CA-2994714-C - WHEAT STEM RUST RESISTANCE GENES AND METHODS OF USE

Abstract

Compositions and methods and for enhancing the resistance of wheat plants to wheat stem rust caused by Puccinia graminis f. sp. tritici are provided. The compositions comprise nucleic acid molecules encoding resistance (R) gene products and variants thereof and plants, seeds, and plant cells comprising such nucleic acid molecules. The methods for enhancing the resistance of a wheat plant to wheat stem rust comprise introducing a nucleic acid molecule encoding an R gene product into a wheat plant cell. Additionally provided are methods for using the wheat plants in agriculture to limit wheat stem rust.

Inventors

• Evans Lagudah
• Sambasivam Periyannan
• Burkhard Steuernagel
• Kamil Witek
• Brande Wulff

Assignees

• COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION
• TWO BLADES FOUNDATION

Dates

Publication Date

20260505

Application Date

20160803

Priority Date

20150804

Claims (1)

1. CLAIMS: 1. A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 19, 22, 5 25, or 34; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, or 35; (c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 21, 24, 27, or 36; 10 ( d) a nucleotide sequence having at least 85% sequence identity to at least one of the nucleotide sequences set forth in (a) or a nucleotide sequence having at least 97% sequence identity to at least one of the nucleotide sequences set forth in (c), wherein the nucleic acid molecule, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant; 15 ( e) a nucleotide sequence encoding an amino acid sequence having at least 94% sequence identity to at least one amino acid sequence set forth in (b ), wherein the nucleic acid molecule, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant; (f) a nucleotide sequence, having at least 85% sequence identity to at 20 least one of the nucleotide sequences set forth in (a) or (c), or encoding an amino acid sequence having at least 85% sequence identity to at least one amino acid sequence set forth in (b), wherein the nucleotide sequence encodes a protein comprising a leucine-rich repeat domain which comprises an amino acid selected from the group consisting of serine (S) at the amino acid position corresponding to amino acid 607 of SEQ ID NO: 2, tyrosine (Y) at 25 the amino acid position corresponding to amino acid 650, of SEQ ID NO: 2, serine (S) at the amino acid position corresponding to amino acid 651 of SEQ ID NO: 2, and glycine (G) at the amino acid position corresponding to amino acid 657 of SEQ ID NO: 2, and wherein the nucleic acid molecule, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant; 30 (g) (h) SEQ ID NO: 50; the nucleotide sequence set forth in SEQ ID NO: 49; a nucleotide sequence encoding the amino acid sequence set forth in Date Re<;ue/Date Received 2023-11-28 84182801 (i) the nucleotide sequence set forth in SEQ ID NO: 51; G) a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence set forth in (g), or a nucleotide sequence having at least 93% sequence identity to the nucleotide sequence set forth in (i ), wherein the nucleic acid molecule, when 5 expressed in a wheat plant, confers resistance to stem rust to the wheat plant; and (k) a nucleotide sequence encoding an amino acid sequence having at least 93% sequence identity to the amino acid sequence set forth in (h), wherein the nucleic acid molecule, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant. 10 2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is an isolated nucleic acid molecule. 3. The nucleic acid molecule of claim 1 or 2, wherein the nucleic acid molecule of ( d), ( e ), (f), U), or (k) encodes a protein comprising a coiled-coil domain, a nucleotidebinding domain, and a leucine-rich repeat domain. 4. The nucleic acid molecule of claim 3, wherein at least one of the coiled-coil domain, the nucleotide-binding domain, and the leucine-rich repeat domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the corresponding domain in SEQ ID NO: 2 or 50. 5. The nucleic acid molecule of claim 3 or 4, wherein the nucleic acid molecule 20 comprises: the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 19, 20, 22, 23, 25, 26, 34, 35, 49 or 50; or a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, 35, or 50. 6. An expression cassette comprising the nucleic acid molecule of any one of claims 1-5 and an operably linked heterologous promoter. 25 7. A vector comprising the nucleic acid molecule of any one of claims 1-5 or the expression cassette of claim 6. 8. A vector of claim 7, further comprising an additional wheat stem rust resistance gene. Date Re<;ue/Date Received 2023-11-28 84182801 9. The vector of claim 8, wherein the additional wheat stem rust resistance gene is selected from the group consisting of Sr26, Sr32, Sr33, Sr39, Sr40, Sr47, and Sr50. 10. A host cell transformed with the nucleic acid molecule of any one of claims 1-5, the expression cassette of claim 6, or the vector of claim 7, 8 or 9. 5 11. The host cell of claim 10, wherein the host cell is a plant cell, a bacterium, a fungal cell, or an animal cell. 12. The host cell of claim 10 or 11, wherein the host cell is not Triticum boeoticum or Triticum monococcum. 13. A plant cell transformed with the nucleic acid molecule of any one of claims 10 1-5, the expression cassette of claim 6, or the vector of claim 7, 8 or 9. 14. The plant cell of claim 13, wherein the plant cell is a wheat plant cell. 15. The plant cell of claim 13 or 14, wherein the wheat plant cell is not a Triticum boeoticum or Triticum monococcum plant cell. 16. A transgenic plant cell comprising stably incorporated in its genome a 15 polynucleotide construct comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 19, 22, 25, 34, or 49; (b) a nucleotide sequence encoding the amino acid sequence set forth in 20 SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, 35, or 50; (c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 21, 24, 27, 36, or 51; ( d) a nucleotide sequence having at least 85% sequence identity to at least one of the nucleotide sequences set forth in (a) or (c), wherein the polynucleotide 25 construct, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant; and ( e) a nucleotide sequence encoding an amino acid sequence having at least 85% sequence identity to at least one amino acid sequence set forth in (c), wherein the Date Re<;ue/Date Received 2023-11-28 84182801 polynucleotide construct, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant. 17. The transgenic plant cell of claim 16, wherein the nucleic acid molecule of (d) or (e) encodes a protein comprising a coiled-coil domain, a nucleotide-binding domain, 5 and a leucine-rich repeat domain. 18. The transgenic plant cell of claim 17, wherein at least one of the coiled-coil domain, the nucleotide-binding domain, and the leucine-rich repeat domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the corresponding domain in SEQ ID NO: 2 or 50. 19. The transgenic plant cell of claim 17 or 18, wherein the leucine-rich repeat domain comprises an amino acid selected from the group consisting of serine (S) at the amino acid position corresponding to amino acid 607 of SEQ ID NO: 2, tyrosine (Y) at the amino acid position corresponding to amino acid 650, of SEQ ID NO: 2, serine (S) at the amino acid position corresponding to amino acid 651 of SEQ ID NO: 2, and glycine (G) at 15 the amino acid position corresponding to amino acid 657 of SEQ ID NO: 2. 20. The transgenic plant cell of any one of claims 16-19, wherein the polynucleotide construct comprises the nucleotide sequence of any one of (b)-(e) and further comprises a promoter operably linked for the expression of the nucleotide sequence in a plant. 21. The transgenic plant cell of claim 20, wherein the promoter is selected from the group consisting of pathogen-inducible, constitutive, tissue-preferred, wound-inducible, and chemical-regulated promoters. 22. The transgenic plant cell of any one of claims 16-21, wherein the transgenic plant cell is a wheat plant cell. 23. The transgenic plant cell of claim 22, wherein a transgenic plant or seed comprising the transgenic plant cell, comprises enhanced resistance to wheat stem rust caused by at least one race of Puccinia graminis f. sp. tritici, relative to a control wheat plant. Date Re<;ue/Date Received 2023-11-28 84182801 24. The transgenic plant cell of claim 22 or 23, wherein the polynucleotide construct comprises at least two nucleotide sequences encoding an R protein for wheat stem rust. 25. The transgenic plant cell of claim 24, wherein each of the at least two 5 nucleotide sequences encoding an R protein for wheat stem rust encodes a different R protein for wheat stem rust. 26. A method for enhancing the resistance of a wheat plant to wheat stem rust, the method comprising introducing a polynucleotide construct into at least one wheat plant cell, the polynucleotide construct comprising a nucleotide sequence selected from the group 1 0 consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 19, 22, 25, 34, or 49; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, 35, or 50; (c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 21, 24, 27, 36, or 51; ( d) a nucleotide sequence having at least 85% sequence identity to at least one of the nucleotide sequences set forth in (a) or (c), wherein the polynucleotide construct, when expressed in a wheat plant, confers resistance to stem rust to the wheat 20 plant; and ( e) a nucleotide sequence encoding an amino acid sequence having at least 85% sequence identity to at least one amino acid sequence set forth in (c), wherein the polynucleotide construct, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant. 27. The method of claim 26, wherein the nucleic acid molecule of (d) or (e) encodes a protein comprising a coiled-coil domain, a nucleotide-binding domain, and a leucine-rich repeat domain. 28. The method of claim 27, wherein at least one of the coiled-coil domain, the nucleotide-binding domain, and the leucine-rich repeat domain comprises an amino acid Date Re<;ue/Date Received 2023-11-28 84182801 sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the corresponding domain in SEQ ID NO: 2 or 50. 29. The method of claim 27 or 28, wherein the leucine-rich repeat domain comprises an amino acid selected from the group consisting of serine (S) at the amino acid 5 position corresponding to amino acid 607 of SEQ ID NO: 2, tyrosine (Y) at the amino acid position corresponding to amino acid 650, of SEQ ID NO: 2, serine (S) at the amino acid position corresponding to amino acid 651 of SEQ ID NO: 2, and glycine (G) at the amino acid position corresponding to amino acid 657 of SEQ ID NO: 2. 30. The method of any one of claims 26-29, wherein the polynucleotide 10 construct is stably incorporated into the genome of the plant cell. 31. The method of any one of claims 26-30, wherein the wheat plant cell is regenerated into a wheat plant comprising in its genome the polynucleotide construct. 32. The method of any one of claims 26-31, wherein the polynucleotide construct comprises the nucleotide sequence of any one of (b )-( e) and further comprises a 15 promoter operably linked for the expression of the nucleotide sequence in a plant. 33. The method of claim 32, wherein the promoter is selected from the group consisting of pathogen-inducible, constitutive, tissue-preferred, wound-inducible, and chemical-regulated promoters. 34. The method of any one of claims 26-33, wherein the wheat plant comprising 20 the polynucleotide construct comprises enhanced resistance to wheat stem rust caused by at least one race of Puccinia graminis f. sp. tritici, relative to a control wheat plant. 35. The method of any one of claims 26-34, wherein the polynucleotide construct comprises at least two nucleotide sequences encoding an R protein for wheat stem rust. 36. The method of claim 35, 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. Date Re<;ue/Date Received 2023-11-28 84182801 37. A wheat plant cell produced by the method of any one of claims 26-36. 38. The wheat plant cell of claim 37, wherein the wheat plant cell is a seed cell. 39. A method of limiting wheat stem rust in agricultural crop production, the method comprising planting a wheat seed and growing a wheat plant therefrom under 5 conditions favorable for the growth and development of the wheat plant, wherein the wheat seed comprises a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 19, 22, 25, 34, or 49; (b) a nucleotide sequence encoding the amino acid sequence set forth in 10 SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, 35, or 50; (c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 21, 24, 27, 36, or 51; (d) a nucleotide sequence having at least 85% sequence identity to at least one of the nucleotide sequences set forth in (a) or (c), wherein the nucleotide sequence, 15 when expressed in a wheat plant, confers resistance to stem rust to the wheat plant; and ( e) a nucleotide sequence encoding an amino acid sequence having at least 85% sequence identity to at least one amino acid sequence set forth in (c), wherein the nucleotide sequence, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant. 20 40. The method of claim 39, further comprising harvesting an at least one seed from the wheat plant. 41. Use of a wheat plant or seed in agriculture, wherein the wheat plant or seed comprises a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 19, 22, 25 25, 34, or 49; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, 35, or 50; (c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 21, 24, 27, 36, or 51; Date Re<;ue/Date Received 2023-11-28 84182801 (d) a nucleotide sequence having at least 85% sequence identity to at least one of the nucleotide sequences set forth in (a) or (c), wherein the nucleotide sequence, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant; and ( e) a nucleotide sequence encoding an amino acid sequence having at 5 least 85% sequence identity to at least one amino acid sequence set forth in (c), wherein the nucleotide sequence, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant. 42. A human or animal food product produced using a wheat plant or seed comprising a nucleotide sequence selected from the group consisting of: 10 (a) the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 19, 22, 25, 34, or 49; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, 35, or 50; (c) the nucleotide sequence set forth in SEQ ID NO: 3, 6, 9, 12, 21, 24, 15 27, 36, or 51; (d) a nucleotide sequence having at least 85% sequence identity to at least one of the nucleotide sequences set forth in (a) or (c), wherein the nucleotide sequence, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant; and ( e) a nucleotide sequence encoding an amino acid sequence having at 20 least 85% sequence identity to at least one amino acid sequence set forth in (c), wherein the nucleotide sequence, when expressed in a wheat plant, confers resistance to stem rust to the wheat plant. 43. A polypeptide comprising an amino acid sequence selected from the group consisting of: 25 (a) the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 19, 22, 25, or 34; (b) the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, or 35; ( c) the amino acid sequence encoded by the nucleotide sequence set forth 30 in SEQ ID NO: 3; Date Re<;ue/Date Received 2023-11-28 84182801 (d) an amino acid sequence having at least 85% sequence identity to at least one amino acid sequence set forth in (b ), wherein a polypeptide comprising the amino acid sequence confers resistance to stem rust to a wheat plant comprising the polypeptide; (e) an amino acid sequence having at least 85% sequence identity to at 5 least one amino acid sequence set forth in (b ), wherein the amino acid sequence comprises a leucine-rich repeat domain which comprises an amino acid selected from the group consisting of serine (S) at the amino acid position corresponding to amino acid 607 of SEQ ID NO: 2, tyrosine (Y) at the amino acid position corresponding to amino acid 650, of SEQ ID NO: 2, serine (S) at the amino acid position corresponding to amino acid 651 of SEQ ID 10 NO: 2, and glycine (G) at the amino acid position corresponding to amino acid 657 of SEQ ID NO: 2, and wherein a polypeptide comprising the amino acid sequence confers resistance to stem rust to a wheat plant comprising the polypeptide; (f) the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 49 or 51; 15 (g) the amino acid sequence set forth in SEQ ID NO: 50; and (h) an amino acid sequence having at least 93% sequence identity to the amino acid sequence set forth in (g), wherein a polypeptide comprising the amino acid sequence confers resistance to stem rust to a wheat plant comprising the polypeptide. 44. The polypeptide of claim 43, wherein the polypeptide of (d) comprises a 20 coiled-coil domain, a nucleotide-binding domain, and a leucine-rich repeat domain. 45. The polypeptide of claim 44, wherein at least one of the coiled-coil domain, the nucleotide-binding domain, and the leucine-rich repeat domain comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the corresponding domain in SEQ ID NO: 2 or 50. 25 46. The polypeptide of claim 44 or 45, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, 5, 8, 11, 20, 23, 26, 35, or 50. 4 7. A method for identifying a wheat plant that displays newly conferred or enhanced resistance to wheat stem rust, the method comprising detecting in the wheat plant the presence of an R gene selected from the group consisting of Sr22 and Sr45, wherein the Date Re<;ue/Date Received 2023-11-28 84182801 presence of the R gene is detected by detecting at least one marker within the R gene or by detecting the R protein encoded by the R gene. 48. The method of claim 47, wherein the presence of the R gene is detected by detecting at least one marker within the R gene. 5 49. The method of claim 47 or 48, wherein the method comprises detecting the presence of both Sr22 and Sr45. 50. The method of claim 47, 48 or 49, wherein Sr22 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1, 4, 7, 10, 19, 22, 25, or 34 and Sr45 comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 49. 51. The method of any one of claims 47-50, 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 an immunological assay for the detection of the R protein encoded by the R gene. Date Re<;ue/Date Received 2023-11-28

Description

WHEAT STEM RUST RESISTANCE GENES AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Patent Application No. 62/200,894, filed August 4, 2015. FIELD OF THE INVENTION The present invention relates to the fields of gene isolation and plant improvement, particularly to enhancing the resistance of plants to plant disease through the use of disease resistance genes. BACKGROUND OF THE INVENTION Plant diseases cause significant yield losses in world-wide wheat production. Among the most damaging diseases of wheat are the rusts. Wheat stem rust caused by Puccinia graminis f. sp. tritici is one of the most devastating diseases affecting wheat production today. While wheat plants comprising resistance (R) genes against Puccinia graminis f. sp. Date Rec;ue/Date Received 2023-01-13 tritici have proven effective in limiting the agronomic losses caused by wheat stem rusted, new races of Puccinia graminis f. sp. tritici have appeared recently for which the R genes are not effective. While pesticides can be used to control wheat stem rust, pesticides are expensive and at odds with the sustainable intensification of agriculture, and in developing 5 countries, pesticides are simply unaffordable for subsistence farmers. The sustainable intensification of agriculture will require increased use of genetic solutions instead of chemical solutions ( e.g. pesticides) to protect crops against pathogens and pests (Jones eta!. (2014)Philos. T. Roy. Soc. B 369:20130087). Wild relatives of domesticated crops, such as wheat, contain an immense diversity of useful R genes that are a 10 valuable resource for sustainable disease control. However, traditional methods for introducing R genes typically involve long breeding timelines to break linkage to deleterious alleles of other genes. Furthermore, R genes can be overcome within a few seasons when deployed one at a time (McDonald and Linde (2002) Annu. Rev. Phytopathol. 40:349-379). Molecular cloning, however, makes it possible to avoid linkage drag and simultaneously 15 introduce multipleR genes (Dangl et al. (2013) Science 341 :746-751), which should delay resistance-breaking pathogen race evolution and thus, provide more durable resistance (McDonald and Linde (2002)Annu. Rev. Phytopathol. 40:349-379). While traditional map-based cloning methods have been employed to isolate R genes from plants, many plant genomes carry large chromosomal regions that are inaccessible to 20 traditional map-based cloning due to suppressed recombination (Gaut et al. (2007) Nature Rev. Genet. 8:77-84) and wheat is no exception. Therefore, new, complementary approaches not relying on recombination need to be applied to identify additional R genes in crop plants and their undomesticated relatives. 25 BRIEF SUMMARY OF THE INVENTION The present invention provides nucleic acid molecules for resistance (R) genes that are known to confer upon a plant resistance to at least one strain of the pathogen that causes wheat stem rust, Puccinia graminis f. sp. tritici. In one embodiment, the present invention provides nucleic acid molecules comprising the R gene Sr22 and variants thereof including, 30 for example, orthologs and non-naturally occurring variants. In another embodiment, the present invention provides nucleic acid molecules comprising the R gene Sr45 and variants thereof including, for example, orthologs and non-naturally occurring variants. The present invention further provides plants, plant cells, and seeds comprising in their genomes one or more polynucleotide constructs of the invention. The polynucleotide constructs comprise a nucleotide sequence encoding a resistance (R) protein of the present invention. Such R proteins are encoded by the R genes of the present invention. In a 5 preferred embodiment, the plants and seeds are transgenic wheat plants and seeds that have been transformed with one or more polynucleotide constructs of the invention. Preferably, such wheat plants comprise enhanced resistance to at least one strain of the pathogen that causes wheat stem rust, Puccinia graminis f. sp. tritici, when compared to the resistance of a control wheat plant that does not comprise the polynucleotide construct. The present invention provides methods for enhancing the resistance of a wheat plant to wheat stem rust. Such methods comprise introducing into at least one wheat plant cell a polynucleotide construct comprising a nucleotide sequence of an R gene of the present invention. In some embodiments, the polynucleotide construct or part thereof is stably incorporated into the genome of the plant cell, and in other embodiments, the polynucleotide 15 construct is not stably incorporated into the genome of the wheat plant cell. The methods for enhancing the resistance of a wheat plant to wheat stem rust can optionally further comprise regenerating the wheat plant cell into a wheat plant that comprises in its genome the polynucleotide construct. Pref