The present invention additionally provides methods for identifying a plant, particularly a wheat or triticale plant, that displays newly conferred or enhanced resistance to stem rust caused by Pgt. The methods find use in breeding plants for resistance to stem rust. Such resistant plants find use in the agricultural production of wheat seeds. The methods comprise detecting in a plant the presence of at least one R gene of the present invention, particularly Sr27. In some embodiments of the invention, detecting the presence of the R gene comprises detecting the entire R gene in genomic DNA isolated from the plant. In preferred embodiments, however, detecting the presence of an R gene comprises detecting the presence of at least one marker within the R gene. In other embodiments of the invention, detecting the presence of an R gene comprises detecting the presence of the R protein encoded by the R gene using, for example, immunological detection methods involving antibodies specific to the R protein.

In the methods for identifying a plant that displays newly conferred or enhanced resistance to stem rust caused by Pgt, detecting the presence of the R gene in the plant can involve one or more of the following molecular biology techniques that are disclosed elsewhere herein or otherwise known in the art including, but not limited to, isolating genomic DNA and/or RNA from the wheat plant, amplifying nucleic acid molecules comprising the R gene and/or marker therein by PCR amplification, sequencing nucleic acid molecules comprising the R gene and/or marker, identifying the R gene, the marker, or a transcript of the R gene by nucleic acid hybridization, and conducting an immunological assay for the detection of the R protein encoded by the R gene. It is recognized that oligonucleotide probes and PCR primers can be designed to identify the R genes of the present invention and that such probes and PCR primers can be utilized in methods disclosed elsewhere herein or otherwise known in the art to rapidly identify in a population of plants one or more plants comprising the presence of an R gene of the present invention. It is further recognized that detecting the presence of the R gene can involve detecting the presence of a fragment of the R gene of the present invention. Such a fragment of an R gene of the present invention can comprise, for example, at least 10, 20, 50, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or more contiguous nucleotides.

Depending on the desired outcome, the polynucleotide constructs of the invention can be stably incorporated into the genome of the plant cell or not stably incorporated into the genome of the plant cell. If, for example, the desired outcome is to produce a stably transformed plant with enhanced resistance to wheat stem rust caused by at least one race of Pgt, then the polynucleotide construct can be, for example, fused into a plant transformation vector suitable for the stable incorporation of the polynucleotide construct into the genome of the plant cell. Typically, the stably transformed plant cell will be regenerated into a transformed plant that comprises in its genome the polynucleotide construct. Such a stably transformed plant is capable of transmitting the polynucleotide construct to progeny plants in subsequent generations via sexual and/or asexual reproduction. Plant transformation vectors, methods for stably transforming plants with an introduced polynucleotide construct and methods for plant regeneration from transformed plant cells and tissues are generally known in the art for both monocotyledonous and dicotyledonous plants or described elsewhere herein.

The present invention provides nucleic acid molecules comprising R genes. Preferably, the R genes are capable of conferring upon a host plant, particularly a wheat or triticale plant, enhanced resistance to at least one race of the pathogen that causes stem rust, Pgt. More preferably, the R genes are capable of conferring upon a host plant, particularly a wheat or triticale plant, enhanced resistance to two, three, four, or more races of Pgt. Thus, such R genes find use in limiting stem rust caused by Pgt in agricultural production. The R genes of the present invention include, but are not limited to, the R genes whose nucleotide sequences are disclosed herein but also include orthologs and other variants that are capable of conferring to a plant resistance to stem rust caused by at least one race of Pgt. Methods are known in the art or otherwise disclosed herein for determining resistance of a plant to stem rust caused by at least one race of Pgt.

The methods of the present invention find use in producing plants, particularly wheat and triticale plants, with enhanced resistance to stem rust caused by at least one race of Pgt. Typically, the methods of the present invention will enhance or increase the resistance of the subject plant to one race of Pgt by at least 25%, 50%, 75%, 100%, 150%, 200%, 250%, 500% or more when compared to the resistance of a control plant to the same race or races of Pgt. Unless stated otherwise or apparent from the context of a use, a control plant for the present invention is a plant that does not comprise the polynucleotide construct of the present invention. Preferably, the control plant is essentially identical (e.g. same species, subspecies, and variety) to the plant comprising the polynucleotide construction of the present invention except the control does not comprise the polynucleotide construct. In some embodiments, the control will comprise a polynucleotide construct but not comprise the one or more R gene sequences that are in a polynucleotide construct of the present invention.

Additionally, the present invention provides transformed plants, seeds, and plant cells produced by the methods of present invention and/or comprising a polynucleotide construct of the present invention. Also provided are progeny plants and seeds thereof comprising a polynucleotide construct of the present invention. The present invention also provides seeds, vegetative parts, and other plant parts produced by the transformed plants and/or progeny plants of the invention as well as food products and other agricultural products produced from such plant parts that are intended to be consumed or used by humans and other animals including, but not limited to pets (e.g., dogs and cats) and livestock (e.g., pigs, cows, chickens, turkeys, and ducks).

The methods of the invention can be used to enhance the resistance of a plant, particularly a wheat or triticale plant, to stem rust, particularly stem rust caused by at least one race of Pgt. As used herein, the term “wheat plant” generally refers to a plant that is a member of the Triticum genus or a member of another genus within the Triticeae tribe, particularly a member of another genus that is capable of producing interspecific hybrids with at least one Triticum sp. Examples of such another genus within the Triticeae tribe are Aegilops and Secale.

The wheat plants of the present invention include, for example, domesticated and non-domesticated plants. The wheat plants of the present invention include, but are not limited to, the following Triticum, Aegilops and Secale species: T. aestivum, T. monococcum, T. turgidum, T. boeoticum, T. timopheevii, and T. urartu, Aegilops tauschii, Secale cereale, and hybrids thereof. Examples of T. aestivum subspecies included within the present invention are aestivum (common wheat), compactum (club wheat), macha (macha wheat), vavilovi (vavilovi wheat), spelta, and sphaecrococcum (shot wheat). Examples of T. turgidum subspecies included within the present invention are turgidum, carthlicum, dicoccom, durum, paleocoichicum, polonicum, turanicum, and dicoccoides. Examples of T. monococcum subspecies included within the present invention are monococcum (einkorn) and aegilopoides. In one embodiment of the present invention, the wheat plant is a member of the Triticum turgidum species; and in particular, a member of the Durum subspecies, for example, a Ciccio, Colosseo, or Utopia cultivar. It is recognized that a wheat plant of the present invention can be a domesticated wheat plant or a non-domesticated wheat plant.

The present invention also encompasses triticale plants, triticale plant parts, and triticale plant cells comprising an R gene of the invention. As used herein, a “triticale plant” refers to a plant that is created by crossing a rye plant (Secale cereale) with either a tetraploid wheat plant (e.g. Triticum turgidum) or a hexaploid wheat plant (e.g. Triticum aestivum). The present invention also includes seeds produced by the triticale plants described herein and methods for controlling weeds in the vicinity of the triticale plants described herein. As used herein, the term “wheat plant” encompasses triticale plants unless stated otherwise or apparent from the context of use.

The term “plant” is intended to encompass plants at any stage of maturity or development, as well as any tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context. As used herein, the term “plant” includes, but is not limited to, seeds, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, tubers, propagules, leaves, flowers, branches, fruits, roots, root tips, anthers, and the like. The present invention also includes seeds produced by the plants of the present invention.

Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides. As used herein, “progeny” and “progeny plant” comprise any subsequent generation of a plant whether resulting from sexual reproduction and/or asexual propagation, unless it is expressly stated otherwise or is apparent from the context of usage.

Plant parts include, but are not limited to, seeds, stems, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, and the like.

In one embodiment of the invention, the nucleotide sequences encoding R proteins have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the entire nucleotide sequence set forth in SEQ ID NO: 1, 2, and/or 4, or to a fragment thereof.

The present invention encompasses isolated or substantially purified polynucleotide (also referred to herein as “nucleic acid molecule”, “nucleic acid” and the like) or protein (also referred to herein as “polypeptide”) compositions including, for example, polynucleotides and proteins comprising the sequences set forth in the accompanying Sequence Listing as well as variants and fragments of such polynucleotides and proteins. An “isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein 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. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 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%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By “fragment” it is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of polynucleotides comprising coding sequences may encode protein fragments that retain biological activity of the full-length or native protein. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.

Polynucleotides that are fragments of a native R polynucleotide comprise at least 16, 20, 50, 75, 100, 125, 150, 175, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, or 3500 contiguous nucleotides, or up to the number of nucleotides present in a full-length R polynucleotide disclosed herein.

“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or 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. 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 R proteins of the invention. 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 outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an R protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein. In certain embodiments of the invention, variants of a particular polynucleotide of the invention will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 2, and 4, and optionally comprises a non-naturally occurring nucleotide sequence that differs from the nucleotide sequence set forth in SEQ ID NO: 1, 2, and/or 4 by at least one nucleotide modification selected from the group consisting of the substitution of at least one nucleotide, the addition of at least one nucleotide, and the deletion of at least one nucleotide.

Variants of a particular polynucleotide of the invention (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. Thus, for example, a polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 3 is disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In certain embodiments of the invention, variants of a particular polypeptide of the invention will have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth SEQ ID NO: 3, and optionally comprises a non-naturally occurring amino acid sequence that differs from the amino acid set forth in SEQ ID NO: 3 by at least one amino acid modification selected from the group consisting of the substitution of at least one amino acid, the addition of at least one amino acid, and the deletion of at least one amino acid.

“Variant” protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of an R protein will have at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein (e.g. the amino acid sequence set forth in SEQ ID NO: 3) as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) PNAS 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.

Thus, the genes and polynucleotides of the invention include both the naturally occurring sequences as well as mutant and other variant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. More preferably, such variants confer to a plant or part thereof comprising the variant enhanced resistance stem rust caused by at least one race of Pgt. In some embodiments, the mutations that will be made in the DNA encoding the variant will not place the sequence out of reading frame. Optimally, the mutations will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by assays that are disclosed herein below.

For example, a wheat plant that is susceptible to wheat stem rust caused by a particular race of Pgt can be transformed with an Sr27 polynucleotide, regenerated into a transformed or transgenic plant comprising the polynucleotide, and tested for resistance to wheat stem rust caused by the particular race of Pgt using standard resistance assays known in the art or described elsewhere herein. Preferred variant polynucleotides and polypeptides of the present invention confer or are capable of conferring upon a wheat plant enhanced resistance to at least one race of Pgt that is known to cause wheat stem rust in a susceptible wheat plant.

Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J Mol. Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The polynucleotides of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology 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 invention. Such sequences include sequences that are orthologs of the disclosed sequences. “Orthologs” is intended to mean 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 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode R proteins and which hybridize under stringent conditions to at least one of the R proteins disclosed herein or otherwise known in the art, or to variants or fragments thereof, are encompassed by the present invention.

In one embodiment, the orthologs of the present invention have coding sequences comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater nucleotide sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOS: 1, 2, and 4, and/or encode proteins comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

Like other NLR proteins, the Sr27 protein comprises certain conserved domains. In Sr27 (comprising the amino acid sequence set forth in SEQ ID NO: 3), the conserved domains include, for example, a coiled-coil domain (amino acids 9 to 160), a nucleotide-binding domain (amino acids 173 to 520) and a leucine-rich repeat domain (amino acids 530 to 940). Preferably, variant Sr27 proteins of the present invention comprise a coiled-coil domain, a nucleotide-binding domain, and a leucine-rich repeat domain corresponding to the domains of Sr27 set forth above.

In some embodiments, variant Sr27 proteins of the present invention comprise a higher percentage of amino acid sequence identity to one, two, or three of such conserved domains than to the full-length amino acid sequence of the Sr27 (SEQ ID NO: 3) or protein disclosed herein. Preferably, such variants comprise a corresponding domain or domains having at least 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one, two, or three of the domains of Sr27 set forth above and further comprise an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 3.

It is recognized that domains in variant Sr27 proteins corresponding to those conserved domains of Sr27 set forth above, as well as any particular conserved amino acids therein, can be identified by methods known to those of skill in the art or disclosed elsewhere herein such as, for example, multiple sequence alignment. It is further recognized that the positions of such conserved domains and conserved amino acids within a particular variant Sr27 protein can vary from the positions in the amino acid sequence set forth in SEQ ID NO: 3 and that through methods such as, for example, multiple sequence alignment, the corresponding positions of such conserved domains and conserved amino acids can be determined for any variant Sr27 protein of the present invention.

Preferably, the variant Sr27 proteins of the present invention and the polynucleotides encoding them confer, or are capable of conferring upon a wheat plant comprising such a protein and/or polynucleotide, enhanced resistance to at least one race of Pgt that is known to cause wheat stem rust in a susceptible wheat plant.

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 plant of interest. Methods for designing PCR primers and PCR cloning are generally 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, New York). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but 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.

It is recognized that the R protein coding sequences of the present invention encompass polynucleotide molecules comprising a nucleotide sequence that is sufficiently identical to the nucleotide sequence of any one or more of SEQ ID NOS: 1, 2, and 4. The term “sufficiently identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 80% or 85% identity, preferably 90% or 91% identity, more preferably 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently identical.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) PNAS 87:2264, modified as in Karlin and Altschul (1993) PNAS 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the polynucleotide molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST; available on the world-wide web at ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the full-length sequences of the invention and using multiple alignment by mean of the algorithm Clustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) using the program AlignX included in the software package Vector NTI Suite Version 7 (InforMax, Inc., Bethesda, MD, USA) using the default parameters; or any equivalent program thereof. By “equivalent program”, it is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by CLUSTALW (Version 1.83) using default parameters (available at the European Bioinformatics Institute website on the world-wide web at: ebi.ac.uk/Tools/clustalw/index.html).

The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides, can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

The polynucleotide constructs comprising R protein coding regions can be provided in expression cassettes for expression in the plant or other organism or non-human host cell of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to the R protein coding region. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide or gene of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the R protein coding region to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a R protein coding region of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants or other organism or non-human host cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the R protein coding region or of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the R protein coding region of the invention may be heterologous to the host cell or to each other.

As used herein, “heterologous” in reference to a nucleic acid molecule or nucleotide sequence is a nucleic acid molecule or nucleotide sequence that originates from a foreign species, or, if from the same species, is modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

The present invention provides host cells comprising at least one of the nucleic acid molecules, expression cassettes, and vectors of the present invention. In preferred embodiments of the invention, a host cell is a plant cell. In other embodiments, a host cell is selected from the group consisting of a bacterium, a fungal cell, and an animal cell. In certain embodiments, a host cell is a non-human animal cell. However, in some other embodiments, the host cell is an in-vitro cultured human cell. In yet some other embodiments, the host cell is a microorganism, particularly a unicellular microorganism. Microorganisms include, but are not limited to, archaebacteria, eubacteria, yeasts, and algae.

While it may be optimal to express the R protein using heterologous promoters, the native promoter of the corresponding R gene may be used.

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

Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

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 may be deleterious to gene expression. The G-C content of the sequence may 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 may additionally contain 5′ leader sequences. 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) (Elroy-Stein et al. (1989) PNAS 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

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

A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants. Such constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, 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.

Tissue-preferred promoters can be utilized to target enhanced expression of the R protein coding sequences 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) PNAS 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

Generally, it will 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. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also WO 99/43819, herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al. (1986) PNAS 83:2427-2430; Somsisch et al. (1988) Mot Gen. Genet. 2:93-98; and Yang (1996) PNAS 93:14972-14977. See also, Chen et al. (1996) Plant J 10:955-966; Zhang et al. (1994) PNAS 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Also of interest are the native promoters from other resistance genes from the target species. These promoters are often pathogen-inducible, and are likely to express the resistance gene at appropriate levels and in appropriate tissues. Examples of such promoters are the Sr57/Lr34, Sr33, Sr35, and Sr22 promoters of wheat (Risk et al. (2012) Plant Biotechnol J 10: 447-487; Periyannan et al. (2013) Science 341: 786-788; Saintenac et al. (2013) Science 341: 783-786; Steuernagel et al. (2016) Nature Biotechnol. 34(6):652-655, doi: 10.1038/nbt.3543).

Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter may be used in the constructions of the invention. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76); MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like, herein incorporated by reference.

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 may 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-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) PNAS 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

The expression cassette can also 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 glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) PNAS 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) PNAS 86:5400-5404; Fuerst et al. (1989) PNAS 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) PNAS 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) PNAS 89:3952-3956; Bairn et al. (1991) PNAS 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) PNAS 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene can be used in the present invention.

Numerous plant transformation vectors and methods for transforming plants are available. See, for example, An et al. (1986) Plant Physiol., 81:301-305; Fry et al. (1987) Plant Cell Rep. 6:321-325; Block (1988) Theor. Appl. Genet. 76:767-774; Hinchee et al. (1990) Stadler Genet. Symp. 203212.203-212; Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee and Slightom (1992) Gene.118:255-260; Christou et al. (1992) Trends Biotechnol. 10:239-246; D'Halluin et al. (1992) Bio/Technol. 10:309-314; Dhir et al. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) PNAS 90:11212-11216; Christou (1993) In Vitro Cell. Dev. Biol.-Plant; 29P:119-124; Davies et al. (1993) Plant Cell Rep. 12:180-183; Dongand Mchughen (1993) Plant Sci. 91:139-148; Franklin et al. (1993) Plant Cell Rep. 12(2):74-79, doi: 10.1007/BF00241938; Golovkin et al. (1993) Plant Sci. 90:41-52; Asano et al. (1994) Plant Cell Rep. 13; Ayeres and Park (1994) Crit. Rev. Plant Sci. 13:219-239; Barcelo et al. (1994) Plant J. 5:583-592; Becker et al. (1994) Plant J. 5:299-307; Borkowska et al. (1994) Acta Physiol. Plant 16:225-230; Christou (1994) Agro. Food Ind. Hi Tech. 5: 17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586; Hartman, et al. (1994) Bio-Technology 12: 919923; Ritala et al. (1994) Plant Mol. Biol. 24:317-325; and Wan and Lemaux (1994) Plant Physiol. 104:3748.

The methods of the invention involve introducing a polynucleotide construct into a plant. By “introducing” is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

By “stable transformation” is intended that the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof. By “transient transformation” is intended that a polynucleotide construct introduced into a plant does not integrate into the genome of the plant.

For the transformation of plants and plant cells, the nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell. The selection of the vector depends on the preferred transformation technique and the target plant species to be transformed.

Methodologies for constructing plant expression cassettes and introducing foreign nucleic acids into plants are generally known in the art and have been previously described. For example, foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors. Other methods utilized for foreign DNA delivery involve the use of PEG mediated protoplast transformation, electroporation, microinjection whiskers, and biolistics or microprojectile bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 to Vasil et al.; Bilang et al. (1991) Gene 100: 247-250; Scheid et al., (1991) Mol. Gen. Genet., 228: 104-112; Guerche et al., (1987) Plant Science 52: 111-116; Neuhause et al., (1987) Theor. Appl Genet. 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlock et al., (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). The method of transformation depends upon the plant cell to be transformed, stability of vectors used, expression level of gene products and other parameters.

Other suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection as Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described by Riggs et al. (1986) PNAS 83:5602-5606, Agrobacterium-mediated transformation as described by Townsend et al., U.S. Pat. No. 5,563,055, Zhao et al., U.S. Pat. No. 5,981,840, direct gene transfer as described by Paszkowski et al. (1984) EMBO J. 3:2717-2722, and ballistic particle acceleration as described in, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl transformation (WO 00/28058). Also see, Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) PNAS 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) PNAS 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

The polynucleotides of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.

If desired, the modified viruses or modified viral nucleic acids can be prepared in formulations. Such formulations are prepared in a known manner (see e.g. for review U.S. Pat. No. 3,060,084, EP-A 707 445 (for liquid concentrates), Browning, “Agglomeration”, Chemical Engineering, Dec. 4, 1967, 147-48, Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and et seq. WO 91/13546, U.S. Pat. Nos. 4,172,714, 4,144,050, 3,920,442, 5,180,587, 5,232,701, 5,208,030, GB 2,095,558, U.S. Pat. No. 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al. Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Germany), 2001, 2. D. A. Knowles, Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, Dordrecht, 1998 (ISBN 0-7514-0443-8), for example by extending the active compound with auxiliaries suitable for the formulation of agrochemicals, such as solvents and/or carriers, if desired emulsifiers, surfactants and dispersants, preservatives, antifoaming agents, anti-freezing agents, for seed treatment formulation also optionally colorants and/or binders and/or gelling agents.

In specific embodiments, the polynucleotide constructs and expression cassettes of the invention can be provided to a plant using a variety of transient transformation methods known in the art. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) PNAS Sci. 91: 2176-2180 and Hush et al. (1994) J. Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and Agrobacterium tumefaciens-mediated transient expression as described elsewhere herein.

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

In certain embodiments of the invention, the nucleotide sequence of a non-functional allele at an R gene locus of the present invention can be modified in planta to a functional allele that provides resistance to at least one race of a plant pathogen. Thus, the present invention provides methods for producing a plant, particularly a triticale plant, with enhanced resistance to stem rust. The methods comprise modifying in a plant or at least one cell thereof, a non-functional allele of the resistance gene Sr27 so as to make a functional allele, whereby the resistance of the plant to stem rust is enhanced. In preferred embodiments, the plant that is produced is a non-transgenic plant, particularly a non-transgenic triticale plant. The methods can further comprise regenerating the plant cell or plant cells comprising the functional allele into a plant comprising the functional allele.

In one embodiment of the invention, a non-functional allele or susceptible allele that is present at the Sr27 locus in a triticale plant can be modified to a functional allele that provides resistance to, for example, at least one, two, three, or four races of Pgt. In another embodiment of the invention, a non-functional allele that is present at the Sr27 locus in a triticale plant can be modified to a functional allele that provides resistance to at least one, two, three, or four races of Pgt that cause stem rust. For example, a non-functional allele at the Sr27 locus can be modified to form whereby the modified allele comprises the nucleotide sequence of Sr27 set forth in SEQ ID NO: 1 and/or a nucleotide sequence encoding the amino sequence set forth SEQ ID NO: 3.

Any methods known in the art for modifying DNA in the genome of a plant can be used to modify the nucleotide sequences of an R gene in planta, e.g. to modify the nucleotide sequence of a non-functional allele to that of a functional allele that provides resistance to a plant pathogen. Such modifications to the DNA in the genome of a plant include, for example, insertions, deletions, substitutions, and combinations thereof. The insertions, deletions, and substitutions can be made using any method known in the art such as, for example, by genome editing techniques as described elsewhere herein or otherwise known in the art.

The insertions comprise an insertion of at least one nucleotide or base pair (bp) in an allele of an R gene of the present invention. The insertion can comprise insertion of any size DNA fragment into the genome. The inserted DNA can be 1 bp in length, 1-5 bp in length, 5-10 bp in length, 10-15 bp in length, 15-20 bp in length, 20-30 bp in length, 30-50 bp in length, 50-100 bp in length, 100-200 bp in length, 200-300 bp in length, 300-400 bp in length, 400-500 bp in length, 500-600 bp in length, 600-700 bp in length, 700-800 bp in length, 800-900 bp in length, 900-1000 by in length, 1000-1500 bp.

The deletions comprise the deletion of at least one bp from an allele of an R gene of the present invention. As used herein, a “deletion” is meant the removal of one or more nucleotides or base pairs from the DNA. Provided herein, a deletion in an allele of an R gene can be the removal of at least 1, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000 or more bp.

The substitutions comprise the replacement of at least one bp from an allele of an R gene of the present invention with another bp. As used herein, a “substitution” is meant the replacement of one or more nucleotides or base pairs from the DNA with non-identical nucleotides or base pairs. When the substitution comprises two or more nucleotides, the two or more nucleotides can be contiguous or non-contiguous within the DNA sequence of the allele. Provided herein, a substitution in an allele of an R gene can be the replacement of at least 1, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 5000 or more base pairs. In some embodiments, the substitution can be the nucleotide sequence of the entire allele or any portion or portions thereof such as, for example, the transcribed region, the 5′ untranslated region, the 3′ untranslated region, an exon, or an intron.

In certain embodiments of the invention, the modification of a non-functional allele at an R gene locus is a homozygous modification. By “homozygous modification” is meant that the modification is in both alleles of the R gene locus in a particular genome of a plant. In other cases, the modification of the R gene locus gene is heterozygous, that is, the modification is only in one allele of the of the R gene locus in the genome of a plant. It is recognized the plants of the invention include, for example, crop plants with genomes that are diploid or polyploid (e.g. tetraploid or hexaploid), including autopolyploids and allopolyploids. An autopolyploid is an organism having more than two sets of chromosomes, all of which were derived from the same species. An allopolyploid is an organism having two or more complete sets of chromosomes that are derived from different species. Depending on the particular the crop plant, 1, 2, 3, 4, 5, 6 or more alleles at an R gene locus of the plant can be modified using the methods disclosed.

Any methods known in the art for modifying DNA in the genome of a plant can be used to alter the coding sequences of an R gene in planta, e.g. to alter the nucleotide sequence of a homologous susceptible allele to that of an allele that provides resistance to at least one race of stem rust. Such methods known in the art for modifying DNA in the genome of a plant include, for example, mutation breeding and genome editing techniques, such as, for example, methods involving targeted mutagenesis, site-directed integration (SDI), and homologous recombination. Targeted mutagenesis or similar techniques are disclosed in U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972, 5,871,984, and 8,106,259; all of which are herein incorporated in their entirety by reference. Methods for gene modification or gene replacement comprising homologous recombination can involve inducing single-strand or double-strand breaks in DNA using zinc-finger nucleases (ZFN), TAL (transcription activator-like) effector nucleases (TALEN), Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas nuclease), or homing endonucleases that have been engineered endonucleases to make double-strand breaks at specific recognition sequences in the genome of a plant, other organism, or host cell. See, for example, Durai et al., (2005) Nucleic Acids Res 33:5978-90; Mani et al. (2005) Biochem Biophys Res Comm 335:447-57; U.S. Pat. Nos. 7,163,824, 7,001,768, and 6,453,242; Arnould et al. (2006) J Mol Biol 355:443-58; Ashworth et al., (2006) Nature 441:656-9; Doyon et al. (2006) J Am Chem Soc 128:2477-84; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; and Smith et al., (2006) Nucleic Acids Res 34:e149; U.S. Pat. App. Pub. No. 2009/0133152; and U.S. Pat. App. Pub. No. 2007/0117128; all of which are herein incorporated in their entirety by reference.

TAL effector nucleases (TALENs) can be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, Fold. The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS 10.1073/pnas.1013133107; Scholze and Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology 29:143-148; all of which are herein incorporated by reference.

The CRISPR/Cas nuclease system can also be used to make single-strand or double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. The CRISPR/Cas nuclease is an RNA-guided (simple guide RNA, sgRNA in short) DNA endonuclease system performing sequence-specific double-stranded breaks in a DNA segment homologous to the designed RNA. It is possible to design the specificity of the sequence (Cho et al. (2013) Nat. Biotechnol. 31:230-232; Cong et al. (2013) Science 339:819-823; Mali et al. (2013) Science 339:823-826; Feng et al. (2013) Cell Res. 23(10): 1229-1232).

In addition, a ZFN can be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. The Zinc Finger Nuclease (ZFN) is a fusion protein comprising the part of the FokI restriction endonuclease protein responsible for DNA cleavage and a zinc finger protein which recognizes specific, designed genomic sequences and cleaves the double-stranded DNA at those sequences, thereby producing free DNA ends (Urnov F. D. et al., Nat Rev Genet. 11:636-46, 2010; Carroll D., Genetics. 188:773-82, 2011).

Breaking DNA using site specific nucleases, such as, for example, those described herein above, can increase the rate of homologous recombination in the region of the breakage. Thus, coupling of such effectors as described above with nucleases enables the generation of targeted changes in genomes which include additions, deletions, substitutions, and other modifications.

Mutation breeding can also be used in the methods provided herein. Mutation breeding methods can involve, for example, exposing the plants or seeds to a mutagen, particularly a chemical mutagen such as, for example, ethyl methanesulfonate (EMS) and selecting for plants that possess a desired modification in the Sr27 gene. However, other mutagens can be used in the methods disclosed herein including, but not limited to, radiation, such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (e.g., product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (e.g., emitted from radioisotopes such as phosphorus 32 or carbon 14), and ultraviolet radiation (preferably from 2500 to 2900 nm), and chemical mutagens such as base analogues (e.g., 5-bromo-uracil), related compounds (e.g., 8-ethoxy caffeine), antibiotics (e.g., streptonigrin), alkylating agents (e.g., sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines. Further details of mutation breeding can be found in “Principals of Cultivar Development” Fehr, 1993 Macmillan Publishing Company the disclosure of which is incorporated herein by reference.

The nucleic acid molecules, expression cassettes, vectors, and polynucleotide constructs of the present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Preferred plants of the present invention are wheat plants. Examples of other plant species of interest include, but are not limited to, peppers (Capsicum spp; e.g., Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens, and the like), tomatoes (Lycopersicon esculentum), tobacco (Nicotiana tabacum), eggplant (Solanum melongena), petunia (Petunia spp., e.g., Petunia x hybrida or Petunia hybrida), pea (Pisum sativum), bean (Phaseolus vulgaris), corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. 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), soybean (Glycine max), teff (Eragrostis tef), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), 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 (Persea 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.), palms, oats, barley, vegetables, ornamentals, and conifers.

In certain embodiments of the invention, the preferred plants are cereal plants. Such cereal plants of the present invention are grass plants (i.e. Poaceae family) cultivated for the edible components of their grain or kernels (i.e. seeds) including, for example, wheat, triticale, rye, barley, oats, maize, sorghum, millet, and rice. In certain other embodiments of the invention, the preferred plants are cereal plants but not including triticale plants.

In some embodiments of the present invention, a plant cell is transformed with a polynucleotide construct encoding an R protein of the present invention. The term “expression” as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. The “expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the “expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide. Examples of polynucleotide constructs and nucleic acid molecules that encode R proteins are described elsewhere herein.

The use of the terms “DNA” or “RNA” herein is not intended to limit the present invention to polynucleotide molecules comprising DNA or RNA. Those of ordinary skill in the art will recognize that the methods and compositions of the invention encompass polynucleotide molecules comprised of deoxyribonucleotides (i.e., DNA), ribonucleotides (i.e., RNA) or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues including, but not limited to, nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). The polynucleotide molecules of the invention also encompass all forms of polynucleotide molecules including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. Furthermore, it is understood by those of ordinary skill in the art that the nucleotide sequences disclosed herein also encompasses the complement of that exemplified nucleotide sequence.

The invention is drawn to compositions and methods for enhancing the resistance of a plant to plant disease, particularly to compositions and methods for enhancing the resistance of a plant to stem rust caused by Pgt. By “disease resistance” is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen are minimized or lessened.

As used herein, a “race” refers to any of a group of laboratory isolates or fungal individuals existing in the field that share a similar virulence phenotype on a range of different resistance gene lines of wheat and are likely derived by clonal reproduction.

As used herein, an “isolate” of Pgt refers to a line of Pgt originally isolated as spores collected from an infected plant in the field or in a laboratory/glasshouse setting. Such an “isolate” is subsequently maintained in pure form by infection and re-isolation of spores from a susceptible plant and storage of said spores.

The present invention encompasses the nucleic acid molecules and polynucleotide constructs disclosed herein or in the accompanying sequence listing and/or drawings including, but not limited to: nucleic acid molecules and polynucleotide constructs comprising the nucleotide sequences set forth in SEQ ID NO: 1, 2, and/or 4; and nucleic acid molecules and polynucleotide constructs encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3. The present invention further encompasses plants, plant cells, host cells, and vectors comprising at least one of such nucleic acid molecules and/or polynucleotide constructs, as well as food products produced from such plants and plant parts. Additionally encompassed by the present invention are uses of plants comprising at least one of such polynucleotide constructs in the methods disclosed elsewhere herein such as, for example, methods for enhancing the resistance of a plant to stem rust caused by Pgt and methods of limiting stem rust in agricultural crop production.

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

EXAMPLES
Example 1: Cloning of the Sr27 Stem Rust Resistance Gene

To clone the Sr27 stem rust resistance gene from triticale cv. Coorong, the inventors utilized the MutRenSeq method (Steuernagel et al. (2016) Nature Biotechnol. 34(6):652-655, doi: 10.1038/nbt.3543; WO 2015/127185; both of which are herein incorporated by reference).

The inventors mutagenized seeds of triticale cv. Coorong with ethyl methanesulfonate-derived (EMS) and identified 27 susceptible ethyl methanesulfonate-derived (EMS) mutants from the Coorong background prepared as described below (FIG. 1). Coorong comprises in its genome the Sr27 stem rust resistance gene and was first released in Australia by the University of Adelaide (Macintosh et al. (1983) Can. J. Plant. Pathol. 5:61-69).

NLR-gene capture and sequencing (RenSeq) was performed on wild-type Coorong and four confirmed susceptible mutants (M2, M3, M4 and M6) as described below. Captured reads from wild-type Coorong were assembled and reads from all lines aligned to this reference to identify sequence changes in the mutant lines. One contig (no. 5723) of 1126 base pairs (bp) was found that contained a mutation in three of the four mutants; one (M2) with a full deletion of the sequence and two (M3, M4) with single base changes causing amino acid substitutions Q264R and G209S (in the conserved p-loop) respectively. This contig contained a coiled-coil (CC) domain and p-loop motif, but not the rest of the NB-ARC or LRR domains, suggesting that it represented only part of a full length NLR gene. To identify the remainder of the gene, the contig was aligned to the Triticum aestivum cv. Chinese Spring reference (CSv1) assembly IWGSC RefSeq v1.0 (Appels et al. (2018) Science 361(6403):eaar7191). The top hit (93.6% identity across the whole DNA sequence) was to the 5′end of a high confidence gene (TraesCS6B01G464400) predicted on chromosome 6B and functionally annotated as a disease resistance gene. The full gene sequence of TraesCS6B01G464400 was then aligned back to the Coorong RenSeq de novo assembly which detected an additional contig (no. 2413) of 2140 bp aligning to the 3′ region of this gene with 93.8% identity across the whole sequence. Contig no. 2413 contained both an NB-ARC and LRR domain. Inspection of the read alignments from the mutant Coorong lines confirmed that Contig no. 2413 was also deleted in mutant M2 and identified an additional single base change in mutant M6 (FIG. 2). PCR amplification confirmed that these two contigs were derived from the same gene in wild-type Coorong which encodes a full-length protein of 956 amino acids that contains a coiled-coil (CC) domain at the N-terminus, followed by the NB-ARC domain and then the LRR motifs at the C-terminus (FIG. 2). Amplification from the four mutants confirmed the nucleotide changes detected by RenSeq in each line. Four additional mutants were also examined by PCR amplification and one (M3) contained a single amino acid change (T211I) in the p-loop motif while the gene sequence failed to amplify from the other three (M7, M8, M9) indicating that they contained deletions of this region. The eight independent mutants containing deletions or amino acid changes in this gene provided strong evidence that it confers Sr27 resistance.

Materials and Methods

Plant Materials and Mutant DNA Preparation

Seeds of Triticale line wild-type Coorong (Coorong carries Sr27) were treated with ethyl methanesulfonate (EMS) following the protocol described by Mago et al. ((2015) Nature Plants 1, 15186). A kill-curve on 20-grains was initially produced with different concentrations, 0.1 0.2, 0.3, 0.4, 0.5 and 0.6% (v/v) to identify the dosage required to achieve 50% mortality. A total of 1960 seeds were treated with 0.3% EthylMethaneSulfonate for 12 hrs then washed thoroughly with water and sown in large pots (40 seeds per 30 cm pot) in a glasshouse with daylight and 23° C. day and 15° C. night temperature. Single heads from each M1 plant were threshed separately and M2 families from each plant were sown in a tray (30 M2 families per tray). Each tray also included resistant (Coorong) and susceptible (Rongcoo, triticale) controls. M2 families obtained as a single spike progeny from each M1 plant were tested for stem rust responses by inoculation with Puccinia graminis f. sp. tritici (Pgt) isolate Pgt21-0. Individual plants from segregating progenies were grown and progeny tested. Homozygous susceptible mutant and resistant sib pairs were recovered from these progenies.

Genomic DNA was extracted from wild-type Coorong triticale and homozygous susceptible mutants following the protocol described by Yu et al. (2017). The quality and quantity of the extracted DNA was first checked with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE) and then on a 0.8% agarose gel.

Resistance Gene Enrichment and Sequencing (RenSeq)

The Target enrichment of NLRs was performed by Arbor Biosciences (Ann Arbor, USA) following the MYbaits protocol using an improved version of the previously published Triticeae bait library available at github.com/steuernb/MutantHunter. Library construction was done by following the TruSeq RNA protocol v2. All enriched libraries were sequenced on a HiSeq 2500 (Illumina, CA, USA) using 250 bp paired end reads and SBS chemistry.

MutantHunter

To identify Sr27 contigs from mutants, the MutantHunter pipeline method of Steuernagel et al. ((2015) Bioinformatics 31:1665-7) was followed. Primary read sequencing data from wild-type Coorong and mutants were first trimmed for quality using Trimmomatic v0.38 (Bolger et al. (2014) Bioinformatics 30:2114-20) with the parameters ILLUMINACLIP: novogene_indexed_adapters.fa: 2:30:10:8: TRUE, LEADING: 28, TRAILING: 28, MINLEN: 20. Data from wild-type Coorong was then de novo assembled using the CLCGW v11.0.1 as paired-end with a length fraction of 0.95 and a similarity fraction of 0.98 and the remaining parameters as default. Contigs shorter than 1 kb were omitted from the final assembly using a custom script. An annotation of NBS-LRR motifs was created using the program NLR-Parser (Steuernagel et al. (2015) Bioinformatics 31:1665-7). Trimmed data of each mutant and wild type was mapped to the de novo assembly using BWA v0.7.15 (Li and Durbin (2009) Bioinformatics 25:1754-1760). Samtools v1.7.0 (Li et al. (2009) Bioinformatics 25:2078-9) was used for processing of resulting SAM files to retain only reads mapping in a proper pair with parameter -f 2, then removing duplicates and generating pileup files using parameters -BQ0 and -aa. SNV calling and subsequent candidate identification was performed using the following scripts from the MuTrigo pipeline (available via https at: github.com/TC-Hewitt/MuTrigo). Contig regions with high levels of SNPs in the wild-type data indicating poor assembly or read alignment were detected using Noisefinder.pyc with default parameters and masked prior to downstream analysis. Potentially mutated nucleotide positions were recorded from pileup files using SNPlogger.pyc with parameter -d 20. SNPtracker.pyc was used to retrieve contigs containing polymorphisms in two or more mutants using default parameters plus parameter -s C>T G>A indel. This translates to considering only polymorphisms with a minimum of 80% mutant allele frequency and selecting only for insertions, deletions, or C-to-T or G-to-A SNVs that do not share an identical position with another mutant or wild-type. Candidate gene contigs were aligned to the chromosome scale reference assembly of Secale cereale inbred line ‘Lo7’ (Rabanus-Wallace et al. (2019) bioRxiv: 2019.12.11.869693) and the Triticum aestivum cv. Chinese Spring reference (CSv1) assembly IWGSC RefSeq v1.0 (Appels et al. (2018) Science 361 (6403): eaar7191) using BLAST v2.7.1 (Altschul et al. (1990) J. Mol. Biol. 215:403-10). A de novo RNA transcript assembly was generated from Illumina RNAseq data from Coorong seedlings infected with Pgt (Upadhyaya et al. (2015) Front Plant Sci. 5:759) (downloaded from NCBI-SRA SAMN07836894, PRJNA415866) using CLCGW v11.0.1 (similarity fraction of 0.98 and the remaining parameters set as default).

Sr27 Gene Structure Confirmation

Primers were designed based on the genomic sequence of the two non-overlapping contigs no. 2413 and no. 5723 (Table 2). Primer pair Sr27c5723ExtF1 and Sr27c2413ExtR1 was used to amplify the non-overlapping region between the contigs no. 5723 and no. 2413 from genomic DNA of wild-type Coorong. Primer pair Sr27F and Sr27R1 was used for amplification for the full-length gene from genomic DNA of the wild-type and mutants for subsequent sequence comparison. All PCRs were performed using Phusion high fidelity DNA polymerase (NEB, USA) according to the manufacturer's instructions. Full-length Sr27 genomic sequence is shown in SEQ ID NO: 1. All mutants used in the RenSeq pipeline were re-confirmed by Sanger sequencing. Predicted exon-intron structures were confirmed by full cDNA amplification from RNA of triticale cv. Coorong. Total RNA was extracted using the PureLink™ RNA Mini Kit (Invitrogen catalog No 12183025, Thermo Fisher Scientific, MA USA) as per the manufacturer's instructions. cDNA synthesis was performed using the protocol described by manufacture (SMART™ PCR cDNA Synthesis kit, Cat No. 634902, Takara Bio USA Inc., (previously Clontech Laboratories Inc.).

Pgt Virulent Mutant Selection

Seedlings of the triticale cultivar Coorong at the 2-3 leaf stage (20 pots [15 cm] with 6-8 plants per pot) were inoculated with −100 g of stem rust isolate Pgt21-0 (Upadhyaya et al. (2015) Front Plant Sci. 5:759) using talc as a carrier (1:4; rust: Talc). Inoculated plants were incubated in a humid chamber maintained at 23° C. for 48 hrs. After this time plants were moved to a glasshouse maintained at 23° C./18° C. (day/night) under natural daylight. Plants were screened twice at 14 and 20 days post inoculation for any susceptible infection sites showing large pustule development typical of a susceptible interaction (infection type 3,4). More than 10 mutant pustules were detected and three of these were collected and re-inoculated onto the susceptible wheat line Morocco for amplification in isolation. After amplification the virulence of each mutant on Sr27 was confirmed by reinfection of wild-type Coorong plants. One mutant was further tested on the full Australian differential set for Pgt comprising a variety of wheat and triticale lines with different known resistance genes (Park (2007) Aust. J. Agric. Res. 58:558-566) and showed an identical virulence profile to Pgt21-0 except for a single additional virulence for Sr27.

Identification of AvrSr27 Gene by Whole Genome Sequence Analysis

DNA was extracted from urediniospores of mutant Pgt lines with a CTAB method (Rogers et al. (1989) Can. J. Bot. 67:1235-1243) with some modifications as described (Upadhyaya et al. (2015) Front Plant Sci. 5:759), quality assessed with a Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE) and quantitated using a broad-range assay in Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). DNA library preparation and Illumina sequencing were performed by the Australian Genome Research Facility (AGRF) on HiSeq2500 with (250 bp PE reads, mutants M1 and M2) or MiSeq (300 bp PE reads, mutant M3) platforms and about 20 million reads obtained for each. Sequence reads were imported to CLC Genomics Workbench (CLCGW) version 10.0.1 or later (QIAGEN), filtered and trimmed to remove low quality ends, sequencing adapters and low-quality reads (Trim using quality score 0.01, maximum number of ambiguities allowed is 2). Reads were mapped to the karyon-phased chromosome level assembled Pgt21-0 reference annotated with 37036 gene models (Li et al. (2019) Nature Commun. 10, 5068) using high stringency settings (similarity fraction 0.98 and length fraction 0.95). Variant calling of each sample against the reference was performed using the “Basic Variant Detection” program with parameters: ignore non-specific matches; minimum coverage 10; significance 1.0%; minimum variant count 2; include broken pairs. The program “Compare variants within group” in CLCGW was used to identify variants specific to each sample as well as shared variants. Non-synonymous variants in secreted protein genes were predicted using the CLCGW tool “Amino Acid Changes” and curated manually by visual inspection of read mapping tracks in CLCGW. Read coverage statistics (mean read depth and percent coverage) for annotated genes were extracted using the “Create Statistics for Target Regions” program.

Example 2: Identifying the AvrSr27 Effector Recognised by Sr27

To identify spontaneous mutants of Pgt with a gain of virulence on Sr27, the inventors inoculated the avirulent isolate Pgt21-0 onto seedlings of the triticale cultivar ‘Coorong’ carrying Sr27. Three large single pustules were selected, purified in isolation and reinoculated onto Coorong to confirm their virulent phenotype (FIG. 3). The inventors extracted genomic DNA from each mutant and obtained Illumina sequence data. Illumina reads were mapped to the Pgt21-0 haplotype-resolved genome assembly (Li et al. (2019) Nature Commun. 10, 5068) to identify potential mutations. The first screen to identify SNPs causing amino-acid changes in secreted protein genes did not find any genes with such mutations in more than one Pgt21-0 mutant line. Next, the inventors looked for loss of read coverage as an indicator of potential deletion mutations. Each of the Pgt21-0 mutant lines showed a large number of genes with zero read coverage, all located on one end of chromosome 2B. Visualisation of read mapping to chromosome 2B revealed the three mutants each contained independent and overlapping deletions covering a portion of chromosome 2B, of which the smallest was 196 kilobase pairs (kbp) in size (FIG. 4). This region contains 50 annotated genes, of which five are predicted to encode secreted proteins. Previously two Australian Pgt isolates (34-2,12 and 34-2,12,13) that belong to a clonal lineage derived from Pgt21-0 and which had evolved virulence for Sr27 in the field were sequenced (Upadhyaya et al. (2015) Front Plant Sci. 5:759; Zhang et al. (2017). Phytopath.107:1032-1038). Analysis of read coverage revealed that these two isolates each contained a small deletion of 13 kbp (FIG. 5A) that spanned two of the secreted protein genes in this region (PGT21_006532 and PGT21_006593) along with a single adjacent gene on the distal side. No other genes in this region contained any changes in these two isolates. The inventors confirmed the presence of this deletion in the genome by PCR amplification of the deletion boundaries in Pgt21-0 and 34-2,12 (FIG. 5B). Sequence data generated from seven South African isolates was also examined (Lewis et al 2018), that are part of the same clonal lineage as Pgt21-0 (Li et al. (2019) Nature Commun. 10, 5068; Visser et al. (2019) Phytopath. 109:133-144) but have evolved independently in South Africa. Four of these isolates are virulent on Sr27 (Visser et al. (2009) Mol. Plant Path. 10:213-222) and must have evolved this phenotype independently of the Australian isolates. Three of these virulent isolates (SA03, SA05 and SA07) contain an identical ~14 kbp deletion that overlaps with the deletion in 34-2,12 and spans the two candidate secreted protein genes plus an additional gene on the proximal side, while the fourth isolate (SA06) contains an independent deletion of 10 kbp that spans just the two secreted protein genes (FIG. 5A). The remaining three avirulent isolates (SA01, SA02, SA04) from South Africa contain a similar sequence to Pgt21-0 in this region, as do three other Australian isolates (PGT098, PGT194, PGT326) of this lineage that are also avirulent on Sr27. A phylogenetic analysis of this clonally derived group of isolates places each of these deletion events in a separate branch of the lineage, consistent with the occurrence of three independent mutations to virulence on Sr27 during the diversification of this lineage (data not shown). The deletion of these two secreted protein gene candidates in three independent field-derived virulent isolates provides strong evidence that at least one of these genes confers the avirulence phenotype. These two genes are closely related to each other and encode predicted secreted proteins of 144 amino acids designated AvrSr27-1 (SEQ ID NO: 6) and AvrSr27-2 (SEQ ID NO: 8) (FIG. 6).

Example 3: Transient Expression Validation of Sr27 Function

To confirm the function of the Sr27 candidate gene, we used a wheat protoplast transfection assay as described below to co-express the gene with the AvrSr27 variants identified above as well as the reporter gene luciferase. Recognition between the resistance and avirulence gene in this assay leads to cell death and therefore a reduction in the expression of the co-transformed luciferase reporter gene which is detected by its bioluminescence (Saur et al. (2019) Plant Methods 15, 118). Co-expression of the Sr27 candidate gene with each of the AvrSr27 gene variants led to a strong reduction in luciferase activity compared to the expression of Sr27 or AvrSr27 genes in the protoplast alone (FIG. 7). This was a specific recognition response as no loss of reporter gene expression was seen when Sr27 was co-expressed with the unrelated Pgt avirulence gene AvrSr50 (Chen et al. (2017) Science 358:1607-1610), nor when AvrSr27-1 was co-expressed with the unrelated wheat resistance gene Sr50 (Mago et al. (2015) Nature Plants 1, 15186). This confirms that the cloned Sr27 sequence confers specific recognition to the AvrSr27 effector from Pgt and hence is responsible for resistance to Pgt isolates expressing the AvrSr27 gene.

Thus, the inventors demonstrated that the Sr27 coding region, when expressed in wheat protoplasts under the control of an operably linked ubiquitin promoter, is sufficient to confer recognition of the corresponding effector AvrSr27 from the wheat stem rust fungus (Pgt). The effector was identified by mutation analysis, which located it within 196 kbp deletion on chromosome 2B of Pgt21-0. Subsequent analysis found two genes that were independently deleted in three different Pgt that acquired virulence on Sr27. The availability of the cloned effector recognised by Sr27 facilitates genetic screening of wheat stem rust isolates for the presence of this effector and therefore their predicted virulence on Sr27.

Materials and Methods

Construct Generation

AvrSr27 gene sequences (shown in SEQ ID NOS: 5, 7, and 9) with the predicted signal peptide excluded and replaced by a single methionine start codon were amplified from cDNA from purified haustoria of Pgt21-0 using Phusion high-fidelity DNA polymerase (Thermo Scientific, Wilmington, DE) and cloned into pENTR™/D-TOPO® according to manufacturer's (Invitrogen™, Thermo Fisher Scientific, MA, USA) instructions. Full-length Sr27 cDNA was also cloned into pENTR™/D-TOPO®. For wheat protoplast transfection Sr27 and AvrSr27 sequences were inserted into the p35s-pUbi-GTW-GFP (Akamatsu et al. (2013) Cell Host Microbe 13:465-476) using Gateway® Technology (Life Techonologies™). Primers used for PCR are shown in Table 2. All plasmids were confirmed by sequencing and analysed using Vector NTI Advance (Life Technologies, Thermo Fisher Scientific, MA, USA) or CodonCode Aligner V.4.0.4 (CodonCode Corporation, MA, USA) software. Plasmids encoding GFP (Arndell et al. (2019) BMC Biotechnol. 19:71) and Luciferase (Saur et al. (2019) Plant Methods 15, 118) were previously described.

Protoplast Expression Assay

Wheat seedlings were grown in a growth chamber under shade at 25° C. degrees with a photoperiod of 16 h of light for 7-9 days. Wheat protoplast isolation and transformation were performed as described (Arndell et al. (2019) BMC Biotechnol. 19:71). High quality plasmid DNA was isolated by using Qiagen Endo-free Plasmid Maxi kits (Cat. No. 12362). DNA concentrations were adjusted to 1 μg/μl and 10 μg of each plasmid was used in co-transformation experiments. After a 24-hour incubation in the dark at 23° C., the luciferase activity assay was performed as described by Saur et al. ((2019) Plant Methods 15, 118) using the Luciferase Assay System (Promega, catalog no. E1501) following the manufacturer's instructions.

Example 4: Comparison of Sr27 to Other Resistance Genes

According to BLAST best hits against IWGSC CS ref v1.0, the location of the closest homologs of the Sr27 candidate in Chinese Spring reference v1.0 was on chromosome 6B (see Example 1). The inventors further extended the list of homologous sequences by performing BLAST against the non-redundant protein data base at NCBI (results not shown). The top hit was an unnamed protein product from Triticum turgidum subsp. durum (GenBank Accession VAI63620.1) and had 90.5% amino acid identity to the Sr27 protein. To determine the evolutionary distance and degree of diversity between Sr27 and other cloned CNL type R genes from wheat at the protein sequence level, the inventors aligned the Sr27 protein to 16 CNL type R genes identified from wheat and performed a phylogenetic analysis as described below (FIG. 8). The closest R gene to Sr27 from the selected group is the wheat stem rust resistance gene Sr13 with 86.7% amino acid identity.

Methods

Phylogenetic Analyses

Phylogenetic and molecular evolutionary analyses were conducted using MEGA version X (Kumar et al. (2018) Mol. Biol. Evol. 35:1547-1549). Protein sequences of Sr27 and other wheat resistance proteins were aligned by CLUSTAL and a Maximum likelihood tree constructed.

Example 5: Confirmation of Sr27 Function in Transgenic Plants

The Sr27 gene construct driven by the ubiquitin promoter (described in Example 3) was transferred into the wheat cultivar Fielder by Agrobacterium-mediated transformation and transgenic lines generated as described below.

Agrobacterium-mediated transformation of wheat was performed as described (Ishida et al. (2015) “Wheat (Triticum aestivum L.) transformation using immature embryos”, In: Wang K (ed), Agrobacterium Protocols, vol. 1, 3rd, Springer, New York, pp. 189-198). Wheat plants of the cultivar Fielder were propagated under glasshouse growth conditions using a 24° C., 16 h light/18° C., 8 h dark growth regime and plants were fertilised fortnightly with Aquasol (Yates, Clayton, Australia). Wheat heads were tagged at anthesis and harvested 12-14 days post anthesis for transformation experiments as described by Ishida et al. (Id.) and as adapted at CSIRO, Canberra, Australia (Richardson et al. (2014) Plant Cell Tiss. Org. 119:647-659). Briefly seeds were surface sterilised for 10 min in a 0.8% sodium hypochlorite solution. Embryos were removed from the seed under aseptic conditions and co-cultivated with Agrobacterium strains containing the Sr22 and Sr45 genomic fragment binary construct for 2 days on WLS-AS medium (Ishida et al. 2015) in the dark. After co-cultivation embryonic axes were excised with a scalpel and explants then transferred to WLS-Res medium and placed in the dark at 24° C. After 5 days explants were transferred to WLS-H15 callus induction media containing 15 mg/ml of hygromycin (Hyg) for callus formation. Two weeks later, the resulting calli were bisected and placed on WLS-H30 (30 mg/l of Hyg) for 3 weeks in the dark. Callus was then regenerated on LSZ-H15 (15 mg/l Hyg) medium in 200 μmols m⁻²s⁻¹light at 24° C. Shoots were transferred to LSF-H15 (15 mg/l Hyg) medium to allow root formation and once robust root systems developed, plants were transferred to soil and maintained in the glasshouse.

T0 lines were screened by PCR with primers for the transgene (Sr27c5723 F×Sr27c5723 R, Table 2) and three positive lines were identified (PC311.3, PC311.17 and PC311.18). T1 seed was harvested from the three positive lines as well as a fourth line lacking the Sr27 transgene (PC311.16). Eleven or twelve progeny of each line were grown in a growth cabinet (23° C., 16 h light) along with control lines (see Table 1). Two-week-old seedlings were inoculated with Pgt race 98-1,2,(3),(5),6 to which Sr27 confers resistance. Rust reactions were assessed after 10-15 days (Table 1). For PC311.3 and PC311.17, all Ti progeny showed strong resistance to Pgt with infection types characterised by tiny necrotic infection spots with no pustule formation, similar to the phenotype observed in wheat line WRT258.5 carrying Sr27 (FIG. 9). T1 progeny of the third Sr27 transgenic line, PC311.18, segregated for resistance with 10 resistant and 1 susceptible progeny (Table 1). PCR analysis confirmed that the single PC311.18 T1 progeny susceptible plant did not contain the Sr27 transgene while the resistant T1 progeny contained Sr27. In contrast the susceptible lines Chinese Spring and Fielder showed high infection types with large uredia and no chlorosis or necrosis (see FIG. 9), as did the PC311.16 line recovered from transformation that did not contain the Sr27 transgene (FIG. 9). These data confirm that the Sr27 transgene confers stem rust resistance in transgenic wheat when expressed by the ubiquitin promoter. T1 progeny of each of the three positive lines were further tested by inoculation with leaf rust (P. triticina) isolate 76-1,3,7,9,10,12,13 and stripe rust (P. striiformis) isolate 198 E16 A+ J+ T+ 17+. All progeny were fully susceptible to the leaf rust and stripe rust isolates confirming that the Sr27 transgene conferred specific resistance to stem rust and this resistance was not due to non-specific activation of defense responses effective against rust pathogens.

TABLE 1

Results of Stripe Rust Resistance Assays

of Sr27 Transgenic Wheat Lines

No. of

progeny

infected with
No. of
No. of

Transgene
Pgt 98-
resistant
susceptible

Family
PCR
1, 2, 3, 5, 6
seedlings
seedlings

PC311-3
+
11
11
0

PC311-16
−
11
0
11

PC311-17
+
12
12