CN116634861A - Rust resistance gene - Google Patents

Abstract

The present application provides compositions and methods for enhancing the resistance of plants, particularly wheat and triticale plants, to stem rust caused by the wheat species rust graminis (Puccinia graminis f.sp.tritici). The compositions comprise nucleic acid molecules encoding resistance (R) gene products and variants thereof, and plants, seeds, and plant cells containing such nucleic acid molecules. The method for enhancing resistance of a plant to stem rust comprises introducing a nucleic acid molecule encoding an R gene product into a plant cell. Methods for using the resistant plants in agriculture to limit stem rust are also provided.

Description

Rust resistance gene

Cross Reference to Related Applications

The application claims the benefits of U.S. provisional patent application number 63/076,153, filed 9/2020, and U.S. provisional patent application number 63/127,220, filed 12/18/2020; both of these U.S. provisional patent applications are hereby incorporated by reference in their entirety.

Reference to sequence Listing submitted as text File

An official copy of the sequence listing is submitted electronically via EFS-Web as a sequence listing in ASCII format with file name 070294-0193seqlst.txt, which was created at 9.7 of 2021 and has a size of 44.3 kilobytes, and is submitted with the present specification. The sequence listing contained in this ASCII formatted document is part of this specification and is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to the field of gene isolation and plant improvement, in particular to enhancing the resistance of plants to plant diseases by using disease resistance genes.

Background

Plant diseases cause significant yield losses in wheat production worldwide. Rust is included among the most damaging wheat diseases. Wheat stem rust caused by the wheat species rust graminis (Puccinia graminis f.sp.tritici) (Pgt) is one of the most devastating diseases affecting wheat production today. While wheat plants comprising a resistance (R) gene against Pgt have proven effective in limiting agronomic losses caused by wheat stem rust, a new variety of Pgt has recently emerged for which the R gene is ineffective. While pesticides can be used to control wheat stem rust, they are expensive and are in conflict with agricultural sustainability and are not fully burdened by self-supporting farmers in developing countries.

Agricultural sustainable intensification would require the increased use of genetic solutions instead of chemical solutions (e.g., pesticides) to protect crops from pathogens and pests (Jones et al, (2014) philios.t. rou.soc.b 369:20130087). However, conventional methods for introducing R genes typically involve long breeding timelines to break linkages to deleterious alleles of other genes. Further, when deployed one at a time, the R gene can be overcome in a few seasons (McDonald and Linde (2002) Annu. Rev. Phytophol. 40:349-379). However, molecular cloning makes it possible to avoid linkage drag (linkage flag) and to introduce multiple R genes simultaneously (Dangl et al, (2013) Science 341:746-751), which should delay the evolution of pathogen varieties that break resistance and thus provide more durable resistance (McDonald and Linde (2002) Annu. Rev. Phytophosol. 40:349-379).

Summary of The Invention

The present invention provides nucleic acid molecules relating to resistance (R) genes known to confer resistance to at least one variety of a pathogen causing wheat stem rust (puccinia graminis wheat race (Pgt)) to plants. In one embodiment, the invention provides nucleic acid molecules comprising R gene Sr27 and variants thereof, including, for example, orthologs and non-naturally occurring variants.

The invention further provides plants, plant cells and seeds comprising in their genome one or more polynucleotide constructs of the invention. The polynucleotide construct comprises a nucleotide sequence encoding a resistance (R) protein of the invention. Such R proteins are encoded by the R genes of the invention. In a preferred embodiment, the plants and seeds are transgenic wheat plants and seeds that have been transformed with one or more polynucleotide constructs of the invention. Preferably, such wheat plants comprise enhanced resistance to at least one variety of a pathogen (Pgt) that causes wheat stem rust when compared to resistance of a control wheat plant that does not comprise the polynucleotide construct.

The present invention provides methods for enhancing the resistance of plants, particularly wheat or triticale plants, to stem rust caused by Pgt. Such methods comprise introducing into at least one plant cell a polynucleotide construct comprising the nucleotide sequence of the R gene of the invention. In some embodiments, the polynucleotide construct or portion thereof is stably incorporated into the genome of the plant cell, while in other embodiments, the polynucleotide construct is not stably incorporated into the genome of the plant cell. The method for enhancing resistance of a plant to stem rust may optionally further comprise regenerating the plant cell into a plant comprising the polynucleotide construct in its genome. Preferably, such plants comprise enhanced resistance to stem rust caused by at least one variety of Pgt relative to control plants.

In addition, the present invention provides methods for identifying plants (particularly wheat or triticale plants) that exhibit newly conferred or enhanced resistance to stem rust caused by Pgt. The method comprises detecting the presence of at least one R gene of the invention, in particular Sr27, in the plant.

Also provided are methods of using the plants of the invention in crop production to limit stem rust caused by Pgt. The method comprises growing a seed produced by a plant of the invention, wherein the seed comprises at least one R gene nucleotide sequence of the invention. The method further comprises growing the plant under conditions conducive to the growth and development of the plant, and optionally harvesting at least one seed or plant part from the plant.

Additionally, plants, plant parts, seeds, plant cells, other host cells, expression cassettes, and vectors are provided comprising one or more of the nucleic acid molecules of the invention.

Brief Description of Drawings

FIG. 1 is a photograph illustrating the identification of Sr27 susceptible mutants. Coorong (upper panel) and EMS-derived susceptibility mutant 1 (lower panel) were inoculated with Pgt-21-0 and photographed 14 days after infection.

FIG. 2 is a schematic representation of Sr27 protein having amino acid changes in the indicated mutants M1, M3, M4 and M6. Two contigs (# 5723 and # 2413) assembled from wild-type cooronig after NB-LRR capture and sequencing contained the 5 'and 3' regions of the gene. Mutants M1 and M4 contained single base changes in contig 5723, while mutant M6 contained single base changes in contig 2413. Amino acid changes in the full length Sr27 protein caused by these mutations are indicated. Mutant M2 does not produce reads specific for these contigs and thus may contain deletions. The mutant M3 was determined to contain another single nucleotide change in the gene by PCR amplification, which resulted in an amino acid change.

FIG. 3 is a graphical illustration of the infection phenotype of Pgt-0 and three spontaneous mutants (21-M1, 21-M2 and 21-M3) on the triticale line Coorong (comprising Sr 27), rongcoo (rust susceptible) and the Coorong-derived mutant line with loss of the Sr27 resistance gene (Sr 27 mutant 1). Images were taken 14 days after seeding of the leaves of seedlings.

FIG. 4 is a schematic representation of the location and size (in kbp) of deletions in three Sr 27-virulence mutants (M1, M2 and M3) of Pgt-0 relative to chromosome 2B (hatched bars). The positions on the chromosome are indicated at 1Mbp intervals from the 5' end, where the Centromere (CM) positions are indicated. The deletions were detected by read mapping of Illumina DNA sequence reads from Pgt21-0 and the three mutants on the Pgt21-0 genomic sequence.

FIGS. 5A and 5B. Field isolates of Pgt, which were virulent to Sr27, contained a small deletion on chromosome 2B. FIG. 5A is a schematic representation of the location and size (in kbp) of deletions in the indicated six Sr 27-virulence isolates from Australia (34-2, 12 and 34-2,12,13) and south Africa (SA 03, SA05, SA06, SA 07) relative to chromosome 2B (grey bars). Deletions were detected by read mapping of Illumina DNA sequence reads from these isolates over the Pgt21-0 genomic sequence. The positions of primers P423, P424 and P426 surrounding the AvrSr27 locus on chromosome 2B are indicated (arrow) with respect to the boundaries of the deleted region in 34-2-12. FIG. 5B shows the confirmation of a 13Kbp deletion in Sr 27-virulent rust isolate 34-2-12. After separation on a 1% agarose gel, PCR amplification products of genomic DNA from Pgt21-0 and 34-2,12 are shown. Primers P383 and P351 were designed to amplify fragments of the AvrSr50 gene as the same control region in both isolates.

FIG. 6 is an amino acid sequence alignment of two related secreted AvrSr27 protein variants from Pgt-0 encoded by the AvrSr27 locus. AvrSr27-1 (SEQ ID NO: 6) and AvrSr27-2 (SEQ ID NO: 8) are encoded on chromosome 2B at avirulence alleles. The amino acid sequence of AvrSr27-1 is shown (single letter code), wherein the same residues in the other variants are indicated by'. The predicted signal peptide region is underlined and in bold.

Fig. 7 is a graphical representation showing the corroboration of Sr27 resistance function. Luciferase activity (luminophore, y-axis) was detected in protoplasts co-expressing Sr27 or AvrSr27-1 or AvrSr27-2 alone, sr27 and AvrSr27 variants in combination, sr50 plus AvrSr27-1, or Sr27 plus AvrSr 50.

FIG. 8 is a maximum likelihood phylogenetic tree comparing the Sr27 amino acid sequence with the protein sequence of a known wheat resistance protein. The scale bar shows amino acid sequence divergence.

FIG. 9 shows phenotypes of exemplary T1 family and control plants containing Sr27 transgenes, which were infected with stem rust variety 98-1,2,3,5,6 and scored 10 days after infection. Representative T1 plants from two transgenic lines containing the Sr27 transgene (PC 311.3 and PC 311.17), one non-transgenic line recovered from tissue culture (PC 311.16), susceptible parent Fielder, chinese Spring WRT 258.5.5 containing the native Sr27 gene, and susceptible parent Chinese Spring (CS) are shown.

Sequence listing

Standard letter abbreviations for nucleotide bases, single-letter or three-letter codes for amino acids are used to show the nucleotide and amino acid sequences listed in the accompanying sequence listing, figures, and those set forth below. The nucleotide sequence follows the standard convention of starting at the 5 'end of the sequence and proceeding toward the 3' end (i.e., left to right in each row). Only one strand of each nucleotide sequence is shown, but it is understood that the complementary strand is included by any reference to the displayed strand. The amino acid sequence follows standard practices starting at the amino terminus of the sequence and proceeding toward the carboxy terminus (i.e., from left to right in each row).

SEQ ID NO. 1 illustrates a nucleotide sequence comprising the R gene (Sr 27) from the triticale (. Times. Triticosecale Wittmack) cultivar Coorong. The nucleotide sequence comprises in the 5 'to 3' direction: the 5 '-untranslated region (5' -UTR) at nucleotides 1-39, the protein coding region 40-872, the intron at nucleotides 873-1779, the protein coding region at nucleotides 1780-3814, the TGA stop codon at nucleotides 3815-3817, and the 3 '-untranslated region (3' -UTR) at nucleotides 3818-3956.

SEQ ID NO. 2 illustrates the nucleotide sequence of the coding region of the cDNA of Sr27 (SEQ ID NO. 1). If desired, a stop codon (e.g., TAA, TAG, or TGA) can be operably linked to the 3' end of a nucleic acid molecule comprising or consisting of SEQ ID NO. 2. The natural stop codon of this cDNA was TGA.

SEQ ID NO. 3 illustrates the amino acid sequence of R protein Sr27 encoded by R gene Sr27 (SEQ ID NO. 1).

SEQ ID NO. 4 illustrates the nucleotide sequence of the open reading frame of Sr27 (SEQ ID NO. 1). The sequence is that part of the genomic sequence of Sr27 that starts at the first nucleotide of the start codon and ends at the last nucleotide of the stop codon. The sequence comprises an intron.

SEQ ID NO. 5 illustrates the coding sequence of AvrSr27-1 from the Amersham wheat species (Pgt) isolate Pgt-0, wherein the predicted signal peptide is excluded and replaced with a single methionine start codon.

SEQ ID NO. 6 illustrates the amino acid sequence of the Avr27-1 protein encoded by AvrSr27-1 (SEQ ID NO: 5) from isolate Pgt, pgt-0.

SEQ ID NO. 7 illustrates the coding sequence of AvrSr27-2 (PGT21_006593_AvrSr27-2) from Pgt isolate Pgt-0, wherein the predicted signal peptide is excluded and replaced with a single methionine start codon.

SEQ ID NO. 8 illustrates the amino acid sequence of the Avr27-2 protein encoded by AvrSr27-2 (SEQ ID NO: 7) from isolate Pgt, pgt-0.

SEQ ID NOS.9-39 are the nucleotide sequences of the primers in Table 2 (example 6) below.

Detailed Description

The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

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

The present invention relates to the isolation of plant resistance (R) genes, in particular R genes that confer resistance to stem rust caused by the wheat species puccinia graminis (Pgt) to plants, in particular wheat or triticale plants. Most rust resistance genes belong to the class of nucleotide-binding leucine-rich repeat receptors (NLR), which are well known as immunoreceptors in plants. TIR-containing NLR (TNL) and CC-Containing NLR (CNL) are two major categories of plant NLR, defined based on the presence of TIR or CC domains at their N-terminus. Most, if not all, of the NLRs in cereal grains belong to the CNL class. Nine full-stage stem rust resistance genes have been cloned, derived from one wheat (t.monococcum) (Sr 21, sr22 and Sr 35), the a genome donor of hexaploid bread wheat, namely, aegilops (Aegilops tauschii) (Sr 33 and Sr 45), the D genome donor of hexaploid bread wheat, namely, diploid rye (Sr 50) and durum (Sr 13), and Sr26 was identified from tall wheat (or elytrigium elongatum).

The present invention relates to Sr27, a locus that provides resistance to many important Pgt isolates in the world, including the Ug99 lineage, and that is present in rye/wheat hybrid cereal triticale (x Triticosecale). As disclosed below, the inventors used a combined chemical mutagenesis and nucleotide-binding leucine-rich repeat receptor (NLR) resistance gene enrichment sequencing method to isolate R gene Sr27 from Pgt-resistant triticale cultivar ('Coorong') that contained Sr27 stem rust resistance genes in its genome.

The present invention provides nucleic acid molecules comprising the nucleotide sequence of an R gene, particularly Sr27, and naturally occurring variants (e.g., orthologs and allelic variants) and synthetic or artificial (i.e., non-naturally occurring) variants thereof. The nucleotide sequence of such R genes (which is also referred to herein as the R gene nucleotide sequence) encodes an R protein. The R gene nucleotide sequences of the present invention include, but are not limited to, wild-type R genes (which comprise a native promoter and a 3' adjacent region comprising a coding region), cDNA sequences, and nucleotide sequences comprising only a coding region. Examples of such R gene nucleotide sequences include the nucleotide sequences shown in SEQ ID NOs 1, 2 and 4 and variants thereof. In embodiments in which the native R gene promoter is not used to drive expression of the nucleotide sequence encoding the R protein, a heterologous promoter may be operably linked to the nucleotide sequence encoding the R protein of the invention to drive expression of the nucleotide sequence encoding the R protein in a plant.

Preferably, the R protein of the invention is a functional R protein capable of conferring on a plant comprising said R protein an increased resistance to stem rust caused by at least one variety of Pgt. In certain embodiments, the R proteins of the invention comprise broad spectrum resistance to multiple varieties of Pgt, such as the R protein encoded by Sr 27.

The invention further provides a transgenic plant comprising a polynucleotide construct comprising an R gene nucleotide sequence of the invention. In some embodiments, the polynucleotide construct is stably incorporated into the genome of the plant, and in other embodiments, the plant is transformed by a transient transformation method and the polynucleotide construct is not stably incorporated into the genome of the plant. Methods for stable and transient transformation of plants are disclosed elsewhere herein or are known in the art. In a preferred embodiment of the invention, the transgenic plant is a wheat plant comprising increased resistance to stem rust caused by at least one variety of Pgt.

In certain embodiments, the transgenic plants of the invention comprise a polynucleotide construct comprising a nucleotide sequence encoding an R protein and a heterologous promoter operably linked for expression of the nucleotide sequence encoding an R protein. The choice of heterologous promoter may depend on many factors, such as the timing, localization and expression pattern desired, and responsiveness to a particular biological or non-biological stimulus. Promoters of interest include, but are not limited to, pathogen-inducible promoters, constitutive promoters, tissue-preferential promoters, wound-inducible promoters, and chemical-regulated promoters.

In certain embodiments of the invention, the transgenic plant, particularly a transgenic wheat plant, may comprise one, two, three, four, five, six or more nucleotide sequences encoding an R protein. Typically, but not necessarily, the two or more R proteins will be different from each other. For the present invention, one R protein is different from another R protein when the two R proteins have different amino acid sequences. In certain embodiments of the invention, each of the different R proteins for stem rust has one or more differences in resistance characteristics, e.g., resistance to different varieties/populations of Pgt. It is recognized that by combining two, three, four, five, six or more nucleotide sequences, each encoding a different R protein for wheat stem rust, wheat plants comprising a wide range of resistance to varieties of Pgt can be produced. In areas where multiple varieties of Pgt are known to occur, such wheat plants can be used in agriculture.

Examples of wheat stem rust R genes that can be combined with the nucleotide sequences of the invention in a single wheat plant include Sr22 (WO 2017/024053), sr26, sr32, sr33 (GenBank accession KF 031299.1), sr35 (GenBank accession KC 573058.1), sr39, sr40, sr45 (WO 2017/024053), sr47, sr50, srTA1662 (WO 2019140351), and adult resistance genes Sr57/Lr34 (GenBank accession FJ 436983.1) and Sr55/Lr67.

Transgenic plants of the invention comprising multiple R genes can be produced by transforming a plant already comprising one or more other R gene nucleotide sequences with a polynucleotide construct comprising an R gene nucleotide sequence of the invention (including, for example, sr27 nucleotide sequences or variants thereof). Such plants that already comprise one or more other R gene nucleotide sequences may comprise an R gene that is native to the genome of the plant, introduced into the plant via sexual propagation, or introduced by transforming the plant or ancestor thereof with an R gene nucleotide sequence. Alternatively, the one or more other R gene nucleotide sequences may be introduced into a transgenic plant of the invention that already comprises a polynucleotide construct of the invention, for example by transformation or sexual reproduction.

In other embodiments, two or more different R gene sequences may be introduced into a plant by stably transforming the plant with a polynucleotide construct or vector comprising two or more R gene nucleotide sequences. It is recognized that such a method may be preferred for plant breeding, as it is expected that the two or more R gene nucleotide sequences will be closely linked and thus isolated as a single locus. Alternatively, the polynucleotide construct of the invention may be incorporated into the genome of a plant immediately adjacent to another R gene nucleotide sequence by using homologous recombination-based genome modification methods described elsewhere herein or known in the art.

The invention further provides methods for enhancing the resistance of a plant (particularly wheat or triticale plants) to stem rust caused by Pgt. The method comprises introducing a polynucleotide construct of the invention into at least one plant cell. In certain embodiments, the polynucleotide construct is stably incorporated into the genome of a plant cell. If desired, the method may further comprise regenerating the plant cell into a plant comprising the polynucleotide construct in its genome. Preferably, such regenerated plants comprise increased resistance to stem rust caused by at least one variety of Pgt relative to the resistance of control plants to stem rust caused by the same Pgt variety. If desired, the method may further comprise producing a plant as described above comprising one, two, three, four, five, six or more nucleotide sequences encoding an R protein, preferably each nucleotide sequence encoding a different R protein.

Especially in areas where stem rust is prevalent, in crop production, the plants disclosed herein may be used in methods for limiting stem rust caused by Pgt. The method of the invention comprises growing a seed produced by a plant of the invention, wherein the seed comprises at least one R gene nucleotide sequence of the invention. The method further comprises growing the plant under conditions conducive to the growth and development of the plant therefrom, and optionally harvesting at least one seed or other plant part from the plant.

In addition, the present invention provides methods for identifying plants (particularly wheat or triticale plants) that exhibit newly conferred or enhanced resistance to stem rust caused by Pgt. The methods can be used in growing plants for stem rust resistance. Such resistant plants may be used in the agricultural production of wheat seeds. The method comprises detecting the presence of at least one R gene of the invention, in particular Sr27, in a plant. 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. However, in a preferred embodiment, 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 an R protein encoded by the R gene by using an immunological detection method, e.g., involving antibodies specific for the R protein.

In the method for identifying a plant exhibiting newly conferred or enhanced resistance to stem rust caused by Pgt, detecting the presence of the R gene in the plant may involve one or more of the following molecular biological techniques disclosed elsewhere herein or known in the art, including, but not limited to: isolating genomic DNA and/or RNA from the wheat plant, amplifying a nucleic acid molecule comprising the R gene and/or a marker therein by PCR amplification, sequencing the nucleic acid molecule 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 performing an immunological assay for detecting 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 used in methods disclosed elsewhere herein or known in the art to rapidly identify one or more plants in a population of plants that contain the presence of the R genes of the present invention. It is further recognized that detecting the presence of the R gene may involve detecting the presence of a fragment of the R gene of the invention. Such fragments of the R genes of the invention may 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 result, the polynucleotide construct of the invention may be stably incorporated into the genome of the plant cell or may be unstable incorporated into the genome of the plant cell. If, for example, the desired result is to produce a stably transformed plant with increased resistance to wheat stem rust caused by at least one variety of Pgt, the polynucleotide construct may, for example, be fused to a plant transformation vector suitable for stably incorporating the polynucleotide construct into the genome of the plant cell. Typically, a stably transformed plant cell will be regenerated into a transformed plant comprising the polynucleotide construct in its genome. Such stably transformed plants are capable of transmitting the polynucleotide construct to progeny plants in subsequent generations by sexual and/or asexual propagation. Plant transformation vectors, methods for stably transforming plants with the introduced polynucleotide constructs, and methods for plant regeneration from transformed plant cells and tissues are generally known in the art for both monocotyledonous and dicotyledonous plants or are described elsewhere herein.

The invention provides nucleic acid molecules comprising an R gene. Preferably, the R gene is capable of conferring on a host plant (in particular wheat or triticale plants) an increased resistance to at least one variety of stem rust causing pathogens Pgt. More preferably, the R gene is capable of conferring enhanced resistance to two, three, four or more varieties of Pgt to a host plant (particularly wheat or triticale plants). Thus, such R genes may be used in agricultural production in restricting stem rust caused by Pgt. The R genes of the invention include, but are not limited to, R genes whose nucleotide sequences are disclosed herein, but also include orthologs and other variants that are capable of conferring resistance to stem rust caused by at least one variety of Pgt to plants. Methods for determining the resistance of a plant to stem rust caused by at least one variety of Pgt are known in the art or disclosed herein.

The methods of the invention are useful for producing plants, particularly wheat and triticale plants, having enhanced resistance to stem rust caused by at least one variety of Pgt. Typically, the methods of the invention will increase or increase the resistance of a test plant to one variety 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 variety of Pgt. Control plants with respect to the present invention are plants that do not comprise the polynucleotide construct of the present invention unless otherwise indicated or apparent from the context of use. Preferably, the control plant is substantially identical (e.g., the same species, subspecies, and variety) to a plant comprising a polynucleotide construct of the invention, except that the control does not comprise the polynucleotide construct. In some embodiments, the control will comprise a polynucleotide construct, but not one or more R gene sequences in the polynucleotide construct of the invention.

In addition, the invention provides transformed plants, seeds and plant cells produced by the methods of the invention and/or comprising the polynucleotide constructs of the invention. Progeny plants and seeds thereof comprising the polynucleotide constructs of the invention are also provided. The 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, which 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, cattle, chickens, turkeys and ducks).

The methods of the invention can be used to enhance the resistance of a plant (particularly wheat or triticale plants) to stem rust (particularly stem rust caused by at least one variety of Pgt). As used herein, the term "wheat plant" generally refers to a plant that is a member of the genus wheat (Triticum), or a member of another genus within the wheat family (Triticum), particularly a member of another genus capable of producing an interspecific hybrid with at least one wheat species. Examples of such other genera within the wheat family are Aegilops (Aegilops) and Secale (Secale).

Wheat plants of the invention include, for example, both domesticated and non-domesticated plants. Wheat plants of the invention include, but are not limited to, the following wheat, aegilops and rye species: wheat (t.aestivum), one-grain wheat, cylindrical wheat (t.targium), wild one-grain wheat (t.booticum), mo Feiwei wheat (t.timoreevii) and uralensis wheat (t.urartu), arthritic wheat, rye (Secale cereale) and hybrids thereof. Examples of common wheat subspecies included in the present invention are common (aestivum), dense ear (compatible um), maca (macha), wars (vavlovivi), spelter (spelta) and spherulites (specrococcum). Examples of cylindrical wheat subspecies included in the present invention are cylindrical (turgium), bos (carthlicum), bi-grain (dicoccom), duromer (durum), paleoichichicam, poland (polonium), hybrid (turanium) and wild bi-grain (t. Examples of a wheat subspecies included in the present invention are a grain (monococcum) and aegilopoides. In one embodiment of the invention, the wheat plant is a member of the species triticum aestivum, and in particular, a member of the subspecies durum, such as Ciccio, colosseo or Utopia cultivars. It is recognized that the wheat plants of the present invention may be either domesticated wheat plants or non-domesticated wheat plants.

The invention also includes triticale plants, triticale plant parts and triticale plant cells comprising the R genes of the invention. As used herein, "triticale plant" refers to a plant created by crossing a rye plant with a tetraploid wheat plant (e.g., triticale) or hexaploidy wheat plant (e.g., triticale). The 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" includes triticale plants unless otherwise indicated or apparent from the context of use.

The term "plant" is intended to include plants at any stage of maturity or development, as well as any tissue or organ (plant part) obtained or derived from any such plant, unless the context clearly indicates otherwise. 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 (which are intact in plants or plant parts such as embryos, pollen, ovules, seeds, tubers, propagules, leaves, flowers, branches, fruits, roots, root tips, anthers, and the like). The invention also includes seeds produced by the plants of the 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 polynucleotide. As used herein, "progeny" and "progeny plants" include any subsequent generation of a plant, whether derived from sexual and/or asexual propagation, unless otherwise specifically indicated or apparent from the context of use.

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 sequence encoding the R protein has 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 includes isolated or substantially purified polynucleotide (also referred to herein as a "nucleic acid molecule," "nucleic acid," etc.) or protein (also referred to herein as a "polypeptide") compositions that include, 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 of components that normally accompany or interact with the polynucleotide or protein (as found in its naturally occurring environment). Thus, the 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 coding 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 may comprise less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb, or 0.1kb of nucleotide sequences that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. Proteins that are substantially free of cellular material include protein preparations having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When the proteins of the invention or biologically active portions thereof are recombinantly produced, optimally, the culture medium has less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-target protein chemicals.

The invention also includes fragments or variants of the disclosed polynucleotides and proteins encoded thereby. "fragment" means a portion of the polynucleotide, or the amino acid sequence encoded thereby and thus a portion of a protein. Fragments of polynucleotides comprising coding sequences may encode protein fragments that retain the biological activity of the full-length or native protein. Alternatively, fragments of polynucleotides useful as hybridization probes typically do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a nucleotide sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full length polynucleotides 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 consecutive nucleotides, or up to the number of nucleotides present in the full-length R polynucleotides disclosed herein.

"variant" is intended to mean a substantially similar sequence. For polynucleotides, variants include polynucleotides having a deletion (i.e., truncation) at the 5 'end and/or the 3' end; deletions and/or additions of one or more nucleotides at one or more internal sites of the native polynucleotide; and/or have substitutions of one or more nucleotides at one or more sites in the natural polynucleotide. As used herein, a "naturally occurring" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that encode the amino acid sequence of one of the R proteins of the invention due to the degeneracy of the genetic code. Naturally occurring allelic variants such as these may be identified by using well known molecular biological techniques, such as the Polymerase Chain Reaction (PCR) and hybridization techniques outlined below. Variant polynucleotides also include synthetically derived polynucleotides such as those generated, for example, by using site-directed mutagenesis, but still encode an R protein of the invention. Typically, 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 the sequence comparison multiple programs and parameters 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 comprise a non-naturally occurring nucleotide sequence differing from the nucleotide sequence set forth in SEQ ID NOs 1, 2 and/or 4 by at least one nucleotide modification selected from the group consisting of: substitution of at least one nucleotide, addition of at least one nucleotide, and deletion of at least one nucleotide.

Variants of a particular polynucleotide of the invention (i.e., a reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between a polypeptide encoded by the variant polynucleotide and a polypeptide encoded by the reference polynucleotide. Thus, for example, polynucleotides encoding polypeptides having a given percentage of sequence identity to the polypeptide of SEQ ID NO. 3 are disclosed. The percent sequence identity between any two polypeptides can be calculated by using the sequence alignment programs and parameters described elsewhere herein. When any given pair of polynucleotides of the invention is evaluated by comparing the percentage of sequence identity shared by the two polypeptides encoded by them, the percentage of 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 greater 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 in SEQ ID NO. 3, and optionally comprise a non-naturally occurring amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO. 3 by at least one amino acid modification selected from the group consisting of: substitution of at least one amino acid, addition of at least one amino acid, and deletion of at least one amino acid.

"variant" protein is intended to mean a protein derived from a natural protein by: deletions of one or more amino acids at the N-terminal and/or C-terminal end of the native protein (so-called truncations); 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 be obtained, for example, from genetic polymorphisms or from artificial manipulation. Biologically active variants of the 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 of the native protein (e.g., the amino acid sequence shown in SEQ ID NO: 3) as determined by the sequence alignment procedure and parameters described elsewhere herein. Biologically active variants of the proteins of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, e.g., 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 a variety of ways, including amino acid substitutions, deletions, truncations and insertions. Methods for such operations are generally known in the art. Methods for mutagenesis and polynucleotide alteration are well known in the art. See, e.g., kunkel (1985) PNAS 82:488-492; kunkel et al, (1987) Methods in enzymol.154:367-382; U.S. Pat. nos. 4,873,192; walker and Gaastra, editors (1983) Techniques in Molecular Biology (MacMillan Publishing Company, new York) and references cited therein. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al, (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., washington, D.C.), incorporated herein by reference. Conservative substitutions, such as the exchange of one amino acid for another with similar properties, may be optimal.

Thus, genes and polynucleotides of the invention include naturally occurring sequences, mutants and other variant forms. Likewise, the proteins of the invention include naturally occurring proteins as well as variants and modified forms thereof. More preferably, such variants confer enhanced resistance to stem rust caused by at least one variety of Pgt to a plant or portion thereof comprising the variants. In some embodiments, the mutation that will be made in the DNA encoding the variant will not place the sequence out of frame. Optimally, the mutation will not create a complementary region that can produce a secondary mRNA structure. See, EP patent application publication No. 75,444.

Deletions, insertions and substitutions of protein sequences included herein are not expected to produce a fundamental change in the characteristics of the protein. However, when it is difficult to predict the exact effect of a substitution, deletion or insertion before doing so, one skilled in the art will appreciate that the effect will be assessed by conventional screening assays. That is, the activity can be assessed by the assays disclosed herein below.

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

Variant polynucleotides and proteins also include sequences and proteins derived from mutagenic and recombinogenic procedures (e.g., DNA shuffling). Strategies for such DNA shuffling are known in the art. See, e.g., 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) Nature391:288-291; and U.S. Pat. nos. 5,605,793 and 5,837,458.

The polynucleotides of the invention may be used to isolate corresponding sequences from other organisms, particularly other plants. In this way, 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. The present invention includes sequences that have been isolated based on their sequence identity to the entire sequences set forth herein or to variants and fragments thereof. Such sequences include sequences that are orthologs of the disclosed sequences. "ortholog" is intended to refer to genes that originate from a common ancestral gene and are found in different species as a result of speciation. Genes found in different species are considered orthologs when: the nucleotide sequences thereof and/or the protein sequences encoded thereby share at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. The function of orthologs is often highly conserved among species. Thus, the present invention includes isolated polynucleotides encoding an R protein and hybridizing under stringent conditions to at least one of the R proteins disclosed herein or known in the art, or to variants or fragments thereof.

In one embodiment, an ortholog of the invention has a coding sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more 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 encodes a protein comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more 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 (which comprises the amino acid sequence shown in SEQ ID NO: 3), the conserved domain includes, 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, the variant Sr27 proteins of the 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, the 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 as compared to the percentage of amino acid sequence identity to the full-length amino acid sequence of Sr27 (SEQ ID NO: 3) or the proteins disclosed herein. Preferably, such variants comprise the corresponding 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 that correspond to those conserved domains of Sr27 set forth above, as well as any particular conserved amino acids therein, can be identified by methods (e.g., multiple sequence alignments) known to those of skill in the art or disclosed elsewhere herein. It is further recognized that the positions of such conserved domains and conserved amino acids within a particular variant Sr27 protein may differ from the positions in the amino acid sequence shown in SEQ ID NO:3, and that the corresponding positions of such conserved domains and conserved amino acids may be determined for any variant Sr27 protein of the invention by methods such as multiple sequence alignment.

Preferably, the variant Sr27 proteins of the invention and the polynucleotides encoding them confer or are capable of conferring on a wheat plant comprising such proteins and/or polynucleotides an increased resistance to at least one variety of Pgt known to cause wheat stem rust in susceptible wheat plants.

In the PCR method, oligonucleotide primers can be designed for use in a PCR reaction to amplify a corresponding DNA sequence from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR clones are generally known in the art and are disclosed in Sambrook et al, (1989) Molecular Cloning: ALaboratory Manual (2 nd edition, cold Spring Harbor Laboratory Press, planview, new York). See also Innis et al, editors (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, new York); innis and Gelfand, editors (1995) PCR Strategies (Academic Press, new York); and Innis and Gelfand, editors (1999) PCR Methods Manual (Academic Press, new York). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, monospecific 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 include polynucleotide molecules comprising nucleotide sequences that are sufficiently identical to the nucleotide sequences 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 comprising a sufficient or minimal number of amino acid residues or nucleotides that are identical or equivalent (e.g., have similar side chains) 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 a common functional activity. For example, amino acid or nucleotide sequences comprising a common 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 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/number of total positions (e.g., overlapping positions) x 100). In one embodiment, the lengths of the two sequences are the same. The percent identity between two sequences can be determined by using techniques similar to those described below, with or without allowing gaps. In calculating the percent identity, exact matches are typically counted.

The determination of the percent identity between two sequences is accomplished by using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) PNAS 87:2264 as modified in Karlin and Altschul (1993) PNAS 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al, (1990) J.mol.biol.215:403. BLAST nucleotide searches can be performed using the NBLAST program (score=100, word length=12) to obtain nucleotide sequences homologous to the polynucleotide molecules of the present invention. BLAST protein searches can be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to protein molecules of the present invention. To obtain a gap alignment for comparison purposes, gap BLAST may be used, as described in Altschul et al, (1997) Nucleic Acids Res.25:3389. Alternatively, PSI-Blast may be used to conduct an iterative search that detects long-range relationships between molecules. See Altschul et al, (1997) supra. When using BLAST, gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST; which are available on the world Wide Web at ncbi.nlm.nih.gov) are used. Another preferred, non-limiting example of a mathematical algorithm for sequence comparison 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 amino acid sequences are compared using the ALIGN program, PAM120 weight residue table, gap length penalty of 12 and gap penalty of 4 can be used. The alignment may also be performed manually by inspection.

Unless otherwise indicated, the sequence identity/similarity values provided herein refer to values obtained by using the full length sequences of the present invention and using multiple alignments by means of the algorithm Clustal W (Nucleic Acid Research,22 (22): 4673-4680, 1994), using the program alignX (using default parameters) included in software package Vector NTI Suite Version (InforMax, inc., bethesda, MD, USA), or any equivalent thereof. "equivalent program" means any sequence comparison program which, when compared to a corresponding alignment generated by CLUSTALW (version 1.83) using default parameters, which is available on the world Wide Web at European bioinformatics institute (European Bioinformatics Institute) website, generates an alignment with the same nucleotide or amino acid residue match and the same percentage of sequence identity for any two sequences in question.

The use of the term "polynucleotide" is not intended to limit the invention to polynucleotides comprising DNA. One of ordinary skill in the art will recognize that polynucleotides may comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. Polynucleotides of the invention also include all forms of sequences, including but not limited to single stranded forms, double stranded forms, hairpins, stem loop structures, and the like.

The polynucleotide construct comprising the R protein coding region may be provided in an expression cassette for expression in a plant or other organism or non-human host cell of interest. The expression cassette will comprise 5 'and 3' regulatory sequences operably linked to the R protein coding region. "operably connected" is intended to mean a functional connection between two or more elements. For example, an operative linkage between a polynucleotide or gene of interest and a regulatory sequence (i.e., a promoter) is a functional linkage that allows expression of the polynucleotide of interest. The operatively connected elements may be contiguous or non-contiguous. When used in reference to the joining of two protein coding regions, "operably linked" means that the coding regions are in the same reading frame. In addition, the cassette may comprise at least one further gene to be co-transformed into the organism. Alternatively, the additional genes may be provided on multiple expression cassettes. Such an expression cassette may be provided with a plurality of restriction sites and/or recombination sites for insertion of the R protein coding region to be under transcriptional control of the regulatory region. In addition, the expression cassette may comprise a selectable marker gene.

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

As used herein, with respect to a nucleic acid molecule or nucleotide sequence, "heterologous" is a nucleic acid molecule or nucleotide sequence that is derived from an external species or, if derived 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 different species than the species from which the polynucleotide is derived, or if from the same/similar species, one or both of them is significantly modified from its original form and/or genomic locus, or the promoter is not a 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 invention provides a host cell comprising at least one of the nucleic acid molecules, expression cassettes and vectors of the invention. In a preferred embodiment, the host cell is a plant cell. In other embodiments, the host cell is selected from the group consisting of: bacterial, fungal and animal cells. In certain embodiments, the host cell is a non-human animal cell. However, in some other embodiments, the host cell is a human cell cultured in vitro. In still other embodiments, the host cell is a microorganism, particularly a unicellular microorganism. Microorganisms include, but are not limited to, archaebacteria, eubacteria, yeasts and algae.

Although the use of heterologous promoters to express the R proteins may be optimal, the native promoters of the corresponding R genes may be used.

The termination region may be native to the transcription initiation region, may be native to an operably linked R protein coding region of interest, may be native to a 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 Ti-plasmids of Agrobacterium tumefaciens (A.tumefaciens), such as octopine synthase and nopaline synthase termination regions. See also Guerineau et al, (1991) mol. Gen. Genet.262:141-144; proudroot (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.

When appropriate, the polynucleotide may be optimized for increased expression in the transformed plant. That is, the polynucleotide may be synthesized for improved expression by using plant-preferred codons. For a discussion of host preferred codon usage see, e.g., campbell and Gowri (1990) Plant Physiol.92:1-11. Methods for synthesizing plant-preferred genes are available in the art. 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, which are incorporated herein by reference.

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

In addition, the expression cassette may comprise a 5' leader sequence. Such leader sequences may function to enhance translation. Translation leader sequences are known in the art and include: picornaviral leader sequences, such as EMCV leader sequences (encephalomyocarditis 5' non-coding region) (Elroy-Stein et al, (1989) PNAS 86:6126-6130); potyvirus leader sequences, such as TEV leader (tobacco plaque virus) (galie et al, (1995) Gene165 (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); an untranslated leader sequence 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) (galie et al, (1989) Molecular Biology of RNA, editor Cech (Lists, new York), pages 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al, (1991) Virology 81:382-385). See also Della-Ciopa et al, (1987) Plant Physiol.84:965-968.

In preparing the expression cassette, various DNA fragments may be manipulated to provide the DNA sequence in the appropriate orientation and (as appropriate) in the appropriate reading frame. For this purpose, adaptors or linkers may be used to ligate the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitution, such as transitions and transversions, may be involved.

Many promoters may be used in the practice of the present invention. Promoters may be selected based on the desired result. The nucleic acid may be combined with a constitutive promoter, a tissue-preferred promoter, 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 may be used to target enhanced expression of the R protein coding sequence 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) therapeutic 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 J.4 (3): 495-505. Such promoters may be modified for weak expression, if desired.

In general, it would be beneficial to express genes from inducible promoters, in particular from pathogen-inducible promoters. Such promoters include those of pathogenic related proteins (PR proteins) induced upon infection by a pathogen; such as PR proteins, SAR proteins, beta-1, 3-glucanase, chitinases, 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, which is incorporated herein by reference.

Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, e.g., 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) mol. 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. patent No. 5,750,386 (nematode inducible); and references cited therein. Of particular interest are inducible promoters for their expression of the maize PRms gene induced by the pathogen Fusarium moniliforme (Fusarium moniliforme) (see, e.g., cordero et al, (1992) Physiol.mol.plant Path.41:189-200).

Also of interest are natural promoters from other resistance genes derived from the target species. These promoters are often pathogen-inducible and may express resistance genes 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 J10:447-487; 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).

In addition, wound-inducible promoters may be used in the constructs of the invention, as pathogens enter plants through wounds or insect lesions. Such wound-inducible promoters include: potato protease inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath.28:425-449; duan et al, (1996) Nature Biotechnology 14:494-498); wun1 and wun, 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), etc., which is incorporated herein by reference.

Chemical regulated promoters can be used to modulate expression of genes in plants by application of exogenous chemical regulators. Depending on the target, the promoter may be a chemical-inducible promoter, wherein application of the chemical induces gene expression; or a chemical repressible promoter, wherein application of the chemical represses gene expression. Chemical inducible promoters are known in the art and include, but are not limited to: a maize In2-2 promoter activated by a benzenesulfonamide herbicide safener; a maize GST promoter activated by a hydrophobic electrophilic compound used as a pre-emergence herbicide; and a tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical regulatory promoters of interest include: steroid responsive promoters (see, e.g., glucocorticoid inducible promoters 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, e.g., gatz et al, (1991) mol. Gen. Genet.227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

The expression cassette may also comprise a selectable marker gene for selecting transformed cells. Selectable marker genes are used to select for 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 herbicide compounds such as glufosinate, bromoxynil, imidazolinones and 2, 4-dichlorophenoxyacetate (2, 4-D). Additional selectable markers include: phenotypic markers, such as beta-galactosidase, and fluorescent proteins, such as Green Fluorescent Protein (GFP) (Su et al, (2004) Biotechnol Bioeng85:610-9 and Fetter et al, (2004) Plant Cell 16:215-28), cyan fluorescent protein (CYP) (Bolte et al, (2004) J.cell Science 117:943-54 and Kato et al, (2002) Plant Physiol 129:913-42) and yellow fluorescent protein (PhiYFP from Evogen) ^TM See Bolte et al, (2004) J.cell Science 117:943-54). For additional selectable markers, see generally yaranton (1992) curr. Opin. Biotech.3:506-511; christophus 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), the operaon, pages 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; the golden (1993) doctor paper, 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; baim et al, (1991) PNAS 88:5072-5076; wyborski et al, (1991) Nucleic Acids Res.19:4647-4653; hillenand-Wissman (1989) Topics mol. Structure. Biol.10:143-162; degenkolb et al, (1991) Antimicrob. Agents chemother35:1591-1595; kleinschnidt et al, (1988) Biochemistry 27:1094-1104; bonin (1993) doctor paper, university of Heidelberg; golden (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, volume 78 (Springer-Verlag, berlin); gill et al, (1988) Nature 334:721-724. These disclosures are incorporated herein by reference.

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

Numerous plant transformation vectors and methods for transforming plants are available. See, e.g., 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 Slight (1992) Gene.118:255-260; christou et al, (1992) Trends Biotechnol.10:239-246; d' Hall et al, (1992) Bio/technology.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; dong and 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.plantain 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 the polynucleotide construct into a plant. By "introduced" is meant that the polynucleotide construct is presented to the plant in such a way that the construct is allowed to enter the interior of the cells of the plant. The methods of the invention do not depend on the particular method used to introduce the polynucleotide construct into the plant, so long as the polynucleotide construct is allowed to enter the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

By "stable transformation" is meant that the polynucleotide construct introduced into the plant is integrated into the genome of the plant and is capable of inheritance by its progeny. "transient transformation" means that the polynucleotide construct introduced into a plant is not integrated into the genome of the plant.

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

Methods for constructing plant expression cassettes and introducing foreign nucleic acids into plants are generally known in the art and have been described previously. For example, foreign DNA may be introduced into plants by using a tumor-inducible (Ti) plasmid vector. Other methods for foreign DNA delivery involve the use of PEG-mediated protoplast transformation, electroporation, microinjection of 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; guearche et al, (1987) Plant Science 52:111-116; neuhuse et al, (1987) Theor. Appl Genet.75:30-36; klein et al, (1987) Nature327:70-73; 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 (Weisbach and Weisbach) Academic Press, inc (1988), and Methods in Plant Molecular Biology (Schulder and Zieladbach) depending on the level of the Gene expression vector, the vector and other parameters to be used.

Other suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into plant genomes include: microinjection as described by 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, for example, in the following documents: sanford et al, U.S. Pat. Nos. 4,945,050; tomes et al, U.S. patent No. 5,879,918; tomes et al, U.S. patent No. 5,886,244; bidney et al, U.S. Pat. nos. 5,932,782; tomes et al, (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," Plant Cell, tissue, and Organ Culture: fundamental Methods, editor Gamborg and Phillips (Springer-Verlag, berlin); mcCabe et al, (1988) Biotechnology 6:923-926; and Lec1 transformation (WO 00/28058). See also Weisineger et al, (1988) Ann.Rev.Genet.22:421-477; sanford et al, (1987) Particulate Science and Technology5:27-37 (onion); christou et al, (1988) Plant Physiol.87:671-674 (Glycine max); mcCabe et al, (1988) Bio/Technology 6:923-926 (Soybean); finer and McMullen (1991) InVitro 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 (corn); klein et al, (1988) Biotechnology 6:559-563 (maize); tomes, U.S. patent 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," Plant Cell, tissue, and Organ Culture: fundamental Methods, editor Gamborg (Springer-Verlag, berlin) (maize); klein et al, (1988) Plant Physiol.91:440-444 (corn); fromm et al, (1990) Biotechnology 8:833-839 (corn); hooykaas-Van Slogeren et al, (1984) Nature (London) 311:311:763-764; bowen et al, U.S. Pat. No. 5,736,369 (cereal); bytebier et al, (1987) PNAS 84:5345-5349 (Liliaceae); de Wet et al, (1985) The Experimental Manipulation of Ovule Tissues, editor Chapman et al, (Longman, new York), pages 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' Hall 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 Botany75:407-413 (rice); osjoda et al, (1996) Nature Biotechnology14:745-750 (maize via Agrobacterium tumefaciens); all of which are incorporated herein by reference.

The polynucleotides of the invention may be introduced into a plant by contacting the plant with a virus or viral nucleic acid. Typically, such methods involve incorporating the polynucleotide constructs of the invention into viral DNA or RNA molecules. Further, it is well recognized that the promoters of the present invention also include promoters for transcription by viral RNA polymerase. Methods for introducing polynucleotide constructs into plants and expressing encoded proteins therein, which involve viral DNA or RNA molecules, are known in the art. See, for example, U.S. patent nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931; which is incorporated herein by reference.

The modified virus or modified viral nucleic acid can be formulated, if desired. Such formulations are prepared in a known manner (see, for example, for reviews, U.S. Pat. No. 3,060,084; EP-A707 445 (for liquid concentrates); browning, "Agglomeration", chemical Engineering, 12/month 4/1967; 147-48;Perry's Chemical Engineer's Handbook, 4 th edition, mcGraw-Hill, new York,1963, pages 8-57; and the following, among others, WO 91/13546;US 4,172,714;US 4,144,050;US 3,920,442;US 5,180,587;US 5,232,701;US 5,208,030;GB 2,095,558;US 3,299,566;Klingman,Weed Control as a Science,John Wiley and Sons,Inc, new York,1961; hance et al, weed Control Handbook, 8 th edition, 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 expanding the active compound with adjuvants suitable for formulating agrochemicals, such as solvents and/or carriers, emulsifying agents, surfactants and dispersants, preservatives, defoamers, anti-freezing agents, and optionally also for seed treatment, and/or binders or gelling agents, if desired.

In particular embodiments, the polynucleotide constructs and expression cassettes of the invention may be provided to plants by using a variety of transient transformation methods known in the art. Such methods include, for example, microinjection or particle bombardment. See, e.g., 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 incorporated herein by reference. Alternatively, the polynucleotide may be transiently transformed into the plant by using techniques known in the art. Such techniques include viral vector systems and agrobacterium tumefaciens-mediated transient expression described elsewhere herein.

The transformed cells may be grown into plants in a conventional manner. See, e.g., mcCormick et al, (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same transformed line or a different line and the resulting constitutively expressed hybrid with 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 that expression of the desired phenotypic characteristic is achieved. In this way, the invention provides transformed seeds (also referred to as "transgenic seeds") having the polynucleotide construct of the invention, e.g., the expression cassette of the invention, stably incorporated into their genome.

In certain embodiments of the invention, the nucleotide sequence of the non-functional allele at the R locus of the invention may be modified in plant in situ (in planta) to a functional allele that provides resistance to at least one variety of plant pathogen. Thus, the present invention provides methods for producing plants (particularly triticale plants) having enhanced resistance to stem rust. The method comprises modifying a non-functional allele of a resistance gene Sr27 in a plant or at least one cell thereof so as to make a functional allele, thereby enhancing the plant's resistance to stem rust. In a preferred embodiment, the plants produced are non-transgenic plants, in particular non-transgenic triticale plants. The method may further comprise regenerating a plant cell comprising the functional allele into a plant comprising the functional allele.

In one embodiment of the invention, the nonfunctional allele or susceptible allele present at the Sr27 locus in triticale plants may be modified to a functional allele that provides resistance to at least one, two, three or four varieties such as Pgt. In another embodiment of the invention, the non-functional allele present at the Sr27 locus in triticale plants may be modified to a functional allele that provides resistance to at least one, two, three or four varieties of Pgt causing stem rust. For example, a non-functional allele at the Sr27 locus may be modified to thereby form a modified allele comprising the nucleotide sequence of Sr27 shown in SEQ ID NO:1 and/or a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3.

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

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

The deletion includes a deletion of at least one bp from the allele of the R gene of the invention. As used herein, "deletion" means the removal of one or more nucleotides or base pairs from DNA. As provided herein, a deletion in an allele of an R gene may 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 substitution includes substitution of at least one bp of the allele from the R gene of the invention with another bp. As used herein, "substitution" means the replacement of one or more nucleotides or base pairs from DNA with non-identical nucleotides or base pairs. When the substitution comprises two or more nucleotides, the two or more nucleotides may be contiguous or non-contiguous within the DNA sequence of the allele. As provided herein, a substitution in an allele of an R gene can be a substitution 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 may be a nucleotide sequence of the entire allele or any portion thereof (e.g., transcribed region, 5 'untranslated region, 3' untranslated region, exon, or intron).

In certain embodiments of the invention, the modification of the nonfunctional allele at the R locus is a homozygous modification. By "homozygous modification" is meant that the modification is among the two alleles of the R gene locus in a particular genome of the plant. In other cases, the modification of the R locus gene is heterozygous, i.e., the modification is in only one allele of the R locus in the genome of the plant. It is recognized that plants of the invention include, for example, crop plants having genomes that are diploid or polyploid (e.g., tetraploid or hexaploid), including homopolyploid and heteropolyploid. An autopolyploid is an organism having more than two sets of chromosomes all originating from the same species. A heteropolyploid is an organism having two or more whole sets of chromosomes from different species. Depending on the particular crop plant, 1, 2, 3, 4, 5, 6 or more alleles at the R gene locus of the plant can be modified by using the disclosed methods.

Any method known in the art for modifying DNA in a plant genome may be used to alter the coding sequence of the R gene in a plant in situ manner, e.g., to alter the nucleotide sequence of a homologous susceptibility allele to a nucleotide sequence of an allele that provides resistance to at least one variety of stem rust. Such methods known in the art for modifying DNA in a plant genome include, for example, mutation breeding and genome editing techniques, such as 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 incorporated herein by reference in their entirety). Methods for genetic modification or gene replacement, including homologous recombination, may involve inducing single-or double-strand breaks in DNA using the following enzymes: zinc Finger Nucleases (ZFNs), TAL (transcription activator-like) effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats/CRISPR-associated nucleases (CRISPR/Cas nucleases) or homing endonucleases (which have been engineered endonucleases to make double strand breaks at specific recognition sequences in the genome of plants, other organisms or host cells). See, e.g., durai et al, (2005) Nucleic Acids Res 33:5978-90; mani et al, (2005) Biochem Biophys Res Comm 335:447-57; U.S. patent 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 Soc128:2477-84; rosen et al, (2006) Nucleic Acids Res 34:4791-800; and Smith et al, (2006) Nucleic Acids Res 34:e149; U.S. patent application publication No. 2009/013514; and U.S. patent application publication No. 2007/017128; all of which are incorporated herein by reference in their entirety.

TAL effector nucleases (TALENs) can be used to make double strand breaks at specific recognition sequences in plant genomes for gene modification or gene replacement by homologous recombination. TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double strand breaks at a specific target sequence in the genome of a plant or other organism. TAL effector nucleases are created by fusing a natural or engineered transcription activator-like (TAL) effector or functional portion thereof to the catalytic domain of an endonuclease (e.g., fokl). The unique, modular TAL effector DNA binding domain allows the design of proteins potentially having any given DNA recognition specificity. Thus, the DNA binding domain of TAL effector nucleases can be engineered to recognize specific DNA target sites and thus be used to make double strand breaks at the desired target sequence. See, WO 2010/079430; morbitzer et al, (2010) PNAS 10.1073/pnas.1013133107; scholze and Boch (2010) Virus 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 Biotechnology29:143-148; all of which are incorporated herein by reference.

CRISPR/Cas nuclease systems can also be used to make single-or double-strand breaks at specific recognition sequences in plant genomes for gene modification or gene replacement by homologous recombination. CRISPR/Cas nuclease is an RNA-guided (single guide RNA, abbreviated sgRNA) DNA endonuclease system that performs sequence-specific double strand breaks in DNA segments homologous to the designed RNA. It is possible to design the specificity of the sequences (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, ZFNs can be used to make double strand breaks at specific recognition sequences in plant genomes for gene modification or gene replacement by homologous recombination. Zinc Finger Nucleases (ZFNs) are fusion proteins that contain a portion of the FokI restriction endonuclease responsible for DNA cleavage and a zinc finger protein that recognizes specific, designed genomic sequences, and cleaves 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).

The use of specific nucleases (e.g., those described hereinabove) to cleave DNA can increase the rate of homologous recombination in the region of cleavage. Thus, coupling of such effectors to nucleases as described above enables the generation of targeted changes in the genome, including additions, deletions, substitutions and other modifications.

Mutation breeding may also be used in the methods provided herein. Mutation breeding methods may involve, for example, exposing plants or seeds to mutagens, particularly chemical mutagens such as Ethyl Methane Sulfonate (EMS), and selecting for plants having the desired modification in the Sr27 gene. However, other mutagens may 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., nuclear fission products generated by uranium 235 in an atomic reactor), beta-radiation (e.g., emitted from a radioisotope such as phosphorus 32 or carbon 14), and ultraviolet radiation (preferably 2500 to 2900 nm); and chemical mutagens, such as base analogs (e.g., 5-bromouracil), related compounds (e.g., 8-ethoxycaffeine), antibiotics (e.g., streptozotocin), alkylating agents (e.g., sulfur mustards, nitrogen mustards, epoxides, ethyleneamines, sulfates, sulfonates, sulfones, lactones), azides, hydroxylamines, nitrous acid, or acridines. Further details of mutation breeding can be found in "Principals of Cultivar Development" Fehr,1993Macmillan Publishing Company (the disclosure of which is incorporated herein by reference).

The nucleic acid molecules, expression cassettes, vectors and polynucleotide constructs of the invention can be used to transform any plant species, including but not limited to monocots and dicots. Preferred plants of the invention are wheat plants. Examples of other plant species of interest include, but are not limited to: capsicum species (Capsicum spp); for example, capsicum (Capsicum annuum), capsicum annuum (c.baccatum), capsicum annuum (c.chinense), capsicum frutescens (c.frutescens), etc., tomato (Lycopersicon esculentum), tobacco (Nicotiana tabacum), eggplant (Solanum melongena), petunia (Petunia species (Petunia spp.), such as Petunia (Petunia x hybrida or Petunia hybrid)), pea (Pisum sativum), bean (Phaseolus vulgaris) corn or maize (Zea mays), brassica species (Brassica sp.) (such as Brassica napus (b.napus), brassica napus (b.rapa), brassica juncea (b.juna)) (particularly those species useful as a source of seed oil), alfalfa (Medicago sativa), rice (Oryza sativa), rye (secaline), sorghum (Sorghum), sorghum (maize), sorghum (38, for example, millet (38, millet (Sorghum), millet (38, etc.) Seed (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), soybean (Glycine max), russian teff (Eragrostis tef), tobacco, potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (island cotton (Gossypium barbadense), upland cotton (Gossypium hirsutum)), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (coffee species (coffee spp.), coconut (coco nucifera), pineapple (Ananas comosus), citrus tree (Citrus species (Citrus spp.)), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.)), avocado (Persea america), fig (Ficus carica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea), papaya (Ca) and papaya (Ca)The plant species may be selected from the group consisting of rica papaya), cashew (Anacardium occidentale), queensland nut (Macadamia integrifolia), almond (Prunus amygdalus), beet (Beta vulgaris), sugarcane (Saccharum spp.), palm, oat, barley, vegetables, ornamental plants and conifers. />

In certain embodiments of the invention, the preferred plant is a cereal plant. Such cereal plants of the invention are herbs (i.e., poaceae) that are planted for edible components of their grain or kernel (i.e., seed), including, for example, wheat, triticale, rye, barley, oats, corn, sorghum, millet, and rice. In certain other embodiments of the invention, the preferred plant is a cereal plant, but does not include a triticale plant.

In some embodiments of the invention, plant cells are transformed with a polynucleotide construct encoding an R protein of the invention. As used herein, the term "expression" refers to biosynthesis of a gene product, including transcription and/or translation of the gene product. "expressing" or "producing" a protein or polypeptide from a DNA molecule refers to the transcription and translation of a coding sequence to produce the protein or polypeptide, while "expressing" or "producing" a protein or polypeptide from an RNA molecule refers to the translation of an RNA coding sequence to produce the protein or polypeptide. Examples of polynucleotide constructs and nucleic acid molecules encoding R proteins are described elsewhere herein.

The use of the term "DNA" or "RNA" herein is not intended to limit the invention to polynucleotide molecules comprising DNA or RNA. One of ordinary skill in the art will recognize that the methods and compositions of the present invention include polynucleotide molecules consisting of deoxyribonucleotides (i.e., DNA), ribonucleotides (i.e., RNA), or a combination of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs, 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 nucleotide. Examples of such analogs include, but are not limited to: phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotides, peptide Nucleic Acids (PNA). The polynucleotide molecules of the present invention also include all forms of polynucleotide molecules including, but not limited to, single stranded forms, double stranded forms, hairpins, stem loop structures, and the like. Further, one of ordinary skill in the art will appreciate that the nucleotide sequences disclosed herein also include complements of the exemplified nucleotide sequences.

The present invention relates to compositions and methods for enhancing resistance of plants to plant diseases, in particular to compositions and methods for enhancing resistance of plants to stem rust caused by Pgt. By "disease resistance" is meant that the plant is free of disease symptoms as a consequence of plant-pathogen interactions. I.e., preventing a pathogen from causing a plant disease and associated disease symptoms, or alternatively, minimizing or alleviating disease symptoms caused by the pathogen.

As used herein, "variety" refers to any one of a group of laboratory isolates or fungal individuals present in the field that share a similar virulence phenotype for a range of different wheat resistance gene lines, and possibly through clonal propagation.

As used herein, an "isolate" of Pgt refers to the Pgt line that was originally isolated as spores collected from infected plants in the field or in a laboratory/greenhouse setting. Subsequently, such "isolates" are maintained in pure form by infection and re-isolation of spores from susceptible plants and storage of the spores.

The present invention includes nucleic acid molecules and polynucleotide constructs disclosed herein or in the accompanying sequence listing and/or figures, including but not limited to: nucleic acid molecules and polynucleotide constructs comprising the nucleotide sequences shown in SEQ ID NO. 1, 2 and/or 4; and nucleic acid molecules and polynucleotide constructs encoding proteins comprising the amino acid sequences shown in SEQ ID NO. 3. The invention further includes 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. In addition, the invention includes the use in a plant comprising at least one of such polynucleotide constructs in the methods disclosed elsewhere herein (e.g., methods for enhancing the resistance of a plant to stem rust caused by Pgt, and methods of restricting stem rust in crop production).

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

Examples

Example 1: cloning of Sr27 stem rust resistance Gene

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

The inventors mutagenized seeds of triticale cultivar coohong with Ethyl Methanesulfonate (EMS), and identified 27 susceptible Ethyl Methanesulfonate (EMS) -derived mutants from the coohong background prepared as described below (fig. 1). Coorong contains the Sr27 stem rust resistance gene in its genome and was first published in Australia by the university of Ardelade (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 susceptibility mutants (M2, M3, M4 and M6) as described below. Captured reads from wild-type Coorong were assembled and reads from all lines were aligned with the reference to identify sequence changes in mutant lines. An contig of 1126 base pairs (bp) was found (accession No. 5723) containing mutations in three of the four mutants; one (M2) has a complete deletion of the sequence, and two (M3, M4) have single base changes that result in amino acid substitutions Q264R and G209S (in the conserved p-loop), respectively. This contig contains a Coiled Coil (CC) domain and a p-cyclic motif, but no NB-ARC or LRR domain remains, suggesting that it is only part of the full length NLR gene. To identify the remainder of the gene, the contig was aligned with the common wheat cultivar Chinese Spring reference (CSv 1) assembly IWGSC RefSeq v1.0 (Appels et al, (2018) Science 361 (6403): ear 7191). The highest hit (93.6% identity across the full DNA sequence) is the 5' end of the high confidence gene (TraesCS 6B01G 464400) predicted on chromosome 6B and functionally annotated as a disease resistance gene. The complete sequence of TraesCS6B01G464400 was then aligned back to the Coorong RenSeq de novo assembly, which detected another 2140bp contig (accession number 2413), which when aligned to the 3' region of the gene, had 93.8% identity across the complete sequence. Contig number 2413 includes both NB-ARC and LRR domains. Examination of the read alignment from mutant Coorong line confirmed that contig number 2413 was also detected in mutant M2 and another single base change was identified in mutant M6 (fig. 2). PCR amplification confirmed that these two contigs were derived from the same gene in wild-type cooroning, which encoded a full-length protein with 956 amino acids comprising a coiled-coil (CC) domain at the N-terminus, followed by an NB-ARC domain, and then an LRR motif 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 in the p-cyclic motif (T211I), whereas the gene sequences could not be amplified from the other three (M7, M8, M9), indicating that they contained a deletion in this region. These eight independent mutants, which contain deletions or amino acid changes in the gene, provide strong evidence that it confers Sr27 resistance.

Materials and methods

Plant material and mutant DNA preparation

Seeds of triticale strain wild-type coohong (coohong carrying Sr 27) were treated with Ethyl Methanesulfonate (EMS) following the protocol described by Mago et al ((2015) Nature Plants 1,15186). Killing curves for 20 grains were initially generated with different concentrations (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6% (v/v)) to identify the dose required to achieve 50% mortality. A total of 1960 seeds were treated with 0.3% ethyl methanesulfonate for 12 hours, then thoroughly washed with water and sown in large pots (40 seeds/30 cm pot) in a greenhouse with sunlight and a temperature of day and night of 23 ℃ and 15 ℃. Individual ears from each M1 plant were threshed separately and the M2 family from each plant was sown in trays (30M 2 families/tray). Each disc also included a resistance (cooong) and susceptibility (Rongcoo) control. The M2 family obtained as single spike progeny from each M1 plant was tested for stem rust response by inoculation with the wheat species of Graptopetalum (Pgt) isolate Pgt-0. Individual plants from the segregating progeny were grown and the progeny tested. Homozygous susceptible mutant and resistant sibling pairs are recovered from these progeny.

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 checked first with a Nanodrop spectrophotometer (Thermo Scientific, wilmington, DE) and then on a 0.8% agarose gel.

Resistance gene enrichment and sequencing (RenSeq)

Target enrichment of NLR by Arbor Biosciences (Ann Arbor, USA) was performed following MYbaits protocol using a modified version of the wheat family bait library previously published available at gitub. Library construction was performed by following the TruSeq RNA protocol v 2. All enriched libraries were sequenced on a HiSeq 2500 (Illumina, CA, USA) using 250bp paired end reads and SBS chemistry.

MutantHunter

To identify Sr27 contigs from mutants, the mutantHunter pipeline method of Steuernagel et al ((2015) Bioinformation 31:1665-7) was followed. Initial read sequencing data from wild-type Coorong and mutants were first trimmed for quality by using Trimmomatic v0.38 (Bolger et al, (2014) Bioinformation 30:2114-20), using the parameter ILLUMINACIP: nonvogene_index_adapters.fa: 2:30:10:8:TRUE, LEADING 28, TRAILING 28, MINLEN 20. Data from wild-type Coorong were then assembled de novo using CLCGW v11.0.1 as the paired end with a length score of 0.95 and a similarity score of 0.98, with the remaining parameters being the default. Using custom scripts, contigs shorter than 1kb were omitted from the final assembly. Annotations of NBS-LRR motifs were created by using the program NLR-Parser (Steuernagel et al, (2015) Bioinformation 31:1665-7). The trimmed data for each mutant and wild type was mapped to the de novo assembly by using BWA v0.7.15 (Li and Durbin (2009) Bioinformation 25:1754-1760). Samtools v1.7.0 (Li et al, (2009) bioenformatics 25:2078-9) is used for the processing of the resulting SAM file to retain only the read map in the correct pairing with parameter-f 2, then duplicate entries are removed and parameters-BQ 0 and-aa are used to generate the pileup file. SNV call and subsequent candidate identification by using the following lines from MuTrigo @ pipelinehttps://github.com/TC-Hewitt/MuTrigo) Is performed by the script of (a). Prior to downstream analysis, the contig region with high SNP levels in wild-type data (which indicates poor assembly or read alignment) was detected using the noiseinder.pyc (with default parameters) and masked. The potentially mutated nucleotide positions were recorded from the pileup file by using SNPlogger. Pyc (with parameter-d 20). SNPtracker. Pyc was used to search for contigs containing polymorphisms in two or more mutants using the default parameter plus the parameter-s C \ >T G\>A index. This translates into only considering polymorphisms with a minimum of 80% mutant allele frequencies, and selecting only for insertions, deletions or C to T or G to a SNV (which do not share the same positions as the other mutant or wild type). Candidate gene contigs were aligned with the chromosome-scale reference assembly of the rye inbred 'Lo7' (Rabanus-Wallace et al, (2019) bioRxiv: 2019.12.11.869693) and the common wheat cultivar Chinese Spring reference (CSv 1) assembly IWSSC RefSeq v1.0 (Appels et al, (2018) Science 361 (6403): ear 7191), using BLAST v2.7.1 (Altschul et al, (1990) J.mol. Biol.215: 403-10). From the Illumina RNAseq data from Coorong seedlings infected with Pgt (Uppaphya et al, (-)2015 Front Plant Sci.5:759) (downloaded from NCBI-SRA SAMN07836894, PRJNA 415866) to generate de novo RNA transcript assemblies using CLCGW v11.0.1 (similarity score of 0.98, and the remaining parameters set as default).

Sr27 Gene Structure confirmation

Primers were designed based on the genomic sequences of the two non-overlapping contig numbers 2413 and 5723 (table 2). Primer pairs Sr27c5723ExtF1 and Sr27c2413ExtR1 were used to amplify the non-overlapping region between contig numbers 5723 and 2413 from genomic DNA of wild-type cooronig. Primer pairs Sr27F and Sr27R1 were used to amplify full-length genes from genomic DNA of wild-type and mutant for subsequent sequence comparison. All PCRs were performed using Phusion high fidelity DNA polymerase (NEB, USA) according to the manufacturer's instructions. The full length Sr27 genomic sequence is shown in SEQ ID NO. 1. All mutants used in the RenSeq pipeline were reconfirmed by sanger sequencing. The predicted exon-intron structure was confirmed by full cDNA amplification of RNA from triticale cultivar Coorong. Total RNA was extracted using the PureLinkTM RNA Mini kit (Invitrogen catalog number 12183025,Thermo Fisher Scientific,MA USA) according to the manufacturer's instructions. cDNA synthesis was performed using protocols described by the manufacturer (SMART ^TM PCR cDNA synthesis kit, catalog No. 634902,Takara Bio USA Inc (formerly Clontech Laboratories inc.).

Pgt virulence mutant selection

Seedlings of triticale cultivar Coorong (20 pots [15cm ],6-8 plants/pot) at the 2-3 leaf stage were inoculated with 100g of stem rust isolate Pgt-0 using Talc as a carrier (1:4; rust: talc) (Uppaphya et al, (2015) Front Plant Sci.5:759). The inoculated plants were incubated in a humid chamber maintained at 23℃for 48 hours. After this time, the plants were moved to a greenhouse maintained at 23 ℃/18 ℃ (day/night) under natural sunlight. Plants were screened twice at any susceptible infection site that showed the development of large rust (pustule) typical for susceptible interactions (infection types 3, 4) 14 and 20 days post inoculation. More than 10 mutant rust spots were detected and three of these were collected and re-inoculated onto susceptible wheat line Morocco for expansion alone. After amplification, virulence of each mutant against Sr27 was confirmed by reinfecting the wild-type cooronig plant. One mutant was further tested on a full Australian set of differences with respect to Pgt, including a variety of wheat and triticale lines with different known resistance genes (Park (2007) Aust.J.Agric.Res.58:558-566), and showed the same virulence profile as Pgt-0, except for a single additional virulence for Sr 27.

Identification of AvrSr27 Gene by Whole genome sequence analysis

DNA was extracted from summer spores of mutant Pgt strain using the CTAB method (Rogers et al, (1989) can. J. Bot. 67:1235-1243) with some of the described modifications (Uppthyaya et al, (2015) Front Plant Sci.5:759), quality was assessed using a Nanodrop spectrophotometer (Thermo Scientific, wilmington, DE) and quantified using a wide range assay in a Qubit 3.0 fluorometer (Invitrogen, carlsbad, calif., USA). DNA library preparation and Illumina sequencing were performed by Australian genomic institute (Australian Genome Research Facility; AGRF) on HiSeq2500 (250 bp PE read, mutants M1 and M2) or MiSeq (300 bp PE read, mutant M3) platforms, and approximately 2 tens of millions of reads were obtained for each mutant. Sequence reads were input to CLC Genomics Workbench (CLCGW) version 10.0.1 or newer version (QIAGEN), filtered, and trimmed to remove low quality ends, where the adaptors and low quality reads were sequenced (Trim uses a quality score of 0.01, maximum number of ambiguities allowed of 2). Reads were mapped to the nuclear phase chromosome horizontal assembled Pgt-0 reference annotated with 37036 gene models (Li et al, (2019) Nature count, 10, 5068) using high stringency settings (similarity score 0.98 and length score 0.95). Variant calls for each sample of the reference were made using the "Basic Variant Detection" procedure, with the following parameters: neglecting non-specific matches; a minimum cover 10; significance 1.0%; minimum variant count 2; including disconnected pairs. The program "Compare variants within group" in CLCGW was used to identify variants specific for each sample as well as shared variants. Non-synonymous variants in secreted protein genes were predicted using the CLCGW tool "Amino Acid Changes" and manually managed by visual inspection of the read mapping trajectories in CLCGW. Read coverage statistics (average read depth and percent coverage) for annotated genes were extracted using the "Create Statistics for Target Regions" program.

Example 2: identification of AvrSr27 effectors recognized by Sr27

To identify spontaneous mutants of Pgt with virulence gain for Sr27, the inventors inoculated an avirulent isolate Pgt-0 onto seedlings of the triticale cultivar 'cooronig' carrying Sr 27. Three large individual rust spots were selected, purified separately, and re-inoculated onto Coorong to confirm their virulence phenotype (fig. 3). The inventors extracted genomic DNA from each mutant and obtained Illumina sequence data. Illumina reads were mapped to Pgt-0 haplotype resolved genomic assemblies (Li et al, (2019) Nature Commun.10, 5068) to identify potential mutations. The first screen for identifying SNPs that cause 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 sought for the loss of read coverage as an indicator of potential deletion mutations. Each of the Pgt-0 mutant lines showed a large number of genes with zero read coverage, all located at one end of chromosome 2B. Visualization of the read map to chromosome 2B revealed that the three mutants each contained independent and overlapping deletions covering a portion of chromosome 2B, with the smallest having a size of 196 kilobase pairs (kbp) (fig. 4). This region contains 50 annotated genes, five of which are predicted to encode secreted proteins. Previously, two Australian Pgt isolates (34-2, 12 and 34-2,12,13) were sequenced, which belong to the clone lineage derived from Pgt-0 and have evolved in the field to virulence for Sr27 (Uppadhyaya 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 13kbp mini-deletion (fig. 5A) spanning two of the secreted protein genes in this region (pgt21_006532 and pgt21_ 006593) together with a single adjacent gene on the distal side. In both isolates, no other genes contained any change in this region. In Pgt, 21-0 and 34-2,12, the inventors confirmed the presence of the deletion in the genome by PCR amplification of the deletion border (FIG. 5B). Sequence data generated from seven south Africa isolates, which were part of the same clonal lineage as Pgt-0 (Li et al, (2019) Nature Commun.10,5068; visser et al, (2019) Phytopath.109:133-144) but evolved independently in south Africa, were also examined (Lewis et al, 2018). Four of these isolates were virulent to Sr27 (Visser et al, (2009) mol. Plant Path.10:213-222) and had to have evolved this phenotype independently of Australian isolates. Three of these virulence isolates (SA 03, SA05 and SA 07) contained the same-14 kbp deletion overlapping the deletions in 34-2,12 and spanning the two candidate secreted protein genes plus another gene on the proximal side, while the fourth isolate (SA 06) contained a single deletion spanning only 10kbp of the two secreted protein genes (FIG. 5A). The remaining three avirulent isolates from south africa (SA 01, SA02, SA 04) contained sequences similar to Pgt-0 in this region, as were three other australian isolates of this lineage (PGT 098, PGT194, PGT 326) that were also avirulent for Sr 27. Phylogenetic analysis of this group of clonally derived isolates placed each of these deletion events in separate branches of the lineage, consistent with the occurrence of three independent mutations regarding virulence to Sr27 during diversification of the lineage (data not shown). The deletion of these two secreted protein gene candidates in three independent field-derived virulence 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 the wheat protoplast transfection assay described below to co-express the gene with the AvrSr27 variant identified above and the reporter luciferase. Recognition between resistance and avirulence genes in this assay results in cell death and thus reduced 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 resulted in a strong decrease in luciferase activity as compared to expression of Sr27 or AvrSr27 gene alone in protoplasts (FIG. 7). This is a specific recognition response because no loss of reporter expression was seen when Sr27 was co-expressed with the unrelated Pgt avirulence gene AvrSr50 (Chen et al, (2017) Science 358:1607-1610) or 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 for the AvrSr27 effector from Pgt and is therefore responsible for resistance to the Pgt isolate expressing the AvrSr27 gene.

Thus, the inventors demonstrated that the Sr27 coding region is sufficient to confer recognition of the corresponding effector AvrSr27 from wheat stem rust fungus (Pgt) when expressed in wheat protoplasts under the control of an operably linked ubiquitin promoter. The effector was identified by mutation analysis that localized it within a 196kbp deletion on chromosome 2B of Pgt 21-0. Subsequent analysis found two genes deleted independently in three different Pgt genes that obtained virulence for Sr 27. The availability of the cloned effector recognized by Sr27 facilitates genetic screening of wheat stem rust isolates for the presence of the effector and thus its predicted virulence for Sr 27.

Materials and methods

Construct generation

cDNA amplification from purified Pgt-0 blots using Phusion high fidelity DNA polymerase (Thermo Scientific, wilmington, DE) where predicted signal peptides are expelledThe AvrSr27 gene sequence (shown in SEQ ID NOS: 5, 7 and 9) was deleted and replaced with a single methionine initiation codon, and was modified according to the manufacturer (Invitrogen ^TM Instructions for Thermo Fisher Scientific, MA, USA) were cloned intoIs a kind of medium. Cloning of full Length Sr27cDNA into +. >Is a kind of medium. For wheat protoplast transfection, use is made ofTechnology(Life Techonologies ^TM ) To insert Sr27 and AvrSr27 sequences into p35s-pUbi-GTW-GFP (Akamatsu et al, (2013) Cell Host Microbe 13:465-476). Primers used for PCR are shown in Table 2. All plasmids were confirmed by sequencing and analyzed by 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) have been previously described.

Protoplast expression assay

Wheat seedlings were grown in the growth chamber at 25℃in the shade for 7-9 days with a photoperiod of 16 hours of light. 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 the Qiagen Endo-free Plasmid Maxi kit (catalog No. 12362). The DNA concentration was adjusted to 1. Mu.g/. Mu.l to 4. Mu.g/. Mu.l, and 10. Mu.g of each plasmid was used in the cotransformation experiments. After incubation in the dark at 23 ℃ for 24 hours, luciferase activity assays were performed as described by Saur et al ((2019) Plant Methods 15,118) using Luciferase Assay System (Promega, catalog No. E1501) following manufacturer's instructions.

Example 4: comparison of Sr27 with other resistance genes

According to the BLAST best hit against IWGSC CS ref v1.0, the closest homolog of the Sr27 candidate in the Chinese Spring reference v1.0 is located on chromosome 6B (see example 1). The inventors further expanded this list of homologous sequences by performing BLAST against the non-abundant protein database at NCBI (data not shown). The highest hit was an unnamed protein product from the cylindrical wheat durum subspecies (GenBank accession No. VAI 63620.1) 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 Sr27 protein with 16 CNL-type R genes identified from wheat and performed 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, which has 86.7% amino acid identity.

Method

Phylogenetic analysis

Phylogenetic and molecular evolution analysis was performed 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 was constructed.

Example 5: confirmation of Sr27 function in transgenic plants

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

Agrobacterium-mediated transformation of Wheat was performed as described (Ishida et al, (2015) "wheel (Triticum aestivum L.) transformation using immature embryos", wang K (editors), agrobacterium Protocols, vol.1, 3 rd edition, springer, new York, pages 189-198). Wheat plants of cultivar Fielder were propagated under greenhouse growth conditions using 24 ℃,16 hour light/18 ℃,8 hour dark growth regime and Aquasol (Yates, clayton, australia) was used once a weekAnd fertilizing the plants. Wheat ears were labeled during anthesis and harvested 12-14 days after anthesis for transformation experiments as described by Ishida et al (supra) and as adapted in CSIRO, canberra, australia (Richardson et al, (2014) Plant Cell tis. Org. 119:647-659). Briefly, seeds were surface sterilized in 0.8% sodium hypochlorite solution for 10 minutes. Embryos are removed from the seeds under sterile conditions and co-cultured with an agrobacterium strain comprising a binary construct of Sr22 and Sr45 genomic fragments in the dark on WLS-AS medium (Ishida et al 2015) for 2 days. After co-cultivation, the hypocotyls were excised with a scalpel, and the explants were transferred to WLS-Res medium and placed in the dark at 24 ℃. After 5 days, the explants were transferred to WLS-H15 callus induction medium containing 15mg/ml hygromycin (Hyg) for callus formation. After two weeks, the resulting calli were cut into two parts and placed on WLS-H30 (30 mg/l Hyg) for 3 weeks in the dark. Then, the callus was grown on LSZ-H15 (15 mg/l Hyg) medium at 200. Mu. Mol m ^-2 s ^-1 Regeneration was performed in light at 24 ℃. Seedlings were transferred to LSF-H15 (15 mg/l Hyg) medium to allow root formation and once a firm root system developed, plants were transferred to soil and maintained in the greenhouse.

T0 lines were screened by PCR with primers for the transgene (Sr 27c5723 FxSr 27c 5723R, table 2) and three positive lines (PC 311.3, PC311.17 and PC 311.18) were identified. T1 seeds were harvested from the three positive lines and a fourth line lacking the Sr27 transgene (PC 311.16). Eleven or twelve progeny of each line were grown in a growth chamber (23 ℃,16 hour light) along with the control line (see table 1). Two week old seedlings were inoculated with Pgt cultivar 98-1,2, (3), (5), 6 (Sr 27 confers resistance thereto). Rust response was assessed after 10-15 days (table 1). For PC311.3 and PC311.17, all T1 progenies showed strong resistance to Pgt, with a type of infection characterized by a tiny necrotic infection spot but no rust formation, similar to the phenotype observed in Sr27 bearing wheat line WRT258.5 (fig. 9). The T1 progeny of the third Sr27 transgenic line PC311.18 segregates for resistance, with 10 resistant progeny and 1 susceptible progeny (table 1). PCR analysis confirmed that the single PC311.18T1 progeny susceptible plant did not contain the Sr27 transgene, whereas resistant T1 progeny contained Sr27. In contrast, the susceptible lines Chinese Spring and Fielder showed high infection types with large summer sporulation and no chlorosis or necrosis (see fig. 9), as done with 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 (trichlamys tritici) isolate 76-1,3,7,9,10,12,13 and stripe rust (trichlamys strigos) isolate 198e16a+j+t+17+. All progeny were completely susceptible to the leaf rust and stripe rust isolates, confirming that the Sr27 transgene confers specific resistance to stem rust and that this resistance is not due to non-specific activation of an effective defensive response against rust pathogens.

TABLE 1 results of stripe rust resistance assay for Sr27 transgenic wheat lines

Example 6: sequence of primer used in cloning Sr27

In table 2 below, the nucleotide sequences of the primers used in cloning Sr27 (described in the above examples) from triticale cultivar cooronig are provided. The nucleotide sequences of these primers are provided as illustrative examples (for purposes of clear understanding) and not as limitations.

TABLE 2 primer sequences

/>

The articles "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one or more elements.

Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

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

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Sequence listing

<110> COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION

<120> stem rust resistance Gene

<130> 070294.0193

<150> US 63/076,153

<151> 2020-09-09

<150> US 63/127,220

<151> 2020-12-18

<160> 39

<170> PatentIn version 3.5

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cctgttcgat cactggtcgt gcattcgagc ttttaggcca tggaggcggc tctggtgacc 60

gtggcgacgg gagtcctcaa acctgtcctg gggaagctgg ccaccctgct cggcgacgag 120

tacaagcgtt ttaagggtgt gcgcaaggag atcaggtctc tcactcatga actcgccgcc 180

atggaggctt ttctcctcaa gatgtcggag gaggaggagg atcccgatgt gcaggataaa 240

gtctggatga atgaggtgcg ggaattgtcc tatgacatgg aggacgccat cgacgacttc 300

atgcaaagca ttggtgacaa agacgaaaag ccggatggct tcactgagaa gatcaaggcc 360

actctaggca agttgggaaa tatgaaggct cgtcatcgaa ttggcaagga gatacatgat 420

ctgaagaaac agatcattga ggtgggcgac aggaatgcaa ggtacaaggg acgcgagatc 480

ttctccaagg ccgtcaatgc gaccgttgac cctagagctc ttgctatctt tgagcatgca 540

tcaaagctcg tcggaattga tgaacccaag gctgagttga tcaagttgtt aactgacgag 600

gatggagttg catcaacaca agaacaagtg aagatggtct gcattgttgg atcgggagga 660

atgggcaaaa caactcttgc aaaccaagtg tatcaagaga tgaaagagga attcaagttt 720

aaggctttca tatcagtgtc acgaaatcca gatatgatga atatcttgag aaccctcctc 780

agtgaaattg ggtgtcaaga ttatgctcac actgaagcag ggagcataca acaactaata 840

agcaagatta ccgattacct agcagaaaaa aggtactatt atatttcttt aaactcactt 900

ctcgcccata gaaagttaaa ttaagaattc tcacatagaa aaaacactcc taataaagaa 960

tcaaaataat tatataatta aattatatac tttttgggtg aaaattaatt gccaaatgta 1020

tggaagccct tatttgcatg tactttacta cttcctccgt tcctaaatat aagtctttgg 1080

agagatttca ctatggacca catacgaagc aaaatgagtg aatctacact ctaaaatgca 1140

tctatataca tccgtatgtg gttcatggtg aaatctctag aaagacttat atttaggaac 1200

ggagggagta gttaactagg ttgttgtatt tggagggaaa ataagtctta tataggtagg 1260

aacatttgat tagtaggtat tcggcatgta tgtgcatctc agaatgcata tagactaaaa 1320

gacaatcttt tccgcaataa agaaatatca tcaatcttca atcaagcaag tatgctactc 1380

cctccgtccc aaaattcttg tcttagattt gtctaaatac agatgtatca agtcacattt 1440

tagtattaga aacatccgta tctgggcaaa tctaagacaa gaattttggg acggagggag 1500

tacatgatat gtaccactct aagtgcttag agctcttttg ctcttatatg gcctatctag 1560

gaaaacatat tttgtttagt aagtgcttag agtagaaaca ctatataggt attttctagc 1620

catgtggccc tgtttaagtt gcatagtacc ctagagccga tccattatct tttgcatgtt 1680

gccaatgaga acatggaaat ttctctttct tcttattttg cttgtacgct tcgttttaac 1740

acatcatact aactattact actaaaaaat catgtgcagg tattttatag tgattgacga 1800

catatgggac gtcaaaacat gggacgttat taagtgcgca ttccccatga ccagatgcgg 1860

tggtgtaata atcaccacca ctcggctgag tgatgttgca tgttcgtgtc attcatcaat 1920

cggtggccat atttataata taaggcctct taatatggag cactcaagac aactattcta 1980

cagaagatta ttcagctccg aagaagattg cccttcatcg ctcgtgaaag tttcttatca 2040

aatcttggaa aaatgtgatg ggttgccttt ggcaatcatt gctatagctg gtttgttggc 2100

taacacagga agatcagagc atcaatggaa ccaagtgaaa gattcaattg gtcgtgcact 2160

tgaaaggaat cctagtgtcg aagtaatgat aaagatattg tcacttagtt actttgatct 2220

tcctccgcat ctaaaaacat gtctcttgta tctcagtata ttcccggaag attctattat 2280

tgagaagaaa acactaatat caagatggat tgctgaagga ttcattcgac aagaaggtag 2340

atatactgca tatgaggtag gagtgaggtg ttttaatgag ctcgtcaaca ggagtttgat 2400

ccaacctgtg aagaaagacg attataaggg gaagagttgt cgagttcacg atataattct 2460

tgatttcata gtatccaagt ccattgaaga gaactttgtt acttttgttg gtgtccccag 2520

tttaactacc gtgacacaag gcaaagtccg ccgtctctcc atgcaagttg aagagaaggt 2580

ggattctatt ttgccaatga gcctgatatt atctcatgtc cgatcactta acatgttcgg 2640

gaatacagtg agtattcctt cgatcatgga gttgaggcat ttgcgtgtcc ttgatttcgg 2700

aggaaacaga ctattggaaa accgtcatct cgcgtatgta gggatgctgt ttcagctaag 2760

gtacctcaac atttacatga cagcagtaag cgagctcccg gaacaaatcg gacacttaca 2820

gtgcttagag atgttggaca tcaggcatac atgggtgtct gagctgccag ccagtattgc 2880

caatctcggc aaactggcac acttacttct tagctcaaat actggcacaa atgttaagtt 2940

tcccgacgga attgctaaga tgcaatcact ggaggctttg catagcgtta acacctgcaa 3000

tcagtcatat aactttctgc aagggcttgg tcagctaaag aatctgagga agctgggcat 3060

taactatcgg ggtgttgccc acgaagacaa ggaagttatt gcttcttctc ttggtaaact 3120

atgcacacaa aacctttgtt ctctaactat gtggaatgat gacgacgact tcttgctaaa 3180

tacatggtgc acttctccgc cgcttaacct ccgaaaactt gtcatatggg gttgtatatt 3240

cccaaaggtt ccgcattggg taggatcact cgtcaaccta cagaagttac acttggaagt 3300

ggggagagga acccggcatg aagatatctg catccttgga gccttacccg ctctgttcac 3360

tctgggtcta cgaggaagcg aaaaacagcc ttcttgtgaa aatagaaggc tggcagttag 3420

tggtgaagct gggttccgat gcctgaggaa gtttaaatac tggaggtggg gggattggat 3480

ggatcttatg tttacggcaa aatgtatgcc caggctagaa aaactgaaga ttatatttta 3540

cggccatgcc gaagatgagg ctcccatcat tcctgctttc gatttcggga tcgaaaacct 3600

gtccagcctc actactttca aatgtcacct aggttatggg cctatggcaa cgaaaattgt 3660

tgacgctgta aaggcttctc tggacagagt agttagcgca catcccaacc accttactct 3720

aatcttcact tattgttgtg tgttttgtaa gagttatgac tgtggtggtc gatgccttct 3780

gtctagagat cttcagtcat cctccgaatc tacttgagta gagtcaagac catgcgtacg 3840

tgcttaattc ttctcaatat taattattta tacaactagt acgagcgcac tatcaacctc 3900

tctaaattcc cttgcccctg tattttcaga tttgtcggac cacggtatat accatc 3956

<210> 2

<211> 2868

<212> DNA

<213> x Triticosecale

<220>

<221> CDS

<222> (1)..(2868)

<400> 2

atg gag gcg gct ctg gtg acc gtg gcg acg gga gtc ctc aaa cct gtc 48

Met Glu Ala Ala Leu Val Thr Val Ala Thr Gly Val Leu Lys Pro Val

1 5 10 15

ctg ggg aag ctg gcc acc ctg ctc ggc gac gag tac aag cgt ttt aag 96

Leu Gly Lys Leu Ala Thr Leu Leu Gly Asp Glu Tyr Lys Arg Phe Lys

20 25 30

ggt gtg cgc aag gag atc agg tct ctc act cat gaa ctc gcc gcc atg 144

Gly Val Arg Lys Glu Ile Arg Ser Leu Thr His Glu Leu Ala Ala Met

35 40 45

gag gct ttt ctc ctc aag atg tcg gag gag gag gag gat ccc gat gtg 192

Glu Ala Phe Leu Leu Lys Met Ser Glu Glu Glu Glu Asp Pro Asp Val

50 55 60

cag gat aaa gtc tgg atg aat gag gtg cgg gaa ttg tcc tat gac atg 240

Gln Asp Lys Val Trp Met Asn Glu Val Arg Glu Leu Ser Tyr Asp Met

65 70 75 80

gag gac gcc atc gac gac ttc atg caa agc att ggt gac aaa gac gaa 288

Glu Asp Ala Ile Asp Asp Phe Met Gln Ser Ile Gly Asp Lys Asp Glu

85 90 95

aag ccg gat ggc ttc act gag aag atc aag gcc act cta ggc aag ttg 336

Lys Pro Asp Gly Phe Thr Glu Lys Ile Lys Ala Thr Leu Gly Lys Leu

100 105 110

gga aat atg aag gct cgt cat cga att ggc aag gag ata cat gat ctg 384

Gly Asn Met Lys Ala Arg His Arg Ile Gly Lys Glu Ile His Asp Leu

115 120 125

aag aaa cag atc att gag gtg ggc gac agg aat gca agg tac aag gga 432

Lys Lys Gln Ile Ile Glu Val Gly Asp Arg Asn Ala Arg Tyr Lys Gly

130 135 140

cgc gag atc ttc tcc aag gcc gtc aat gcg acc gtt gac cct aga gct 480

Arg Glu Ile Phe Ser Lys Ala Val Asn Ala Thr Val Asp Pro Arg Ala

145 150 155 160

ctt gct atc ttt gag cat gca tca aag ctc gtc gga att gat gaa ccc 528

Leu Ala Ile Phe Glu His Ala Ser Lys Leu Val Gly Ile Asp Glu Pro

165 170 175

aag gct gag ttg atc aag ttg tta act gac gag gat gga gtt gca tca 576

Lys Ala Glu Leu Ile Lys Leu Leu Thr Asp Glu Asp Gly Val Ala Ser

180 185 190

aca caa gaa caa gtg aag atg gtc tgc att gtt gga tcg gga gga atg 624

Thr Gln Glu Gln Val Lys Met Val Cys Ile Val Gly Ser Gly Gly Met

195 200 205

ggc aaa aca act ctt gca aac caa gtg tat caa gag atg aaa gag gaa 672

Gly Lys Thr Thr Leu Ala Asn Gln Val Tyr Gln Glu Met Lys Glu Glu

210 215 220

ttc aag ttt aag gct ttc ata tca gtg tca cga aat cca gat atg atg 720

Phe Lys Phe Lys Ala Phe Ile Ser Val Ser Arg Asn Pro Asp Met Met

225 230 235 240

aat atc ttg aga acc ctc ctc agt gaa att ggg tgt caa gat tat gct 768

Asn Ile Leu Arg Thr Leu Leu Ser Glu Ile Gly Cys Gln Asp Tyr Ala

245 250 255

cac act gaa gca ggg agc ata caa caa cta ata agc aag att acc gat 816

His Thr Glu Ala Gly Ser Ile Gln Gln Leu Ile Ser Lys Ile Thr Asp

260 265 270

tac cta gca gaa aaa agg tat ttt ata gtg att gac gac ata tgg gac 864

Tyr Leu Ala Glu Lys Arg Tyr Phe Ile Val Ile Asp Asp Ile Trp Asp

275 280 285

gtc aaa aca tgg gac gtt att aag tgc gca ttc ccc atg acc aga tgc 912

Val Lys Thr Trp Asp Val Ile Lys Cys Ala Phe Pro Met Thr Arg Cys

290 295 300

ggt ggt gta ata atc acc acc act cgg ctg agt gat gtt gca tgt tcg 960

Gly Gly Val Ile Ile Thr Thr Thr Arg Leu Ser Asp Val Ala Cys Ser

305 310 315 320

tgt cat tca tca atc ggt ggc cat att tat aat ata agg cct ctt aat 1008

Cys His Ser Ser Ile Gly Gly His Ile Tyr Asn Ile Arg Pro Leu Asn

325 330 335

atg gag cac tca aga caa cta ttc tac aga aga tta ttc agc tcc gaa 1056

Met Glu His Ser Arg Gln Leu Phe Tyr Arg Arg Leu Phe Ser Ser Glu

340 345 350

gaa gat tgc cct tca tcg ctc gtg aaa gtt tct tat caa atc ttg gaa 1104

Glu Asp Cys Pro Ser Ser Leu Val Lys Val Ser Tyr Gln Ile Leu Glu

355 360 365

aaa tgt gat ggg ttg cct ttg gca atc att gct ata gct ggt ttg ttg 1152

Lys Cys Asp Gly Leu Pro Leu Ala Ile Ile Ala Ile Ala Gly Leu Leu

370 375 380

gct aac aca gga aga tca gag cat caa tgg aac caa gtg aaa gat tca 1200

Ala Asn Thr Gly Arg Ser Glu His Gln Trp Asn Gln Val Lys Asp Ser

385 390 395 400

att ggt cgt gca ctt gaa agg aat cct agt gtc gaa gta atg ata aag 1248

Ile Gly Arg Ala Leu Glu Arg Asn Pro Ser Val Glu Val Met Ile Lys

405 410 415

ata ttg tca ctt agt tac ttt gat ctt cct ccg cat cta aaa aca tgt 1296

Ile Leu Ser Leu Ser Tyr Phe Asp Leu Pro Pro His Leu Lys Thr Cys

420 425 430

ctc ttg tat ctc agt ata ttc ccg gaa gat tct att att gag aag aaa 1344

Leu Leu Tyr Leu Ser Ile Phe Pro Glu Asp Ser Ile Ile Glu Lys Lys

435 440 445

aca cta ata tca aga tgg att gct gaa gga ttc att cga caa gaa ggt 1392

Thr Leu Ile Ser Arg Trp Ile Ala Glu Gly Phe Ile Arg Gln Glu Gly

450 455 460

aga tat act gca tat gag gta gga gtg agg tgt ttt aat gag ctc gtc 1440

Arg Tyr Thr Ala Tyr Glu Val Gly Val Arg Cys Phe Asn Glu Leu Val

465 470 475 480

aac agg agt ttg atc caa cct gtg aag aaa gac gat tat aag ggg aag 1488

Asn Arg Ser Leu Ile Gln Pro Val Lys Lys Asp Asp Tyr Lys Gly Lys

485 490 495

agt tgt cga gtt cac gat ata att ctt gat ttc ata gta tcc aag tcc 1536

Ser Cys Arg Val His Asp Ile Ile Leu Asp Phe Ile Val Ser Lys Ser

500 505 510

att gaa gag aac ttt gtt act ttt gtt ggt gtc ccc agt tta act acc 1584

Ile Glu Glu Asn Phe Val Thr Phe Val Gly Val Pro Ser Leu Thr Thr

515 520 525

gtg aca caa ggc aaa gtc cgc cgt ctc tcc atg caa gtt gaa gag aag 1632

Val Thr Gln Gly Lys Val Arg Arg Leu Ser Met Gln Val Glu Glu Lys

530 535 540

gtg gat tct att ttg cca atg agc ctg ata tta tct cat gtc cga tca 1680

Val Asp Ser Ile Leu Pro Met Ser Leu Ile Leu Ser His Val Arg Ser

545 550 555 560

ctt aac atg ttc ggg aat aca gtg agt att cct tcg atc atg gag ttg 1728

Leu Asn Met Phe Gly Asn Thr Val Ser Ile Pro Ser Ile Met Glu Leu

565 570 575

agg cat ttg cgt gtc ctt gat ttc gga gga aac aga cta ttg gaa aac 1776

Arg His Leu Arg Val Leu Asp Phe Gly Gly Asn Arg Leu Leu Glu Asn

580 585 590

cgt cat ctc gcg tat gta ggg atg ctg ttt cag cta agg tac ctc aac 1824

Arg His Leu Ala Tyr Val Gly Met Leu Phe Gln Leu Arg Tyr Leu Asn

595 600 605

att tac atg aca gca gta agc gag ctc ccg gaa caa atc gga cac tta 1872

Ile Tyr Met Thr Ala Val Ser Glu Leu Pro Glu Gln Ile Gly His Leu

610 615 620

cag tgc tta gag atg ttg gac atc agg cat aca tgg gtg tct gag ctg 1920

Gln Cys Leu Glu Met Leu Asp Ile Arg His Thr Trp Val Ser Glu Leu

625 630 635 640

cca gcc agt att gcc aat ctc ggc aaa ctg gca cac tta ctt ctt agc 1968

Pro Ala Ser Ile Ala Asn Leu Gly Lys Leu Ala His Leu Leu Leu Ser

645 650 655

tca aat act ggc aca aat gtt aag ttt ccc gac gga att gct aag atg 2016

Ser Asn Thr Gly Thr Asn Val Lys Phe Pro Asp Gly Ile Ala Lys Met

660 665 670

caa tca ctg gag gct ttg cat agc gtt aac acc tgc aat cag tca tat 2064

Gln Ser Leu Glu Ala Leu His Ser Val Asn Thr Cys Asn Gln Ser Tyr

675 680 685

aac ttt ctg caa ggg ctt ggt cag cta aag aat ctg agg aag ctg ggc 2112

Asn Phe Leu Gln Gly Leu Gly Gln Leu Lys Asn Leu Arg Lys Leu Gly

690 695 700

att aac tat cgg ggt gtt gcc cac gaa gac aag gaa gtt att gct tct 2160

Ile Asn Tyr Arg Gly Val Ala His Glu Asp Lys Glu Val Ile Ala Ser

705 710 715 720

tct ctt ggt aaa cta tgc aca caa aac ctt tgt tct cta act atg tgg 2208

Ser Leu Gly Lys Leu Cys Thr Gln Asn Leu Cys Ser Leu Thr Met Trp

725 730 735

aat gat gac gac gac ttc ttg cta aat aca tgg tgc act tct ccg ccg 2256

Asn Asp Asp Asp Asp Phe Leu Leu Asn Thr Trp Cys Thr Ser Pro Pro

740 745 750

ctt aac ctc cga aaa ctt gtc ata tgg ggt tgt ata ttc cca aag gtt 2304

Leu Asn Leu Arg Lys Leu Val Ile Trp Gly Cys Ile Phe Pro Lys Val

755 760 765

ccg cat tgg gta gga tca ctc gtc aac cta cag aag tta cac ttg gaa 2352

Pro His Trp Val Gly Ser Leu Val Asn Leu Gln Lys Leu His Leu Glu

770 775 780

gtg ggg aga gga acc cgg cat gaa gat atc tgc atc ctt gga gcc tta 2400

Val Gly Arg Gly Thr Arg His Glu Asp Ile Cys Ile Leu Gly Ala Leu

785 790 795 800

ccc gct ctg ttc act ctg ggt cta cga gga agc gaa aaa cag cct tct 2448

Pro Ala Leu Phe Thr Leu Gly Leu Arg Gly Ser Glu Lys Gln Pro Ser

805 810 815

tgt gaa aat aga agg ctg gca gtt agt ggt gaa gct ggg ttc cga tgc 2496

Cys Glu Asn Arg Arg Leu Ala Val Ser Gly Glu Ala Gly Phe Arg Cys

820 825 830

ctg agg aag ttt aaa tac tgg agg tgg ggg gat tgg atg gat ctt atg 2544

Leu Arg Lys Phe Lys Tyr Trp Arg Trp Gly Asp Trp Met Asp Leu Met

835 840 845

ttt acg gca aaa tgt atg ccc agg cta gaa aaa ctg aag att ata ttt 2592

Phe Thr Ala Lys Cys Met Pro Arg Leu Glu Lys Leu Lys Ile Ile Phe

850 855 860

tac ggc cat gcc gaa gat gag gct ccc atc att cct gct ttc gat ttc 2640

Tyr Gly His Ala Glu Asp Glu Ala Pro Ile Ile Pro Ala Phe Asp Phe

865 870 875 880

ggg atc gaa aac ctg tcc agc ctc act act ttc aaa tgt cac cta ggt 2688

Gly Ile Glu Asn Leu Ser Ser Leu Thr Thr Phe Lys Cys His Leu Gly

885 890 895

tat ggg cct atg gca acg aaa att gtt gac gct gta aag gct tct ctg 2736

Tyr Gly Pro Met Ala Thr Lys Ile Val Asp Ala Val Lys Ala Ser Leu

900 905 910

gac aga gta gtt agc gca cat ccc aac cac ctt act cta atc ttc act 2784

Asp Arg Val Val Ser Ala His Pro Asn His Leu Thr Leu Ile Phe Thr

915 920 925

tat tgt tgt gtg ttt tgt aag agt tat gac tgt ggt ggt cga tgc ctt 2832

Tyr Cys Cys Val Phe Cys Lys Ser Tyr Asp Cys Gly Gly Arg Cys Leu

930 935 940

ctg tct aga gat ctt cag tca tcc tcc gaa tct act 2868

Leu Ser Arg Asp Leu Gln Ser Ser Ser Glu Ser Thr

945 950 955

<210> 3

<211> 956

<212> PRT

<213> x Triticosecale

<400> 3

Met Glu Ala Ala Leu Val Thr Val Ala Thr Gly Val Leu Lys Pro Val

1 5 10 15

Leu Gly Lys Leu Ala Thr Leu Leu Gly Asp Glu Tyr Lys Arg Phe Lys

20 25 30

Gly Val Arg Lys Glu Ile Arg Ser Leu Thr His Glu Leu Ala Ala Met

35 40 45

Glu Ala Phe Leu Leu Lys Met Ser Glu Glu Glu Glu Asp Pro Asp Val

50 55 60

Gln Asp Lys Val Trp Met Asn Glu Val Arg Glu Leu Ser Tyr Asp Met

65 70 75 80

Glu Asp Ala Ile Asp Asp Phe Met Gln Ser Ile Gly Asp Lys Asp Glu

85 90 95

Lys Pro Asp Gly Phe Thr Glu Lys Ile Lys Ala Thr Leu Gly Lys Leu

100 105 110

Gly Asn Met Lys Ala Arg His Arg Ile Gly Lys Glu Ile His Asp Leu

115 120 125

Lys Lys Gln Ile Ile Glu Val Gly Asp Arg Asn Ala Arg Tyr Lys Gly

130 135 140

Arg Glu Ile Phe Ser Lys Ala Val Asn Ala Thr Val Asp Pro Arg Ala

145 150 155 160

Leu Ala Ile Phe Glu His Ala Ser Lys Leu Val Gly Ile Asp Glu Pro

165 170 175

Lys Ala Glu Leu Ile Lys Leu Leu Thr Asp Glu Asp Gly Val Ala Ser

180 185 190

Thr Gln Glu Gln Val Lys Met Val Cys Ile Val Gly Ser Gly Gly Met

195 200 205

Gly Lys Thr Thr Leu Ala Asn Gln Val Tyr Gln Glu Met Lys Glu Glu

210 215 220

Phe Lys Phe Lys Ala Phe Ile Ser Val Ser Arg Asn Pro Asp Met Met

225 230 235 240

Asn Ile Leu Arg Thr Leu Leu Ser Glu Ile Gly Cys Gln Asp Tyr Ala

245 250 255

His Thr Glu Ala Gly Ser Ile Gln Gln Leu Ile Ser Lys Ile Thr Asp

260 265 270

Tyr Leu Ala Glu Lys Arg Tyr Phe Ile Val Ile Asp Asp Ile Trp Asp

275 280 285

Val Lys Thr Trp Asp Val Ile Lys Cys Ala Phe Pro Met Thr Arg Cys

290 295 300

Gly Gly Val Ile Ile Thr Thr Thr Arg Leu Ser Asp Val Ala Cys Ser

305 310 315 320

Cys His Ser Ser Ile Gly Gly His Ile Tyr Asn Ile Arg Pro Leu Asn

325 330 335

Met Glu His Ser Arg Gln Leu Phe Tyr Arg Arg Leu Phe Ser Ser Glu

340 345 350

Glu Asp Cys Pro Ser Ser Leu Val Lys Val Ser Tyr Gln Ile Leu Glu

355 360 365

Lys Cys Asp Gly Leu Pro Leu Ala Ile Ile Ala Ile Ala Gly Leu Leu

370 375 380

Ala Asn Thr Gly Arg Ser Glu His Gln Trp Asn Gln Val Lys Asp Ser

385 390 395 400

Ile Gly Arg Ala Leu Glu Arg Asn Pro Ser Val Glu Val Met Ile Lys

405 410 415

Ile Leu Ser Leu Ser Tyr Phe Asp Leu Pro Pro His Leu Lys Thr Cys

420 425 430

Leu Leu Tyr Leu Ser Ile Phe Pro Glu Asp Ser Ile Ile Glu Lys Lys

435 440 445

Thr Leu Ile Ser Arg Trp Ile Ala Glu Gly Phe Ile Arg Gln Glu Gly

450 455 460

Arg Tyr Thr Ala Tyr Glu Val Gly Val Arg Cys Phe Asn Glu Leu Val

465 470 475 480

Asn Arg Ser Leu Ile Gln Pro Val Lys Lys Asp Asp Tyr Lys Gly Lys

485 490 495

Ser Cys Arg Val His Asp Ile Ile Leu Asp Phe Ile Val Ser Lys Ser

500 505 510

Ile Glu Glu Asn Phe Val Thr Phe Val Gly Val Pro Ser Leu Thr Thr

515 520 525

Val Thr Gln Gly Lys Val Arg Arg Leu Ser Met Gln Val Glu Glu Lys

530 535 540

Val Asp Ser Ile Leu Pro Met Ser Leu Ile Leu Ser His Val Arg Ser

545 550 555 560

Leu Asn Met Phe Gly Asn Thr Val Ser Ile Pro Ser Ile Met Glu Leu

565 570 575

Arg His Leu Arg Val Leu Asp Phe Gly Gly Asn Arg Leu Leu Glu Asn

580 585 590

Arg His Leu Ala Tyr Val Gly Met Leu Phe Gln Leu Arg Tyr Leu Asn

595 600 605

Ile Tyr Met Thr Ala Val Ser Glu Leu Pro Glu Gln Ile Gly His Leu

610 615 620

Gln Cys Leu Glu Met Leu Asp Ile Arg His Thr Trp Val Ser Glu Leu

625 630 635 640

Pro Ala Ser Ile Ala Asn Leu Gly Lys Leu Ala His Leu Leu Leu Ser

645 650 655

Ser Asn Thr Gly Thr Asn Val Lys Phe Pro Asp Gly Ile Ala Lys Met

660 665 670

Gln Ser Leu Glu Ala Leu His Ser Val Asn Thr Cys Asn Gln Ser Tyr

675 680 685

Asn Phe Leu Gln Gly Leu Gly Gln Leu Lys Asn Leu Arg Lys Leu Gly

690 695 700

Ile Asn Tyr Arg Gly Val Ala His Glu Asp Lys Glu Val Ile Ala Ser

705 710 715 720

Ser Leu Gly Lys Leu Cys Thr Gln Asn Leu Cys Ser Leu Thr Met Trp

725 730 735

Asn Asp Asp Asp Asp Phe Leu Leu Asn Thr Trp Cys Thr Ser Pro Pro

740 745 750

Leu Asn Leu Arg Lys Leu Val Ile Trp Gly Cys Ile Phe Pro Lys Val

755 760 765

Pro His Trp Val Gly Ser Leu Val Asn Leu Gln Lys Leu His Leu Glu

770 775 780

Val Gly Arg Gly Thr Arg His Glu Asp Ile Cys Ile Leu Gly Ala Leu

785 790 795 800

Pro Ala Leu Phe Thr Leu Gly Leu Arg Gly Ser Glu Lys Gln Pro Ser

805 810 815

Cys Glu Asn Arg Arg Leu Ala Val Ser Gly Glu Ala Gly Phe Arg Cys

820 825 830

Leu Arg Lys Phe Lys Tyr Trp Arg Trp Gly Asp Trp Met Asp Leu Met

835 840 845

Phe Thr Ala Lys Cys Met Pro Arg Leu Glu Lys Leu Lys Ile Ile Phe

850 855 860

Tyr Gly His Ala Glu Asp Glu Ala Pro Ile Ile Pro Ala Phe Asp Phe

865 870 875 880

Gly Ile Glu Asn Leu Ser Ser Leu Thr Thr Phe Lys Cys His Leu Gly

885 890 895

Tyr Gly Pro Met Ala Thr Lys Ile Val Asp Ala Val Lys Ala Ser Leu

900 905 910

Asp Arg Val Val Ser Ala His Pro Asn His Leu Thr Leu Ile Phe Thr

915 920 925

Tyr Cys Cys Val Phe Cys Lys Ser Tyr Asp Cys Gly Gly Arg Cys Leu

930 935 940

Leu Ser Arg Asp Leu Gln Ser Ser Ser Glu Ser Thr

945 950 955

<210> 4

<211> 3778

<212> DNA

<213> x Triticosecale

<400> 4

atggaggcgg ctctggtgac cgtggcgacg ggagtcctca aacctgtcct ggggaagctg 60

gccaccctgc tcggcgacga gtacaagcgt tttaagggtg tgcgcaagga gatcaggtct 120

ctcactcatg aactcgccgc catggaggct tttctcctca agatgtcgga ggaggaggag 180

gatcccgatg tgcaggataa agtctggatg aatgaggtgc gggaattgtc ctatgacatg 240

gaggacgcca tcgacgactt catgcaaagc attggtgaca aagacgaaaa gccggatggc 300

ttcactgaga agatcaaggc cactctaggc aagttgggaa atatgaaggc tcgtcatcga 360

attggcaagg agatacatga tctgaagaaa cagatcattg aggtgggcga caggaatgca 420

aggtacaagg gacgcgagat cttctccaag gccgtcaatg cgaccgttga ccctagagct 480

cttgctatct ttgagcatgc atcaaagctc gtcggaattg atgaacccaa ggctgagttg 540

atcaagttgt taactgacga ggatggagtt gcatcaacac aagaacaagt gaagatggtc 600

tgcattgttg gatcgggagg aatgggcaaa acaactcttg caaaccaagt gtatcaagag 660

atgaaagagg aattcaagtt taaggctttc atatcagtgt cacgaaatcc agatatgatg 720

aatatcttga gaaccctcct cagtgaaatt gggtgtcaag attatgctca cactgaagca 780

gggagcatac aacaactaat aagcaagatt accgattacc tagcagaaaa aaggtactat 840

tatatttctt taaactcact tctcgcccat agaaagttaa attaagaatt ctcacataga 900

aaaaacactc ctaataaaga atcaaaataa ttatataatt aaattatata ctttttgggt 960

gaaaattaat tgccaaatgt atggaagccc ttatttgcat gtactttact acttcctccg 1020

ttcctaaata taagtctttg gagagatttc actatggacc acatacgaag caaaatgagt 1080

gaatctacac tctaaaatgc atctatatac atccgtatgt ggttcatggt gaaatctcta 1140

gaaagactta tatttaggaa cggagggagt agttaactag gttgttgtat ttggagggaa 1200

aataagtctt atataggtag gaacatttga ttagtaggta ttcggcatgt atgtgcatct 1260

cagaatgcat atagactaaa agacaatctt ttccgcaata aagaaatatc atcaatcttc 1320

aatcaagcaa gtatgctact ccctccgtcc caaaattctt gtcttagatt tgtctaaata 1380

cagatgtatc aagtcacatt ttagtattag aaacatccgt atctgggcaa atctaagaca 1440

agaattttgg gacggaggga gtacatgata tgtaccactc taagtgctta gagctctttt 1500

gctcttatat ggcctatcta ggaaaacata ttttgtttag taagtgctta gagtagaaac 1560

actatatagg tattttctag ccatgtggcc ctgtttaagt tgcatagtac cctagagccg 1620

atccattatc ttttgcatgt tgccaatgag aacatggaaa tttctctttc ttcttatttt 1680

gcttgtacgc ttcgttttaa cacatcatac taactattac tactaaaaaa tcatgtgcag 1740

gtattttata gtgattgacg acatatggga cgtcaaaaca tgggacgtta ttaagtgcgc 1800

attccccatg accagatgcg gtggtgtaat aatcaccacc actcggctga gtgatgttgc 1860

atgttcgtgt cattcatcaa tcggtggcca tatttataat ataaggcctc ttaatatgga 1920

gcactcaaga caactattct acagaagatt attcagctcc gaagaagatt gcccttcatc 1980

gctcgtgaaa gtttcttatc aaatcttgga aaaatgtgat gggttgcctt tggcaatcat 2040

tgctatagct ggtttgttgg ctaacacagg aagatcagag catcaatgga accaagtgaa 2100

agattcaatt ggtcgtgcac ttgaaaggaa tcctagtgtc gaagtaatga taaagatatt 2160

gtcacttagt tactttgatc ttcctccgca tctaaaaaca tgtctcttgt atctcagtat 2220

attcccggaa gattctatta ttgagaagaa aacactaata tcaagatgga ttgctgaagg 2280

attcattcga caagaaggta gatatactgc atatgaggta ggagtgaggt gttttaatga 2340

gctcgtcaac aggagtttga tccaacctgt gaagaaagac gattataagg ggaagagttg 2400

tcgagttcac gatataattc ttgatttcat agtatccaag tccattgaag agaactttgt 2460

tacttttgtt ggtgtcccca gtttaactac cgtgacacaa ggcaaagtcc gccgtctctc 2520

catgcaagtt gaagagaagg tggattctat tttgccaatg agcctgatat tatctcatgt 2580

ccgatcactt aacatgttcg ggaatacagt gagtattcct tcgatcatgg agttgaggca 2640

tttgcgtgtc cttgatttcg gaggaaacag actattggaa aaccgtcatc tcgcgtatgt 2700

agggatgctg tttcagctaa ggtacctcaa catttacatg acagcagtaa gcgagctccc 2760

ggaacaaatc ggacacttac agtgcttaga gatgttggac atcaggcata catgggtgtc 2820

tgagctgcca gccagtattg ccaatctcgg caaactggca cacttacttc ttagctcaaa 2880

tactggcaca aatgttaagt ttcccgacgg aattgctaag atgcaatcac tggaggcttt 2940

gcatagcgtt aacacctgca atcagtcata taactttctg caagggcttg gtcagctaaa 3000

gaatctgagg aagctgggca ttaactatcg gggtgttgcc cacgaagaca aggaagttat 3060

tgcttcttct cttggtaaac tatgcacaca aaacctttgt tctctaacta tgtggaatga 3120

tgacgacgac ttcttgctaa atacatggtg cacttctccg ccgcttaacc tccgaaaact 3180

tgtcatatgg ggttgtatat tcccaaaggt tccgcattgg gtaggatcac tcgtcaacct 3240

acagaagtta cacttggaag tggggagagg aacccggcat gaagatatct gcatccttgg 3300

agccttaccc gctctgttca ctctgggtct acgaggaagc gaaaaacagc cttcttgtga 3360

aaatagaagg ctggcagtta gtggtgaagc tgggttccga tgcctgagga agtttaaata 3420

ctggaggtgg ggggattgga tggatcttat gtttacggca aaatgtatgc ccaggctaga 3480

aaaactgaag attatatttt acggccatgc cgaagatgag gctcccatca ttcctgcttt 3540

cgatttcggg atcgaaaacc tgtccagcct cactactttc aaatgtcacc taggttatgg 3600

gcctatggca acgaaaattg ttgacgctgt aaaggcttct ctggacagag tagttagcgc 3660

acatcccaac caccttactc taatcttcac ttattgttgt gtgttttgta agagttatga 3720

ctgtggtggt cgatgccttc tgtctagaga tcttcagtca tcctccgaat ctacttga 3778

<210> 5

<211> 435

<212> DNA

<213> wheat seed of rust graminearum (Puccinia graminis fsp. Tritici)

<220>

<221> CDS

<222> (1)..(435)

<400> 5

atg cat tac atc acc ccc ata atc ctt atg tca att gga caa ttt ctt 48

Met His Tyr Ile Thr Pro Ile Ile Leu Met Ser Ile Gly Gln Phe Leu

1 5 10 15

ggc ata tta ttg gga gca gga ggt ctt gtg ggt gca atg aca cca cat 96

Gly Ile Leu Leu Gly Ala Gly Gly Leu Val Gly Ala Met Thr Pro His

20 25 30

cac caa agc aat tgc aac tcc cca tct ttg aca ttt ccc agg ttc att 144

His Gln Ser Asn Cys Asn Ser Pro Ser Leu Thr Phe Pro Arg Phe Ile

35 40 45

gga aaa tgt gac tcc tgc cag ctc cat acc aaa gct acc aac ctg gtg 192

Gly Lys Cys Asp Ser Cys Gln Leu His Thr Lys Ala Thr Asn Leu Val

50 55 60

agc tgc acc tct tgt agg aaa tcc tca ttg gta tat gaa gaa tgt tcc 240

Ser Cys Thr Ser Cys Arg Lys Ser Ser Leu Val Tyr Glu Glu Cys Ser

65 70 75 80

acc aaa ggc tgt cct gct aat tgg cac aaa agc acc tgt caa gaa ccc 288

Thr Lys Gly Cys Pro Ala Asn Trp His Lys Ser Thr Cys Gln Glu Pro

85 90 95

aag ttc aat aga ggt att ctg tcc tgt tac tgt gag aac tgc cag cag 336

Lys Phe Asn Arg Gly Ile Leu Ser Cys Tyr Cys Glu Asn Cys Gln Gln

100 105 110

cac acc aag gaa aaa cag aca att tcc tgc aaa aat tgt aag aat tca 384

His Thr Lys Glu Lys Gln Thr Ile Ser Cys Lys Asn Cys Lys Asn Ser

115 120 125

gcc acc acc ttc tca cat tgt tca agc cca gag tgt cac agc aga tgg 432

Ala Thr Thr Phe Ser His Cys Ser Ser Pro Glu Cys His Ser Arg Trp

130 135 140

taa 435

<210> 6

<211> 144

<212> PRT

<213> wheat seed of Puccinia graminearum

<400> 6

Met His Tyr Ile Thr Pro Ile Ile Leu Met Ser Ile Gly Gln Phe Leu

1 5 10 15

Gly Ile Leu Leu Gly Ala Gly Gly Leu Val Gly Ala Met Thr Pro His

20 25 30

His Gln Ser Asn Cys Asn Ser Pro Ser Leu Thr Phe Pro Arg Phe Ile

35 40 45

Gly Lys Cys Asp Ser Cys Gln Leu His Thr Lys Ala Thr Asn Leu Val

50 55 60

Ser Cys Thr Ser Cys Arg Lys Ser Ser Leu Val Tyr Glu Glu Cys Ser

65 70 75 80

Thr Lys Gly Cys Pro Ala Asn Trp His Lys Ser Thr Cys Gln Glu Pro

85 90 95

Lys Phe Asn Arg Gly Ile Leu Ser Cys Tyr Cys Glu Asn Cys Gln Gln

100 105 110

His Thr Lys Glu Lys Gln Thr Ile Ser Cys Lys Asn Cys Lys Asn Ser

115 120 125

Ala Thr Thr Phe Ser His Cys Ser Ser Pro Glu Cys His Ser Arg Trp

130 135 140

<210> 7

<211> 435

<212> DNA

<213> wheat seed of Puccinia graminearum

<220>

<221> CDS

<222> (1)..(435)

<400> 7

atg cat tac atc acc ccc ata atc ctt atg tca att gga aaa ttt ctt 48

Met His Tyr Ile Thr Pro Ile Ile Leu Met Ser Ile Gly Lys Phe Leu

1 5 10 15

gga atg ata ttg gga gca gga agt ctt gtg ggt gca atg aca cca cat 96

Gly Met Ile Leu Gly Ala Gly Ser Leu Val Gly Ala Met Thr Pro His

20 25 30

cac caa agc aat tgc aac tcc cca tgt ttg gta ttt gtc aca ttc acc 144

His Gln Ser Asn Cys Asn Ser Pro Cys Leu Val Phe Val Thr Phe Thr

35 40 45

aaa aaa tgt gac tcc tgc cag ttc aat aca aaa ttc act aac ctg atg 192

Lys Lys Cys Asp Ser Cys Gln Phe Asn Thr Lys Phe Thr Asn Leu Met

50 55 60

agc tgc acc tct tgt agg aaa tcc tca gtg gta tat gaa gaa tgt tcc 240

Ser Cys Thr Ser Cys Arg Lys Ser Ser Val Val Tyr Glu Glu Cys Ser

65 70 75 80

acc aaa ggc tgt cct gct aat tgg cac aaa agt acc tgt caa gaa ccc 288

Thr Lys Gly Cys Pro Ala Asn Trp His Lys Ser Thr Cys Gln Glu Pro

85 90 95

aag ttt gag aga ggt gtt cta cac agc ctc tgt gca aac tgc cag aag 336

Lys Phe Glu Arg Gly Val Leu His Ser Leu Cys Ala Asn Cys Gln Lys

100 105 110

cac aca aag gca aca ccg aca att tcc tgc aaa aat tgt aag aat tca 384

His Thr Lys Ala Thr Pro Thr Ile Ser Cys Lys Asn Cys Lys Asn Ser

115 120 125

gcc agc acc tac cca tat tgt tca agc cca gag tgt cac aga aga tgg 432

Ala Ser Thr Tyr Pro Tyr Cys Ser Ser Pro Glu Cys His Arg Arg Trp

130 135 140

taa 435

<210> 8

<211> 144

<212> PRT

<213> wheat seed of Puccinia graminearum

<400> 8

Met His Tyr Ile Thr Pro Ile Ile Leu Met Ser Ile Gly Lys Phe Leu

1 5 10 15

Gly Met Ile Leu Gly Ala Gly Ser Leu Val Gly Ala Met Thr Pro His

20 25 30

His Gln Ser Asn Cys Asn Ser Pro Cys Leu Val Phe Val Thr Phe Thr

35 40 45

Lys Lys Cys Asp Ser Cys Gln Phe Asn Thr Lys Phe Thr Asn Leu Met

50 55 60

Ser Cys Thr Ser Cys Arg Lys Ser Ser Val Val Tyr Glu Glu Cys Ser

65 70 75 80

Thr Lys Gly Cys Pro Ala Asn Trp His Lys Ser Thr Cys Gln Glu Pro

85 90 95

Lys Phe Glu Arg Gly Val Leu His Ser Leu Cys Ala Asn Cys Gln Lys

100 105 110

His Thr Lys Ala Thr Pro Thr Ile Ser Cys Lys Asn Cys Lys Asn Ser

115 120 125

Ala Ser Thr Tyr Pro Tyr Cys Ser Ser Pro Glu Cys His Arg Arg Trp

130 135 140

<210> 9

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 9

cctgttcgat cactggtcg 19

<210> 10

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 10

gtgaagatgg tctgcattgt tggatcg 27

<210> 11

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 11

gatggtatat accgtggtcc gacaaat 27

<210> 12

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 12

cggaggttaa gcggcggaga 20

<210> 13

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 13

ggttttgtgt gcatagttta ccaagag 27

<210> 14

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 14

atgaacccaa ggctgagttg 20

<210> 15

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 15

acatgcaaat aagggcttcc 20

<210> 16

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 16

gtaaggctcc aaggatgcag 20

<210> 17

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 17

taagtttccc gacggaattg 20

<210> 18

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 18

aagaacaagt gaagatggtc tgc 23

<210> 19

<211> 28

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 19

ttctgtagaa tagttgtctt gagtgctc 28

<210> 20

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 20

tacgaggaag cgaaaaacag c 21

<210> 21

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 21

atcttctcag tgaagccatc 20

<210> 22

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 22

aaatgtgact tgatacatct g 21

<210> 23

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 23

atagtgattg acgacatatg g 21

<210> 24

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 24

gattcattcg acaagaaggt 20

<210> 25

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 25

attcagattt aagagtcttg attgagtccc catg 34

<210> 26

<211> 29

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 26

caccatgcaa ttagccagtg tcttatgtg 29

<210> 27

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 27

gtcttcctac ctgtgttggc gccttgcaaa atg 33

<210> 28

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 28

caccatgatg cattcaatta tctttcaaac actcc 35

<210> 29

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 29

ttaccatctt ctgtgacact ctggg 25

<210> 30

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 30

ttaccatctg ctgtgacact ctgg 24

<210> 31

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 31

caccatggca atgacaccac atcaccaaag caat 34

<210> 32

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 32

ccatcttctg tgacactctg ggcttg 26

<210> 33

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 33

ccatctgctg tgacactctg ggcttg 26

<210> 34

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 34

caccatgcat tacatcaccc ccataatcct t 31

<210> 35

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 35

caccatggca ggaagtcttg tgggtgcaat gac 33

<210> 36

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 36

caccatggca ggaggtcttg tgggtgcaat gac 33

<210> 37

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 37

aagtggataa cgtactctgc acaac 25

<210> 38

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 38

agtgactgca attcaccaat atttcg 26

<210> 39

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 39

aacattcagt gcgaggaatg gggag 25

Claims (54)

1. A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

(a) The nucleotide sequence shown in SEQ ID NO. 1, 2 or 4;

(b) A nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO. 3, and optionally wherein the nucleotide sequence is not naturally occurring;

(c) A nucleotide sequence having at least 87% sequence identity to at least one of the nucleotide sequences set forth in (a), wherein the nucleic acid molecule is capable of conferring resistance to stem rust to a wheat plant comprising the nucleic acid molecule, and optionally wherein the nucleotide sequence is not naturally occurring; and

(d) A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 91% sequence identity to at least one of the amino acid sequences set forth in (b), wherein the nucleic acid molecule is capable of conferring resistance to stem rust to a wheat plant comprising the nucleic acid molecule, and optionally wherein the nucleotide sequence does not occur naturally.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is an isolated nucleic acid molecule.

3. The nucleic acid molecule of claim 1 or 2, wherein the nucleic acid molecule of (c) or (d) encodes a protein comprising a coiled-coil domain, a nucleotide binding domain, and a leucine-rich repeat domain.

4. The nucleic acid molecule of claim 3, wherein at least one of the coiled-coil domain, the nucleotide binding domain, and the leucine-rich repeat domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the corresponding domain in SEQ ID No. 3.

5. An expression cassette comprising the nucleic acid molecule of any one of claims 1-4 and an operably linked heterologous promoter.

6. A vector comprising the nucleic acid molecule of any one of claims 1-4 or the expression cassette of claim 5.

7. The vector of claim 6, further comprising an additional wheat stem rust resistance gene.

8. The vector of claim 7, wherein the additional wheat stem rust resistance gene is selected from the group consisting of: sr22, sr26, sr32, sr33, sr39, sr40, sr45, sr47, and Sr50.

9. A host cell comprising the nucleic acid molecule of any one of claims 1-5, the expression cassette of claim 5, or the vector of any one of claims 6-8.

10. The host cell of claim 9, wherein the host cell is a plant cell, a bacterium, a fungal cell, or an animal cell.

11. The host cell of claim 9 or 10, wherein the host cell is a wheat plant cell.

12. A plant comprising the nucleic acid molecule of any one of claims 1-5, the expression cassette of claim 5, or the vector of any one of claims 6-8.

13. The plant of claim 12, wherein the plant is a cereal plant.

14. The plant of claim 12 or 13, wherein the plant is wheat.

15. A transgenic plant or seed comprising a polynucleotide construct stably incorporated into its genome, the polynucleotide construct comprising a nucleotide sequence selected from the group consisting of:

(a) The nucleotide sequence shown in SEQ ID NO. 1, 2 or 4;

(b) A nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO. 3, and optionally wherein the nucleotide sequence is not naturally occurring;

(c) A nucleotide sequence having at least 87% sequence identity to at least one of the nucleotide sequences set forth in (a), wherein the nucleic acid molecule is capable of conferring resistance to stem rust to a wheat plant comprising the nucleic acid molecule, and optionally wherein the nucleotide sequence is not naturally occurring; and

(d) A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 91% sequence identity to at least one of the amino acid sequences set forth in (b), wherein the nucleic acid molecule is capable of conferring resistance to stem rust to a wheat plant comprising the nucleic acid molecule, and optionally wherein the nucleotide sequence does not occur naturally.

16. The transgenic plant or seed of claim 15, wherein the nucleic acid molecule of (c) or (d) encodes a protein comprising a coiled-coil domain, a nucleotide binding domain, and a leucine-rich repeat domain.

17. The transgenic plant or seed of claim 16, wherein at least one of the coiled-coil domain, the nucleotide binding domain, and the leucine-rich repeat domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the corresponding domain in SEQ ID No. 3.

18. The transgenic plant or seed of any one of claims 15-17, wherein the polynucleotide construct further comprises a promoter operably linked for expression of the nucleotide sequence in a plant.

19. The transgenic plant or seed of claim 18, wherein the promoter is selected from the group consisting of: pathogen inducible promoters, constitutive promoters, tissue-preferred promoters, wound-inducible promoters and chemical regulated promoters.

20. The transgenic plant or seed of any one of claims 15-19, wherein the transgenic plant is a wheat plant and the transgenic seed is a wheat seed.

21. The transgenic plant or seed of claim 20, wherein the transgenic plant or seed comprises increased resistance to wheat stem rust caused by at least one variety of a puccinia graminis wheat species (Puccinia graminis f.sp.tritici) relative to a control wheat plant.

22. The transgenic plant or seed of claim 20 or 21, wherein the polynucleotide construct comprises at least two nucleotide sequences encoding an R protein for wheat stem rust.

23. The transgenic plant or seed of claim 22, wherein each of the at least two nucleotide sequences encoding an R protein for wheat stem rust encodes a different R protein for wheat stem rust.

24. A method for enhancing the resistance of a wheat plant to wheat stem rust, the method comprising introducing into at least one wheat plant cell a polynucleotide construct comprising a nucleotide sequence selected from the group consisting of:

(a) The nucleotide sequence shown in SEQ ID NO. 1, 2 or 4;

(b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 3;

(c) A nucleotide sequence having at least 87% sequence identity to at least one of the nucleotide sequences set forth in (a), wherein the nucleic acid molecule is capable of conferring resistance to stem rust to a wheat plant comprising the nucleic acid molecule; and

(d) A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having at least 91% sequence identity to at least one of the amino acid sequences set forth in (b), wherein said nucleic acid molecule is capable of conferring resistance to stem rust to a wheat plant comprising said nucleic acid molecule.

25. The method of claim 24, wherein the nucleic acid molecule of (c) or (d) encodes a protein comprising a coiled-coil domain, a nucleotide binding domain, and a leucine-rich repeat domain.

26. The method of claim 25, wherein at least one of the coiled-coil domain, the nucleotide binding domain, and the leucine-rich repeat domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the corresponding domain in SEQ ID No. 3.

27. The method of any one of claims 24-26, wherein the polynucleotide construct is stably incorporated into the genome of the plant cell.

28. The method of any one of claims 24-27, wherein the wheat plant cell is regenerated into a wheat plant comprising the polynucleotide construct in its genome.

29. The method of any one of claims 24-28, wherein the polynucleotide construct further comprises a promoter operably linked for expression of the nucleotide sequence in a plant.

30. The method of claim 29, wherein the promoter is selected from the group consisting of: pathogen inducible promoters, constitutive promoters, tissue-preferred promoters, wound-inducible promoters and chemical regulated promoters.

31. The method of any one of claims 21-30, wherein the wheat plant comprising the polynucleotide construct comprises increased resistance to wheat stem rust caused by at least one variety of a puccinia graminis wheat species relative to a control wheat plant.

32. The method of any one of claims 24-31, wherein the polynucleotide construct comprises at least two nucleotide sequences encoding an R protein for wheat stem rust.

33. The method of claim 32, wherein each of the at least two nucleotide sequences encoding R proteins for wheat stem rust encodes a different R protein for wheat stem rust.

34. A wheat plant produced by the method of any one of claims 24-33.

35. A seed of the wheat plant of claim 34, wherein the seed comprises the polynucleotide construct.

36. A method of limiting wheat stem rust in crop production, the method comprising planting the wheat seed according to any one of claims 20-23 and 35, and cultivating the wheat plant under conditions conducive to the growth and development of the wheat plant.

37. The method of claim 36, further comprising harvesting at least one seed from the wheat plant.

38. Use of the wheat plant or seed of any one of claims 20-23, 34 and 35 in agriculture.

39. A human or animal food product produced by using the wheat plant or seed of any one of claims 20-23, 34 and 35.

40. A polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) An amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO. 1, 2 or 4;

(b) The amino acid sequence shown in SEQ ID NO. 3; and

(c) An amino acid sequence having at least 91% sequence identity to the amino acid sequence set forth in SEQ ID No. 3, wherein a polypeptide comprising said amino acid sequence is capable of conferring resistance to stem rust to a wheat plant comprising said polypeptide, and optionally wherein said polypeptide is not naturally occurring.

41. The polypeptide of claim 40, wherein the polypeptide of (c) comprises a coiled-coil domain, a nucleotide binding domain, and a leucine rich repeat domain.

42. The polypeptide of claim 41, wherein at least one of the coiled-coil domain, the nucleotide binding domain, and the leucine-rich repeat domain comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the corresponding domain in SEQ ID NO. 3.

43. A method for identifying a wheat plant exhibiting newly conferred or enhanced resistance to wheat stem rust, the method comprising detecting the presence of R gene Sr27 in the wheat plant.

44. The method of claim 43, wherein the presence of said R gene is detected by detecting at least one marker within Sr 27.

45. The method of any one of claims 43 or 44, wherein Sr27 comprises or consists of the nucleotide sequence of seq id no: the nucleotide sequence shown in SEQ ID No. 1, 2 or 4, or the nucleotide sequence encoding SEQ ID No. 3.

46. The method of any one of claims 43-45, wherein detecting the presence of the R gene comprises a member selected from the group consisting of: PCR amplification, nucleic acid sequencing, nucleic acid hybridization, and immunological assays for detecting the R protein encoded by the R gene.

47. The method of any one of claims 43-46, wherein detecting the presence of the R gene comprises detecting the presence of a fragment of the R gene.

48. A wheat plant identified by the method of any one of claims 43-47.

49. The seed of the wheat plant of claim 48.

50. A method for producing a non-transgenic triticale plant having increased resistance to stem rust, the method comprising modifying a non-functional allele of a resistance gene Sr27 in the triticale plant or at least one cell thereof so as to produce a functional allele, thereby increasing the resistance of the triticale plant to stem rust.

51. The method of claim 50, wherein said modifying a non-functional allele comprises introducing into said non-functional allele at least one genetic modification selected from the group consisting of: insertions, deletions and substitutions of at least one base pair.

52. The method of claim 50 or 51, wherein the modified nonfunctional allele comprises gene editing or mutation breeding.

53. The method of any one of claims 50-52, wherein the functional allele comprises a nucleotide sequence selected from the group consisting of:

(a) The nucleotide sequence shown in SEQ ID NO. 1; and

(b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 3.

54. A non-transgenic triticale plant produced by the method of claim 50.