When used in reference to two or more amino acid sequences, the term “operably linked” means that the first amino acid sequence is in a functional relationship with at least one of the additional amino acid sequences.

The term “resistance” is used herein to mean an absence or reduction of one or more disease symptoms in a plant caused by a plant pathogen. Resistance can mean that disease symptoms, such as, for example, number of lesions, defoliation, and associated yield loss, are reduced, minimized or lessened, when compared to a plant that is susceptible to the disease or a plant that does not contain an effective resistance gene, such as, for example, CcRpp2-R1 and CcRpp2-R3 genes that reduce one or more disease symptom. Further, resistance can include the prevention or delay of proliferation of a pathogen (e.g., fungi).

“Plant pathogen” or “fungal pathogen” can be used herein to mean fungal pathogens of, for example, the genus Phakopsora, including the species Phakopsora pachyrhizi and Phakopsora meibomide. These species are known to cause ASR in plants. A plant disease or a legume crop species disease, for example, can be ASR.

The term “disease resistance gene” or “resistance gene” is used herein to mean a gene that encodes a protein or polypeptide capable of enhancing or improving a defense or immune system response in a plant.

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

The term “encode” is used herein to mean that the nucleic acid comprises the required information, specified by the use of codons to direct translation of the nucleotide sequence (e.g., a legume sequence) into a specified protein. A nucleic acid encoding a protein can comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or can lack such intervening non-translated sequences (e.g., as in cDNA).

Aspects of the disclosure encompass isolated or recombinant polynucleotide or protein compositions. An “isolated” or “recombinant” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a heterologous recombinant bacterial or plant host cell. An isolated or recombinant nucleic acid molecule, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

The terms “inhibit,” “inhibition,” “inhibiting”, “reduced”, “reduction” and the like as used herein to mean any decrease in the expression or function of a target gene product, including any relative decrease in expression or function up to and including complete abrogation of expression or function of the target gene product.

The terms “increase,” “increasing,” “enhance,” “enhancing” and the like are used herein to mean any boost or gain or rise in the expression, function or activity of a target gene (e.g., TIR gene) product providing an increased resistance to one or more pathogens (e.g., Phakopsora spp.) or to a disease (e.g., ASR) compared to a susceptible plant. Further, the terms “induce” or “increase” as used herein can mean higher expression of a target gene product, such that the level is increased 10% or more, 50% or more or 100% relative to a cell or plant lacking the target gene or protein of the present disclosure.

The term “expression” as used herein refers to the biosynthesis or process by which a polynucleotide, for example, is produced, including the transcription and/or translation of a gene product. For example, a polynucleotide of the present disclosure can be transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into a polypeptide or protein.

The term “gene product” can refer to for example, transcripts and encoded polypeptides. Inhibition of (or increase in) expression or function of a gene product (i.e., a gene product of interest) can be in the context of a comparison between any two plants, for example, expression or function of a gene product in a genetically altered plant versus the expression or function of that gene product in a corresponding, but susceptible wild-type plant or other susceptible plant. The expression level of a gene product in a wild-type plant can be absent. For example, a “wild-type” plant can be a plant, plant cell or plant part that does not express an exogenous CcRpp2-R1 and/or CcRpp2-R3 nucleic acid or exogenous CcRpp2-R1 and/or CcRpp2-R3 protein.

Alternatively, inhibition of (or increase in) expression or function of the target gene product can be in the context of a comparison between plant cells, organelles, organs, tissues, or plant parts within the same plant or between plants, and includes comparisons between developmental or temporal stages within the same plant or between plants. Any method or composition that down-regulates expression of a target gene product, either at the level of transcription or translation, or down-regulates functional activity of the target gene product can be used to achieve inhibition of expression or function of the target gene product. Similarly, any method or composition that induces or up-regulates expression of a target gene product, either at the level of transcription or translation, or increases or activates or up-regulates functional activity of the target gene product can be used to achieve increased expression or function of the target gene or protein. Methods for inhibiting or enhancing gene expression are well known in the art.

The term “introducing” as used herein defines a process of altering the content of a cell/plant through the use of traditional breeding or recombinant transformation techniques. When using recombinant transformation techniques a nucleic acid or protein is passed across a plant cell membrane or cell wall into the interior of a plant cell. Methods for introducing polynucleotides into plants are known in the art, including procedures resulting in stable transformation methods or transient transformation methods. 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, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell.

“Stable transformation” or “stably transformed” means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.

The term “transformation” is used herein to mean the transfer of, for example, a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. The term “host cell” refers to the cell into which transformation of the recombinant DNA construct takes place and can include a yeast cell, a bacterial cell, and/or a plant cell. Examples of methods of plant transformation include Agrobacterium-mediated transformation and particle-bombardment. Transformed plant cells can then be used to regenerate a transformed plant by methods known to one skilled in the art.

The term “transgenic” is used herein to refer to a plant, including any part derived from a plant, such as a cell, tissue, or organ in which an exogenous nucleic acid (e.g., recombinant construct, vector or expression cassette including one or more nucleic acids) is integrated into the genome by a genetic engineering method, such as Agrobacteria transformation. By carrying out a gene technology method, the exogenous nucleic acid is stably integrated into a chromosome, so that successive generations may also be transgenic. As used herein, “transgenic” also encompasses biological processes including the crossing of plants and/or natural recombination.

EMBODIMENTS

Crop diseases cause serious crop management issues and can sometimes lead to total crop failure. Asian soybean rust is a threat to world soybean production and is currently addressed by the use of foliar fungicides. Stable and reliable genetic resistance in commercial plant lines is an important feature associated with soybean crop yields, and presently, commercially grown soybean cultivars that are fully resistant to Asian soybean rust caused by Phakopsora pachyrhizi, are not available. The causal agents of ASR, Phakopsora pachyrhizi and Phakopsora meibomiae, infect leaf tissue from a broad range of leguminous plants (at least 31 species in 17 genera; Slaminko et al. (2008) Plant Dis., 92:797-771 ; and at least 42 species in 19 genera; Frederick et al. (2002) Mycology, 92:217-227, respectively). In total, a further 152 species in other genera have been described to be potential hosts of Phakopsora pachyrhizi (Bonde et al. (2008) Plant Dis., 92:30-38; Goellner et al. (2010) Molecular Plant Pathology, 11 : 169-177; Ono et al. (1992) Mycol. Res., 96(10):825-850; and Slaminko et al. (2008) Plant Dis., 92:797-771). Currently, fungicide applications are the only commercially-available method to mitigate ASR. Besides fungicides, another management strategy can be used in South American countries such as Brazil for mitigating ASR. In particular, the use of short-cycle varieties planted at the beginning of the growing season (allowing crops to avoid disease favorable conditions) and host-free period, decreases the amount of primary inoculum.

Presently, no commercially grown soybean (Glycine max) cultivars are available that are fully resistant to Phakopsora pachyrhizi. Resistance to Phakopsora pachyrhizi in soybeans is rare; USDA evaluated the entire USA soybean germplasm collection and found that fewer than 5% were resistant or partially resistant to Phakopsora pachyrhizi. Furthermore, the genes available in these soybean accessions only provide resistance that is isolate-specific; therefore these sources are not able to provide durable resistance under field conditions such as where multiple races are present.

Given that ASR is a major threat to soybean production, it is beneficial to identify sources of resistance genes and incorporate these transgenic genes into legume germplasm, such as Glycine max, for enhanced protection. To identify novel resistance genes, several non-Glycine max legume species were screened for variation in resistance to Phakopsora pachyrhizi. Dominant resistance genes were identified and confirmed to be members of the TIR-TIR class of resistance (R) genes. When transferred to soybeans, the binary CcRpp2-R1 and CcRpp2-R3 resistance genes disclosed herein can provide resistance to Phakopsora pachyrhizi via heterologous expression.

Plants can defend themselves through a variety of cellular mechanisms. It is currently understood that the plant immune system is made up of receptors on the outside (often called the first tier immunity) and the inside of a cell (often referred to as the second tier immunity). Both sets of receptors can detect and respond to a pathogen. The first tier responds to primary elements of a pathogen resulting in activation of pathogen-associated molecular pattern (PAMP)-triggered immunity. Successful pathogens overcome PAMP-triggered immunity by secreting molecules called “effector proteins” or “effectors” that are either localized to the plant apoplast or are taken up into the plant cell. Effectors manipulate host cell functions to suppress host immune responses in order to facilitate the establishment of infection or to otherwise enhance growth conditions for the pathogen, e.g. by ensuring availability to nutrients. Plants have, in some cases, evolved a second tier of immunity in which R gene products recognize the activity of specific effectors resulting in an effector-triggered immunity. R genes typically encode proteins that feature C-terminal leucine-rich repeats (LRRs) and nucleotide-binding site (NBS) domains. Such nucleic acid binding LRRs are designated nucleotide-binding LRR (NLR) proteins. The NBS domain functions as a molecular switch depending on the bound nucleotide: ADP-bound in the resting state and ATP-bound in the active state. The LRR domain is generally thought to be involved in effector recognition and autoinhibition (Ting et al., Immunity, 28 (2008), pp. 285-287). Typical plant NLRs almost universally feature the additional coiled-coil (CC) or Toll/interleukin-1 receptor (TIR) N-terminal domain. These N-terminal domains are used to sort plant NLRs into two main groups termed CNLs (CC-NLRs) and TNLs (TIR-NLRs). Both CC and TIR domains have been demonstrated to play key roles in the formation of dimers and oligomers.

In plants, the TIR domain occurs at the N terminus of a major subclass of the (TIR-NLR) family resistance receptors (R proteins), which trigger defense responses after perception of pathogen effectors (Dodds and Rathjen, Nat Rev Genet. 2010;11:539-548). Recent findings indicate that TIR domain in R proteins functions as an NAD+-cleaving enzyme to trigger localized cell death, known as the hypersensitive response (HR). See: TIR-only protein regulates cell death in plants Proceedings of the National Academy of Sciences March 2017, 114 (10) E2053-E2062; DOI: 10.1073/pnas.1620973114 2) and TIR domains of plant immune receptors are NAD+-cleaving enzymes that promote cell death. SCIENCE 23 AUG 2019: 799-803. The LRR domain of plant R proteins appears to be the major determinant of recognition specificity. The NB domain is shared with mammalian nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which also function as regulators of innate immune responses and apoptosis. Evidence suggests the NB domain can bind and hydrolyse nucleotides, and the presence of bound ATP or ADP may determine whether the R protein is in an active or inactive signaling state. However, in plant R proteins, the mechanism by which effector recognition is linked to the activation of defense signaling is poorly understood.

As a result of the “arms race” between host and pathogen, pathogen effectors can have either an avirulence or virulence effect. The virulence activity of effectors is associated with the manipulation of normal host cell functions or the suppression of host immune responses by the pathogen in order to establish successful infection. In avirulence, recognition by the corresponding plant R protein activates a host immune or defense response, resulting in programmed cell death and resistance to the pathogen.

The nucleic acids and polypeptides disclosed herein are useful in generating transgenic plants exhibiting fungal resistance and in methods for conferring or enhancing or increasing fungal resistance to a plant (e.g., a legume crop species). Methods and compositions disclosed herein may comprise the following polypeptide and polynucleotides sequences:

SEQ ID NO: 1 CcRpp2-R1Aa coding sequence from Cajanus cajan (polynucleotide sequence).

SEQ ID NO: 2: CcRpp2-R1Aa (polypeptide sequence).

SEQ ID NO: 3: CcRpp2-R3Aa coding sequence from Cajanus cajan (polynucleotide sequence).

SEQ ID NO: 4: CcRpp2-R3Aa (polypeptide sequence).

In another embodiment the CcRpp2-R1 polynucleotides of SEQ ID NOs: 5-20 and the CcRpp2-R3 polynucleotides of SEQ ID NOs: 37-47, and the respective CcRpp2-R1 polypeptides of SEQ ID NOs: 21-36 and CcRpp2-R3 polypeptides of SEQ ID NOs: 48-58 disclosed herein are useful in generating transgenic plants exhibiting fungal resistance and in methods for conferring or enhancing or increasing fungal resistance to a plant (e.g., a legume crop species).

In some embodiments, a CcRpp2-R1 polypeptide is provided that has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 2 and 21-36, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

In some embodiments, the CcRpp2-R3 polypeptide is provided that has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 4 and 48-58, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

Polypeptides of the present disclosure can be produced either from a nucleic acid disclosed herein, or by the use of standard molecular biology techniques. For example, a CcRpp2-R1 and/or CcRpp2-R3 protein of the present disclosure can be produced by expression of a recombinant nucleic acid of the embodiments in an appropriate host cell, or alternatively by a combination of ex vivo procedures.

Compositions and methods disclosed herein are useful in protecting plants from fungal pathogens. The interactions between a host and a pathogen can be described in a continuum of “immunity” to “susceptibility.” The terms “immunity” or “immune” are used herein to mean the absence of any macroscopically visible disease symptom(s). The term “ resistance” is used herein to mean the presence of macroscopically visible lesions with no or limited sporulation, and/or a reduction in the extent or degree and/or a delay in the progression of any disease symptom(s) and can be, for example, manifested as reduced number of lesions or lesions with a reduction in sporulation. As used herein, the term “susceptibility” or the phrase “lack of resistance” to ASR refers to the occurrence of lesions with sporulation levels equal to or above the sporulation level observed in a reference standard, such as, for example, cultivars Williams or Peking.

Methods of the present disclosure can be carried out, for example, to provide enhanced resistance by Glycine max to the obligate biotrophic fungus Phakopsora pachyrhizi, the main causal agent of ASR, or to Phakopsora meibomiae. For example, increased or enhanced resistance to a fungal pathogen may be compared to the response of a susceptible plant, such as, for example, Williams or Peking. Resistance can vary and is related to the proportion (i.e., percent) of disease symptoms (e.g., lesions) observed on a plant or plant part (e.g., leaf). A numerical score or value for immunity, resistance and susceptibility can be given. For example, a numerical score for resistance represents the degree of resistance a plant exhibits to a plant disease (e.g., ASR). The numerical scores can also be used to compare the degree of resistance, for example, between a plant of interest (e.g., a transgenic legume crop plant) to that of a susceptible plant (e.g., Williams or Peking) or a reference standard.

Methods and compositions for resistance disclosed herein relate to the isolation of one or more resistance genes from a legume species, and the subsequent transfer of one or more of these resistance genes to another plant, soybeans, for example, to provide resistance to Phakopsora spp. via homologous or heterologous expression. An aspect of the present disclosure comprises the transfer of functioning TIR genes to a sexually compatible or incompatible species to produce disease resistance. Polypeptides and TIR genes (e.g., CcRpp2-R1 and CcRpp2-R3 polypeptides and CcRpp2-R1 and CcRpp2-R3 genes) described herein can be used alone or in a stack with other resistance genes such as R genes (including NB-LRR resistance genes) or in a stack with non-R genes (including non-NB-LRR resistance genes) to provide resistance to ASR.

The transgenic approach of the present disclosure therefore can be used alone or in combination with other strategies to produce or confer ASR resistance in plants. Other useful strategies include, but are not limited to, blocking the functional activity of effectors, inhibiting the uptake of a pathogen or pathogen factors (e.g., fungi) into the host cell (e.g., plant cell) and/or conventional breeding for resistance.

In one embodiment, the transgenic approach of the present disclosure may be used in combination with the transgenic expression of a CcRpp1 polynucleotide (for example SEQ ID NO: 59) and the polypeptide encoded thereby (SEQ ID NO: 60) (See also the NB-LRR2 polynucleotide and the polypeptide encoded thereby as disclosed in U.S. Patent Application Publication No. US2018-0103600, incorporated herein by reference in its entirety). Such approach may be by way of either a breeding stack or a molecular stack containing a CcRpp1 gene and a binary of the CcRpp2-R1 and CcRpp2-R3 genes disclosed herein.

Methods of the present disclosure can provide or enhance the resistance of a plant, such that the causal agents of a disease, such as ASR, can no longer reproduce. The term “enhance” means to improve, increase, amplify, multiply, elevate and/or raise, thereby reducing one or more disease symptoms. Accordingly, plants (e.g., soybean) exhibit an increased resistance to a disease (e.g., ASR) when compared to plants that are susceptible or tolerant to Phakopsora spp. In an aspect, methods described herein can reduce one or more symptoms (i.e., disease symptoms) of a legume plant disease (e.g., ASR). A method can comprise exposing a transgenic legume crop plant (e.g., soybean) to a legume plant disease resulting in the transgenic legume crop plant having enhanced resistance to the plant disease. In some aspects, the transgenic legume crop plant comprises a CcRpp2-R1 and CcRpp2-R3 polynucleotide. One or more legume-derived CcRpp2-R1 and CcRpp2-R3 polynucleotides may have at least 90% sequence identity to a sequence as disclosed herein.

In an aspect, the plant, plant part, or plant cell is derived from a plant including but not limited to, alfalfa, clover, peas, beans, lentils, lupins, mesquite, carob, soybeans, peanuts, and tamarind. Progeny, variants, and mutants of disease resistant plants disclosed herein are within the scope of the present disclosure, provided that these progeny, variants, and mutants comprise the original/modified polynucleotides of the parent plant.

In one embodiment, the plant is a legume. In another embodiment, the CcRpp2-R1 and CcRpp2-R3 polypeptides, CcRpp2-R1 and CcRpp2-R3 polynucleotides, and/or CcRpp2-R1 and CcRpp2-R3 resistance genes are derived from a legume. Examples of legumes include, but are not limited to, the genus Phaseolus (e.g., French bean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean (Phaseolus lunatus), Tepary bean (Phaseolus acutifolius), runner bean (Phaseolus coccineus)); the genus Glycine (e.g., Glycine soja, soybeans (Glycine max (L.))); pea (Pisum) (e.g., shelling peas (sometime called smooth or roundseeded peas; Pisum sativum); marrowfat pea (Pisum sativum), sugar pea (Pisum sativum), also called snow pea, edible-podded pea or mangetout, (Pisum granda)); peanut (Arachis hypogaea), clover (Trifolium spp.), medick (Medicago), kudzu vine (Pueraria lobata), common lucerne, alfalfa (Medicago sativa), chickpea (Cicer), lentils (Lens culinaris), lupins (Lupinus); vetches (Vicia), field bean, broad bean (Vicia faba), vetchling (Lathyrus) (e.g., chickling pea (Lathyrus sativus), heath pea (Lathyrus tuberosus)); genus Vigna (e.g., moth bean (Vigna aconiti folia), adzuki bean (Vigna angularis), urd bean (Vigna mungo), mung bean (Vigna radiata), bambara groundnut (Vigna subterrane), rice bean (Vigna umbellata), Vigna vexillata, Vigna unguiculata (also known as asparagus bean, cowpea)); pigeon pea (Cajanus cajari; Cajanus cajan), the genus 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 the genus Canavalia (e.g., jack bean (Canavalia ensiformis)), sword bean (Canavalia gladiata).

Compositions and methods described herein can result in an agronomically desirable line or variety. Agronomic characteristics or traits include, but are not limited to, herbicide tolerance, increased yield, insect control, weed control, pest control, pathogen disease resistance (e.g., fungal, virus, bacterial), high protein production, germination and seedling growth control, enhanced nutrition, environmental stress resistance, increased digestibility, male sterility, flowering time, or transformation technology traits such as cell cycle regulation and/or gene targeting.

The present disclosure provides a method for screening or assaying legume plants for resistance, immunity, or susceptibility to a plant disease. General methods for determination of resistance, immunity, or susceptibility of a plant to a particular pathogen are known to one skilled in the art. For example, a method for screening or assaying legume plants for resistance, immunity or susceptibility to a plant disease may comprise exposing a plant cell, tissue or organ (e.g., leaf) to a pathogen (e.g., Phakopsora pachyrhizi) and then determining and/or measuring in the exposed plant, the degree of resistance, immunity and/or susceptibility to a plant disease (e.g., ASR) caused by the pathogen. The method can further comprise measuring any observable plant disease symptoms on the plant exposed to the plant pathogen and then comparing the plant disease symptoms to a reference standard to determine the degree or extent of disease resistance.

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

In one embodiment an isolated or recombinant nucleic acid is provided that is free of sequences (optimally protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in some embodiments of the disclosure, the isolated polynucleotide sequence encoding the resistance proteins disclosed herein can contain less than about 5 kb, about 4 kb, about 3 kb, about 2 kb, about 1 kb, about 0.5 kb, or about 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, about 20%, about 10%, about 5%, or about 1% (by dry weight) of contaminating protein. When the protein of the embodiments, or a biologically active portion thereof, is recombinantly produced, optimally culture medium represents less than about 30%, about 20%, about 10%, about 5%, or about 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants relating to the nucleotide sequences and proteins encoded are within the scope of the present disclosure. A “fragment” refers to a portion of the nucleotide sequence or a portion of the amino acid sequence and thus the protein encoded thereby. Fragments of a nucleotide sequence can encode protein fragments that retain the biological activity of the native protein and have the ability to confer resistance (i.e., fungal resistance) upon a plant. Alternatively, fragments of a nucleotide sequence, that are useful as hybridization probes, do not necessarily encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence can range from at least about 15 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the polypeptides of the present disclosure. “Functional fragment,” “fragment that is functionally equivalent,” and “functionally equivalent fragment” are used interchangeably herein. These terms refer to a portion or subsequence of a polypeptide sequence of the present disclosure in which its native ability is retained.

A fragment of a nucleotide sequence that encodes a biologically active portion of a polypeptide of the present disclosure can encode at least about 15, about 25, about 30, about 40, or 45 to about 50 contiguous amino acids, or up to the total number of amino acids present in a full-length polypeptide of the embodiments (for example, 341 amino acids for the peptide encoded by SEQ ID NO: 2).

Fragments of a nucleotide sequence that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a protein.

In some embodiments, the CcRpp2-R1 polypeptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more amino acids from the N-terminus of CcRpp2-R1 polypeptides of SEQ ID NOs: 2 and 21-36.

In some embodiments, the CcRpp2-R3 polypeptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more amino acids from the N-terminus of CcRpp2-R3 polypeptides of SEQ ID NOs: 4 and 48-58.

In some embodiments, the CcRpp2-R1 polypeptide fragment is an N-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N-terminus and/or C-terminus relative to CcRpp2-R1 polypeptides of SEQ ID NOs: 2 and 21-36.

In some embodiments, the CcRpp2-R3 polypeptide fragment is an N-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N-terminus and/or C-terminus relative to CcRpp2-R3 polypeptides of SEQ ID NOs: 4 and 48-58.

In some embodiments, a CcRpp2-R1 polypeptide comprises an amino acid sequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of any one of the CcRpp2-R1 polypeptides of SEQ ID NOs: 2 and 21-36, wherein the CcRpp2-R1 polypeptide has fungal resistance activity.

In some embodiments, a CcRpp2-R3 polypeptide comprises an amino acid sequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the amino acid sequence of any one of the CcRpp2-R3 polypeptides of SEQ ID NOs: 4 and 48-58, wherein the CcRpp2-R3 polypeptide has fungal resistance activity.

In some embodiments, a CcRpp2-R1 polypeptide comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entire length of the amino acid sequence of any one of the CcRpp2-R1 polypeptides of SEQ ID NOs: 2 and 21-36.

In some embodiments, a CcRpp2-R3 polypeptide comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entire length of the amino acid sequence of any one of the CcRpp2-R3 polypeptides of SEQ ID NOs: 4 and 48-58.

In some embodiments, the polypeptide fragment is an N-terminal and/or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids from the N-terminus and/or C-terminus, by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon, and/or insertion of a stop codon.

The term “full-length sequence,” when referring to a specified polynucleotide, means having the entire nucleic acid sequence of a native sequence. In one embodiment fragments of the polynucleotide sequences disclosed herein, including SEQ ID NOs: 1 and 3 are provided. Such fragments can be used as hybridization probes or PCR primers, and do not necessarily encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence can range from at least about 15 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the polypeptides of the present disclosure.

In accordance with one embodiment a method of identifying plants comprising a CcRpp2-R1 and/or CcRpp2-R3 gene of the disclosure is provided. The method comprises obtaining a nucleic acid sample from one or more plants, and contacting said nucleic acid sample with a nucleic acid sequence that specifically binds to a CcRpp2-R1 and/or CcRpp2-R3 gene of the disclosure, and detecting the specific binding of the nucleic acid to its target sequence. For example, the method can detect the target sequence through the use of a labeled probe or by conducting a PCR reaction with suitable PCR primers that only produce an amplicon in the presence of the target sequence. In one embodiment the method comprises obtaining a nucleic acid sample from one or more plants, and contacting the nucleic acid sample with either

- i) a polynucleotide that comprises a sequence of at least 8 nucleotides that are identical or have at least 90-95% sequence identity to a contiguous sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5-20 and 37-47, or complements thereof; wherein said method further comprises subjecting said sample and said polynucleotide to stringent hybridization conditions; and assaying said sample for hybridization of said polynucleotide to said DNA; or
- ii) a pair of PCR primers, wherein a first and second PCR primer each specifically bind to a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5-20 and 37-47, wherein said first and second PCR primers are capable of producing an amplicon when bound to their target complementary sequences and subjected to standard PCR reaction conditions; subjecting said sample to polymerase chain reaction conditions; and assaying for an amplicon generated between said first and second primers.

In some embodiments, fusion proteins are provide comprising a CcRpp2-R1 polypeptide and/or CcRpp2-R3 polypeptide of the disclosure represented by a formula selected from the group consisting of:

R1-L-R2, R2-L-R1, R1-R2 or R2-R1

- wherein RI is a CcRpp2-R1 polypeptide, chimeric CcRpp2-R1 polypeptide of the disclosure, or a protein of interest and R2 is a CcRpp2-R3 polypeptide, chimeric CcRpp2-R3 polypeptide of the disclosure, or a protein of interest. The R1 polypeptide is fused either directly or through a linker (L) segment to the R2 polypeptide. The term “directly” defines fusions in which the polypeptides are joined without a peptide linker. Thus “L” represents a chemical bound or polypeptide segment to which both R1 and R2 are fused in frame, most commonly L is a linear peptide to which R1 and R2 are bound by amide bonds linking the carboxy terminus of R1 to the amino terminus of L and carboxy terminus of L to the amino terminus of R2. By “fused in frame” is meant that there is no translation termination or disruption between the reading frames of R1 and R2. The linking group (L) is generally a polypeptide of between 1 and 500 amino acids in length.

A fragment of a nucleotide sequence of the present disclosure can encode a biologically active portion of a polypeptide, or it can be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a polypeptide conferring resistance can be prepared by isolating a portion of one of the nucleotide sequences of the embodiments, expressing the encoded portion of the protein and assessing the ability of the encoded portion of the protein to confer or enhance fungal resistance in a plant. Nucleic acid molecules that are fragments of a nucleotide sequence of the embodiments comprise at least about 15, about 20, about 50, about 75, about 100, or about 150 nucleotides, or up to one less than the total number of nucleotides present in a full-length nucleotide sequence disclosed herein (for example, 5210 nucleotides for SEQ ID NO: 8).

One source of polynucleotides that encode CcRpp2-R1 and/or CcRpp2-R3 polypeptides or related proteins is a species selected from, but not limited to, Arachis, Cercis, Cajanus, Glycine, Medicago, Phaseolus, Pisum or Vigna species, which contains a homologous CcRpp2-R1 polynucleotide or CcRpp2-R3 polynucleotide.

The polynucleotides of SEQ ID NOs: 1 and 5-20 and 3 and 37-47 can be used to express CcRpp2-R1 and CcRpp2-R3 polypeptides, respectively, in legume host plants that include but are not limited to alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, peanut or tamarind.

The polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode CcRpp2-R1 and CcRpp2-R3 polypeptides or related proteins. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from species selected from, but not limited to, Arachis, Cercis, Cajanus, Glycine, Medicago, Phaseolus, Pisum or Vigna.

Polynucleotides that encode CcRpp2-R1 and CcRpp2-R3 polypeptides can also be synthesized de novo from a CcRpp2-R1 or CcRpp2-R3 polypeptide sequence. The sequence of the polynucleotide gene can be deduced from a CcRpp2-R1 or CcRpp2-R3 polypeptide sequence through use of the genetic code. Computer programs such as “BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can be used to convert a peptide sequence to the corresponding nucleotide sequence encoding the peptide. Examples of CcRpp2-R1 or CcRpp2-R3 polypeptide sequences that can be used to obtain corresponding nucleotide encoding sequences include, but are not limited to the CcRpp2-R1 or CcRpp2-R3 polypeptides of SEQ ID NOs: 2, 4, 21-36 and 48-58.

In some embodiments, the nucleic acid molecule encoding a CcRpp2-R1 or CcRpp2-R3 polypeptide is a polynucleotide having the sequence set forth in one of SEQ ID NOs: 1, 3, 5-20 and 37-47, and variants, fragments and complements thereof. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also encompassed. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a plant.

In some embodiments, the nucleic acid molecule encoding a CcRpp2-R1 or CcRpp2-R3 polypeptide is a non-genomic nucleic acid sequence.

In some embodiments, the nucleic acid molecule encoding a CcRpp2-R1 or CcRpp2-R3 polypeptide is a non-genomic polynucleotide having a nucleotide sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity, to any one of the nucleic acid sequences of SEQ ID NOs: 1, 3, 5-20 and 37-47, wherein the encoded CcRpp2-R1 or CcRpp2-R3 polypeptide has fungal resistance activity.

In some embodiments, the CcRpp2-R1 polynucleotide encodes a CcRpp2-R1 polypeptide having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 2 and 21-36, and has at least one amino acid substitution, deletion, insertion or combination therefore, compared to the native sequence.

In some embodiments, the CcRpp2-R3 polynucleotide encodes a CcRpp2-R3 polypeptide having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 4 and 48-58, and has at least one amino acid substitution, deletion, insertion or combination therefore, compared to the native sequence.

In some embodiments, the nucleic acid molecule encodes a CcRpp2-R1 polypeptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entire length of any one of the amino acid sequences of SEQ ID NOs: 2 and 21-36.

In some embodiments, the nucleic acid molecule encodes a CcRpp2-R3 polypeptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entire length of any one of the amino acid sequences of SEQ ID NOs: 4 and 48-58.

In some embodiments, the nucleic acid molecule encodes a CcRpp2-R1 polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 2 and 21-36 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acid substitutions, deletions and/or insertions compared to the amino acid at the corresponding position of the respective SEQ ID NO: 2 and 21-36.

In some embodiments, the nucleic acid molecule encodes a CcRpp2-R3 polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 4 and 48-58 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more amino acid substitutions, deletions and/or insertions compared to the amino acid at the corresponding position of the respective SEQ ID NO: 4 and 48-58.

The polynucleotide coding sequences can be modified to add a codon at the position following the methionine start codon to create a restriction enzyme site for recombinant cloning purposes and/or for expression purposes. In some embodiments, the CcRpp2-R1 and/or CcRpp2-R3 polypeptide further comprises an alanine residue at the position after the translation initiator methionine.

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

Variants of a particular polynucleotide of the embodiments (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs known in the art. Where any given pair of polynucleotides of the present disclosure is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, wherein the percent sequence identity between the two encoded polypeptides is at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity.

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

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

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

Variants may be made by making random mutations or the variants may be designed. In the case of designed mutants, there is a high probability of generating variants with similar activity to the native polypeptide when amino acid identity is maintained in critical regions of the polypeptide which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. A high probability of retaining activity will also occur if substitutions are conservative. Amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type are least likely to materially alter the biological activity of the variant. Table 1 provides a listing of examples of amino acids belonging to each class.

TABLE 1

Classes of amino acids

Class of Amino Acid
Examples of Amino Acids

Nonpolar Side Chains
Ala (A), Val (V), Leu (L), Ile (I),

Pro (P), Met (M), Phe (F), Trp (W)

Uncharged Polar
Gly (G), Ser (S), Thr (T), Cys (C),

Side Chains
Tyr (Y), Asn (N), Gln (Q)

Acidic Side Chains
Asp (D), Glu (E)

Basic Side Chains
Lys (K), Arg (R), His (H)

Beta-branched Side
Thr, Val, Ile

Chains

Aromatic Side Chains
Tyr, Phe, Trp, His

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

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). Known methods of PCR include, and are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be labeled with a detectable group such as ³²P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the polynucleotides of the embodiments. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are known in the art.

Various procedures can be used to check for the presence or absence of a particular sequence of DNA, RNA, or a protein. These include, for example, Southern blots, northern blots, western blots, and ELISA analysis. These techniques are well known in the art.

The compositions and methods of the present disclosure are useful for modulating the levels of one or more proteins in a plant. The term “modulate” is used herein to mean an increase or decrease in the level of a protein within a genetically altered (i.e., transformed) plant relative to the level of that protein from the corresponding non-transformed plant (i.e., a plant not genetically altered in accordance with the methods of the present disclosure).

The genes and polynucleotides of the present disclosure include naturally occurring sequences as well as mutant or altered forms. The proteins disclosed herein also encompass naturally occurring proteins as well as variations, fragments and modified forms thereof. Such variants and fragments will continue to possess the desired ability to confer or enhance plant fungal pathogen resistance. In an aspect, mutations made in the DNA encoding the variant or fragments thereof generally do not place the sequence out of the reading frame and optimally will not create complementary regions that could produce secondary mRNA structure.

The gene or genes of the present disclosure can be expressed as a transgene in order to make plants resistant to ASR. The use of different promoters described herein or known to those of skill in the art will allow the gene's expression to be modulated in different circumstances (i.e., the promoters can be selected based on the desired outcome). For instance, higher levels of expression in a particular tissue system or organ (e.g., leaves) may be desired to enhance resistance. The entire gene can be inserted (e.g., both native promoter and coding sequence), as a transgene, permitting quick combination with other traits, such as insect or herbicide resistance.

In accordance with one embodiment a polynucleotide encoding a polypeptide having at least 85%, 90%, 95% or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 21-36 and 48-58 is provided, wherein the polypeptide when expressed in the cells of a plant confers resistance to Asian Soybean Rust (ASR) disease for said plant. In a further embodiment the polynucleotide is selected from the group consisting of SEQ ID NOs: 1, 3, 5-20 and 37-47 or a polynucleotide having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NOs: 1, 3, 5-20 and 37-47. In one embodiment these polynucleotide sequences can be operably linked to heterologous regulatory elements necessary for expressing the encoded CcRpp2-R1 and CcRpp2-R3 gene products in a plant cell. For example, the regulatory elements can include promoters; translation leader sequences; enhancers; termination sequences; and polyadenylation recognition sequences. In one embodiment a recombinant polynucleotide is provided wherein a heterologous plant promoter is operably linked to a CcRpp2-R1 or CcRpp2-R3 coding sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5-20 and 37-47 or a polynucleotide having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NOs: 1, 3, 5-20 and 37-47. In one embodiment a recombinant polynucleotide is provided wherein a heterologous plant promoter is operably linked to a CcRpp2-R1 or CcRpp2-R3 coding sequence selected from the group consisting of SEQ ID NOs: 1 or 3 or a polynucleotide having at least 85%, 90%, 95% or 99% sequence identity to SEQ ID NOs: 1 or 3. In one embodiment a recombinant polynucleotide is provided wherein a heterologous plant promoter is operably linked to a CcRpp2-R1 or CcRpp2-R3 coding sequence selected from the group consisting of SEQ ID NOs: 1 or 3.

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

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

In one embodiment, the stacked combination includes one or more genes encoding pesticidal proteins including, but not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLOS Pathogens 7:1-13); from Pseudomonas protegens strain CHA0 and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58:12343-12349) and from Pseudomonas pseudoalcaligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal, 3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069); U.S. Pat. Nos. 6,048,838, and 6,379,946; a PIP-1 polypeptide of U.S. Pat. No. 9,688,730; an AfIP-1A and/or AfIP-1B polypeptide of U.S. Pat. No. 9,475,847; a PIP-47 polypeptide of US Publication Number US20160186204; an IPD045 polypeptide, an IPD064 polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077 polypeptide of International Patent Application Publication Number WO 2016/114973; an IPD080 polypeptide of PCT Serial Number PCT/US17/56517; an IPD078 polypeptide, an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, an IPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide of Serial Number PCT/US17/54160; PIP-72 polypeptide of US Patent Publication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide of US Publication Number US20170166921; an IPD098 polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109 polypeptide of US Serial number 62/521084; a PtIP-83 polypeptide of US Publication Number US20160347799; a PtIP-96 polypeptide of US Publication Number US20170233440; an IPD079 polypeptide of PCT Publication Number WO2017/23486; an IPD082 polypeptide of PCT Publication Number WO 2017/105987, an IPD090 polypeptide of Serial Number PCT/US17/30602, an IPD093 polypeptide of U.S. Ser. No. 62/434,020; an IPD103 polypeptide of Serial Number PCT/US17/39376; an IPD101 polypeptide of U.S. Ser. No. 62/438,179; an IPD121 polypeptide of U.S. Ser. No. 62/508,514; and δ-endotoxins including, but not limited to a Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry 72 classes of δ-endotoxin polypeptides and the B. thuringiensis cytolytic cyt1 and cyt2 genes. Members of these classes of B. thuringiensis insecticidal proteins can be found in Crickmore, et al., “Bacillus thuringiensis toxin nomenclature” (2011), at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed on the world-wide web using the “www” prefix).

In another embodiment, the stacked combination includes a polynucleotide encoding resistance to an herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J. 7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449, respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and 5,378,824; U.S. patent application Ser. No. 11/683,737 and International Publication WO 1996/33270.

In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to Glyphosate (resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar) genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS which can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes. See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 5,094,945, 4,940,835; 5,866,775; 6,225,114; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 and 5,491,288 and International Publications EP 1173580; WO 2001/66704; EP 1173581 and EP 1173582, which are incorporated herein by reference for this purpose. Glyphosate resistance is also imparted to plants that express a gene encoding a glyphosate oxido-reductase enzyme as described more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose. In addition, glyphosate resistance can be imparted to plants by the over expression of genes encoding glyphosate N-acetyltransferase. See, for example, U.S. Pat. Nos. 7,462,481; 7,405,074 and US Patent Application Publication Number US 2008/0234130. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC® Accession Number 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. EP Application Number 0 333 033 to Kumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in EP Application Numbers 0 242 246 and 0 242 236 to Leemans, et al.; De Greef, et al., (1989) Bio/Technology 7:61, describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616 and 5,879,903, which are incorporated herein by reference for this purpose. Exemplary genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

In another embodiment, the stacked combination includes a polynucleotide encoding a protein for resistance to herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+genes) and a benzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell 3:169, describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA molecules containing these genes are available under ATCC® Accession Numbers 53435, 67441 and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes, et al., (1992) Biochem. J. 285:173.

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

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

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

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

In another embodiment, the stacked combination includes a polynucleotide encoding bromoxynil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance.

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

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

- (1) Down-regulation of stearoyl-ACP to increase stearic acid content of the plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).
- (2) Elevating oleic acid via FAD-2 gene modification and/or decreasing linolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos. 6,063,947; 6,323,392; 6,372,965 and WO 1993/11245).
- (3) Altering conjugated linolenic or linoleic acid content, such as in WO 2001/12800.
- (4) Altering LEC1, AGP, Dek1, Supera11, mi1 ps, and various Ipa genes such as Ipa1, Ipa3, hpt or hggt. For example, see, WO 2002/42424, WO 1998/22604, WO 2003/011015, WO 2002/057439, WO 2003/011015, U.S. Pat. Nos. 6,423,886, 6,197,561, 6,825,397 and US Patent Application Publication Numbers US 2003/0079247, US 2003/0204870 and Rivera-Madrid, et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.
- (5) Genes encoding delta-8 desaturase for making long-chain polyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and 8,338,152), delta-9 desaturase for lowering saturated fats (U.S. Pat. No. 8,063,269), Primula Δ6-desaturase for improving omega-3 fatty acid profiles.
- (6) Isolated nucleic acids and proteins associated with lipid and sugar metabolism regulation, in particular, lipid metabolism protein (LMP) used in methods of producing transgenic plants and modulating levels of seed storage compounds including lipids, fatty acids, starches or seed storage proteins and use in methods of modulating the seed size, seed number, seed weights, root length and leaf size of plants (EP 2404499).
- (7) Altering expression of a High-Level Expression of Sugar-Inducible 2 (HSI2) protein in the plant to increase or decrease expression of HSI2 in the plant. Increasing expression of HSI2 increases oil content while decreasing expression of HSI2 decreases abscisic acid sensitivity and/or increases drought resistance (US Patent Application Publication Number 2012/0066794).
- (8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oil content in plant seed, particularly to increase the levels of omega-3 fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (US Patent Application Publication Number 2011/0191904).
- (9) Nucleic acid molecules encoding wrinkled1-like polypeptides for modulating sugar metabolism (U.S. Pat. No. 8,217,223).

A feature of the present disclosure are methods comprising introducing a polynucleotide into a plant. In some aspects of the present disclosure, the polynucleotide can be presented in such a manner that the sequence gains access to the interior of a cell of the plant, including its potential insertion into the genome of a plant. The methods of the present disclosure do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide gains access to the interior of at least one cell of the plant.

A polynucleotide can be transiently or stably introduced into a host cell and can be maintained non-integrated, for example, as a plasmid.

Transformation methods as well as methods for introducing polynucleotide sequences into plants can depend on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include, but are not limited to, microinjection, electroporation, direct gene transfer, Lecl transformation and ballistic particle acceleration. As newer methods become available, they can also be applied to the present disclosure as the method of transformation or transfection is not critical.

The cells that have been transformed can be grown into plants in accordance with conventional ways. These plants can then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations can be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In some aspects of the present disclosure, the transformed seed or transgenic seed having a nucleotide construct or an expression cassette is stably incorporated into their genome.

In an aspect, the present disclosure encompasses seeds comprising a polynucleotide sequence disclosed herein that can develop into or can be used to develop a plant or plants with increased or enhanced resistance to a pathogen (e.g., fungi) or infection caused by a pathogen as compared to, for example, a wild-type variety of the plant seed. In an aspect, the present disclosure features seeds from transgenic legume crop plants wherein the seed comprises a polynucleotide disclosed herein.

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

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

In an aspect, constructs or vectors or expression cassettes are not present in the genome of the original plant or are present in the genome of the transgenic plant, but not at their natural locus of the genome of the original plant.

The compositions disclosed herein can be generated or maintained through the process of introgressing. Introgressing is sometimes called “backcrossing” when the process is repeated two or more times. In introgressing or backcrossing, the “donor” parent refers to the parental plant with 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 parental plant into which the gene or locus is being introgressed. The initial cross gives rise to the FI generation; the term “BC1” then refers to the second use of the recurrent parent, and “BC2” refers to the third use of the recurrent parent, and so on.

Accordingly, an aspect of the present disclosure is a method of enhancing plant resistance to a plant disease, such as ASR. The method can comprise conferring resistance to a pathogen, for example, a pathogen that causes ASR, by introgression of legume-derived CcRpp2-R1 and CcRpp2-R3 binary resistance genes, or homologs thereof, into germplasm in a breeding program (i.e., a breeding program for resistance to ASR).

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

Aspects of the present disclosure comprise methods for identification of germplasm as a source of resistance including, but not limited to, germplasm in one or more of the following genus: Glycine, Vigna, and Lablab.

In one embodiment, the legume crop species or legume-derived gene is derived from the genus Glycine. Examples of Glycine species include, but are not limited to, Glycine arenaria, Glycine argyrea, Glycine cyrtoloba, Glycine canescens, Glycine clandestine, Glycine curvata, Glycine falcata, Glycine latifolia, Glycine microphylla, Glycine pescadrensis, Glycine stenophita, Glycine syndetica, Glycine soja, Glycine tabacina and Glycine tomentella.

In another embodiment, the legume crop species or legume-derived gene is derived from the genus Vigna. Vigna is a pantropic genus that comprises approximately 100 species. It is a taxonomic group subdivided into the subgenera Vigna, Haydonia, Plectotropis (African), Ceratotropis (Asian), Sigmoidotropis, and Lasiopron. The genus includes economically relevant species such as Vigna unguiculata (L.) Walp (cowpea), Vigna radiata (L.) Wilczek (mung bean), Vigna angularis (Willd.) Ohwi and Ohashi (azuki bean), Vigna mungo (L.) Hepper (black gram), and Vigna umbellata (Thunb.) Ohwi and Ohashi (rice bean). Four subspecies are recognized within Vigna unguiculata: dendtiana, a wild relative of cultivated subspecies; cylindrica, cultivated catjang; sesquipedalis, cultivated yardlong bean; and unguiculata, cultivated black-eyed pea. Vigna unguiculata ssp. unguiculata is further divided into cultivar groups Unguiculata, grown as a pulse; Biflora or Cilindrica (catjang), mainly used as a forage; Sesquipedalis (yardlong or asparagus bean), grown as a vegetable; Textilis, cultivated for the fibres of its long floral peduncles; and Melanophthalmus (black-eyed pea). Susceptibility of several Vigna species, including Vigna radiata, Vigna mungo and Vigna unguiculata to Phakopsora pachyrhizi has been reported under field and greenhouse conditions.

In another embodiment, the legume crop species or legume-derived gene is derived from the genus Lablab. Lablab purpureus (L.) Sweet is a leguminous species (Verdcourt (1971) Flora of Tropical East Africa, pp. 696-699, Crown Agents, London, UK; and Duke et al. (1981) Handbook of Legumes of World Economic Importance, pp. 102-106, Plenum Press, New York, USA and London, UK) native to Asia and Africa (Pengelly and Maass, (2001) Gen. resour. crop ev. 48: 261-272). It is commonly known as lablab bean, hyacinth bean, bonavist bean, field bean, Egyptian bean, poor man's bean, Tonga bean (English) and by at least 20 additional vernacular names. It is grown in Africa, Asia, and the Caribbean as either a pulse crop or as a green vegetable (Duke et al. (1981) Handbook of Legumes of World Economic Importance, pp. 102-106, Plenum Press, New York, USA and London, UK); and Pengelly and Maass, (2001) Gen. resour. crop ev. 48: 261-272).

Lablab purpureas has been reported as an alternative host for Phakopsora pachyrhizi (Perez-Hernandez, (2007) Alternative hosts of Phakopsora pachyrhizi in the Americas: An analysis of their role in the epidemiology of Asian soybean rust in the continental U.S. M.Sc. thesis. Iowa State University. Ames, Iowa. U.S.A.; Vakili (1981) Plant Dis. 65: 817-819; and Poonpolgul and Surin, (1980) Soybean Rust Newsletter, 3: 30-31).

In an aspect, the legume crop species or legume-derived gene is derived from the genus Cicer, Cajanus, Medicago, Phaseolus, Pisum, Pueraria, or Trifolium. Examples of Cicer species include, but are not limited to, Cicer arietinum, Cicer echinospermum, Cicer reticulatum and Cicer pinnatifldum. An example of the Cajanus species include, but is not limited to Cajanus cajan. Examples of the Medicago species include, but are not limited to, Medicago truncatula and Medicago sativa. Examples of the Phaseolus species include, but are not limited to, Phaseolus vulgaris, Phaseolus lunatus, Phaseolus acutifolius and Phaseolus coccineus. Examples of the Pisum species include, but are not limited to, Pisum abyssinicum, Pisum sativum, Pisum elatius, Pisum fulvum, Pisum transcaucasium and Pisum humile. An example of the Pueraria species includes, but is not limited to, Pueraria lobata. Examples of the Trifolium species include, but are not limited to, Trifolium aureum and Trifolium occidentale.

The present disclosure also comprises sequences described herein that can be provided in expression cassettes or DNA constructs for expression in the plant of interest. In an aspect, the cassette can include 5′ and 3′ heterologous regulatory sequences operably linked to a sequence disclosed herein. “Operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. Such regulatory sequences are well known in the art and include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence in certain host cells or under certain conditions. The design of the vector can depend on, for example, the type of the host cell to be transformed or the level of expression of nucleic acid desired. The cassette can contain one or more additional genes to be co-transformed into the plant. And, any additional gene(s) can be provided on multiple expression cassettes.

Expression cassettes of the present disclosure can include many restriction sites for insertion of the nucleotide sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette can also contain selectable marker genes.

An expression cassette can further include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region, a DNA sequence of the disclosure, and a transcriptional and translational termination region functional in plants. The transcriptional initiation region, the promoter, can be native or analogous or foreign or heterologous to the plant host. Additionally, the promoter can be the natural sequence or alternatively a synthetic sequence. The term “foreign” means that the transcriptional initiation region is not found in the native plant into which the transcriptional initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Examples of promoters include, but are not limited to, the Cauliflower Mosaic Virus 35S and soybean Ubiquitin 6.

While it may be preferable to express the sequences using heterologous promoters, homologous promoters or native promoter sequences can be used. Such constructs would change expression levels in the host cell (i.e., plant or plant cell). Thus, the phenotype of the host cell (i.e., plant or plant cell) is altered.

A termination region can be native with the transcriptional initiation region, native with the operably linked DNA sequence of interest, or derived from another source. Convenient termination regions are available from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

In an aspect, endogenous or transgenic resistance orthologs can be altered by homologous or non-homologous recombinatory methods, such as, for example, by genome editing. Such alterations refer to a nucleotide sequence having at least one modification when compared to its non-modified sequence and include, for example: (i) replacement of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

In some embodiments, the disclosed CcRpp2-R1 and CcRpp2-R3 polynucleotide compositions can be introduced into the genome of a plant using genome editing technologies, or previously introduced CcRpp2-R1 and CcRpp2-R3 polynucleotides in the genome of a plant may be edited using genome editing technologies.

For example, the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. For example, the disclosed polynucleotides can be introduced into a desired location in a genome using a CRISPR-Cas system, for the purpose of site-specific insertion. The desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with an existing trait of interest. Existing traits of interest could be either an endogenous trait or a previously introduced trait.

“Target site,” “target sequence,” “target DNA,” “target locus,” “genomic target site,” “genomic target sequence,” and “genomic target locus” are used interchangeably herein and refer to a polynucleotide sequence, for example in the genome (including chloroplastic and mitochondrial DNA) of a cell, to which an endonuclease is recruited, and optionally nicks or cleaves the DNA of 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 thereby not be naturally occurring in the genome, or the target site can be found in a heterologous genomic location compared to where it occurs in nature.

In some embodiments, where the disclosed CcRpp2-R1 and CcRpp2-R3 polynucleotides have previously been introduced into a genome or are endogenous homologs of CcRpp2-R1 and CcRpp2-R3 in other legume species, genome editing technologies may be used to alter or modify the introduced polynucleotide sequences or the endogenous homologs. Site specific modifications that can be introduced into the disclosed CcRpp2-R1 and CcRpp2-R3 polynucleotide compositions include those produced using any method for introducing site specific modification, including, but not limited to, through the use of gene repair oligonucleotides (e.g. U.S. Patent Application Publication No. 2013/0019349), or through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Such technologies can be used to modify the previously introduced polynucleotides through the insertion, deletion or substitution of nucleotides within the introduced polynucleotides. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotides. Additional sequences that may be added include, additional expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing technologies may be used to position coding sequences for additional ASR resistance proteins in close proximity to the disclosed CcRpp2-R1 and CcRpp2-R3 polynucleotide compositions disclosed herein within the genome of a plant, in order to generate molecular stacks of ASR-resistance proteins.

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

The gene(s) can be optimized for increased expression in the transformed plant as needed. In other words, the genes can be synthesized using plant-preferred codons for improved expression. Methods for synthesizing plant-preferred genes are known in the art.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that can be deleterious to gene expression. The G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

The expression cassettes can additionally contain 5′ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), and human immunoglobulin heavy chain binding protein (BiP); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4); tobacco mosaic virus leader (TMV); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382 385). Other methods known to enhance translation can also be utilized, such as, introns.

The various DNA fragments can be manipulated while preparing the expression cassette, to ensure that the DNA sequences are in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers can be employed to join the DNA fragments. Alternatively, other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, or removal of restriction sites. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, can be involved.

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

For expression of a target gene and/or protein (e.g., one or more CcRpp2-R1 and CcRpp2-R3 genes and/or one or more CcRpp2-R1 and CcRpp2-R3 proteins) of the present disclosure in a plant or plant cell, the methods described herein comprise transforming a plant or plant cell with a polynucleotide, for example, as disclosed herein, that encodes the target protein. The polynucleotides described herein can be operably linked to a promoter that drives expression in a plant cell. Any promoter known in the art can be used in the methods of the present disclosure including, but not limited to, constitutive promoters, pathogen-inducible promoters, wound-inducible promoters, tissue-preferred promoters, and chemical-regulated promoters. The choice of promoter may depend on the desired timing and location of expression in the transformed plant as well as other factors, which are known to those of skill in the art. Transformed cells or plants can be grown or bred to generate a plant comprising one or more of polynucleotides that were introduced into the cell or plant that, for example, encodes CcRpp2-R1 and CcRpp2-R3 proteins.

A number of promoters can be used in the practice of the disclosure. The promoters can be selected based on the desired outcome. That is, the nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in the host cell of interest. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS; and the like. Other constitutive promoters include, for example, those disclosed in U.S. Pat. Nos. 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 known in the art, and can be contemplated for use in the present disclosure.

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

Of interest are promoters that are expressed locally at or near the site of pathogen infection. Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter can be used in the constructions of the disclosure. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene, wun1 and wun2, win1 and win2, systemin, WIP1, MPI gene, and the like.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter can be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (e.g., the glucocorticoid-inducible promoter, and tetracycline-inducible and tetracycline-repressible promoters).

Tissue-preferred promoters can be utilized to target enhanced expression of the target genes or proteins (e.g., polynucleotide sequences encoding legume-derived CcRpp2-R1 and CcRpp2-R3 polypeptides) within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2): 255 -265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified.

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

“Seed-preferred” promoters include both “seed-specific” promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as “seed-germinating” promoters (those promoters active during seed germination). Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message), cZ19B1 (maize 19 kDa zein), milps (myo-inositol-1-phosphate synthase), and celA (cellulose synthase) (see WO 00/11177, herein incorporated by reference). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference.

Expression of the polynucleotides of the present disclosure can involve the use of the intact, native CcRpp2-R1 and CcRpp2-R3 genes, wherein the expression is driven by a cognate 5′ upstream promoter sequence(s). Alternatively, expression can be generated using constructs assembled with 5′ transcriptional control sequences provided by heterologous CcRpp2-R1 and CcRpp2-R3 disease resistance genes expressed in the host legume. One skilled in the art will be able to identify genes encoding CcRpp2-R1 and CcRpp2-R3 proteins following the teachings of this application, to evaluate their expression level, and to select preferred promoter sequences that can be used for expression of the CcRpp2-R1 and/or CcRpp2-R3 gene of interest. The use of either cognate or heterologous CcRpp2-R1 and CcRpp2-R3 promoter sequences provides an option to regulate protein expression to avoid or minimize any potential undesired outcomes associated with inappropriate or unwanted expression and plant defense activation.

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

In another embodiment, transgenic plants expressing polynucleotides and polypeptides disclosed herein (i.e. the CcRpp2-R1 and CcRpp2-R3 resistance gene and polypeptide sequences) may also have one or more fungicides applied to the transgenic plants as a method of further preventing ASR associated damage to a legume crop species. These fungicidal compounds may also be applied to supplement the protection of a transgenic legume crop species comprising the CcRpp2-R1 and CcRpp2-R3 resistance gene sequences to a wider variety of undesirable diseases. These fungicides may be formulated or tank-mixed with other fungicide(s) disclosed herein or applied sequentially with the other fungicide(s). Such fungicides may include 2-(thiocyanatomethylthio)-benzothiazole, 2-phenylphenol, 8-hydroxyquinoline sulfate, ametoctradin, aminopyrifen, amisulbrom, antimycin, Ampelomyces quisqualis, azaconazole, azoxystrobin, Bacillus subtilis, Bacillus subtilis strain QST713, benalaxyl, benomyl, benthiavalicarb-isopropyl, benzovindiflupyr, benzylaminobenzene-sulfonate (BABS) salt, bicarbonates, biphenyl, bismerthiazol, bitertanol, bixafen, blasticidin-S, borax, Bordeaux mixture, boscalid, bromuconazole, bupirimate, calcium polysulfide, captafol, captan, carbendazim, carboxin, carpropamid, carvone, chlazafenone, chloroinconazide, chloroneb, chlorothalonil, chlozolinate, Coniothyrium minitans, copper hydroxide, copper octanoate, copper oxychloride, copper sulfate, copper sulfate (tribasic), cuprous oxide, cyazofamid, cyflufenamid, cymoxanil, cyproconazole, cyprodinil, dazomet, debacarb, diammonium ethylenebis-(dithiocarbamate), dichlofluanid, dichlorophen, diclocymet, diclomezine, dichloran, diethofencarb, difenoconazole, difenzoquat ion, diflumetorim, dimethomorph, dimoxystrobin, diniconazole, diniconazole-M, dinobuton, dinocap, diphenylamine, dithianon, dodemorph, dodemorph acetate, dodine, dodine free base, edifenphos, enestrobin, enestroburin, epoxiconazole, ethaboxam, ethoxyquin, etridiazole, famoxadone, fenamidone, fenarimol, fenbuconazole, fenfuram, fenhexamid, fenoxanil, fenpiclonil, fenpropidin, fenpropimorph, fenpyrazamine, fentin, fentin acetate, fentin hydroxide, ferbam, ferimzone, fluazinam, fludioxonil, fluindapyr, flumorph, fluopicolide, fluopyram, fluoroimide, fluoxapiprolin, fluoxastrobin, fluquinconazole, flusilazole, flusulfamide, flutianil, flutolanil, flutriafol, fluxapyroxad, folpet, formaldehyde, fosetyl, fosetyl-aluminium, fuberidazole, furalaxyl, furametpyr, guazatine, guazatine acetates, GY-81, hexachlorobenzene, hexaconazole, hymexazol, imazalil, imazalil sulfate, imibenconazole, iminoctadine, iminoctadine triacetate, iminoctadine tris(albesilate), inpyrfluxam, iodocarb, ipconazole, ipfenpyrazolone, iprobenfos, iprodione, iprovalicarb, isofetamide, isoflucypram, isoprothiolane, isopyrazam, isotianil, kasugamycin, kasugamycin hydrochloride hydrate, kresoxim-methyl, laminarin, mancopper, mancozeb, mandipropamid, maneb, mefenoxam, mepanipyrim, mepronil, meptyl-dinocap, mercuric chloride, mercuric oxide, mercurous chloride, metalaxyl, metalaxyl-M, metam, metam-ammonium, metam-potassium, metam-sodium, metconazole, methasulfocarb, methyl iodide, methyl isothiocyanate, metiram, metominostrobin, metrafenone, mildiomycin, myclobutanil, nabam, nitrothal-isopropyl, nuarimol, octhilinone, ofurace, oleic acid (fatty acids), orysastrobin, oxadixyl, oxathiapiprolin, oxine-copper, oxpoconazole fumarate, oxycarboxin, pefurazoate, penconazole, pencycuron, penflufen, pentachlorophenol, pentachlorophenyl laurate, penthiopyrad, phenylmercury acetate, phosphonic acid, phthalide, picoxystrobin, polyoxin B, polyoxins, polyoxorim, potassium bicarbonate, potassium hydroxyquinoline sulfate, probenazole, prochloraz, procymidone, propamocarb, propamocarb hydrochloride, propiconazole, propineb, proquinazid, prothioconazole, pydiflumetofen, pyrametostrobin, pyraoxystrobin, pyraclostrobin, pyraziflumid, pyrazophos, pyribencarb, pyributicarb, pyrifenox, pyrimethanil, pyriofenone, pyroquilon, quinoclamine, quinoxyfen, quintozene, Reynoutria sachalinensis extract, sedaxane, silthiofam, simeconazole, sodium 2-phenylphenoxide, sodium bicarbonate, sodium pentachlorophenoxide, spiroxamine, sulfur, SYP-Z048, tar oils, tebuconazole, tebufloquin, tecnazene, tetraconazole, thiabendazole, thifluzamide, thiophanate-methyl, thiram, tiadinil, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazoxide, tricyclazole, tridemorph, trifloxystrobin, triflumizole, triforine, triticonazole, validamycin, valifenalate, valiphenal, vinclozolin, zineb, ziram, zoxamide, Candida oleophila, Fusarium oxysporum, Gliocladium spp., Phlebiopsis gigantea, Streptomyces griseoviridis, Trichoderma spp., (RS)-N-(3,5-dichlorophenyl)-2-(methoxymethyl)-succinimide, 1,2-dichloropropane, 1,3-dichloro-1,1,3,3-tetrafluoroacetone hydrate, 1-chloro-2,4-dinitronaphthalene, 1-chloro-2-nitropropane, 2-(2-heptadecyl-2-imidazolin-1-yl)ethanol, 2,3-dihydro-5-phenyl-1,4-dithi-ine 1,1,4,4-tetraoxide, 2-methoxyethylmercury acetate, 2-methoxyethylmercury chloride, 2-methoxyethylmercury silicate, 3-(4-chlorophenyl)-5-methylrhodanine, 4-(2-nitroprop-1-enyl)phenyl thiocyanateme, ampropylfos, anilazine, azithiram, barium polysulfide, Bayer 32394, benodanil, benquinox, bentaluron, benzamacril, benzamacril-isobutyl, benzamorf, binapacryl, bis(methylmercury) sulfate, bis(tributyltin) oxide, buthiobate, cadmium calcium copper zinc chromate sulfate, carbamorph, CECA, chlobenthiazone, chloraniformethan, chlorfenazole, chlorquinox, climbazole, copper bis(3-phenylsalicylate), copper zinc chromate, coumoxystrobin, cufraneb, cupric hydrazinium sulfate, cuprobam, cyclafuramid, cypendazole, cyprofuram, decafentin, dichlobentiazox, dichlone, dichlozoline, diclobutrazol, dimethirimol, dinocton, dinosulfon, dinoterbon, dipymetitrone, dipyrithione, ditalimfos, dodicin, drazoxolon, EBP, enoxastrobin, ESBP, etaconazole, etem, ethirim, fenaminstrobin, fenaminosulf, fenapanil, fenitropan, fenpicoxamid, florylpicoxamid, flubeneteram, flufenoxystrobin, fluopimomide, fluotrimazole, furcarbanil, furconazole, furconazole-cis, furmecyclox, furophanate, glyodine, griseofulvin, halacrinate, Hercules 3944, hexylthiofos, ICIA0858, ipfentrifluconazole, ipflufenoquin, isopamphos, isovaledione, mandestrobin, mebenil, mecarbinzid, mefentrifluconazole, metazoxolon, methfuroxam, methylmercury dicyandiamide, metsulfovax, metyltetraprole, milneb, mucochloric anhydride, myclozolin, N-3,5-dichlorophenyl-succinimide, N-3-nitrophenylitaconimide, natamycin, N-ethylmercurio-4-toluenesulfonanilide, nickel bis(dimethyldithiocarbamate), OCH, phenylmercury dimethyldithiocarbamate, phenylmercury nitrate, phosdiphen, prothiocarb; prothiocarb hydrochloride, pyracarbolid, pyrapropoyne, pyridachlometyl, pyridinitril, pyrisoxazole, pyroxychlor, pyroxyfur, quinacetol; quinacetol sulfate, quinazamid, quinconazole, quinofumelin, rabenzazole, salicylanilide, SSF-109, sultropen, tecoram, thiadifluor, thicyofen, thiochlorfenphim, thiophanate, thioquinox, tioxymid, triamiphos, triarimol, triazbutil, trichlamide, triclopyricarb, triflumezopyrim, urbacid, zarilamid, (2S,3S)-3-(o-tolyl)butan-2-yl (4-methoxy-3-(propionyloxy)picolinoyl)-L-alaninate, and any combinations thereof.

The present disclosure also includes kits for the assays described herein. The polypeptide sequences and polynucleotides can be packaged as a component of a kit with instructions for completing the assay disclosed herein. The kits of the present disclosure can include any combination of the polypeptides and/or polynucleotides described herein and suitable instructions (written and/or provided as audio-, visual-, or audiovisual material). In one embodiment, the kit relates to a DNA detection kit for identifying TIR genes (e.g., CcRpp2-R1 and CcRpp2-R3 genes) or CcRpp2-R1 and CcRpp2-R3 proteins against ASR. Kits utilizing any of the sequences disclosed herein for the identification of a transgenic event (e.g., CcRpp2-R1 and CcRpp2-R3) in a plant for efficacy against ASR are provided. For example, the kits can comprise a specific probe having a sequence corresponding to or is complementary to a sequence having between 80% and 100% sequence identity with a specific region of the transgenic event. The kits can include any reagents and materials required to carry out the assay or detection method.

In accordance with embodiment 1 an ASR resistance polypeptide is provided selected from:

- a) a CcRpp2-R1 polypeptide comprising an amino acid sequence having greater than 60% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 2 and 21-36; or
- b) a CcRpp2-R3 polypeptide comprising an amino acid sequence having greater than 60% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 4 and 48-58.

In accordance with embodiment 2 an ASR resistance composition is provided comprising a CcRpp2-R1 polypeptide of embodiment 1 and a CcRpp2-R3 polypeptide of embodiment 1.

In accordance with embodiment 3 a polynucleotide encoding an ASR resistance polypeptide is provided wherein the encoded polypeptide is selected from:

- a) a CcRpp2-R1 polypeptide comprising an amino acid sequence having greater than 60% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 2 and 21-36, optionally wherein the polynucleotide encoding an ASR resistance polypeptide is operably linked to a heterologous regulatory element such as a heterologous plant promoter; or
- b) a CcRpp2-R3 polypeptide comprising an amino acid sequence having greater than 60% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 4 and 48-58, optionally wherein the polynucleotide encoding an ASR resistance polypeptide is operably linked to a heterologous regulatory element such as a heterologous plant promoter.

In accordance with embodiment 4 the recombinant polynucleotide of embodiment 3 is provided, wherein the recombinant polynucleotide is selected from:

- a) a polynucleotide having at least 70% sequence identity to the polynucleotide of any one of SEQ ID NOs: 1 and 5-20; and
- b) the polynucleotide having at least 70% sequence identity to the polynucleotide of any one of SEQ ID NOs: 3 and 37-47.

In accordance with embodiment 5 a DNA construct is provided comprising, the recombinant polynucleotide of embodiment 3 or 4 and a heterologous regulatory sequence operably linked to the recombinant polynucleotide.

In accordance with embodiment 6 a transgenic plant or plant cell is provided comprising the DNA construct of any one of embodiments 1-5.

In accordance with embodiment 7 the transgenic plant of claim 6 is provided, wherein the plant is a legume crop plant.

In accordance with embodiment 8 a transgenic legume crop plant of embodiment 7 is provided, wherein transgenic legume crop plant is soybean.

In accordance with embodiment 9 a method of conferring disease resistance in a legume crop species is provided, wherein the method comprises transforming a legume crop species with a heterologous legume-derived CcRpp2-R1 gene and a heterologous legume-derived CcRpp2-R3 gene that confer disease resistance to a legume crop species disease.

In accordance with embodiment 10 a method of embodiment 9 is provided, wherein the legume crop species disease is caused by a plant pathogen.

In accordance with embodiment 11 a method of embodiment 9 or 10 is provided, wherein the plant pathogen is Phakopsora pachyrhizi or Phakopsora meibomiae.

In accordance with embodiment 12 a method of any one of embodiments 9-11 is provided, wherein the legume crop species disease is Asian soybean rust.

In accordance with embodiment 13 a method of any one of embodiments 9, 10, 11 or 12 is provided, wherein the legume crop species is an alfalfa, clover, pea, bean lentil, lupin, mesquite, carob, soybean, peanut or tamarind.

In accordance with embodiment 14 a method of any one of embodiments 9, 10, 11, 12 or 13 is provided, wherein the legume crop species is soybean.

In accordance with embodiment 15 a method of any one of embodiments 9, 10, 11, 12, 13 or 14 is provided, wherein the legume-derived CcRpp2-R1 or CcRpp2-R3 genes are derived from genus Arachis, Cercis, Cajanus, Glycine, Medicago, Phaseolus, Pisum or Vigna.

In accordance with embodiment 16 a transgenic legume crop plant of any one of embodiments 6-8 is provided, further comprising one or more additional resistance genes, optionally wherein the additional resistance gene is a CcRpp1 gene.

In accordance with embodiment 17 a transgenic legume crop plant of any one of embodiments 6-8 or 16 is provided, further comprising an improved agronomic trait.

In accordance with embodiment 18 a seed from the transgenic legume crop plant of embodiments 16 or 17, wherein the seed has a DNA construct of any one of embodiments 1-5.

In accordance with embodiment 19 a method of reducing one or more symptoms of a legume plant disease is provided, wherein the method comprises exposing the transgenic legume crop plant of any one of claims 6-8 to the legume plant disease wherein the transgenic legume crop plant has an enhanced resistance to the plant disease.

In accordance with embodiment 20 the method of embodiment 19 is provided, wherein the plant disease is Asian soybean rust.

In accordance with embodiment 21 a method of producing an Asian soybean rust resistant plant is provided, wherein the method comprising transforming a plant cell with a legume-derived CcRpp2-R1 gene and a legume-derived CcRpp2-R3 gene.

In accordance with embodiment 22 the method of embodiment 21 is provided, further comprising regenerating the transformed plant from the transformed plant cell.

In accordance with embodiment 22 the method of embodiment 22 is provided, further comprising the step of growing the transformed plant wherein the expression of the legume-derived CcRpp2-R1 gene and the legume-derived CcRpp2-R3 gene results in enhanced resistance to Asian soybean rust disease in the transformed plant.

In accordance with embodiment 23 the method of any one of embodiments 21-23 is provided, wherein the Asian soybean rust resistant plant is a legume species.

In accordance with embodiment 24 a legume plant is provided that is a progeny from a cross between a transgenic legume plant comprising a legume-derived CcRpp2-R1 gene and a legume-derived CcRpp2-R3 gene disclosed herein and a similar legume plant that is not transformed with the legume-derived CcRpp2-R1 gene and the legume-derived CcRpp2-R3 gene.

In accordance with embodiment 25 the plant of embodiment 24 is provided, wherein the legume plant is an alfalfa, clover, pea, bean, lentil, lupin, mesquite, carob, soybean, peanut or tamarind species.

In accordance with embodiment 26 a method of assaying a legume plant for disease resistance to a plant disease is provided, wherein the method comprises exposing a portion of the legume plant comprising a legume-derived CcRpp2-R1 gene and a legume-derived CcRpp2-R3 gene to a plant pathogen; measuring plant disease symptoms on the legume plant exposed to the plant pathogen; and comparing the plant disease symptoms to a reference standard for disease resistance, optionally wherein the plant disease is caused by a plant pathogen, optionally wherein the plant pathogen is caused by Phakopsora pachyrhizi or Phakopsora meibomiae, optionally wherein the plant disease is Asian soybean rust

In accordance with embodiment 27 a method of enhancing plant resistance to Asian soybean rust (ASR) disease is provided, wherein the method comprises conferring resistance to an ASR pathogen by introgression of a legume-derived CcRpp2-R1 gene and a legume-derived CcRpp2-R3 gene into germplasm in a breeding program for resistance to ASR.

In accordance with embodiment 28 the method of embodiments 27 is provided, wherein the legume-derived CcRpp2-R1 gene encodes a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 2 and the legume-derived CcRpp2-R3 gene encodes a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 4.

In accordance with embodiment 29 the method of any one of embodiments 27 or 28 is provided, wherein the CcRpp2-R1 gene encodes the polypeptide of SEQ ID NO: 2 and the CcRpp2-R3 gene encodes the polypeptide of SEQ ID NO: 4, optionally wherein the germplasm is a legume crop species, optionally wherein the legume crop species is an alfalfa, clover, pea, bean, lentil, lupin, mesquite, carob, soybean, peanut or tamarind species, optionally wherein the legume crop species is soybean.

In accordance with embodiment 30 the method of embodiment 29 is provided, wherein a plant transformed with the polypeptide displays enhanced resistance to ASR when compared to a susceptible plant.

In accordance with embodiment 31 a recombinant DNA construct of embodiment 5 is provided, further comprising one or more NB-LRR polynucleotides or a fragment thereof.

In accordance with embodiment 32 a recombinant DNA construct of embodiment 5 or 31 is provided, The recombinant DNA construct of claim 5, further comprising one or more resistance genes.

In accordance with embodiment 32 a seed comprising the recombinant DNA construct of any one of embodiments 5, 31 or 32 is provided.

In accordance with embodiment 33 a plant comprising the recombinant DNA construct of any one of embodiments 5, 31 or 32 is provided.

In accordance with embodiment 34 a seed of embodiment 32 or a plant of embodiment 33 is provided wherein said seed or plant comprises a nucleic acid sequence encoding a polypeptide selected from:

- a) a CcRpp2-R1 polypeptide comprising an amino acid sequence having greater than 90% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 2 and 21-36; or
- b) a CcRpp2-R3 polypeptide comprising an amino acid sequence having greater than 90% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 4 and 48-58.

In accordance with embodiment 34, the seed or plant of embodiment 33 is provided wherein the CcRpp2-R1 polypeptide is SEQ ID NO: 2 and the CcRpp2-R3 polypeptide is SEQ ID NO: 4, optionally wherein the CcRpp2-R1 polynucleotide is SEQ ID NO: 1 and the CcRpp2-R3 polynucleotide is SEQ ID NO: 3.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1
Mapping and Cloning of CcRpp2 Genes

Pigeon pea (Cajanus cajan) is a diploid legume, with a genome size of approximately 830 Mbp (Varshney et al. (2012) Nat. Biotechnol., 30:83-89). The plant is self-fertile and has a life cycle between 2-3 months seed-to-seed. C. cajan (accession G108-99) was previously characterized as exhibiting resistance to Asian Soybean Rust (ASR) disease. As disclosed herein this variety was further screened to investigate if additional uncharacterized resistance genes to Asian Soybean Rust (ASR) disease, other than the known CcRpp1 locus are present in the plant's genome. More particularly, several segregating populations were generated by crossing C. cajan (accession G108-99) with accessions that show full susceptibility, including Ra, Rb, Rc, Rd, Re and Rf. Segregation analysis indicated that a single major resistance gene confers resistance in these populations, except for the Rd population that showed a 15:1 segregation pattern. Further analysis showed that the resistances observed in these accessions map to the same locus, except for a potential second locus in the Rd population.

Specifically, segregation and marker analysis were conducted and demonstrated the existence of a second resistance locus in the accession G108-99 (Rd). 292 F2 plants from population CG8-1 (G48-95 x G108-99) were screened with ASR isolate PPUFV-02. 266 plants were classified as resistant and 24 as susceptible. The observed numbers fit a 15:1 segregation ratio expected for two independent genes. The same population was screened using markers SSR10581 and dCAPS239615 that flank the interval that contains the previously described CcRpp1. Based on this screening, 56 plants were selected that were homozygous susceptible for this interval, i.e., do not contain the resistance allele at the CcRpp1 locus. At least twelve plants homozygous for the susceptible allele at the interval displayed an immune phenotype (class 0) corroborating the hypothesis on the existence of a new resistance locus in accession G108-99. These 56 F2 plants were selfed to obtain F3 seed. The F3 families were inoculated with isolate PPUFV-02 and genotyped using markers SSR10581 and dCAPS239615. The resistance in some of these F3 families displayed a 3:1 segregation and markers analysis confirmed the presence of G48-95 DNA at CcRpp1 locus, corroborating the segregation of a single resistance gene elsewhere in the Cajanus genome.

Two segregating F3 families were selected for mapping of the new resistance locus. DNA was isolated and sent to The DNA Facility at Iowa State University for SNP genotyping using the Sequenom MassARRAY iPLEX platform. The SNPs to be screened span the Cajanus genome at 10-20 cM intervals. They were selected after comparison of G48-95 and G108-99 genomic Illumina data.

Fine mapping and BAC library screens were used to identify the physical interval of the new resistance gene identified herein as CcRpp2 (See FIG. 1). Three BAC clones were identified from a Rd library: BACP6 (116,744 bp), BACJ17 (139,830 bp) and xcajanus-2 (161,443 bp). All BACs include the markers Rdint_264620 and dCAPS_393933, but they don't contain the marker Rdint_385686. BACJ17 includes the full BACP6 and the third BAC and neither of the BACs fully covers the interval of 121 kb (FIG. 1). Using suitable markers one loss and one gain of function recombinant on the distal side (marker position Rdint_264620) and three loss of function recombinants on the proximal side of the interval on the marker position Rdint_385686, were identified narrowing down the interval to 121,252 bp. Rd BAC library were constructed and screened using three markers located in this interval: Rdint_264620; dCAPS_393933 and Rdint_385686.

The region between the contig_153610 and contig_135277 harbours a sequence that resembles a TIR-NB-LRR gene (homolog to Glyma14g024500). However, after a more detailed observation by checking the C. cajan TIR-NB-LRR sequence on the SMART (Simple Modular Architecture Research Tool; a web resource that provides a simple identification and extensive annotation of protein domains and the exploration of protein domain architectures), the sequence was not a full NB-LRR gene but a TIR-TIR domain. Four copies of the TIR domain are present twice in the interval of 121,252 bp and appear to be duplicated.

To visualize the expressed genes the programs Geneious and TopHat (fast splice junction mapper for RNAseq reads) were used and the mapping results were analysed to identify splice junctions between exons. The first TIR-TIR domain set on the BAC did not show any polymorphism between the resistant and susceptible transcriptome reads. Interestingly though, a clear frameshift caused by a two nucleotide deletion was observed in the TIR-TIR sequence of the susceptible parent. In addition, TopHat analysis showed a slight induction in the transcriptome dataset of the resistant parent. Therefore this TIR-TIR sequence was a prime candidate for conferring the CcRpp2 resistance. The second TIR-TIR domain did not show an induction in the resistant transcriptome reads and no polymorphisms were detected between the resistant and susceptible reads in the expressed contigs. However, again a frame shift in the gene variant present in the susceptible parent is intriguing.

The TIR-TIR gene found in BACJ17 (BACJ17 position: 63,676 to 66,081) is upregulated in the transcriptome of the resistant parent (G108-99) in relation to the susceptible transcriptome parent (G48-95). The TIR-TIR domains encoded by this gene belong to a rare TIR-2 superfamily in which the number of family members in the dicots is restricted to 2-5 genes per species (Sarris et al., 2016). A CT deletion on the second exon was present only in the susceptible allele (position on the BACJ17: 64,691) causing a frameshift on the gene, which results in an early stop codon, creating a short polypeptide sequence that will most likely not be functional and contains only one TIR domain. This CT deletion found in the susceptible allele S48 is fixed in other four C. cajan accessions that do not convey resistance at the CcRpp2 locus, indicating that this gene is likely to be the CcRpp2 resistant gene candidate. A comparison of the TIR-TIR gene present at the CcRpp2 locus of G108-99 to six other accessions of C. cajan reveals that only G108-99 conveys resistance via the CcRpp1 and CcRpp2 locus. G48-95 is the susceptible mother plant used in the cross. G59-95, G119-99, G127-97 and G146-97 contain resistance at the CcRpp1 locus but do not convey resistance via the CcRpp2 locus.

RNA Ligase Mediated Rapid Amplification of cDNA Ends.

(RLM-RACE) was conducted on the first TIR-TIR gene present in the resistant accession to obtain the whole TIR-TIR gene sequence including the 5′ and 3′ UTRs. The RNA used for the RACE experiments was isolated from uninfected tissue and from non-etiolated leaf material. Interestingly, two different full length transcripts were observed, with one of them showing a deletion of 51 nucleotides at the end of the second exon. The two splice variants observed in the first TIR-TIR resistant transcript are caused by an alternative splicing event resulting in two variations of peptide sequences, with one losing 17 aa from their sequence but this event did not change the function of these two variants.

To determine the relative abundance of the full TIR-TIR transcript versus the splice variant RACE products the program TopHat was used to align the transcriptome of both parents against the updated version of the BACJ17. The relative abundance of the full TIR-TIR resistant transcript with a coverage of 220×the number of reads versus 9×the number of reads on the variant carrying the 51 nt deletion.

To identify the minimal effective promoter of the TIR-TIR transcript it was assumed that a shorter promoter length, not containing reads from the upstream RNA Helicase gene, has all the transcription elements to drive both TIRs gene expression in the soybean transformants.

EXAMPLE 2
Transformation of Soybean with the Cajanus cajan Genes, CcRpp2-R1Aa (SEQ ID NO: 1) and CcRpp2-R3Aa (SEQ ID NO: 3)

A plant transformation construct was designed to provide high-level constitutive expression of CcRpp2-R1Aa (SEQ ID NO: 1) and moderate-level constitutive expression of CcRpp2-R3Aa (SEQ ID NO: 3) in soybean. A slot vector was produced with a 1026 bp SfiI fragment containing the CcRpp2-R1Aa coding region that was ligated at the 5′ end to a 1948 bp soybean ubiquitin promoter+IntronI fragment and on the 3′ end to a 1163 bp phaseolin terminator fragment. The entire promoter-coding region-terminator cassette was flanked by attR1 and attL4 Gateway® recombination sites. A second slot vector was generated with a 1035 bp BamHI+SnaBI fragment containing the CcRpp2-R3Aa coding region that was ligated at the 5′ end to a 2,576 bp maize histone 2B promoter+IntronI fragment and on the 3′ end to a 902 bp soybean ubiquitin 14 (UBQ14) terminator fragment. The entire promoter-coding region-terminator cassette was located between Gateway attL3 and attR2 recombination sites. The final stacked gene construct was created by recombining the two promoter-coding region-terminator cassettes, separated by a 1,531 bp attL2 and attL1 flanked buffer fragment, in a Gateway based plant expression vector between compatible attR4 and attR3 recombination sites. This vector, in addition to the above elements, contained a spectinomycin resistance gene for bacterial selection and an herbicide resistant soybean ALS gene as a plant selectable marker.

The final CcRpp2Aa binary-containing plant expression vector was electroporated into Escherichia coli. Transformants were then selected and pDNA were isolated by standard miniprep methods. Transformants were characterized by diagnostic restriction enzyme digestions of miniprep DNA. A positive clone containing the expected pattern of digestion bands was selected and subsequently transformed into Agrobacterium tumefaciens. Transformants were selected, sequence verified to contain the binary CcRpp2Aa plant transformation construct, and submitted for Agrobacterium-mediated transformation.

Agrobacterium-mediated transformation of soybean. Transgenic soybean lines were produced from immature seed cultures following the Agrobacterium-mediated transformation protocol (Finer and McMullen 1991; Stewart et al. 1996; Cho et al. 2015). Briefly, immature seeds were harvested from soybean pods of plants grown in the greenhouse under standard conditions. Seeds were surface sterilized, immature cotyledons were aseptically excised and the cultures were maintained in 250 ml flasks containing 50 ml of liquid media on rotary shakers at 26° C. under cool white fluorescent lights with a 16/8 h day/night photoperiod (Samoylov et al. 1998; Cho et al. 2011). Agrobacterium tumefaciens carrying plasmids with genes of interest were used to transform the immature cotyledons. Transgenic events were selected and regenerated to maturity. These plants were grown under the same conditions as the wild type plants but in separate growth chambers.

Additional molecular stack constructs were assembled to express the genomic and predicted cDNA of both CcRpp2-R1Aa and CcRpp2-R3Aa behind either a soybean ubiquitin promoter or a maize histone 2B promoter for high-level or moderate-level expression, respectively. Events were generated from each of the additional constructs and confirmed to express the CcRpp2 transgenes. Homozygous and hemizygous plants displayed the red-brown (RB) phenotype when challenged with Phakopsora pachyrhizi; however, in contrast to event 1-2 (see below), the additional construct designs resulted in a reduced resistance profile with no significant decrease in sporulation detected.

EXAMPLE 3
Testing Transgenic Plants for Efficacy Against ASR

The molecular stack of CcRpp2-R1Aa (SEQ ID NO: 1) and CcRpp2-R3Aa (SEQ ID NO: 3) genes was tested for efficacy against ASR by transformation of the plant expression construct into soybean, followed by inoculation of transgenic plants with Phakopsora pachyrhizi and scoring of plant disease symptoms.

One transgenic event, event 1-2, was recovered from the soy transformation experiment and confirmed by qPCR to contain the CcRpp2-R1Aa and CcRpp2-R3Aa genes. The event was additionally shown by qRT-PCR to express a diagnostic 197 bp fragment of the CcRpp2-R1Aa transcript and a 202 bp fragment of the CcRpp2-R3Aa transcript.

T1 transgenic testing for efficacy of binary CcRpp2Aa against Phakopsora pachyrhizi. Seeds from one T1 event were planted and grown under growth chamber conditions for 15 days until Vc. The plants were sampled at V1 for qPCR to determine the transgene copy number and inoculated with a suspension of Phakopsora pachyrhizi spores. The inoculation was performed with urediniospores collected from a susceptible variety. Freshly harvested spores were suspended in an aqueous solution of 0.01% Tween 20 and mixed thoroughly; the spore concentration was then adjusted to 4×10³sp/ml with a hemocytometer. Plants were spray-inoculated with the urediniospore suspension, incubated at 100% relative humidity in the dark for 22 hours and then transferred to a growth chamber optimized for disease development (23° C., 70% RH, 16 hr photoperiod) where they were allowed to grow and develop symptoms for 15 days. New growth was excised regularly in order to keep the unifoliates for the duration of the experiment.

In order to assess the effect of the binary CcRpp2-R1Aa and CcRpp2-R3Aa, plants were scored qualitatively as Resistant (R; red-brown (RB), low or non-sporulating lesions), and Susceptible (S; tan, highly sporulating lesions) and quantitatively, by excising and scanning leaves followed by determination of lesion counts. The null, heterozygous and homozygous plants were scored 15 days after inoculation. In order to determine the effect of the binary gene, the transgenic plants were compared to the null plants from the same event.

ASR infection assay results were summarized in Table 2. These results showed that the binary CcRpp2Aa (CcRpp2-R1Aa and CcRpp2-R3Aa) in homozygous samples provided red-brown type resistance to ASR with little to no sporulation. Plant leaves were visually assessed for the presence of lesions and microscopically evaluated to detect the presence of uredinia. Low to no sporulation was observed on 95.45% of homozygous plants representing event 1-2 that were confirmed to express CcRpp2-R1Aa and CcRpp2-R3Aa, with one plant showing mid-level sporulation. Heterozygous plants also displayed resistance, red-brown lesions with low to no sporulation observed on 89.47% of plants; however, mid-level sporulation was observed on three plants and one heterozygous plant displayed high-level sporulation. Null plants contained tan, highly sporulating lesions, typical of a susceptible reaction to the pathogen.

These ASR infection assay results show that the binary CcRpp2 genes CcRpp2-R1Aa and CcRpp2-R3Aa were able to provide resistance to Phakopsora pachyrhizi when transgenically transferred from the host legume, Cajanus cajan to Glycine max plants.

Table 2. Measured traits for event 1-2 carrying binary CcRpp2Aa. Zygosity was used as transgene copy number (null=0, hemiz=1, homoz=2); R=resistant, S=susceptible; Avg LC/cm²=average lesion count per area unit (cm²); Phenotype pct=percent of plants with observed phenotype

Lesion

Phenotype
Avg

Event
Zygosity
n
Reaction
type
Sporulation
(pct)
(LC/cm²)

1-2
Homoz
22
R
RB
None-Low
95.45
12.37

Hemiz
38
R
RB
None-Low
89.47
13.84

Null
15
S
Tan
High
100
16.78

EXAMPLE 4
Identification of Homologous Proteins

Gene identities may be determined by conducting BLAST (Basic Local Alignment 20 Search Tool; Altschul, et al., (1993) J. Mol. Biol. 215:403-410; see also ncbi.nlm.nih.gov/BLAST/, which can be accessed using the www prefix) searches under default parameters for similarity to sequences contained in the publicly available BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the 25 SWISS-PROT protein sequence database, EMBL, and DDBJ databases). In addition to public databases, proprietary internal databases were also searched. Certain polynucleotide sequences were analyzed. The resulting percent identity values of CcRpp2-R1Aa (SEQ ID NO: 1) and certain homologous proteins are presented in Table 3. The resulting percent identity values of CcRpp2-R3Aa (SEQ ID NO: 3) and certain homologous proteins are presented in Table 4.

TABLE 3

CcRpp2-R1Aa homologous proteins and their origins

Percent

Amino

Acid

Sequence

Identity to

full length

NT
AA
CcRpp2-

SEQ
SEQ
R1Aa

ID
ID
(SEQ ID

Reference
NO
NO
NO: 2)
Variety
Public Database Gene Reference
Source Organism

CcRpp2-
1
2
—
—
—
Cajanus cajan

R1Aa

CcRpp2-
5
21
45.22
V14167
aradu.V14167.gnm1.ann1.Aradu.L0FGB.1 gene =
Arachis duranensis

R1Ad*

aradu.Aradu.L0FGB

CcRpp2-
6
22
44.08
Tifrunner
arahy.Tifrunner.gnm2.ann1.SGE08U.1 gene =
Arachis hypogaea

R1Ah*

arahy.SGE08U

CcRpp2-
6
22
44.08
K30076
araip.K30076.gnm1.ann1.Araip.FPQ85.1 gene =
Arachis ipaensis

R1Ai*

araip.Araip.FPQ85

CcRpp2-
7
23
39.88
ICPL87119
cajca.ICPL87119.gnm1.ann1.C.cajan_05869.1 gene =
Cajanus cajan

R1Cc*

cajca.C.cajan_05869

CcRpp2-
8
24
43.12
ISC453364
cerca.ISC453364.gnm1.ann1.Cerca571S26910
Cercis canadensis

R1Cc2*

CcRpp2-
9
25
66.57
PI483463
glyso.PI483463.gnm1.ann1.GlysoPI483463.14G021900.1 gene =
Glycine soja

R1Gs1

glyso.PI483463.gnm1.ann1.GlysoPI483463.14G021900

CcRpp2-
10
26
49.86
A17
medtr.A17_HM341.gnm4.ann2.Medtr5g092630.1 gene =
Medicago truncatula

R1Mt*

medtr.A17_HM341.Medtr5g092630

CcRpp2-
11
27
65.11
G19833
phavu.G19833.gnm2.ann1.Phvul.008G267700.1 gene =
Phaseolus vulgaris

R1Pv1

phavu.Phvul.008G267700

CcRpp2-
12
28
30.50
VC1973A
pissa.Cameor.gnm1.ann1.Psat2g013520.1 gene =
Pisum sativum

R1Ps1

pissa.Cameor.gnm1.ann1.Psat2g013520

CcRpp2-
13
29
67.64
93B86
Internal
Glycine max

R1Gm1

CcRpp2-
13
29
67.64
93Y21
Internal
Glycine max

R1Gm2

CcRpp2-
13
29
67.64
Wm82
glyma.Wm82.gnm2.ann1.Glyma.14G024400.1 gene =
Glycine max

R1Gm3

glyma.Glyma.14G024400

CcRpp2-
14
30
34.02
Milvus
tripr.MilvusB.gnm2.ann1.mRNA23019 gene =
Trifolium pratense

R1Tp

tripr.gene22257

CcRpp2-
15