samples may alternatively or additionally be collected when the relevant plants are at a particular growth stage.

[0377] Furthermore, the present disclosure is not limited to an initial sample and a single subsequent sample. Several subsequent samples can be collected at different time points and then processed to generate resistance characteristics and / or virulence characteristics.

[0378] It is also envisioned that the method for determining at least one pesticide or other disease control measure (as described above) for a certain location as described herein can be applied to adjacent locations. This is based on the following assumption: the variant of the plant pathogen present in this location is also present in the adjacent location. For environmental ground samples of plants or crops that have not been treated with pesticides, this may be the case. It is well known that different situations can be found in adjacent locations, which is usually due to the cultivation of different resistant plants or crops and / or due to the different treatment procedures of pesticides (e.g., fungicides) that have been adopted in the past few years. In this case, before disease control measures are applied to a location that has been determined to be an adjacent location, additional factors need to be considered.

[0379] A method for determining resistance traits and / or virulence traits of a plant pathogen at a location is provided. The method includes receiving a sample of the plant pathogen from the location, wherein the sample can be a field sample or an environmental sample. The method further includes performing polynucleotide sequencing, polynucleotide amplification, polynucleotide detection and / or polypeptide detection on the sample to determine the presence of one or more ASR effectors within the ASR population. The presence of at least one ASR effector within the ASR plant pathogen population is determined, and the at least one ASR effector is quantified. Thereafter, the method further includes generating resistance traits and / or virulence traits of the plant pathogen or pathogen population.

[0380] 10. Methods for identifying variant polypeptides encoding R genes or other plant pathogen resistance traits

[0381] Variants of polypeptides encoding R genes or other plant pathogen resistance traits (e.g., ASR resistance traits) (including sequences from other organisms) can be identified based on their sequence identity and / or functional identity to a given RG31 and / or RG35 polypeptide and gene disclosed herein. In one example, variant polypeptides and polynucleotides of RG31 and / or RG35 polypeptides and polynucleotide sequences encoding such polypeptides comprise proteins that interact with the same set of effector proteins (see Table C), thereby generating a localized hypersensitive response via a common mode of action when expressed in plants (see also Example 3). Variant polypeptides and polynucleotides comprising orthologs and allelic variants of the RG31 and / or RG35 polypeptides of SEQ ID NOs: 1 or 2 or 20 and / or the RG31 and / or RG35 genes of SEQ ID NOs: 3-6 or 21-22 are expected to interact with effector proteins to generate a localized hypersensitive cell death response. On the other hand, polypeptides that are hypothesized to interact with a different set of effector proteins than those recognized by RG31 and RG35 and / or polypeptides that do not produce a local hypersensitivity response with effectors recognized by RG31 and / or RG35 (e.g., SPE-35 or an active variant or fragment thereof), as well as polynucleotides encoding such polypeptides, do not have the same mode of action as RG31 and / or RG35, regardless of whether they have a high or low degree of sequence identity with RG31 and / or RG35. In a particular embodiment, as illustrated in Example 3, even though RG31 and RG35 have a high degree of sequence identity with the R protein RG1 (disclosed as SEQ ID NO: 47 in WIPO Publication No. WO2019103918 A1 and encoded by a polynucleotide disclosed as SEQ ID NO: 6), they do not interact with the same set of effector proteins, indicating that they have different modes of action in conferring disease resistance.

[0382] Thus, in some embodiments, methods of identifying novel disease resistance polypeptides are disclosed, the methods comprising: identifying a set of plant pathogen effector proteins that interact with the RG31 polypeptide of SEQ ID NO: 1 or the RG35 polypeptide of SEQ ID NO: 2 or 20; assaying the identified set of plant pathogen effector proteins for interaction with a putative RG31 polypeptide, or an RG31 polypeptide having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 1, or a putative RG35 polypeptide, or an RG35 polypeptide having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 2 or 20; and, in response to detecting an interaction between the putative RG31 or RG35 polypeptide and the identified effector, indicating, e.g., via the presence of a hypersensitive response or localized cell death, that the putative RG31 or RG35 polypeptide has the same mode of action as the corresponding RG31 or RG35 polypeptide.

[0383] In some embodiments, the effector protein is expressed in cells, purified, and infiltrated into plant cells for interaction assays with putative RG31 or RG35 polypeptides. In other embodiments, a transient expression system in plants (e.g., an Agrobacterium-mediated transient expression system) can be used to observe a hypersensitive response cell death phenotype (HR) triggered by co-expression of a putative RG31 or RG35 gene system comprising (a) an RG31 or RG35 variant (e.g., an ortholog or homolog or allelic variant of RG31 or RG35) and its putative cognate effector (e.g., an effector protein recognized by the RG31 or RG35 polypeptide of SEQ ID NO: 1 or 2 or 20). The presence of an interaction, such as an HR response and localized cell death (e.g., as shown in Example 3), indicates that the protein encoded by the putative Rg31 or Rg35 gene has functional identity and a common mode of action with the RG31 or RG35 polypeptides of the present disclosure, respectively, even if the sequence identity is low (e.g., less than 60% sequence identity, such as 50% identity or 40% identity or less). The absence of interactions, such as the absence of a HR response and local cell death (e.g., as shown in Example 3), indicates that the protein encoded by the polypeptide does not share a common mode of action with the RG31 or RG35 polypeptides of the present disclosure, respectively, even if the sequence identity is high (e.g., greater than 60% sequence identity, such as 80% identity or 90% identity or more).

[0384] Non-limiting examples include:

[0385] Example Examples of Nucleic Acids and Polypeptides for Controlling Disease Resistance

[0386] A1. A nucleic acid molecule comprising a nucleotide sequence operably linked to a heterologous regulatory element, wherein the nucleotide sequence comprises a polynucleotide encoding an RG31 or RG35 polypeptide or an active variant thereof, wherein the polypeptide or active variant thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1 or 2 or 20,

[0387] wherein increased expression of said RG31 or RG35 polypeptide or an active variant thereof confers increased disease resistance to said plant compared to a control plant cell.

[0388] A2. The nucleic acid molecule as described in Example A1,

[0389] wherein the polynucleotide comprises:

[0390] (i) a nucleotide sequence encoding an RG31 polypeptide having at least 80% sequence identity to SEQ ID NO: 1 or an RG35 polypeptide having at least 80% sequence identity to SEQ ID NO: 2 or 20;

[0391] (ii) a nucleotide sequence encoding the RG31 polypeptide of SEQ ID NO: 1 or the RG35 polypeptide of SEQ ID NO: 2 or 20; or

[0392] (iii) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22.

[0393] A3. The nucleic acid molecule of embodiment A1 or A2, wherein the heterologous regulatory element is a heterologous promoter active in a plant, and wherein the heterologous promoter is a constitutive promoter, an inducible promoter, or an endogenous promoter, and wherein the inducible promoter is optionally a rust-inducible promoter.

[0394] A4. The nucleic acid molecule of embodiment A3, wherein the heterologous promoter has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 7, 8, 10, 11, 12, 14, and 15.

[0395] A5. The nucleic acid molecule of embodiment A3, wherein the rust-inducible promoter has a nucleotide sequence that has at least 95% sequence identity to SEQ ID NO: 14 or 15, or wherein the rust-inducible promoter comprises SEQ ID NO: 14 or 15.

[0396] A6. The nucleic acid molecule of embodiment A3, wherein the endogenous promoter has a nucleotide sequence with at least 95% sequence identity to any one of SEQ ID NOs: 7, 8, and 10, or wherein the rust-inducible promoter comprises SEQ ID NOs: 7, 8, or 10.

[0397] A7. The nucleic acid molecule of any one of embodiments A1-A3, wherein the heterologous regulatory element comprises a terminator sequence, an intron, a 5'UTR, or a 3'UTR.

[0398] A8. The nucleic acid molecule of any one of embodiments A1-A7, wherein the polynucleotide encoding the RG31 or RG35 polypeptide or active variant thereof comprises at least one native intron or at least one heterologous intron.

[0399] A9. The nucleic acid molecule of any one of embodiments A1-A8, wherein the RG31 or RG35 polypeptide, or active variant or fragment thereof, is tagged with a detectable label.

[0400] A10. A vector comprising the nucleic acid molecule of any one of embodiments A1-A9.

[0401] A11. A vector comprising a polynucleotide encoding:

[0402] (a) an RG31 polypeptide or an active variant thereof, comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1; or

[0403] (b) an RG35 polypeptide or an active variant thereof, comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 20,

[0404] Wherein increased expression of the RG31 or RG35 polypeptide or an active variant thereof in a plant confers to the plant enhanced disease resistance compared to control plant cells.

[0405] A12. The vector of embodiment A11, wherein the polynucleotide comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22.

[0406] A13. A transgenic cell comprising the nucleic acid molecule of any one of embodiments A1-A9 or the vector of any one of embodiments A10-A12.

[0407] A14. A transgenic cell comprising a heterologous polynucleotide encoding an RG31 polypeptide or an RG35 polypeptide, or an active variant of an RG31 polypeptide or an RG35 polypeptide, wherein the polypeptide or its active variant comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 1, 2 or 20.

[0408] A15. The transgenic cell of embodiment A14, wherein the polynucleotide comprises a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 3-6, 21 or 22.

[0409] A16. The transgenic cell of any one of embodiments A13-A15, wherein the transgenic cell is a transgenic plant cell.

[0410] A17. The transgenic plant cell of embodiment A16, wherein the polynucleotide is stably integrated into the genome of the cell.

[0411] A18. The transgenic plant cell of embodiment A16 or A17, wherein the transgenic plant cell has an increased expression level of the RG31 polypeptide or the RG35 polypeptide or an active variant thereof compared to a control plant cell, and wherein the transgenic plant cell has increased disease resistance.

[0412] A19. The transgenic plant cell of embodiment A19, wherein the transgenic plant cell has increased resistance to Asian soybean rust compared to a control plant cell.

[0413] A20. The transgenic plant cell of any one of embodiments A16-A19, wherein the transgenic plant cell is a transgenic plant cell of a legume, optionally wherein the legume is selected from the group comprising alfalfa, clover, pea, lentil, lupine, mesquite, carob, soybean, pigeon pea, peanut, and tamarind.

[0414] A21. The transgenic plant cell of embodiment A20, wherein the transgenic plant cell is a transgenic soybean plant cell.

[0415] A22. The transgenic plant cell of any one of embodiments A16-A19, wherein the transgenic plant cell is a monocot cell, a dicot cell, a legume cell, a soybean cell, a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugarcane cell, a wheat cell, a sunflower cell, a tomato cell, a cotton cell, a sugar beet cell, or a tobacco cell.

[0416] A23. A plant, or plant part, derived from the transgenic cell of any one of embodiments A13-A22.

[0417] A24. The plant of embodiment A23, wherein said plant is a soybean plant, and wherein said soybean plant is an elite soybean plant.

[0418] A25. The plant part of embodiment A24, wherein said plant part is a transgenic seed, wherein said transgenic seed has stably integrated said polynucleotide into its genome.

[0419] A26. A harvested product derived from the transgenic seed of embodiment A25, and wherein the harvested product comprises the polynucleotide.

[0420] A27. A processed product derived from the harvested product of embodiment A26, wherein the processed product is flour, meal, oil,...

Claims

1. A DNA construct comprising a polynucleotide operably linked to a heterologous regulatory element, wherein the polynucleotide encodes a polypeptide comprising: (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2 or 20, wherein increased expression of the polypeptide in a plant increases disease resistance of the plant; (b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or 2 or 20, wherein increased expression of the polypeptide in a plant increases disease resistance of the plant; or (c) an amino acid sequence comprising SEQ ID NO: 1, 2, or 20.

2. The DNA construct of claim 1 , wherein the polynucleotide encoding the polypeptide comprises: (a) a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22; (b) a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs. 3, 4, 5, 6, 21 or 22; or (c) a nucleotide sequence comprising any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22.

3. The DNA construct of any one of claims 1 to 2, wherein the heterologous regulatory element comprises a promoter active in plants.

4. DNA construct as claimed in claim 3, wherein said promoter is a tissue-specific promoter or a constitutive promoter or a rust-responsive promoter.

5. The DNA construct of claim 1, wherein the heterologous regulatory element comprises a terminator sequence, an intron, a 5'UTR, or a 3'UTR.

6. A DNA construct as claimed in claim 5, wherein the polynucleotide encoding the polypeptide is operably linked to a promoter sequence comprising: (a) a nucleotide sequence that is at least 95% identical to any one of SEQ ID NO: 7, 8, 10, 12, 14 or 15, wherein the nucleotide sequence is capable of driving expression of a polypeptide of interest in a plant cell; or (b) a nucleotide sequence comprising any one of SEQ ID NO: 7, 8, 10, 12, 14 or 15.

7. A DNA construct as described in any one among claims 1-6, wherein the polynucleotide encoding the polypeptide comprises at least one natural intron or at least one heterologous intron. A vector comprising the DNA construct according to any one of claims 1 to 7.

9. A vector comprising a polynucleotide encoding a polypeptide comprising: (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2 or 20, wherein increased expression of the polypeptide in a plant increases disease resistance of the plant; (b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or 2 or 20, wherein increased expression of the polypeptide in a plant increases disease resistance of the plant; or (c) an amino acid sequence comprising SEQ ID NO: 1, 2, or 20.

10. The vector of claim 9, wherein the polynucleotide comprises (a) a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22; (b) a nucleotide sequence that is at least 95% identical to any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22; or (c) a nucleotide sequence comprising any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22.

11. A cell comprising the DNA construct according to any one of claims 1 to 7, or the vector according to any one of claims 8 to 10.

12. A cell comprising a heterologous polynucleotide encoding a polypeptide comprising: (a) an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2 or 20, wherein increased expression of the polypeptide in a plant increases disease resistance of the plant; (b) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or 2 or 20, wherein increased expression of the polypeptide in a plant increases disease resistance of the plant; or (c) an amino acid sequence comprising SEQ ID NO: 1, 2, or 20.

13. The cell of claim 12, wherein the polynucleotide comprises: (a) a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22; (b) a nucleotide sequence that is at least 95% identical to any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22; or (c) a nucleotide sequence comprising any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22.

14. The cell of any one of claims 11-13, wherein the cell is a plant cell, optionally wherein the plant cell is (a) a monocot cell, (b) a dicot cell, (c) a legume cell, (d) a soybean cell, (e) a barley cell, a maize cell, an oat cell, a rice cell, a sorghum cell, a sugarcane cell, or a wheat cell; or (f) a sunflower cell, a tomato cell, a cotton cell, a sugar beet cell, or a tobacco cell.

15. The plant cell of claim 14, wherein the polynucleotide is stably integrated into the genome of the cell, and wherein the plant cell has increased expression levels of the polypeptide compared to a control plant cell and the plant cell has increased disease resistance.

16. A plant comprising the plant cell according to any one of claims 14-15.

17. The plant of claim 16, wherein the transgenic plant has increased resistance to Asian soybean rust, wherein the transgenic plant is a legume plant, optionally wherein the legume plant is a soybean plant.

18. A method of producing a plant with increased disease resistance, the method comprising: (a) introducing into the genome of a plant cell a heterologous polynucleotide encoding a polypeptide comprising: i) an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2 or 20, wherein increased expression of the polypeptide in a plant increases disease resistance of the plant; ii) an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or 2 or 20, wherein increased expression of the polypeptide in a plant increases disease resistance of the plant; or iii) an amino acid sequence comprising SEQ ID NO: 1 or 2 or 20; and (b) Regenerating the soybean plant cell of (a) into a soybean plant wherein expression of the polypeptide in the plant increases disease resistance of the plant.

19. The method of claim 18, wherein the heterologous polynucleotide comprises: (a) a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO: 3, 4, 5, 6, 21 or 22; (b) a nucleotide sequence that is at least 95% identical to any one of SEQ ID NO: 3, 4, 5, 6 or 21; or (c) a nucleotide sequence comprising any one of SEQ ID NOs. 3, 4, 5, 6, 21 or 22.

20. The method of claim 18 or 19, wherein the plant is a leguminous plant.

21. The method of claim 20, wherein the leguminous plant is a soybean plant.

22. The method of any one of claims 18-21, wherein the increase in disease resistance comprises increased resistance to Asian soybean rust.

23. The method of any one of claims 18 to 22, wherein the heterologous polynucleotide is introduced into the plant genome by transforming the plant cell with a DNA construct as described in any one of claims 1 to 7 or a vector as described in any one of claims 8 to 10.

24. A method of reducing Asian soybean rust damage or controlling an ASR pathogen in an area under cultivation, the method comprising planting the plant of any one of claims 16-17 in the area under cultivation.

25. A method for producing an Asian soybean rust (ASR)-resistant soybean plant, the method comprising the steps of: (a) selecting a soybean plant from a plurality of soybean plants by detecting the presence of a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 90%, 95%, or 100% sequence identity to SEQ ID NO: 1 or 2 or 20; and (b) generating ASR-resistant progeny soybean plants from the selected soybean plants in a breeding program.

26. A method of controlling disease resistance in an area under cultivation, said method comprising the step of planting in said area under cultivation a leguminous plant, plant part or seed having stably integrated into its genome: (a) a nucleotide sequence encoding an RG31 polypeptide or an active variant thereof, which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1; or a heterologous nucleotide sequence encoding an RG35 polypeptide or an active variant thereof, which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 20; and (b) a heterologous promoter operably coupled to said nucleotide sequence, The expression of the RG31 polypeptide, RG35 polypeptide or an active variant thereof confers disease resistance to the legume, plant or seed.

27. The method of claim 28, wherein one or more of the RG31 polypeptide, RG35 polypeptide, or active variant thereof is tagged with a detectable label.

28. The method of any one of claims 26-27, wherein the heterologous promoter comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 7, 8, 10, 12, 14, and 15, or wherein the promoter comprises the sequence of any one of SEQ ID NOs: 7, 8, 10, 12, 14, and 15.

29. A leguminous plant produced by the method of any one of claims 25-28.

30. The legume plant of claim 29, wherein the legume plant is a legume crop plant, and optionally wherein the legume crop plant is alfalfa, clover, pea, bean, lentil, lupine, mesquite, carob, soybean, pigeon pea, peanut or tamarind.

31. The leguminous plant of claim 30, wherein the leguminous crop plant is a soybean plant.

32. The leguminous plant of claim 31 , wherein the soybean plant is an elite soybean plant.

33. The leguminous plant of any one of claims 30-32, wherein the plant is resistant to one or more of the following: soybean cyst nematode, bacterial pustule, root knot nematode, soybean gray spot, Phytophthora, brown stem rot, nematodes, Asian soybean rust, powdery mildew, graminis bisporus, powdery mildew of chrysanthemum family, powdery mildew of wheat, graminis cucurbitae, powdery mildew of cucumber, ultimum pythium, Uncinaria vitis, Mycosphaeria pisum, rice blast, chorionella oryzae, rice blast, Rhizoctonia solani, Phytophthora sojae, Schizaphis graminis, Bemisia tabaci, corn aphid, net-shaped wild slug, small sugarcane borer, Schizaphis graminis, and green peach aphid.

34. The leguminous plant of claim 33, wherein the plant is resistant to Asian soybean rust.

35. A composition comprising: (a) an RG31 polypeptide or an active variant thereof, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1; or (b) an RG35 polypeptide or an active variant thereof, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 20, wherein the presence of said composition in a plant confers increased disease resistance to said plant relative to a control plant not comprising said composition.

36. A composition as described in claim 35, wherein the RG31 polypeptide or an active variant thereof has an amino acid sequence comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1; and wherein the RG35 polypeptide or an active variant thereof has an amino acid sequence comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2 or 20 or the amino acid sequence of SEQ ID NO: 2 or 20.

37. A plant comprising in its genome a stably integrated nucleic acid molecule comprising a heterologous promoter operably coupled to: (a) a polynucleotide encoding an RG31 polypeptide or an active variant thereof, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1; or (b) a polynucleotide encoding an RG35 polypeptide or an active variant thereof, comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 20; wherein expression of said nucleic acid molecule in said plant confers increased disease resistance to said plant relative to a control plant not comprising said nucleic acid molecule.

38. The plant of claim 37, wherein the polynucleotide encoding the RG31 polypeptide or an active variant thereof comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 3-4; and wherein the polynucleotide encoding the RG35 polypeptide or an active variant thereof comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 5-6 or 21-22.

39. The plant of any one of claims 37-38, wherein the plant is resistant to Asian soybean rust.

40. A method for identifying a novel disease resistance polypeptide, the method comprising: (a) providing a set of plant pathogen effector proteins that interact with the RG31 polypeptide of SEQ ID NO: 1 or the RG35 polypeptide of SEQ ID NO: 2 or 20; (b) determining the interaction of the identified panel of plant pathogen effector proteins with a putative RG31 polypeptide having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO: 1 or a putative RG35 polypeptide having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO: 2 or 20; and (c) identifying the putative RG31 polypeptide or putative RG35 polypeptide from step (b), thereby identifying a novel disease-resistance polypeptide.

41. The method of claim 40, wherein step (b) comprises measuring a hypersensitive response or localized cell death in the plant or plant cell or plant tissue, thereby indicating that the putative RG31 polypeptide has the same mode of action as the RG31 polypeptide or indicating that the putative RG35 polypeptide has the same mode of action as the RG35 polypeptide.

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