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

Stem rust resistance gene

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/076,153, filed on September 9, 2020, and U.S. Provisional Patent Application No. 63/127,220, filed on December 18, 2020; both of which are hereby incorporated by reference into this document in their entirety.

References to sequence listings submitted as text files

An official copy of the sequence listing is submitted electronically via EFS-Web as a sequence listing in ASCII format with the file name 070294-0193SEQLST.TXT (created on September 7, 2021 and having a size of 44.3 kilobytes), and is submitted together with this specification. The sequence listing contained in this ASCII formatted document is part of this specification and is incorporated herein in its entirety by reference.

Field of the Invention

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

Background of the Invention

Plant diseases cause significant yield losses in wheat production worldwide. Among the most damaging wheat diseases is rust. Wheat stem rust, caused by Puccinia graminis f.sp.tritici (Pgt), is one of the most devastating diseases affecting wheat production today. Although wheat plants containing resistance (R) genes to Pgt have been shown to be effective in limiting agronomic losses caused by wheat stem rust, new varieties of Pgt have recently emerged for which the R gene is ineffective. Although pesticides can be used to control wheat stem rust, pesticides are expensive and conflict with sustainable intensification of agriculture, and in developing countries, pesticides are completely unaffordable for subsistence farmers.

Sustainable intensification of agriculture will require an increased use of genetic solutions to replace chemical solutions (e.g., pesticides) to protect crops from pathogens and pests (Jones et al., (2014) Philos. T. Roy. Soc. B 369: 20130087). However, traditional methods for introducing R genes typically involve long breeding timelines to break linkage with harmful alleles of other genes. Further, when deployed one at a time, R genes can be overcome in just a few seasons (McDonald and Linde (2002) Annu. Rev. Phytopathol. 40: 349-379). However, molecular cloning has made it possible to avoid linkage drag and introduce multiple R genes simultaneously (Dangl et al., (2013) Science 341:746-751), which should delay the evolution of resistance-breaking pathogen species and thus provide more durable resistance (McDonald and Linde (2002) Annu. Rev. Phytopathol. 40:349-379).

SUMMARY OF THE INVENTION

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

The present invention further provides plants, plant cells and seeds comprising one or more polynucleotide constructs of the present invention in its genome. The polynucleotide constructs comprise nucleotide sequences encoding resistance (R) proteins of the present invention. Such R proteins are encoded by R genes of the present 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 present invention. Preferably, such wheat plants comprise enhanced resistance to at least one variety of the pathogen (Pgt) causing wheat stem rust when compared to the resistance of a control wheat plant that does not comprise the polynucleotide constructs.

The present invention provides a method for enhancing the resistance of plants (particularly wheat or triticale plants) to stem rust caused by Pgt. Such a method comprises introducing a polynucleotide construct comprising the nucleotide sequence of the R gene of the present invention into at least one plant cell. In some embodiments, the polynucleotide construct or a 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 the resistance of plants to stem rust may optionally further comprise regenerating the plant cell into a plant comprising the polynucleotide construct in its genome. Preferably, such a plant comprises enhanced resistance to stem rust caused by at least one variety of Pgt relative to a control plant.

In addition, the present invention provides a method 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 present invention (particularly 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 methods include planting seeds produced by the plants of the invention, wherein the seeds comprise at least one R gene nucleotide sequence of the invention. The methods further include cultivating the plants under conditions conducive to the growth and development of the plants, and optionally harvesting at least one seed or plant part from the plants.

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 THE DRAWINGS

Figure 1 is a photographic illustration of the identification of the Sr27 susceptible mutant. Coorong (upper panels) and EMS-derived susceptible mutant 1 (lower panels) were inoculated with Pgt21-0 and photographed 14 days after infection.

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

Figure 3 is a photographic illustration of the infection phenotype of Pgt21-0 and three spontaneous mutants (21-M1, 21-M2 and 21-M3) on triticale lines Coorong (containing Sr27), Rongcoo (rust susceptible) and a mutant line derived from Coorong with a loss of the Sr27 resistance gene (Sr27 mutant 1). Images were taken 14 days after seedling leaf inoculation.

Fig. 4 is a schematic diagram of the position and size (in kbp) of the deletions in the three Sr27-virulence mutants (M1, M2 and M3) of Pgt21-0 relative to chromosome 2B (bar with hatching). The position on the chromosome is indicated at intervals of 1Mbp from the 5' end, wherein the centromere (CM) position is indicated. The deletion is detected by mapping the reads of the Illumina DNA sequence reads from Pgt21-0 and the three mutants on the Pgt21-0 genome sequence.

Fig. 5A and 5B. field isolates of Pgt with virulence for Sr27 include small deletions on chromosome 2B. Fig. 5A is a schematic diagram of the position and size (in kbp) of the deletions in the six Sr27-virulence isolates indicated from Australia (34-2,12 and 34-2,12,13) and South Africa (SA03, SA05, SA06, SA07) relative to chromosome 2B (grey bars). The deletion is detected by mapping the reads of the Illumina DNA sequence reads from these isolates on the Pgt21-0 genomic sequence. Relative to the border of the missing region in 34-2-12, the positions of primers P423, P424 and P426 around the AvrSr27 seat on chromosome 2B are indicated (arrows). Fig. 5B shows the confirmation of the 13Kbp deletion in the Sr27-virulence rust isolate 34-2-12. PCR amplification products from genomic DNA of Pgt21-0 and 34-2,12 are shown after separation on a 1% agarose gel. Primers P383 and P351 were designed to amplify a fragment of the AvrSr50 gene as a control region that was identical in both isolates.

Figure 6 is an amino acid sequence alignment of two related secreted AvrSr27 protein variants from Pgt21-0 encoded by the AvrSr27 locus. AvrSr27-1 (SEQ ID NO: 6) and AvrSr27-2 (SEQ ID NO: 8) are encoded on chromosome 2B at the avirulence allele. The amino acid sequence of AvrSr27-1 is shown (single letter code), where identical residues in other variants are indicated by '.' The predicted signal peptide region is underlined and in bold.

Figure 7 is a graphical representation showing confirmation of the resistance function of Sr27. Luciferase activity (luminescence units, y-axis) was measured in protoplasts co-expressing Sr27 alone or AvrSr27-1 or AvrSr27-2, combined Sr27 and AvrSr27 variants, Sr50 plus AvrSr27-1, or Sr27 plus AvrSr50.

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

Figure 9 shows the phenotype of exemplary T1 families containing Sr27 transgenes and control plants infected with stem rust varieties 98-1, 2, 3, 5, 6 and scored 10 days after infection. Images show representative T1 plants from two transgenic lines containing the Sr27 transgene (PC311.3 and PC311.17), one non-transgenic line recovered from tissue culture (PC311.16), the susceptible parent Fielder, Chinese Spring WRT258.5 containing the native Sr27 gene, and the susceptible parent Chinese Spring (CS).

Sequence Listing

The nucleotide and amino acid sequences listed in the accompanying sequence table, the accompanying drawings, and those described below are shown using standard letter abbreviations for nucleotide bases and single-letter or three-letter codes for amino acids. The nucleotide sequences follow the standard convention of starting at the 5' end of the sequence and advancing to the 3' end (i.e., from left to right in each row). Only one chain of each nucleotide sequence is shown, but it should be understood that the complementary chain is included by any reference to the displayed chain. The amino acid sequences follow the standard convention of starting at the amino-terminal end of the sequence and advancing to the carboxyl-terminal end (i.e., from left to right in each row).

SEQ ID NO: 1 sets forth a nucleotide sequence comprising an R gene (Sr27) from triticale (×Triticosecale Wittmack) cv. Coorong. The nucleotide sequence comprises, in a 5' to 3' direction: a 5'-untranslated region (5'-UTR) at nucleotides 1-39, a protein coding region 40-872, an intron at nucleotides 873-1779, a protein coding region at nucleotides 1780-3814, a TGA stop codon at nucleotides 3815-3817, and a 3'-untranslated region (3'-UTR) at nucleotides 3818-3956.

SEQ ID NO:2 sets forth 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) may 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 the cDNA is TGA.

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

SEQ ID NO: 4 sets forth the nucleotide sequence of the open reading frame of Sr27 (SEQ ID NO: 1). This sequence is that portion of the genomic sequence of Sr27 that begins at the first nucleotide of the start codon and ends at the last nucleotide of the stop codon. This sequence contains introns.

SEQ ID NO:5 sets forth the coding sequence of AvrSr27-1 from Puccinia graminearum race (Pgt) isolate Pgt21-0, wherein the predicted signal peptide is excluded and replaced with a single methionine start codon.

SEQ ID NO:6 sets forth the amino acid sequence of the Avr27-1 protein encoded by AvrSr27-1 (SEQ ID NO:5) from the Pgt isolate Pgt21-0.

SEQ ID NO:7 sets forth the coding sequence of AvrSr27-2 from Pgt isolate Pgt21-0 (PGT21_006593_AvrSr27-2), wherein the predicted signal peptide is excluded and replaced with a single methionine start codon.

SEQ ID NO:8 sets forth the amino acid sequence of the Avr27-2 protein encoded by AvrSr27-2 (SEQ ID NO:7) from Pgt isolate Pgt21-0.

SEQ ID NOs: 9-39 are the nucleotide sequences of the primers in Table 2 below (Example 6).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now 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 occur to those skilled in the art having the benefit of the teachings given in the foregoing description and the associated drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and modifications and other embodiments are also readily included within the scope of the appended claims. Although specific terms are used herein, they are used only in a general and descriptive sense 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 Puccinia graminearum (Pgt) in plants, in particular wheat or triticale plants. Most rust resistance genes belong to the class of nucleotide-binding leucine-rich repeat receptors (NLRs), which are well known as immune receptors in plants. TIR-containing NLRs (TNLs) and CC-containing NLRs (CNLs) are the two main classes of plant NLRs, which are defined based on the presence of a TIR or CC domain at their N-termini. Most, if not all, NLRs in cereals belong to the CNL class. Nine all-stage stem rust resistance genes have been cloned from einkorn wheat (T. monococcum) (Sr21, Sr22 and Sr35), the A genome donor of hexaploid bread wheat, Aegilops tauschii (Sr33 and Sr45), the D genome donor of hexaploid bread wheat, diploid rye (Sr50) and durum wheat (Sr13), and Sr26 was identified from tall wheatgrass (T. ponticum).

The present invention relates to Sr27, a locus that confers resistance to many important Pgt isolates worldwide, including the Ug99 lineage, and is present in the rye/wheat hybrid cereal triticale (×Triticosecale). As disclosed hereinafter, the inventors used a combined chemical mutagenesis and nucleotide-bound leucine-rich repeat receptor (NLR) resistance gene enrichment sequencing approach to isolate the R gene Sr27 from a Pgt-resistant triticale cultivar (‘Coorong’) that contains the Sr27 stem rust resistance gene in its genome.

The present invention provides nucleic acid molecules comprising nucleotide sequences of R genes, in particular Sr27 and naturally occurring variants thereof (e.g., orthologs and allelic variants) and synthetic or artificial (i.e., non-naturally occurring) variants thereof. The nucleotide sequences of such R genes (which are also referred to herein as R gene nucleotide sequences) encode R proteins. The R gene nucleotide sequences of the present invention include, but are not limited to, wild-type R genes (which include a natural promoter and a 3′ adjacent region containing a coding region), cDNA sequences, and nucleotide sequences containing only coding regions. 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 natural R gene promoter is not used to drive the 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 present invention to drive the nucleotide sequence encoding the R protein to be expressed in plants.

Preferably, the R protein of the present invention is a functional R protein that can confer to the plant comprising the R protein enhanced resistance to stem rust caused by at least one variety of Pgt. In certain embodiments, the R protein of the present invention comprises broad-spectrum resistance to multiple varieties of Pgt, such as an R protein encoded by Sr27.

The present invention further provides transgenic plants comprising a polynucleotide construct comprising an R gene nucleotide sequence of the present 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 present invention, the transgenic plant is a wheat plant comprising enhanced resistance to stem rust caused by at least one variety of Pgt.

In certain embodiments, the transgenic plant of the present invention comprises a polynucleotide construct comprising a nucleotide sequence encoding an R protein and a heterologous promoter operably connected to express the nucleotide sequence encoding the R protein. The selection of a heterologous promoter can depend on many factors, such as the desired timing, localization and expression pattern and the responsiveness to a specific biological or abiotic stimulus. Interesting promoters include, but are not limited to, pathogen-inducible promoters, constitutive promoters, tissue-preferred promoters, wound-inducible promoters, and chemical-regulated promoters.

In certain embodiments of the present invention, the transgenic plant, particularly the transgenic wheat plant, may comprise one, two, three, four, five, six or more nucleotide sequences encoding R proteins. Typically, but not necessarily, the two or more R proteins will be different from each other. For the present invention, an R protein is different from another R protein when the two R proteins have different amino acid sequences. In certain embodiments of the present invention, each of the different R proteins for stem rust has one or more differences in resistance characteristics, such as resistance to different varieties/variety groups for Pgt. It is recognized that by combining two, three, four, five, six or more nucleotide sequences (each nucleotide sequence encodes a different R protein for wheat stem rust), a wheat plant containing a broad spectrum resistance to multiple varieties of Pgt can be produced. In regions 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 sequence of the present invention in a single wheat plant include Sr22 (WO 2017/024053), Sr26, Sr32, Sr33 (GenBank Accession No. KF031299.1), Sr35 (GenBank Accession No. KC573058.1), Sr39, Sr40, Sr45 (WO 2017/024053), Sr47, Sr50, SrTA1662 (WO2019140351) and adult plant resistance genes Sr57/Lr34 (GenBank Accession No. FJ436983.1) and Sr55/Lr67.

Transgenic plants of the present 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 present invention (including, for example, an Sr27 nucleotide sequence or a variant thereof). Such plants already comprising one or more other R gene nucleotide sequences can comprise such R genes that are native to the genome of the plant, introduced into the plant via sexual reproduction, or introduced by transforming the plant or its ancestor with an R gene nucleotide sequence. Alternatively, the one or more other R gene nucleotide sequences can be introduced into a transgenic plant of the present invention that already comprises a polynucleotide construct of the present invention, for example, by transformation or sexual reproduction.

In other embodiments, two or more different R gene sequences can be introduced into the 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 can be preferred for plant breeding because it is expected that the two or more R gene nucleotide sequences will be closely linked and thus separated as a single locus. Alternatively, the polynucleotide construct of the present invention can be incorporated into the genome of the plant in the immediate vicinity of another R gene nucleotide sequence by using a genome modification method based on homologous recombination described elsewhere herein or known in the art.

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

The plants disclosed herein can be used in methods for limiting stem rust caused by Pgt in crop production, particularly in regions where stem rust is prevalent. The methods of the invention comprise planting seeds produced by the plants of the invention, wherein the seeds comprise at least one R gene nucleotide sequence of the invention. The methods further comprise cultivating the plants under conditions conducive to the growth and development of plants therefrom, and optionally harvesting at least one seed or other plant part from the plants.

In addition, the present invention provides a method for identifying plants (particularly wheat or triticale plants) that exhibit newly conferred or enhanced resistance to stem rust caused by Pgt. The method can be used in cultivating plants for resistance to stem rust. Such resistant plants can be used in the agricultural production of wheat seeds. The method includes detecting the presence of at least one R gene of the present invention (particularly Sr27) in a plant. In some embodiments of the present invention, detecting the presence of the R gene includes detecting the entire R gene in genomic DNA isolated from the plant. However, in preferred embodiments, detecting the presence of the R gene includes detecting the presence of at least one marker within the R gene. In other embodiments of the present invention, detecting the presence of the R gene includes detecting the presence of an R protein encoded by the R gene by using, for example, an immunological detection method involving antibodies specific for the R protein.

In the method for identifying plants that exhibit 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 biology 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 nucleic acid molecules comprising the R gene and/or markers therein by PCR amplification, sequencing nucleic acid molecules comprising the R gene and/or markers, identifying the R gene, the marker or transcripts of the R gene by nucleic acid hybridization, and performing immunological assays 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 gene of the present invention, and such probes and PCR primers can be used in methods disclosed elsewhere herein or known in the art to quickly identify one or more plants comprising the presence of the R gene of the present invention in a plant population. 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 present invention. Such fragments of the R gene of the invention may contain, 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 consecutive nucleotides.

Depending on the desired result, the polynucleotide construct of the present invention can be stably incorporated into the genome of the plant cell or unstably incorporated into the genome of the plant cell. If, for example, the desired result is to produce a stably transformed plant with enhanced resistance to wheat stem rust caused by at least one variety of Pgt, then the polynucleotide construct can, for example, be fused to a plant transformation vector suitable for stably incorporating the polynucleotide construct into the genome of the plant cell. Typically, the stably transformed plant cell will be regenerated into a transformed plant comprising the polynucleotide construct in its genome. Such stably transformed plants can transmit the polynucleotide construct to progeny plants in subsequent generations by sexual and/or asexual reproduction. Plant transformation vectors, methods for stably transforming plants with introduced polynucleotide constructs, and methods for regenerating plants from transformed plant cells and tissues are generally known in the art for both monocots and dicots or described elsewhere in this article.

The present invention provides nucleic acid molecules comprising R genes. Preferably, the R gene is capable of conferring enhanced resistance to at least one variety of the pathogen Pgt that causes stem rust on a host plant (particularly a wheat or triticale plant). More preferably, the R gene is capable of conferring enhanced resistance to two, three, four or more varieties of Pgt on a host plant (particularly a wheat or triticale plant). Therefore, such an R gene can be used in agricultural production to limit the stem rust caused by Pgt. The R gene of the present invention includes but is not limited to the R gene whose nucleotide sequence is disclosed herein, but also includes orthologs and other variants that can confer resistance to stem rust caused by at least one variety of Pgt on a plant. 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 present invention can be used to produce plants, particularly wheat and triticale plants, with enhanced resistance to stem rust caused by at least one variety of Pgt. Typically, the methods of the present invention will enhance or increase the resistance of the test plant to a variety of Pgt by at least 25%, 50%, 75%, 100%, 150%, 200%, 250%, 500% or more when compared to the resistance of the control plant to the same variety of Pgt. Unless otherwise specified or obvious from the context of use, the control plant of the present invention is a plant that does not contain the polynucleotide construct of the present invention. Preferably, the control plant is substantially the same (e.g., the same species, subspecies and variant) as the plant containing the polynucleotide construct of the present invention, except that the control does not contain the polynucleotide construct. In some embodiments, the control will contain a polynucleotide construct, but not one or more R gene sequences in the polynucleotide construct of the present invention.

In addition, the present invention provides transformed plants, seeds and plant cells produced by the method of the present invention and/or comprising the polynucleotide construct of the present invention. Progeny plants and seeds thereof comprising the polynucleotide construct of the present invention are also provided. The present invention also provides seeds, nutrient parts and other plant parts produced by the transformed plants and/or progeny plants of the present invention, and 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 method of the present invention can be used to enhance the resistance of plants, 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 Triticum, or a member of another genus within the Triticeae, particularly a member of another genus that is capable of producing interspecific hybrids with at least one Triticeae species. Examples of such another genus within the Triticeae are Aegilops and Secale.

The wheat plants of the present invention include, for example, domesticated and undomesticated plants. The wheat plants of the present invention include, but are not limited to, the following Triticum, Aegilops and Secale species: common wheat (T.aestivum), einkorn wheat, cylindrical wheat (T.turgidum), wild einkorn wheat (T.boeoticum), Timopheevii wheat (T.timopheevii) and Urartu wheat (T.urartu), Aegilops tauschii, Secale cereale and hybrids thereof. Examples of common wheat subspecies included in the present invention are common (aestivum), compactum (compactum), macha (macha), vavilovi (vavilovi), spelta (spelta) and sphaecrococcum (sphaecrococcum). Examples of cylindrical wheat subspecies included in the present invention are cylindrical (turgidum), Persian (carthlicum), two grains (dicoccom), hard grains (durum), paleocoichicum, Polish (polonicum), mixed (turanicum) and wild two grains (T.dicoccoides). Examples of E. triticum subspecies included in the present invention are monococcum and aegilopoides. In one embodiment of the present invention, the wheat plant is a member of the species Triticum cylindrum, and in particular, a member of the subspecies durum, such as the cultivars Ciccio, Colosseo or Utopia. It is recognized that the wheat plant of the present invention can be a domesticated wheat plant or an undomesticated wheat plant.

The present invention also includes triticale plants, triticale plant parts and triticale plant cells comprising R gene of the present invention. As used in this article, "triticale plants" refer to plants created by hybridizing rye plants with tetraploid wheat plants (e.g., cylindrical wheat) or hexaploid wheat plants (e.g., common wheat). The present invention also includes seeds produced by triticale plants described in this article, and methods for controlling weeds near triticale plants described in this article. As used in this article, the term "wheat plant" includes triticale plants, unless otherwise noted or apparent from the context of use.

The term "plant" is intended to include plants at any stage of maturity or development, and 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 callus, plant pieces 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, etc.). The present invention also includes seeds produced by the plants of the present invention.

Progeny, variants and mutants of the regenerated plants are also included within the scope of the present invention, provided that these parts comprise the introduced polynucleotides. As used herein, "progeny" and "progeny plants" include any subsequent generations of plants, whether derived from sexual reproduction and/or asexual reproduction, unless otherwise expressly stated or apparent from the context of use.

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

In one embodiment of the present 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 greater sequence identity to the entire nucleotide sequence shown in SEQ ID NO:1, 2 and/or 4, or a fragment thereof.

The present invention includes isolated or substantially purified polynucleotide (also referred to herein as "nucleic acid molecules," "nucleic acids," etc.) or protein (also referred to herein as "polypeptides") compositions, including, for example, polynucleotides and proteins comprising the sequences set forth in the accompanying sequence listing, as well as variants and fragments of such polynucleotides and proteins. An "isolated" or "purified" polynucleotide or protein, or a biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein (as found in the environment in which it naturally occurs). Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium (when produced by recombinant techniques), or substantially free of chemical precursors or other chemicals (when chemically synthesized). Optimally, an "isolated" polynucleotide is free of sequences (optimally, protein 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 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the 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 protein. When a protein of the invention, or a biologically active portion thereof, is 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 present invention also includes fragments or variants of the disclosed polynucleotides and proteins encoded therefrom. "Fragment" means a part of the polynucleotide, or a part of the amino acid sequence and thus protein encoded therefrom. The fragment of the polynucleotide comprising the coding sequence can encode a protein fragment that retains the biological activity of the full-length or native protein. Alternatively, the fragment of the polynucleotide useful as a hybridization probe does not usually encode a protein that retains biological activity or does not retain promoter activity. Therefore, the fragment of the nucleotide sequence can vary in the range of from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides and up to the full-length polynucleotide of the present invention.

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

"Variant" is intended to refer to substantially similar sequences. For polynucleotides, variants include polynucleotides having a deletion (i.e., truncation) at the 5' end and/or the 3' end; having a deletion and/or addition of one or more nucleotides at one or more internal sites of a natural polynucleotide; and/or having a substitution of one or more nucleotides at one or more sites in a natural polynucleotide. As used herein, "natural (of)" polynucleotides or polypeptides include naturally occurring nucleotide sequences or amino acid sequences, respectively. For polynucleotides, conservative variants include those sequences that encode the amino acid sequence of one of the R proteins of the present invention due to the degeneracy of the genetic code. Naturally occurring allelic variants such as these can be identified by using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated by using site-directed mutagenesis but still encoding the R protein of the present 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 greater sequence identity to that particular polynucleotide as determined by the sequence comparison 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 greater sequence identity to at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2 and 4, and optionally, a non-naturally occurring nucleotide sequence comprising a nucleotide sequence shown in SEQ ID NO: 1, 2 and/or 4 that differs by at least one nucleotide modification selected from the group consisting of: a substitution of at least one nucleotide, an addition of at least one nucleotide, and a deletion of at least one nucleotide.

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

"Variant" protein is intended to refer to a protein derived from a native protein by: deletion of one or more amino acids at the N-terminus and/or C-terminus of the native protein (so-called truncation); 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 derived, 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 greater sequence identity with respect 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 programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or even 1 amino acid residue.

The proteins of the present invention can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) PNAS 82: 488-492; Kunkel et al., (1987) Methods in Enzymol. 154: 367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein. Guidance on 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.) (which is incorporated herein by reference). Conservative substitutions, such as exchanging one amino acid for another with similar properties, may be optimal.

Therefore, genes and polynucleotides of the present invention include naturally occurring sequences and mutants and other variant forms. Similarly, proteins of the present invention include naturally occurring proteins and variations and modified forms thereof. More preferably, such variants confer enhanced resistance to stem rust caused by at least one variety of Pgt to the plant or part thereof comprising the variant. In some embodiments, the mutations made in the DNA encoding the variant will not place the sequence outside the reading frame. Optimally, the mutations will not create complementary regions capable of producing secondary mRNA structures. See, EP Patent Application Publication No. 75,444.

It is expected that deletions, insertions and substitutions of the protein sequences included herein will not produce fundamental changes in the characteristics of the protein. However, while it is difficult to predict the precise effect of a substitution, deletion or insertion before doing so, those skilled in the art will appreciate that the effect will be evaluated by conventional screening assays. That is, the activity can be evaluated by the assays disclosed herein below.

For example, wheat plants susceptible to wheat stem rust caused by a particular variety of Pgt can be transformed with an Sr27 polynucleotide, regenerated into transformed or transgenic plants comprising the polynucleotide, and tested for resistance to wheat stem rust caused by a particular variety of Pgt 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 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, for example, Stemmer (1994) PNAS 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al., (1997) Nature Biotech. 15: 436-438; Moore et al., (1997) J. Mol. Biol. 272: 336-347; Zhang et al., (1997) PNAS 94: 4504-4509; Crameri et al., (1998) Nature 391: 288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458.

The polynucleotides of the present invention can be used to separate corresponding sequences from other organisms (particularly other plants). In this way, methods such as PCR, hybridization, etc. can be used to identify such sequences based on their sequence homology with the sequences set forth in this article. The present invention includes sequences separated based on their entire sequences set forth in this article or with their variants and fragments' sequence identity. Such sequences include sequences as orthologs of disclosed sequences." orthologs" are intended to refer to genes derived from common ancestral genes and found in different species due to speciation. When the following circumstances, genes found in different species are considered to be orthologs: their nucleotide sequences and/or their encoded protein sequences share at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity. The functions of orthologs are often highly conserved between species. Thus, the present invention includes isolated polynucleotides that encode an R protein and hybridize under stringent conditions to at least one of the R proteins disclosed herein or known in the art, or variants or fragments thereof.

In one embodiment, an ortholog of the present 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 greater nucleotide sequence identity to a nucleotide sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 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 greater amino acid sequence identity to the amino acid sequence shown in SEQ ID NO:3.

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

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

It is recognized that domains in variant Sr27 proteins corresponding to those conserved domains of Sr27 set forth above, and any particular conserved amino acids therein, can be identified by methods known to those skilled in the art or disclosed elsewhere herein (e.g., multiple sequence alignment). 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 can be determined for any variant Sr27 protein of the invention by methods such as multiple sequence alignment.

Preferably, the variant Sr27 proteins of the present invention and polynucleotides encoding them confer or are capable of conferring to wheat plants comprising such proteins and/or polynucleotides enhanced resistance to at least one species 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 the corresponding DNA sequence from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Plainview, 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, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, etc.

It is recognized that the R protein coding sequence of the present invention includes such a polynucleotide molecule, which comprises a nucleotide sequence that is sufficiently identical to any one or more of the nucleotide sequences of SEQ ID NO: 1, 2 and 4. The term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence that comprises a sufficient or minimum 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, an amino acid or nucleotide sequence comprising a common structural domain having at least about 80% or 85% identity, preferably 90% or 91% identity, more preferably 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is defined herein as being sufficiently identical.

In order to measure the identity percentage of two amino acid sequences or two nucleic acids, the sequences are compared for the best comparison purpose. The identity percentage between the two sequences is a function of the number of identical positions shared by the sequences (i.e., identity percentage=identical position number/total position number (e.g., overlapping positions) x 100). In one embodiment, the length of the two sequences is identical. The identity percentage between the two sequences can be measured by using techniques similar to those described below, wherein gaps are allowed or not allowed. In calculating the identity percentage, typically accurate matching is counted.

The determination of the percent identity between two sequences is achieved 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 an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the polynucleotide molecules of the present invention. BLAST protein searches can be performed with the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. In order to obtain gap alignments for comparison purposes, gap BLAST can be utilized, as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterative search that detects long-range relationships between molecules. See Altschul et al., (1997) supra. When using BLAST, gap BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST; 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 using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Alignments can also be performed manually by inspection.

Unless otherwise indicated, the sequence identity/similarity values provided herein refer to the values obtained by using the full-length sequences of the invention and using a multiple alignment by means of the algorithm Clustal W (Nucleic Acid Research, 22 (22): 4673-4680, 1994), using the program AlignX (using default parameters) included in the software package Vector NTI Suite Version 7 (InforMax, Inc., Bethesda, MD, USA), or any equivalent program thereof. "Equivalent program" means any sequence comparison program that generates an alignment having identical nucleotide or amino acid residue matches and identical sequence identity percentages for any two sequences in question when compared with the corresponding alignment generated by CLUSTALW (version 1.83) using default parameters (which is available on the World Wide Web at the European Bioinformatics Institute website: ebi.ac.uk/Tools/clustalw/index.html).

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

The polynucleotide construct comprising the R protein coding region can be provided in an expression cassette for expression in a plant or other organism or non-human target host cell. The expression cassette will include 5' and 3' regulatory sequences operably connected to the R protein coding region. "Operably connected" is intended to refer to the functional connection between two or more elements. For example, the operably connected between the target polynucleotide or gene and the regulatory sequence (i.e., promoter) is a functional connection that allows the expression of the target polynucleotide. The operably connected element can be adjacent or non-adjacent. When used to refer to the connection of two protein coding regions, "operably connected" means that the coding region is in the same reading frame. In addition, the box can include at least one other gene to be co-transformed into the organism. Alternatively, the other gene can be provided on multiple expression cassettes. Such an expression cassette can be equipped with multiple restriction sites and/or recombination sites, which are used to insert the R protein coding region to be under the transcriptional regulation of the regulatory region. In addition, the expression cassette can include a selection marker gene.

The expression cassette will contain, in a 5'-3' transcriptional direction: a transcription and translation initiation region (i.e., a promoter), an R protein coding region of the present invention, and a transcription and translation termination region (i.e., a termination region) that is functional in a plant or other organism or non-human host cell. The regulatory region (i.e., a promoter, a transcription regulatory region, and a translation termination region) and/or the R protein coding region of the present invention may be native/similar to the host cell or to each other. Alternatively, the regulatory region and/or the R protein coding region of the present invention may be heterologous to the host cell or to each other.

As used in this article, with respect to nucleic acid molecules or nucleotide sequences, "heterologous" is such a nucleic acid molecule or nucleotide sequence, which is derived from an alien species, or if derived from the same species, is modified from its native form in composition and/or genomic locus by intentional human interference. For example, the promoter operably connected to a heterologous polynucleotide is from a species different from the species from which the polynucleotide is derived, or if from the same/similar species, one or both of them are significantly modified from its original form and/or genomic locus, or the promoter is not a native promoter for the polynucleotide operably connected. As used in this article, a chimeric gene comprises a coding sequence operably connected to a transcription initiation region heterologous to the coding sequence.

The present invention provides a host cell comprising at least one of the nucleic acid molecules, expression cassettes and vectors of the present invention. In preferred embodiments, the host cell is a plant cell. In other embodiments, the host cell is selected from the group consisting of bacteria, fungal cells 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, archaea, eubacteria, yeast and algae.

Although it may be optimal to use a heterologous promoter to express the R protein, the native promoter of the corresponding R gene may be used.

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

When appropriate, the polynucleotides can be optimized for increased expression in transformed plants. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. 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, e.g., U.S. Patent 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.

Other sequence modifications are known to enhance gene expression in cellular hosts. These include sequences that eliminate coding false polyadenylation signals, exon-intron splice site signals, transposon-like repetitive sequences and other such well-characterized sequences, which may be harmful for gene expression. The G-C content of the sequence can be adjusted to an average level for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

In addition, the expression cassette may include a 5' leader sequence. Such leader sequences may function to enhance translation. Translation leader sequences are known in the art and include: small RNA virus leader sequences, such as the EMCV leader sequence (encephalomyocarditis 5' non-coding region) (Elroy-Stein et al., (1989) PNAS 86: 6126-6130); Potyvirus leader sequences, such as the TEV leader sequence (tobacco etch virus) (Gallie et al., (1995) Gene 165 (2): 233-238), MDMV leader sequence (maize dwarf mosaic virus) (Virology 154: 9-20), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al., (1991) Nature 353: 90-94); non-translated 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 sequence (TMV) (Gallie et al., (1989) Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader sequence (MCMV) (Lommel et al., (1991) Virology 81:382-385). See also, Della-Cioppa et al., (1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may be manipulated to provide the DNA sequence in the proper orientation and, as appropriate, in the proper reading frame. For this purpose, adapters or linkers may be used to join 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-displacement, such as transitions and transversions, may be involved.

Many promoters can be used in the practice of the present invention. Promoters can be selected based on the desired results. The nucleic acid can 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), etc. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, and 6,177,611.

Tissue-preferred promoters can be used to target the enhanced expression of R protein coding sequences in specific plant tissues. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include: Yamamoto et al., (1997) Plant J. 12(2):255-265; Kawamata et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell et al., (1997) Transgenic Res. 6(2):157-168; Rinehart et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp et al., (1996) Plant Physiol. 112(2):525-535; Canevascini et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et al., (1993) PNAS 90(20):9586-9590; and Guevara-Garcia et al., (1993) Plant J. 4(3):495-505. If desired, such promoters can be modified for weak expression.

Generally, it will be beneficial to express genes from inducible promoters, particularly from pathogen-inducible promoters. Such promoters include those from pathogen-related proteins (PR proteins) induced after infection by pathogens; for example, PR proteins, SAR proteins, β-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al., (1983) Neth. J. Plant Pathol. 89: 245-254; Uknes et al., (1992) Plant Cell 4: 645-656; and Van Loon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819, 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. Pat. No. 5,750,386 (nematode inducible); and references cited therein. Of particular interest is the inducible promoter of the maize PRms gene whose expression is induced by the pathogen 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 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 wheat Sr57/Lr34, Sr33, Sr35, and Sr22 promoters (Risk et al., (2012) Plant Biotechnol J 10:447-487; Periyannan et al., (2013) Science 341:786-788; Saintenac et al., (2013) Science 341:783-786; Steuernagel et al., (2016) Nature Biotechnol. 34 (6): 652-655, doi: 10.1038/nbt.3543).

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

Chemical-regulated promoters can be used to modulate gene expression in plants by applying exogenous chemical regulators. Depending on the target, the promoter can be a chemical-inducible promoter, in which the application of the chemical induces gene expression; or a chemical-repressible promoter, in which the application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to: the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners; the maize GST promoter, which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides; and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemically-regulatable promoters of interest include: steroid-responsive promoters (see, e.g., the 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 include a selectable marker gene for selecting transformed cells. Selectable marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicidal compounds such as glufosinate, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetic acid salts or esters (2,4-D). Additional selectable markers include: phenotypic markers, such as β-galactosidase, and fluorescent proteins, such as green fluorescent protein (GFP) (Su et al., (2004) Biotechnol Bioeng 85:610-9 and Fetter et al., (2004) Plant Cell 16:215-28), cyan 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 Evrogen, see Bolte et al., (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al., (1992) PNAS 89:6314-6318; Yao et al., (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al., (1980) The Operon, pp. 177-220; Hu et al., (1987) Cell 48:555-566; Brown et al., (1987) Cell 49:603-612; Figge et al., (1988) Cell 52:713-722; Deuschle et al., (1989) PNAS 86:5400-5404; Fuerst et al., (1989) PNAS 86:2549-2553; Deuschle et al., (1990) Science 248:480-483; Gossen (1993) PhD thesis, University of Heidelberg; Reines et al., (1993) PNAS 90:1917-1921; Labow et al., (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al., (1992) PNAS 89:3952-3956; Baim et al., (1991) PNAS 88:5072-5076; Wyborski et al., (1991) Nucleic Acids Res. 19: 4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al., (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al., (1988) Biochemistry 27: 1094-1104; Bonin (1993) PhD thesis, University of Heidelberg; Gossen et al., (1992) PNAS 89: 5547-5551; Oliva et al., (1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al., (1988) Nature 334:721-724. The disclosures of these are incorporated herein by reference.

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

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

The methods of the present invention involve introducing a polynucleotide construct into a plant. "Introducing" means presenting the polynucleotide construct to the plant in such a way that the construct gains access to the interior of a cell of the plant. The methods of the present invention do not rely on a specific method for introducing a polynucleotide construct into a plant, as long as the polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing a polynucleotide construct into a plant are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

"Stable transformation" means that the polynucleotide construct introduced into the plant is integrated into the genome of the plant and can be inherited by its progeny. "Transient transformation" means that the polynucleotide construct introduced into the plant is not integrated into the genome of the plant.

For transformation of plants and plant cells, the nucleotide sequence of the present invention is inserted into any vector known in the art suitable for expressing the nucleotide sequence 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 can be introduced into plants by using tumor-inducing (Ti) plasmid vectors. Other methods for foreign DNA delivery involve the use of PEG-mediated protoplast transformation, electroporation, microinjection whiskers, and biolistic or microparticle bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 to Vasil et al.; Bilang et al., (1991) Gene 100:247-250; Scheid et al., (1991) Mol. Gen. Genet., 228:104-112; Guerche et al., (1987) Plant Science 52:111-116; Neuhause et al., (1987) Theor. Appl Genet. 75:30-36; Klein et al., (1987) Nature 327:70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227:1229-1231; DeBlock et al., (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). The transformation method depends on the plant cell to be transformed, the stability of the vector used, the expression level of the gene product and other parameters.

Other suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include: microinjection as described by Crossway et al., (1986) Biotechniques 4:320-334; by Riggs et al., (1986) PNAS electroporation as described in 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 in Paszkowski et al. (1984) EMBO J. 3:2717-2722; and ballistic particle acceleration as described in, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al., (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," Plant Cell, Tissue, and Organ Culture: Fundamental Methods, eds. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al., (1988) Biotechnology 6:923-926; and Lec1 transformation (WO 00/28058). See also, Weissinger et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al., (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al., (1990) Biotechnology 8:736-740 (rice); Klein et al., (1988) PNAS 85:4305-4309 (maize); Klein et al., (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al., (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al., (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al., (1987) PNAS 84:5345-5349 (Liliaceae); De Wet et al., (1985) The Experimental Manipulation of Ovule Tissues, ed. Chapman et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler et al., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are incorporated herein by reference.

The polynucleotides of the present invention can be introduced into plants by contacting the plants with viruses or viral nucleic acids. Typically, such methods involve incorporating the polynucleotide constructs of the present invention into viral DNA or RNA molecules. Further, it is recognized that the promoters of the present invention also include promoters for transcription by viral RNA polymerases. 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 are incorporated herein by reference.

If desired, the modified virus or modified viral nucleic acid can be prepared into a formulation. Such preparations are prepared in a known manner (see, for example, for a review, US 3,060,084; EP-A 707 445 (for liquid concentrates); Browning, "Agglomeration", Chemical Engineering, December 4, 1967, 147-48; Perry's Chemical Engineer's Handbook, 4th edition, McGraw-Hill, New York, 1963, pp. 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, 1996). Sons, Inc., New York, 1961; Hance et al., Weed Control Handbook, 8th edition, Blackwell Scientific Publications, Oxford, 1989; and Mollet, H., Grubermann, A., Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Germany), 2001, 2; D. A. Knowles, Chemistry and Technology of Agrochemical Formulations, Kluwer Academic Publishers, Dordrecht, 1998 (ISBN 0-7514-0443-8)), for example by extending the active compound with adjuvants suitable for formulating agrochemicals, such as solvents and/or carriers, if desired, emulsifiers, surfactants and dispersants, preservatives, defoamers, antifreeze agents, and, for seed treatment formulations, optionally, colorants and/or binders and/or gelling agents.

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

Transformed cells can be grown into plants in a conventional manner. See, for example, McCormick et al., (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same transformed strain or different strains, and the resulting hybrids with the constitutive expression of the desired phenotypic characteristics can be identified. Two or more generations can be grown to ensure that the expression of the desired phenotypic characteristics is stably maintained and inherited, and then seeds are harvested to ensure that the expression of the desired phenotypic characteristics is obtained. In this way, the present invention provides transformed seeds (also referred to as "transgenic seeds") having polynucleotide constructs of the present invention stably incorporated into their genome, such as expression cassettes of the present invention.

In certain embodiments of the present invention, the nucleotide sequence of the non-functional allele at the R gene locus of the present invention can be modified to a functional allele in a plant in situ manner (in planta), which provides resistance to at least one variety of plant pathogens. Therefore, the present invention provides a method for producing a plant (particularly a triticale plant) with enhanced resistance to stem rust. The method includes modifying the non-functional allele of the resistance gene Sr27 in a plant or at least one of its cells so as to make a functional allele, thereby enhancing the resistance of the plant to stem rust. In a preferred embodiment, the plant produced is a non-transgenic plant, particularly a non-transgenic triticale plant. The method may further include regenerating a plant cell comprising the functional allele into a plant comprising the functional allele.

In one embodiment of the invention, a non-functional allele or susceptible allele present at the Sr27 locus in a triticale plant can be modified to a functional allele that provides resistance to, for example, at least one, two, three, or four varieties of Pgt. In another embodiment of the invention, a non-functional allele present at the Sr27 locus in a triticale plant can be modified to a functional allele that provides resistance to at least one, two, three, or four varieties of Pgt that causes stem rust. For example, a non-functional allele at the Sr27 locus can 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 the DNA in the plant genome can be used to modify the nucleotide sequence of the R gene in a plant in situ manner, such as modifying the nucleotide sequence of a non-functional allele to the nucleotide sequence of a functional allele, which provides resistance to plant pathogens. Such modifications of the DNA in the plant genome include, for example, insertions, deletions, substitutions, and combinations thereof. The insertions, deletions, and substitutions can be made using any method known in the art, such as by genome editing techniques described elsewhere herein or known in the art.

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

Deletion includes deletion of at least one bp from an allele of an R gene of the present invention. As used herein, "deletion" means removing one or more nucleotides or base pairs from DNA. As provided herein, deletion in an allele of an R gene can be 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 displacement comprises replacing at least one bp from the allele of R gene of the present invention with another bp. As used in this article, "displacement" means replacing one or more nucleotides or base pairs from DNA with unequal nucleotides or base pairs. When the displacement comprises two or more nucleotides, the two or more nucleotides can be adjacent or non-adjacent in the DNA sequence dna of the allele. As provided in this article, the displacement in the allele of R gene can be for replacing 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 displacement can be the nucleotide sequence of whole allele or any part thereof (for example, transcription region, 5 ' untranslated region, 3 ' untranslated region, exon or intron).

In certain embodiments of the present invention, the modification of the non-functional allele at the R locus is a homozygous modification. "Homozygous modification" means that the modification is among the two alleles of the R locus in the specific genome of the plant. In other cases, the modification of the R locus gene is heterozygous, that is, the modification is only among one allele of the R locus in the genome of the plant. It is recognized that the plant of the present invention includes, for example, a crop plant having a genome that is diploid or polyploid (for example, tetraploid or hexaploid) (including homopolyploid and allopolyploid). Homopolyploid is an organism with more than two sets of chromosomes all derived from the same species. Allopolyploid is an organism with two or more sets of complete sets of chromosomes derived from different species. Depending on the specific crop plant, 1, 2, 3, 4, 5, 6 or more alleles at the R locus of the plant can be modified by using the disclosed method.

Any method known in the art for modifying the DNA in the plant genome can be used to change the coding sequence of the R gene in a plant in situ manner, such as changing the nucleotide sequence of the homologous susceptible allele to the nucleotide sequence of the allele providing resistance to at least one variety of stem rust. Such methods known in the art for modifying the DNA in the 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. Patent 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 gene modification or gene replacement including homologous recombination can involve inducing single-stranded or double-stranded 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 are endonucleases that have been engineered to make double-stranded 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. Pat. Nos. 7,163,824, 7,001,768, and 6,453,242; Arnould et al., (2006) J Mol Biol 355:443-58; Ashworth et al., (2006) Nature 441:656-9; Doyon et al., (2006) J Am Chem Soc 128:2477-84; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; and Smith et al., (2006) Nucleic Acids Res 34:el49; U.S. Patent Application Publication No. 2009/0133152; and U.S. Patent Application Publication No. 2007/0117128; all of which are incorporated herein by reference in their entirety.

TAL effector nucleases (TALENs) can be used to create 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 create double-strand breaks at specific target sequences in the genomes of plants or other organisms. TAL effector nucleases are created by fusing natural or modified transcription activator-like (TAL) effectors or their functional parts to the catalytic domain of an endonuclease (e.g., FokI). Unique, modular TAL effector DNA binding domains allow the design of proteins that potentially have any given DNA recognition specificity. Therefore, the DNA binding domains of TAL effector nucleases can be modified to recognize specific DNA target sites, and therefore used to create double-strand breaks at desired target sequences. See, WO 2010/079430; Morbitzer et al., (2010) PNAS 10.1073/pnas.1013133107; Scholze and Boch (2010) Virulence 1:428-432; Christian et al., Genetics (2010) 186:757-761; Li et al., (2010) Nuc. Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et al., (2011) Nature Biotechnology 29:143-148; all of which are incorporated herein by reference.

CRISPR/Cas nuclease systems can also be used to create single-strand 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 a DNA segment homologous to the designed RNA. It is possible to design the specificity of the sequence (Cho et al., (2013) Nat. Biotechnol. 31: 230-232; Cong et al., (2013) Science 339: 819-823; Mali et al., (2013) Science 339: 823-826; Feng et al., (2013) Cell Res. 23 (10): 1229-1232).

In addition, ZFNs can be used to create 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 cuts double-stranded DNA at those sequences, thereby generating free DNA ends (Urnov F.D. et al., Nat Rev Genet. 11:636-46, 2010; Carroll D., Genetics. 188:773-82, 2011).

Using specific nucleases (e.g., those described above in this article) to break DNA can increase the homologous recombination rate in the break region. Therefore, the coupling of such effectors and nucleases described above enables the generation of targeted changes in the genome, including additions, deletions, substitutions and other modifications.

Mutation breeding can also be used in the methods provided herein. Mutation breeding methods can involve, for example, exposing plants or seeds to a mutagen, particularly a chemical mutagen such as ethyl methanesulfonate (EMS), and selecting plants with the desired modification in the Sr27 gene. However, other mutagens can be used in the methods disclosed herein, including, but not limited to: radiation, such as X-rays, gamma-rays (e.g., cobalt 60 or cesium 137), neutrons (e.g., nuclear fission products produced by uranium 235 in an atomic reactor), beta-radiation (e.g., emitted from radioisotopes such as phosphorus 32 or carbon 14), and ultraviolet radiation (preferably, 2500 to 2900 nm); and chemical mutagens, such as base analogs (e.g., 5-bromouracil), related compounds (e.g., 8-ethoxycaffeine), antibiotics (e.g., streptomycin), alkylating agents (e.g., sulfur mustard, nitrogen mustard, epoxides, ethyleneamines, sulfates, sulfonates, sulfones, lactones), azides, hydroxylamines, nitrites, or acridines. Further details of mutation breeding can be found in "Principals of Cultivar Development" Fehr, 1993 Macmillan Publishing Company (the disclosure of which is incorporated herein by reference).

The nucleic acid molecules, expression cassettes, vectors and polynucleotide constructs of the present invention can be used to transform any plant species, including but not limited to monocots and dicots. The preferred plant of the present invention is a wheat plant. Examples of other plant species of interest include, but are not limited to, peppers (Capsicum spp.; e.g., Capsicum annuum, C. baccatum, C. chinense, C. frutescens, C. pubescens, etc.), tomatoes (Lycopersicon esculentum), tobacco (Nicotiana tabacum), eggplant (Solanum melongena), petunias (Petunia spp., e.g., Petunia xhybrida or Petunia hybrida), peas (Pisum sativum), beans (Phaseolus vulgaris), corn or maize (Zea mays), Brassica species (Brassica sp.) (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species that can be used as a source of seed oils), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millets (e.g., pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), foxtail millet (Setaria italica), seeds (Eleusinecoracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), soybean (Glycinemax), Ethiopian teff (Eragrostis tef), tobacco, potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus carica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), queensland nut (Macadamia integrifolia), almond (Prunusamygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), palm, oats, barley, vegetables, ornamentals and conifers.

In certain embodiments of the invention, the preferred plants are cereal plants. Such cereal plants of the invention are herbaceous plants (i.e., Poaceae) grown for the edible components of their grains or kernels (i.e., seeds), including, for example, wheat, triticale, rye, barley, oats, corn, sorghum, millet, and rice. In certain other embodiments of the invention, the preferred plants are cereal plants, but do not include triticale plants.

In some embodiments of the present invention, plant cells are transformed with polynucleotide constructs encoding the R protein of the present invention. As used in this article, the term "expression" refers to the biosynthesis of a gene product, including the transcription and/or translation of the gene product. "Expression" or "production" of 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 "expression" or "production" of 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 an R protein are described elsewhere in this article.

The use of the term "DNA" or "RNA" herein is not intended to limit the present 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 composed 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 to reference nucleic acids, and which are metabolized in a manner similar to reference nucleotides. Examples of such analogs include but are not limited to: phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs). 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, etc. Further, one of ordinary skill in the art will understand that the nucleotide sequences disclosed herein also include complements of the exemplified nucleotide sequences.

The present invention relates to compositions and methods for enhancing plant resistance to plant diseases, and in particular to compositions and methods for enhancing plant resistance to stem rust caused by Pgt. "Disease resistance" means that the plant avoids disease symptoms as a consequence of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and associated disease symptoms, or alternatively, disease symptoms caused by the pathogens are minimized or mitigated.

As used herein, "race" refers to any one of a group of laboratory isolates or fungal individuals found in the field that share a similar virulence phenotype for a range of different wheat resistance lines and may have been derived through clonal propagation.

As used herein, an "isolate" of Pgt refers to a Pgt line that was initially isolated as spores collected from infected plants in the field or in a laboratory/greenhouse setting. Subsequently, such an "isolate" is maintained in pure form by infecting and re-isolating spores from susceptible plants and storing the spores.

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

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

Example

Example 1: Cloning of the Sr27 stem rust resistance gene

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

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

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

Materials and methods

Plant material and mutant DNA preparation

Seeds of the triticale line wild-type Coorong (Coorong carries Sr27) were treated with ethyl methanesulfonate (EMS) following the experimental protocol described by Mago et al. ((2015) Nature Plants 1, 15186). Kill 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 washed thoroughly with water and sown in large pots (40 seeds/30 cm pot) in a greenhouse with daylight and 23°C day and 15°C night temperatures. Individual ears from each M1 plant were threshed separately, and the M2 families from each plant were sown in trays (30 M2 families/tray). Each tray also included resistant (Coorong) and susceptible (Rongcoo, triticale) controls. M2 families obtained from each M1 plant as single ear progeny were tested for stem rust response by inoculation with Puccinia graminearum wheat race (Pgt) isolate Pgt21-0. Individual plants from the segregating progeny were grown and the progeny were tested. Homozygous susceptible mutants and resistant sibling pairs were recovered from these progenies.

Genomic DNA was extracted from wild-type Coorong triticale and homozygous susceptible mutants following the protocol described by Yu et al. (2017). The quality and quantity of the extracted DNA were 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 NLRs was performed by Arbor Biosciences (Ann Arbor, USA) following the MYbaits protocol using an improved version of the previously published Triticeae bait library available at github.com/steuernb/MutantHunter. Library construction was performed by following the TruSeq RNA protocol v2. All enriched libraries were sequenced on a HiSeq 2500 (Illumina, CA, USA) using 250 bp paired-end reads and SBS chemistry.

MutantHunter

To identify Sr27 contigs from mutants, the MutantHunter pipeline method of Steuernagel et al. ((2015) Bioinformatics 31: 1665-7) was followed. Initial read sequencing data from wild-type Coorong and mutants were first trimmed for quality using Trimmomatic v0.38 (Bolger et al., (2014) Bioinformatics 30: 2114-20) with parameters ILLUMINACLIP:novogene_indexed_adapters.fa:2:30:10:8:TRUE,LEADING:28,TRAILING:28,MINLEN:20. Then, data from wild-type Coorong were assembled from scratch using CLCGW v11.0.1 as paired ends with a length score of 0.95 and a similarity score of 0.98, and the remaining parameters were default. Overlaps shorter than 1 kb were ignored from the final assembly using a custom script. The annotation of NBS-LRR motifs was created by using the program NLR-Parser (Steuernagel et al., (2015) Bioinformatics 31: 1665-7). By using BWA v0.7.15 (Li and Durbin (2009) Bioinformatics 25: 1754-1760), the trimmed data of each mutant and wild type were mapped to the de novo assembly. Samtoolsv1.7.0 (Li et al., (2009) Bioinformatics 25: 2078-9) was used for the processing of the resulting SAM files to retain only the read mappings in the correct pairing with parameter -f 2, then duplicates were removed and pileup files were generated using parameters -BQ0 and -aa. SNV calls and subsequent candidate identification were performed by using the following script from the MuTrigo pipeline ( https://github.com/TC-Hewitt/MuTrigo ). Before downstream analysis, Noisefinder.pyc (with default parameters) was used to detect overlapping group regions with high SNP levels in wild-type data (which indicated poor assembly or read alignment) and masked. Potentially mutated nucleotide positions were recorded from pileup files using SNPlogger.pyc (with parameter -d20). SNPtracker.pyc was used to retrieve overlapping groups containing polymorphisms in two or more mutants, wherein the default parameters were used plus parameter -s C\>TG\>A indel. This was converted into considering only polymorphisms with a minimum of 80% mutant allele frequency, and only with respect to insertion, deletion, or C to T or G to A SNV (which does not share the same position with another mutant or wild type) selection was performed. Candidate gene contigs were aligned to the chromosome-scale reference assembly of rye inbred line 'Lo7' (Rabanus-Wallace et al., (2019) bioRxiv:2019.12.11.869693) and the common wheat cv. Chinese Spring reference (CSv1) assembly IWGSCRefSeq v1.0 (Appels et al., (2018) Science 361(6403):eaar7191) using BLAST v2.7.1 (Altschul et al., (1990) J. Mol. Biol. 215:403-10). De novo RNA transcript assemblies were generated from Illumina RNAseq data from Pgt-infected Coorong seedlings (Upadhyaya et al., (2015) Front Plant Sci. 5:759) (downloaded from NCBI-SRA SAMN07836894, PRJNA415866) using CLCGW v11.0.1 (similarity score of 0.98, and remaining parameters set to default).

Confirmation of Sr27 gene structure

Primers were designed based on the genomic sequences of two non-overlapping contigs numbered 2413 and 5723 (Table 2). Primers were used to amplify the non-overlapping region between contigs numbered 5723 and numbered 2413 from the genomic DNA of wild-type Coorong with Sr27c5723ExtF1 and Sr27c2413ExtR1. Primers were used to amplify the full-length gene from the genomic DNA of wild-type and mutants for subsequent sequence comparison. According to the manufacturer's instructions, Phusion high-fidelity DNA polymerase (NEB, USA) was used to perform all PCRs. The full-length Sr27 genomic sequence is shown in SEQ ID NO: 1. All mutants used in the RenSeq pipeline were re-confirmed by Sanger sequencing. The predicted exon-intron structure was confirmed by amplifying the full cDNA from the RNA of the black wheat cultivar Coorong. Total RNA was extracted using the PureLink™ RNA Mini Kit (Invitrogen Cat. No. 12183025, Thermo Fisher Scientific, MA USA) according to the manufacturer's instructions. cDNA synthesis was performed using the protocol described by the manufacturer (SMART ^™ PCR cDNA Synthesis Kit, Cat. No. 634902, Takara Bio USA Inc. (formerly Clontech Laboratories Inc.)).

Selection of Pgt virulence mutants

Seedlings of black wheat cultivar Coorong (20 pots [15 cm], 6-8 plants/pot) at the 2-3 leaf stage were inoculated with ~100 g of stem rust isolate Pgt21-0 (Upadhyaya et al., (2015) Front Plant Sci. 5:759). The inoculated plants were incubated in a humid chamber maintained at 23°C for 48 hours. After this period, the plants were moved to a greenhouse maintained at 23°C/18°C (day/night) under natural daylight. Plants were screened twice at 14 and 20 days after inoculation for any susceptible infection sites that showed large rust spots (pustule) development (infection types 3, 4) typical for susceptible interactions. More than 10 mutant rust spots were detected, and three of these were collected and re-inoculated on susceptible wheat line Morocco for individual amplification. After amplification, the virulence of each mutant to Sr27 was confirmed by reinfecting wild-type Coorong plants. One mutant was further tested on a full Australian differential set for 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 spectrum of virulence properties as Pgt21-0, except for a single additional virulence to Sr27.

Identification of the AvrSr27 gene by whole-genome sequence analysis

DNA was extracted from uredia of mutant Pgt lines using the CTAB method (Rogers et al., (1989) Can. J. Bot. 67: 1235-1243) with some described modifications (Upadhyaya 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, CA, USA). DNA library preparation and Illumina sequencing were performed by the Australian Genome Research Facility (AGRF) on a HiSeq2500 (250 bp PE reads, mutants M1 and M2) or MiSeq (300 bp PE reads, mutant M3) platform, and approximately 20 million reads were obtained for each mutant. Sequence reads are input to CLCGenomics Workbench (CLCGW) version 10.0.1 or later (QIAGEN), filtered, and trimmed to remove low-quality ends, where adapters and low-quality reads are sequenced (Trim uses a quality score of 0.01, and the maximum number of ambiguities allowed is 2). Reads are mapped to the nuclear phase chromosome level assembly type Pgt21-0 reference (Li et al., (2019) Nature Commun.10, 5068) annotated with 37036 gene models, where high stringency settings (similarity score 0.98 and length score 0.95) are used. Variant calls for each sample of the reference are performed using the "Basic Variant Detection" program, with the following parameters: ignoring nonspecific matches; minimum coverage 10; significance 1.0%; minimum variant count 2; including disconnected pairs. The program "Compare variants withingroup" in CLCGW is used to identify specific variants and shared variants for each sample. The CLCGW tool "AminoAcid Changes" was used to predict non-synonymous variants in secreted protein genes and was manually curated by visual inspection of read mapping tracks in CLCGW. The "Create Statistics for Target Regions" program was used to extract read coverage statistics (average read depth and coverage percentage) for annotated genes.

Example 2: Identification of AvrSr27 effectors recognized by Sr27

In order to identify spontaneous mutants of Pgt with virulence gain for Sr27, the inventors inoculated the avirulent isolate Pgt21-0 into the seedlings of the black wheat cultivar 'Coorong' carrying Sr27. Three large single rust spots were selected, purified individually, and then inoculated into Coorong to confirm its virulence phenotype (Fig. 3). The inventors extracted genomic DNA from each mutant and obtained Illumina sequence data. Illumina reads were mapped to Pgt21-0 haplotype-analyzed genome assemblies (Li et al., (2019) Nature Commun.10, 5068) to identify potential mutations. The first screening for identifying SNPs causing amino acid changes in secreted protein genes did not find any genes with such mutations in more than one Pgt21-0 mutant strain. Then, the inventors looked for the loss of read coverage, which served as an indicator of potential deletion mutations. Each of the Pgt21-0 mutant strains shows a large number of genes with zero read coverage, all located at one end of chromosome 2B. Visualization of the read mapping to chromosome 2B reveals that each of the three mutants contains independent and overlapping deletions that cover a portion of chromosome 2B, with the smallest size being 196 kilobase pairs (kbp) (Fig. 4). This region contains 50 annotated genes, five of which are predicted to encode secretory proteins. Previously, two Australian Pgt isolates (34-2,12 and 34-2,12,13) were sequenced, belonging to a clonal lineage derived from Pgt21-0 and having evolved toxicity for Sr27 in the wild (Upadhyaya et al., (2015) Front Plant Sci.5:759; Zhang et al., (2017). Phytopath.107:1032-1038). The analysis of read coverage reveals that the two isolates each include a small deletion of 13kbp (Fig. 5A), which spans two (PGT21_006532 and PGT21_006593) of the secretory protein gene in the region together with a single adjacent gene on the distal side. In the two isolates, no other genes in the region include any changes. In Pgt21-0 and 34-2,12, the inventors confirmed the presence of the deletion in the genome by PCR amplification of the missing border (Fig. 5B). The sequence data generated from seven South African isolates (Lewis et al., 2018) were also checked, and these seven South African isolates were part of the clonal pedigree identical to Pgt21-0 (Li et al., (2019) Nature Commun.10,5068; Visser et al., (2019) Phytopath.109:133-144) but evolved independently in South Africa. Four of these isolates are virulent for Sr27 (Visser et al., (2009) Mol. Plant Path. 10: 213-222) and must have evolved this phenotype independently of Australian isolates. Three of these virulent isolates (SA03, SA05 and SA07) include the same ~ 14kbp deletion overlapping with the deletion in 34-2,12 and spanning the two candidate secretory protein genes plus another gene on the proximal side, while the fourth isolate (SA06) includes an independent deletion of 10kbp spanning only the two secretory protein genes (Fig. 5A). The remaining three avirulent isolates (SA01, SA02, SA04) from South Africa include sequences similar to Pgt21-0 in this region, as also done for three other Australian isolates (PGT098, PGT194, PGT326) of this pedigree that are avirulent for Sr27. The phylogenetic analysis of the clone-derived isolate group places each of these deletion events in the separate branches of the pedigree, which is consistent with the appearance of three independent mutations for the virulence of Sr27 during the diversification of the pedigree (data not shown). The disappearance of these two secreted protein gene candidates in three independent field-derived virulence isolates provides strong evidence to prove that at least one of these genes confer the avirulence phenotype. These two genes are closely related to each other, and the secreted proteins with 144 amino acids predicted by encoding are named AvrSr27-1 (SEQ ID NO:6) and AvrSr27-2 (SEQ ID NO:8) (Fig. 6).

Example 3: Transient expression verification of Sr27 function

In order 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 gene luciferase. In this assay, recognition between resistance and avirulence genes leads to 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). Compared with the expression of Sr27 or AvrSr27 genes alone in protoplasts, the co-expression of each of the Sr27 candidate gene and the AvrSr27 gene variant leads to a strong reduction in luciferase activity (Fig. 7). This is a specific recognition response, because 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), no loss of reporter gene expression was seen. This confirms that the cloned Sr27 sequence confers specific recognition of the AvrSr27 effector from Pgt and is therefore responsible for resistance to Pgt isolates expressing the AvrSr27 gene.

Therefore, the inventors have 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, which located it within a 196 kbp deletion on chromosome 2B of Pgt21-0. Subsequent analysis found two genes that were independently deleted in three different Pgts that conferred virulence for Sr27. The availability of cloned effectors recognized by Sr27 facilitates genetic screening of wheat stem rust isolates for the presence of the effector and therefore its predicted virulence for Sr27.

Materials and methods

Construct generation

The AvrSr27 gene sequence (shown in SEQ ID NOs: 5, 7, and 9) in which the predicted signal peptide was excluded and replaced with a single methionine start codon was amplified from cDNA from purified Pgt21-0 haustoria using Phusion ^High -Fidelity DNA Polymerase (Thermo Scientific, Wilmington, DE) and cloned into The full-length Sr27 cDNA was also cloned into For wheat protoplast transfection, use Technology (Life Technologies ^TM ) was used 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 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 described previously.

Protoplast expression assay

Wheat seedlings were grown in a growth chamber in the shade at 25 ° C with a photoperiod of 16 hours of light for 7-9 days. Wheat protoplast isolation and transformation were performed as described (Arndell et al., (2019) BMC Biotechnol. 19: 71). High-quality plasmid DNA was isolated by using the Qiagen Endo-free Plasmid Maxi kit (Cat. No. 12362). DNA concentration was adjusted to 1 μg/μl to 4 μg/μl, and 10 μg of each plasmid was used in the co-transformation experiment. After incubation at 23 ° C in the dark for 24 hours, the manufacturer's instructions were followed, using the Luciferase Assay System (Promega, Catalog No. E1501), as described by Saur et al. ((2019) Plant Methods 15, 118) to perform luciferase activity assays.

Example 4: Comparison of Sr27 with other resistance genes

According to the best BLAST hit against IWGSC CS ref v1.0, the position of the closest homolog of the Sr27 candidate in the Chinese Spring reference v1.0 is on chromosome 6B (see Example 1). The inventors further expanded the list of homologous sequences by performing BLAST against the non-redundant protein database at NCBI (data not shown). The top hit was an unnamed protein product from Triticum cylindraceum durum subsp. (GenBank accession number VAI63620.1) and had 90.5% amino acid identity with the Sr27 protein. In order 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 compared the Sr27 protein with 16 CNL-type R genes identified from wheat and performed a phylogenetic analysis as described below (Figure 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 evolutionary analyses were 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 wheat cv. 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) "Wheat (Triticum aestivum L.) transformation using immature embryos", Wang K (ed.), Agrobacterium Protocols, Vol. 1, 3rd edition, Springer, New York, pp. 189-198). Wheat plants of the cultivar Fielder were propagated under greenhouse growth conditions using a 24°C, 16-hour light/18°C, 8-hour dark growth regime, and the plants were fertilized biweekly with Aquasol (Yates, Clayton, Australia). Wheat ears were tagged during the flowering period and harvested 12-14 days after the flowering period for transformation experiments, as described by Ishida et al. (supra) and as adapted at CSIRO, Canberra, Australia (Richardson et al., (2014) Plant Cell Tiss. Org. 119: 647-659). In short, the seeds were surface sterilized in 0.8% sodium hypochlorite solution for 10 minutes. The embryos were removed from the seeds under sterile conditions and co-cultured on WLS-AS medium (Ishida et al., 2015) in the dark with the Agrobacterium strain containing the binary construct of Sr22 and Sr45 genomic fragments for 2 days. After co-cultivation, the embryo axis was excised with a scalpel, and then the explant was transferred to WLS-Res medium and placed in the dark at 24 ° C. After 5 days, the explant was transferred to WLS-H15 callus induction medium containing 15mg/ml hygromycin (Hyg) for callus formation. After two weeks, the resulting callus was cut into two parts and placed on WLS-H30 (30mg/l Hyg) in the dark for 3 weeks. Then, the callus was regenerated on LSZ-H15 (15mg/l Hyg) medium at 200μmol m ^-2 s ^-1 light at 24 ° C. Shoots were transferred to LSF-H15 (15 mg/l Hyg) medium to allow root formation, and once a strong root system had developed, the plants were transferred to soil and maintained in the greenhouse.

The TO lines were screened by PCR with primers for the transgene (Sr27c5723 F x Sr27c5723 R, Table 2), and three positive lines (PC311.3, PC311.17, and PC311.18) were identified. T1 seeds were harvested from the three positive lines and a fourth line (PC311.16) lacking the Sr27 transgene. Eleven or twelve progeny of each line were grown in a growth chamber (23°C, 16 hours of light) with a control line (see Table 1). Two-week-old seedlings were inoculated with Pgt varieties 98-1, 2, (3), (5), 6 (to which Sr27 confers resistance). Rust responses were evaluated after 10-15 days (Table 1). For PC311.3 and PC311.17, all T1 progenies showed strong resistance to Pgt, with infection types characterized by tiny necrotic infection spots but without rust formation, which is similar to the phenotype observed in the wheat line WRT258.5 carrying Sr27 (Figure 9). The T1 progeny of the third Sr27 transgenic line PC311.18 segregated for resistance, with 10 resistant progenies and 1 susceptible progeny (Table 1). PCR analysis confirmed that the single PC311.18 T1 progeny susceptible plant did not contain the Sr27 transgene, while the resistant T1 progeny contained Sr27. In contrast, the susceptible lines Chinese Spring and Fielder showed high infection types with large summer spores and no chlorosis or necrosis (see Figure 9), as did the PC311.16 lines recovered from transformation that did not contain the Sr27 transgene (Figure 9). These data confirm that the Sr27 transgene confers stem rust resistance in transgenic wheat when expressed via the ubiquitin promoter. T1 progeny of each of the three positive lines were further tested by inoculation with leaf rust (P. triticina) isolates 76-1, 3, 7, 9, 10, 12, 13 and stripe rust (P. striiformis) isolate 198E16 A+J+T+17+. All progeny were fully susceptible to the leaf rust and stripe rust isolates, confirming that the Sr27 transgene confers specific resistance to stem rust and that the resistance is not due to nonspecific activation of an effective defense response against the rust pathogen.

Table 1. Results of stripe rust resistance assay for Sr27 transgenic wheat lines

Example 6: Sequences of primers used in cloning Sr27

In Table 2 below, the nucleotide sequences of the primers used in cloning Sr27 from triticale cv. Coorong (described in the Examples above) are provided. The nucleotide sequences of these primers are provided as illustrative examples (for the purpose of clarity of understanding) and not as limitations.

Table 2. Primer sequences

The articles "a" and "an" are used herein to refer to one or to more than one (ie, 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 "comprising" or "including" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

All publications and patent applications mentioned in this specification indicate the level of expertise in the field to which the invention pertains. All publications and patent applications are incorporated herein 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 atcggggagga 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 tacttacta 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

gaaaacatattttgtttagt 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 tttctttatca 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 aagtcacattttagtattag 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 gtttctttatc 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 attattatttt acggccatgc cgaagatgag gctcccatca ttcctgcttt 3540

cgatttcggg atcgaaaacc tgtccagcct cactactttc aaatgtcacc taggttatgg 3600

gcctatggca acgaaaattg ttgacgctgt aaaggcttct ctggacagag tagttagcgc 3660

acatcccaac caccttatactc taatcttcac ttattgttgt gtgttttgta agagttatga 3720

ctgtggtggt cgatgccttc tgtctagaga tcttcagtca tcctccgaat ctacttga 3778

<210> 5

<211> 435

<212> DNA

<213> 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> 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> 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> 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

atgaacccaaggctgagttg 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 tctttatgtg 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 shown in SEQ ID NO:3, and optionally, wherein said 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 said nucleic acid molecule is capable of conferring resistance to stem rust comprising said A wheat plant of a nucleic acid molecule, and optionally, wherein said nucleotide sequence does not occur naturally; 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 imparting resistance to stem rust Sex conferring to a wheat plant comprising said nucleic acid molecule, and optionally, wherein said nucleotide sequence does not occur naturally. 2. The nucleic acid molecule of claim 1, wherein said 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 said coiled-coil domain, said nucleotide binding domain and said leucine-rich repeat domain comprises the corresponding Domains have amino acid sequences that are at least 95%, 96%, 97%, 98%, 99% or 100% identical. 5. An expression cassette comprising a nucleic acid molecule according to any one of claims 1-4 and an operably linked heterologous promoter. 6. A vector comprising a nucleic acid molecule according to any one of claims 1-4 or an expression cassette according to 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 a nucleic acid molecule according to any one of claims 1-5, an expression cassette according to claim 5 or a vector according to any one of claims 6-8. 10. The host cell of claim 9, wherein said 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 said 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 said plant is a cereal plant. 14. The plant of claim 12 or 13, wherein said plant is wheat. 15. A transgenic plant or seed comprising a polynucleotide construct stably incorporated into its genome, said 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, and optionally, wherein said 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 said nucleic acid molecule is capable of conferring resistance to stem rust comprising said A wheat plant of a nucleic acid molecule, and optionally, wherein said nucleotide sequence does not occur naturally; 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 imparting resistance to stem rust Sex conferring to a wheat plant comprising said nucleic acid molecule, and optionally, wherein said 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 said coiled-coil domain, said nucleotide binding domain, and said leucine-rich repeat domain is comprised as in SEQ ID NO:3 The corresponding domains have amino acid sequences that are at least 95%, 96%, 97%, 98%, 99% or 100% identical. 18. The transgenic plant or seed of any one of claims 15-17, wherein said polynucleotide construct further comprises a promoter operably linked for expression of said 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 promoter. 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 said transgenic plant or seed comprises enhanced resistance to at least one variety of Puccinia graminis f.sp. tritici relative to control wheat plants resistance to wheat stem rust. 22. The transgenic plant or seed of claim 20 or 21, wherein said 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 increasing the resistance of wheat plants to wheat stem rust, said method comprising introducing into at least one cell of a wheat plant a polynucleotide construct comprising Nucleotide sequences selected from the group: (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 said nucleic acid molecule is capable of conferring resistance to stem rust comprising said the wheat plant of 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 imparting resistance to stem rust Sex confers 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 said coiled-coil domain, said nucleotide binding domain, and said leucine-rich repeat domain comprises the corresponding structure in SEQ ID NO:3 Domains have amino acid sequences that are at least 95%, 96%, 97%, 98%, 99% or 100% identical. 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 said polynucleotide construct further comprises a promoter operably linked for expression of said nucleotide sequence in plants. 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 son. 31. The method of any one of claims 21-30, wherein the wheat plant comprising the polynucleotide construct comprises an enhanced expression for at least one species of Puccinia graminearum racee tritici relative to control wheat plants. resistance to wheat stem rust. 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 an R protein 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. The seed of the wheat plant of claim 34, wherein said seed comprises said polynucleotide construct. 36. A method for limiting wheat stem rust in crop production, said method comprising planting a wheat seed according to any one of claims 20-23 and 35, and cultivating said wheat seed under conditions favorable to the growth and development of the wheat plant. wheat plant. 37. The method of claim 36, further comprising harvesting at least one seed from said 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 shown 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 shown 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 said coiled-coil domain, said nucleotide binding domain and said leucine-rich repeat domain comprises the corresponding structure in SEQ ID NO:3 Domains have amino acid sequences that are at least 95%, 96%, 97%, 98%, 99% or 100% identical. 43. A method for identifying wheat plants displaying newly conferred or enhanced resistance to wheat stem rust, said method comprising detecting in said wheat plants the presence of the R gene Sr27. 44. The method of claim 43, wherein the presence of the R gene is detected by detecting at least one marker within Sr27. 45. The method of any one of claims 43 or 44, wherein Sr27 comprises or consists of the following nucleotide sequence: 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 for detection by Immunological assays for 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 non-transgenic triticale plants with enhanced resistance to stem rust, said method comprising modifying a non-functional allele of the resistance gene Sr27 in a triticale plant or at least one cell thereof so that A functional allele is produced, 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 at least one base Base pair insertions, deletions and substitutions. 52. The method of claim 50 or 51, wherein said modifying a non-functional 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) 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.