WO2023152742A1 - Pm69 and use thereof - Google Patents

Pm69 and use thereof Download PDF

Info

Publication number
WO2023152742A1
WO2023152742A1 PCT/IL2023/050137 IL2023050137W WO2023152742A1 WO 2023152742 A1 WO2023152742 A1 WO 2023152742A1 IL 2023050137 W IL2023050137 W IL 2023050137W WO 2023152742 A1 WO2023152742 A1 WO 2023152742A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
plant
seq
acid molecule
gene
Prior art date
Application number
PCT/IL2023/050137
Other languages
French (fr)
Inventor
Tzion Fahima
Yinghui Li
Zhenzhen WEI
Liubov GOVTA
Original Assignee
Carmel Haifa University Economic Corporation Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Carmel Haifa University Economic Corporation Ltd. filed Critical Carmel Haifa University Economic Corporation Ltd.
Publication of WO2023152742A1 publication Critical patent/WO2023152742A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the present invention is in the field of wheat genomics and disease resistance.
  • Powdery mildew (Pm) caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgf) is one of the most destructive wheat diseases worldwide.
  • Pm3b/Pm8 Pm2a, Pm21, Pm60IMlIW172, Pm5e, Pm41 and Pmla encode nucleotide -binding leucine-rich repeat (NLR) protein.
  • Pm24 encodes a tandem kinase protein and Pm4 encodes a putative chimeric protein of a serine/threonine kinase, multiple C2 domains, and transmembrane regions.
  • Pm38 Lr34IYrl8ISr57, ABC transporter
  • Pm46 Lr67IYr46ISr55, hexose transporter
  • R-genes novel disease resistance genes
  • next-generation sequencing To overcome the limitations of positional cloning R-genes and reduce the genomic complexity of wheat, several methods based on next-generation sequencing (NGS) have been developed and their efficiency was demonstrated for cloning of novel R-genes. These methods include Mutagenesis and Resistance gene Enrichment and Sequencing (MutRenSeq), Association genetics with R gene enrichment Sequencing (AgRenSeq), Mutant Chromosome flow sorting and short-read Sequencing (MutChromSeq), and Targeted Chromosome-based Cloning via long-range Assembly (TACCA). However, these methods still have some limitations.
  • MutRenSeq and AgRenSeq only identify NLR genes that can be captured by hybridization, while TACCA and MutChromSeq rely on the purification of individual chromosomes that carry mutations in the target genes.
  • BSR-Seq Bulked segregant RNA sequencing
  • CCT-Seq bulked segregant Core Genome Targeted sequencing
  • More than 20 Pm resistance genes have been identified and mapped in WEW. Among them, only two were cloned, which were Pm41 localized on chromosome arm 3BL and TdPm60 localized on chromosome arm 7 AL.
  • Pm.41 and TdPm60 showed abundance of 1.81% and 25.6% among the tested natural
  • Pm69 (PmG3M), identified from WEW accession G305- 3M, is a dominant gene conferring a wide-spectrum resistance to Bgt isolates from around the globe. Pm69 is the only gene derived from WEW that was mapped to the telomeric region of chromosome arm 6BL. The full sequence of PM69, which can be integrated into cereal strains that lack it and thus confer Pm resistance is greatly needed.
  • the present invention provides isolated nucleic acid molecules comprising a nucleic acid sequence that encodes a functional Pm69 protein that confers resistance to powdery milder (Pm).
  • Functional Pm69 protein as well as artificial vectors, and transgenic plants expressing the nucleic acid molecule or protein are also provided. Methods of conferring resistance to Pm and detecting a functional Pm69 gene are also provided.
  • an isolated nucleic acid molecule comprising a nucleic acid sequence that encodes a protein with at least 85% homology to SEQ ID NO: 17 and that confers resistance to powdery mildew (Pm).
  • the nucleic acid sequence encodes a protein with at least 85% identity to SEQ ID NO: 17 and that confers resistance to Pm.
  • the nucleic acid sequence comprising at least 85% homology to SEQ ID NO: 15 or 16.
  • the nucleic acid sequence comprises at least 85% identity to SEQ ID NO: 15 or 16.
  • the protein that confers resistance comprises methionine 55, serine 507, glycine 606, serine 200 and at least one of glycine 11 and arginine 553 within SEQ ID NO: 17.
  • the protein that confers resistance comprises a functional Rx_N domain, LRR domain and NB-ARC domain such that the protein confers resistance to PM.
  • the functional Rx_N domain comprises SEQ ID NO: 34
  • the functional LRR domain comprises SEQ ID NO: 35
  • the functional NB-ARC domain comprises SEQ ID NO: 36.
  • the nucleic acid molecule is a DNA molecule or an RNA molecule.
  • conferring Pm resistance is conferring Pm resistance to cereal plant.
  • the cereal plant is a wheat plant.
  • the nucleic acid sequence comprises at least 85% identity to SEQ ID NO: 16.
  • the nucleic acid sequence consists of SEQ ID NO: 16.
  • the isolated nucleic acid molecule further comprises a transcription regulatory element operatively linked to the nucleic acid sequence.
  • resistance comprises a post-haustorial immune responses to the Pm.
  • the post-haustorial immune response comprises intracellular reactive oxidation species (ROS) production in response to Pm.
  • ROS reactive oxidation species
  • an artificial vector comprising the isolated nucleic acid molecule of the invention.
  • the artificial vector further comprises at least one nucleic acid sequence of a pathogen-resistance gene.
  • the pathogen is Stripe Rust (Pst).
  • the pathogen-resistance gene is selected from Yrl5, Yr5, Yr36, Yrl8 and Yr46.
  • the pathogen-resistance gene is Yr 15.
  • the artificial vector comprises at least one transcriptional regulatory element active in plant cells and operatively linked to the nucleic acid sequence.
  • the transcriptional regulatory element is a promoter
  • the promoter is a heterologous promoter of the Pm69 endogenous promoter.
  • the plant is a cereal plant.
  • the cereal plant is selected from wheat, barley, rye, triticale, oat, rice and maize.
  • the artificial vector is for use in conferring resistance to Pm to a cell of a plant.
  • resistance comprises a post-haustorial immune responses to the Pm.
  • the post-haustorial immune response comprises intracellular reactive oxidation species (ROS) production in response to Pm.
  • ROS reactive oxidation species
  • transgenic plant or cell thereof comprising a nucleic acid molecule of the invention or an artificial vector of the invention.
  • the plant is a cereal plant.
  • the cereal plant is any one of barley, rye, triticale, oat, wheat, rice and maize.
  • the cereal plant is wheat.
  • an isolated protein comprising at least 85% homology to SEQ ID NO: 17 and comprising the ability to confer resistance to Pm.
  • the isolated protein comprises at least 85% identity to SEQ ID NO: 17.
  • the isolated protein consists of an amino acid sequence with at least 85% identity to SEQ ID NO: 17.
  • the isolated protein consists of SEQ ID NO: 17.
  • a method of conferring resistance to Pm to a plant or a cell thereof comprising at least one of: a. expressing in the cell of the plant at least one of an isolated nucleic acid molecule of the invention, an artificial vector of the invention, and an isolated protein of the invention; and b. converting at least one pm69 non-functional allele of the cell of the plant into a functional Pm69 gene, thereby conferring resistance to Pm to a plant or cell thereof.
  • a method for detecting a functional Pm69 gene in a sample, which a functional Pm69 gene confers resistance to Pm comprising: a. providing nucleic acid molecules from the sample; b. detecting a DNA molecule comprising a sequence that encodes an mRNA with at least 80% homology to SEQ ID NO: 16 or an mRNA comprising a sequence with at least 80% homology to SEQ ID NO: 16; and c.
  • steps b-c comprise detecting a nucleic acid molecule comprising SEQ ID NO: 16 in the provided nucleic acid molecules.
  • the sample is from a plant.
  • the plant is a cereal plant.
  • the cereal plant is selected from wheat, barley, oat, triticale, rye, rice and maize.
  • the sample is from any one of cultivated plant germplasm, pre-breeding materials, and elite plant cultivars.
  • Figures 1A-B Fine mapping of Pm69 from WEW G305-3M.
  • (1A) Macroscopic observation of the response of G305-3M, LDN and Fl leaves to Bgt # 70 infection at 7 days post-infection (dpi); DAB staining of leaves at 3 dpi to detect ROS accumulation visualized as Reddish-brown coloration; Trypan blue staining of leaves at 3 dpi to visualize fungal structures and plant cell death as blue coloration; MH: mature haustorium; HP: haustorial primordium; Hy: hyphae. Scale bars 50 pm.
  • IB Genomic region containing Pm69 genetic region on the long arm of wheat chromosome 6B (top row).
  • the locations of Pm69 flanking genetic markers on the WEW _v2.0 reference genome (second row).
  • the green-colored markers were developed based on WEW _v2.0
  • the blue-colored markers were developed based on ONT contigs, (third row).
  • the physical map of the Pm69 region is marked in blue.
  • Figures 2A-B Comparison of the G305-3M ONT contigs with the (2A) 6B pseudomolecule of WEW_v2.0 and (2B) durum wheat Svevo RefSeq Rel. 1.0 around the Pm69 genetic region.
  • X-axis the cumulative length of G305-3M contigs;
  • Y-axis the physical location of 6B pseudomolecule in the reference genome.
  • Contigs utg380-utgl7163 marked with different colors belong to the ONT assembly of G305-3M
  • Figures 3A-D The workflow of identification of Pm69 by ONT contigs by MutRNAseq.
  • (3A) The phenotypes of EMS-derived mutants (M-40, M-22, M-12, and M- 8) and wild type (G305-3M) infected with Bgt #10. M-40, M-22, M-12 and M-8 and G305- 3M were analyzed by RNA-seq to identify SNPs among the three ONT contigs.
  • FIG. 4A-C Functional validation of Pm69 candidate through VIGS.
  • 4A VIGS of Pm69 candidate gene is in G305-3M.
  • 4B VIGS of Pm69 candidate gene in the introgression line (Ruta+Pm69).
  • BSMV GFP was used as a negative control.
  • BSMV wild type, the wild type of pCa-BSMV- vector, was used as a negative control.
  • BSMV: PDS is the PDS gene silencing construct used to check the efficiency of the VIGS system;
  • BMSV:Pm69-l and Pm69-2 were the constructs that targeted 5’UTR- Rx_N domain and NB-ARC domain, respectively.
  • Asterisks indicate the level of significance by t-test of the Pm69 expression levels in the target plants compared with the negative control of BSMV: wild type, p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***).
  • Figures 5A-D The geographic distribution of the Pm69 allele in WEW populations.
  • (5A) The geographic distribution of wild emmer wheat (WEW) natural populations (black circles) which were screened for the presence of Pm69 functional allele.
  • (5B) The geographic distribution of WEW individual plants collected near the recorded G305-3M collection site (marked by a blue triangle, south of Kadita, Northern Israel).
  • (5C) A submap of 5B showing the three accessions contain Pm69. The maps were obtained from Google Maps.
  • FIG. 6 Micro-Collinearity analysis of the Pm69 genetic region among different Triticeae genomes. Lines indicate genes with high collinearity. The numbers on the yellow-colored NLRs present different NLR clades based on the evolutionary analysis, which means the same number of NLRs may have evolved by duplication events. The dark blue marks Pm69 and baby blue marks Pm69 homologs. The red triangle marks the locations of Pm69 or its homologs with highest similarity. The pink color marks OPR11 homologs which are more conserved among different genomes than the Pm69 homologs. The green color marks other non-NLR proteins. The NLRs show more genetic diversity than their flanking non-NLR proteins.
  • FIGS 7A-B Introgression of the Pm69 into the cultivated wheat and pyramided with yellow rust resistance genes.
  • (7A) The phenotypes of different wheat parental and introgression lines to Bgt #70 and Pst #5006.
  • WEW: G305-3M contains Pm69 and G25 contains Yrl5.
  • Figure 8 The genetic map of Pm69.
  • the deletion bin map of wheat chromosome arm 6BL (left); Primary genetic map of the Pm69 by using a small population (2500 F2 individuals) (middle); High-resolution genetic map of Pm69 on wheat chromosome 6BL (right).
  • FIG. 9 The phenotypic response of different wheat accessions to Bgt #70.
  • Bgt #70 showed a strong virulence to several wheat species.
  • the WEW accession: G305-3M and G18-16 that contained functional Pm genes (Pm69 and TdPm60. respectively) showed high resistance to Bgt #70.
  • the Zavitan, Svevo and CS were highly susceptible to Bgt #70, suggesting that the three reference genomes in Figure S2 did not contain the functional Pm69 allele.
  • Figure 10 The phenotypic response of EMS-derived mutants to Bgt #70 and Bgt #15.
  • the resistant plant is the sister line of each mutant, which was derived from the same M o plant.
  • Bgt #15 is another isolate used for Pm69 genetic mapping.
  • Figure 11 The expression patterns of Pm69 in G305-3M.
  • the expression levels of Pm69 fold change as compared to ubiquitin levels
  • the time points ranged from 0-72 hpi in both non-inoculated (mock) and Bgt #70-inoculated plants.
  • Asterisks indicate the level of significance by t-test, p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***), of the difference in Pm69 gene expression in non-inoculated and By/-inoculatcd plants.
  • Figure 12 Sequence alignments of Pm69 protein and five representative susceptible Pm69 homologs. The red colored region presents the conserved part of the protein. The sequence of those homologs could be found on the website of WheatOmics 1.0 and EnsemblPlants by using their ID names. The alignments were constructed by the multiple alignment tool CLUSTALW from the NCBI.
  • Figure 13 Three Pm69-similar sequences in the whole genome of WEW_ v2.0. Blasting the Pm69 protein sequence against Zavitan reference genome WEW_v2.0 revealed three copies in 716-719 Mbp with different lengths of introns, 615, 15676 and 3390 bp, respectively. The blue color marks exon 1 and green color marks exon 2. Identity to Pm69 is provided.
  • the present invention provides isolated nucleic acid molecules comprising a nucleic acid sequence that encodes a functional Pm69 protein that confers resistance to powdery milder (Pm).
  • Isolated nucleic acid molecule comprising a nucleic acid sequence that encodes a protein with at least 85% homology to SEQ ID NO: 17 and that confers resistance to Pm are also provided.
  • the functional Pm69 protein as well as artificial vectors, and transgenic plants expressing the nucleic acid molecule or protein are provided as are methods of conferring resistance to Pm and detecting a functional Pm69 gene.
  • the invention is based, at least in part, of the surprising discovery of the sequence of Pm69 which confers resistance to powdery mildew (Pm). Further, when Pm69 was deployed into cultivated wheat breeding lines in conferred resistance to Pm and pyramiding of Pm69 with a yellow rust gene, Yrl5, enriching the wheat disease resistance breeding resource.
  • the Rx_N domain, LRR domain and NB-ARC domain were found to be required for this resistance and multiple pm69 non-functional alleles with mutations in the coding region lack a Pm-resistance phenotype and are found in many domesticated species that are susceptible to Pm. Furthermore, by comparing these non-functional alleles to the functional allele it is possible to distinguish between non-functional alleles and/or pseudogenes and the functional allele in the future.
  • nucleic acid molecule comprising a nucleic acid sequence that encodes a protein that confers resistance to Pm.
  • Pm refers to powdery mildew, which is also called Blumeria graminis f. sp. tritici (Bgf). It is a biotrophic fungus that is one of the most destructive wheat diseases worldwide. In some embodiments, Pm comprises all strains of powdery mildew. In some embodiments, Pm is cereal plant Pm. In some embodiments, Pm is wheat Pm. [071] In some embodiments, the protein that confers resistance is Pm69. Pm69 is also known as nucleotide-binding leucine-rich repeat 6 (NLR6). The nucleotide and protein sequence for Pm(59/Pm69 is first disclosed herein.
  • NLR6 nucleotide-binding leucine-rich repeat 6
  • Pm69 is a functional Pm69. In some embodiments, functional is functional in conferring Pm resistance. In some embodiments, functional is functional in inducing a post-haustorial immune response. In some embodiments, the immune response is in response to Pm infection. In some embodiments, a post-hausorial immune response comprises intracellular reactive oxidation species (ROS) production. In some embodiments, the production is in response to Pm infection.
  • Pm69 refers to a functional Pm69 gene, which confers resistance to Pm.
  • pm69 refers to a non-functional pm69 gene, which does not confer resistance to Pm.
  • gene is used broadly to refer to any nucleic acid associated with a biological function.
  • the term “gene” includes coding sequences (CDS) and/or regulatory sequences required for expression.
  • CDS coding sequences
  • the term “gene” can also apply to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
  • the term “confers resistance” refers to increasing the survival of a plant or cell when challenged with a pathogen i.e., Pm. In some embodiments, increasing survival is a decrease in the growth of the pathogen. In some embodiments, increasing survival is a decrease in the spread of the pathogen. In some embodiments, increasing survival comprises lack of infection by the pathogen. In some embodiments, resistance comprises not being able to be infected by the pathogen. In some embodiments resistance comprises increased survival as compared to survival without the vector, nucleic acid moleucle, or protein of the invention.
  • the increase refers to at least 10% increase, 20% increase, 30% increase, 40% increase, 50% increase, 60% increase, 70% increase, 80% increase, 90% increase, or 100% increase in survival.
  • the decrease refers to at least 10% decrease, 20% decrease, 30% decrease, 40% decrease, 50% decrease, 60% decrease, 70% decrease, 80% decrease, 90% decrease, or 100% decrease.
  • the increase is at least a 50% increase.
  • the decrease is at least a 50% decrease.
  • conferring resistance comprises conferring a post- haustorial immune response to the pathogen.
  • conferring resistance comprises conferring an intracellular ROS response to the pathogen.
  • the functional Pm69 gene comprises the Pm69 CDS.
  • the Pm69 CDS comprises the nucleic acid sequence atggaggcggctctcgtgagtgtggccacgggggtcctgaagcccgtcctggagaagctggcagctctgcttggtgacgagtac aagcggttcaagggagtgcgcaaggatatcaagtccatcgctcgtgagctcgctgccatggaggcttttctctcaagatgtccga ggaggaggatccagatgtacaagacaaattttggatgaatgaggtgcgggagctctcctatgatatggaggatgccatgacgact tcatgcaaagcgttggtgaca
  • the Pm69 CDS consists of SEQ ID NO: 16.
  • SEQ ID NO: 16 is a cDNA sequence devoid of introns. In some embodiment SEQ ID NO: 16 is a DNA sequence not found in nature.
  • the functional Pm69 gene comprises the Pm69 genomic sequence which comprises the nucleic acid sequence cgctgcacaagtctcagttgctgcacttgcctcacgccgtcacgtgtcgccggagtctggtcgacggacgacacggctggacacc gtctacgctccagccacacaaaattaagcgagctagcacgtcctgcaacccatggaggcggctctctcgtgagtgtggccacgggg gtcctgaagcccctggagaagctggcagctctctgcttggtgacgagtacaagcggttcaagggatatcaagt ccatgg
  • the Pm69 genomic sequence consists of SEQ ID NO: 15.
  • SEQ ID NO: 15 is a DNA sequence of the full transcript sequence of the Pm69 mRNA. It will be understood by a skilled artisan that SEQ ID NO: 15 can also be an RNA sequence in which the thymine residues are uracil.
  • the Pm69 genomic sequence comprises the genomic sequence of Pm69 and 2 kilobases upstream and/or downstream from that sequence.
  • SEQ ID NO: 14 comprises the Pm69 genomic sequence with 2 kilobases upstream and two kilobases downstream from SEQ ID NO: 15.
  • the Pm69 CDS is a cDNA reverse transcribed from an RNA which codes for the amino acid sequence MEAALVSVATGVLKPVLEKLAALLGDEYKRFKGVRKDIKSIARELAAMEAFLLK MSEEEDPDVQDKFWMNEVRELSYDMEDAIDDFMQSVGDKDEKPDDFIEKIKNTL EKLGKMKARRRIGKEIQDLKKQIIQVGDRNARYKGRQTFSSTKNEIVDPRILARFE HASKLVGIDETKAEIIKLLGEENGQVPRQQQLKILSIVGFGGMGKTTLANQVYQDL KGEFQYRAFISVSQNPDLMKILRTILSEITGISYPGTEAGCIEQLIDKIKDFLADKRYL IVIDDIWDIKHWEVIRCALANNHYENRVITTTRDRDVARKVGGAYELKPLPDETSK ILFFGRIFGINNDCPDDLVEVSETIMKKCGGVPLAIITIAGL
  • the Pm69 protein comprises SEQ ID NO: 17. In some embodiments, the Pm69 protein consists of SEQ ID NO: 17. [076] In some embodiments, Pm69 comprises a functional Rx_N domain. An Rx_N domain is also known as an Rx N-terminal domain. Rx_N domains are found in many plant resistance proteins at the protein’s N-terminus. It is predicted to be a coiled-coil, but some work has shown that it adopts a four helical bundle fold. In some embodiments, the Rx_N domain of Pm69 comprises or consists of amino acids 12-79 of SEQ ID NO: 17. In some embodiments, the Rx_N domain comprises a methionine at position 55 in SEQ ID NO: 17.
  • the Rx_N domain does not comprise an alanine at position 55 in SEQ ID NO: 17.
  • a functional Pm69 comprises a methionine at position 55 of SEQ ID NO: 17.
  • a functional Pm69 does not comprise an alanine at position 55 of SEQ ID NO: 17.
  • the Rx_N domain comprises a glycine at position 11 of SEQ ID NO: 17.
  • the Rx_N domain does not comprise an arginine at position 11 of SEQ ID NO: 17.
  • the functional Pm69 comprises a glycine at position 11 of SEQ ID NO: 17.
  • the functional Pm69 does not comprise an arginine at position 11 of SEQ ID NO: 17.
  • the Rx_N domain comprises an arginine at position 553 of SEQ ID NO: 17.
  • the Rx_N domain does not comprise a lysine at position 553 of SEQ ID NO: 17.
  • the functional Pm69 comprises an arginine at position 553 of SEQ ID NO: 17.
  • the functional Pm69 does not comprise a lysine at position 553 of SEQ ID NO: 17.
  • the Rx_N domain comprises a glycine at position 11 and an arginine at position 553 of SEQ ID NO: 17.
  • the Rx_N domain does not comprise an arginine at position 11 and a lysine at position 553 of SEQ ID NO: 17.
  • the functional Pm69 comprises a glycine at position 11 and an arginine at position 553 of SEQ ID NO: 17. In some embodiments, the functional Pm69 does not comprise an arginine at position 11 and a lysine at position 553 of SEQ ID NO: 17.
  • Pm69 comprises a functional leucine rich repeat (LRR) domain.
  • LRR domains generally consists of 2-45 leucine-rich repeats with each repeat being about 20-30 residues long. Structurally these domains adopt an arc or horseshoe shape, with the concave face consisting of parallel P-strands and the convex face representing a more variable region of secondary structures including helices.
  • the LRR domain of Pm69 comprises or consists of amino acids 549-708 of SEQ ID NO: 17.
  • the LRR domain comprises a glycine at position 606 of SEQ ID NO: 17.
  • the LRR domain does not comprise a glutamic acid at position 606 of SEQ ID NO: 17.
  • the functional Pm69 comprises a glycine at position 606 of SEQ ID NO: 17. In some embodiments, the functional Pm69 does not comprise a glutamic acid at position 606 of SEQ ID NO: 17.
  • Pm69 comprises a functional NB-ARC domain.
  • NB-ARC domains are functional ATPase domains that generally regulate signaling. In particular, its nucleotide-binding state is proposed to regulate activity of the R protein.
  • the NB-ARC domain of Pm69 comprises or consists of amino acids 172-416 of SEQ ID NO: 17.
  • the NB-ARC domain comprises a serine at position 200 of SEQ ID NO: 17.
  • the NB-ARC domain does not comprise a phenylalanine at position 200 of SEQ ID NO: 17.
  • the nucleic acid molecule is DNA. In some embodiments, the DNA is cDNA. In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the DNA or RNA is double stranded. In some embodiments, the DNA or RNA is single stranded. In some embodiments, the DNA molecule is a vector.
  • the nucleic acid molecule is an isolated nucleic acid molecule.
  • isolated nucleic acid molecule refers to a nucleic acid molecule that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the nucleic acid in nature.
  • a preparation of isolated DNA or RNA contains the nucleic acid in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • an isolated nucleic acid molecule is at least 90% pure.
  • an isolated nucleic acid molecule is at least 95% pure. In some embodiments, pure is with respect to other nucleic acid molecule. In some embodiments, pure is with respect to other non-nucleic acid cellular components.
  • the isolated DNA is cDNA. In some embodiments, the isolated nucleic acid is any one of DNA, RNA, and cDNA. In some embodiments, the isolated nucleic acid molecule is a synthesized nucleic acid molecule. Synthesis of nucleic acid molecules is well known in the art and may be performed, for example, by ligating or covalently linking by primer linkers multiple nucleic acid molecules together or by in vitro amplification (e.g., PCR) or reversetranscription.
  • nucleic acid is well known in the art.
  • a “nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising nucleotides. Nucleotides are comprised of nucleosides and phosphate groups.
  • the nitrogenous bases of nucleosides include, for example, naturally occurring purine or pyrimidine nucleosides as found in DNA (e.g., an adenine "A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” or a C). Sequences given herein are given as DNA sequences comprising thymine but it will be understood that they can also refer to an RNA with the same sequence but with the thymines replaced by uracils.
  • nucleic acid molecule includes but is not limited to singlestranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNAs, circular nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, amplification products, modified nucleic acids, plasmid or organellar nucleic acids, and artificial nucleic acids such as oligonucleotides.
  • ssRNA singlestranded RNA
  • dsRNA double-stranded RNA
  • ssDNA single-stranded DNA
  • dsDNA double-stranded DNA
  • small RNAs circular nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, amplification products, modified nucleic acids, plasmid or organellar nucleic acids, and artificial nucleic acids such as oligonucleotides.
  • the nucleic acid sequence comprises at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to the Pm69 coding sequence. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleic acid sequence comprises at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 16. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleic acid sequence comprises at least 80% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 85% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 90% homology to SEQ ID NO: 16.
  • the nucleic acid sequence comprises at least 95% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 97% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 99% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleic acid sequence comprises at least 85% homology to SEQ ID NO: 15. In some embodiments, homology is identity.
  • the nucleic acid sequence comprises SEQ ID NO: 14. In some embodiments, the nucleic acid sequence consists of SEQ ID NO: 14. In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 15. In some embodiments, the nucleic acid sequence consists of SEQ ID NO: 15. In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 16. In some embodiments, the nucleic acid sequence consists of SEQ ID NO: 16. [006] In some embodiments, the nucleic acid sequence encodes a protein that confers resistance to Pm.
  • a protein that confers resistance comprises at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 17 or a fragment analog or homolog thereof. Each possibility represents a separate embodiment of the invention.
  • a protein that confers resistance comprises at least 85% homology to SEQ ID NO: 17.
  • homology is identity.
  • a fragment is a fragment that confers resistance.
  • a fragment is a fragment comprising an Rx_N domain, a LRR domain or a NB-ARC domain.
  • a fragment is a fragment comprising an Rx_N domain, a LRR domain and a NB-ARC domain.
  • a fragment comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 amino acids of SEQ ID NO: 17.
  • amino acids of SEQ ID NO: 17 are consecutive amino acids of SEQ ID NO: 17.
  • an analog or homolog is an analog or homolog that confers resistance.
  • analog or “homolog” includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities to confer resistance to Pm.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another
  • one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine
  • substitution of one basic residue such as lysine, arginine or histidine for another
  • substitution of one acidic residue such as aspartic acid or glutamic acid for another
  • the terms “peptide”, “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • the terms “peptide”, “polypeptide” and “protein” as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof.
  • the peptides, polypeptides and proteins described have modifications rendering them more stable while in the organism or more capable of penetrating into cells.
  • the terms “peptide”, “polypeptide” and “protein” apply to naturally occurring amino acid polymers.
  • the terms “peptide”, “polypeptide” and “protein” apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • conferring resistance is conferring resistance to a plant.
  • conferring resistance is conferring resistance to a plant cell.
  • a plant is a cell of a plant.
  • the plant is a cereal plant.
  • the plant is a grain plant.
  • the grain/cereal plant is wheat.
  • the grain plant is selected from barley, rye, oat, triticale, spelt and wheat.
  • the grain plant is selected from barley, rye, rice, maize, triticale, oat, spelt and wheat.
  • the term “wheat” refers to a plant of the genus Triticum. Wheat can be used for the production of grain such as is used for bread, cereal or pasta for non-limiting examples. In some embodiments, wheat is bread wheat or duram wheat. In some embodiments, wheat is duram wheat. In some embodiments, wheat comprises spelt. In some embodiments, wheat is Triticum turgidum.
  • the isolated nucleic acid molecule comprises a fragment, a homolog or an analog to the Pm69 gene, wherein the fragment, homoIgo or analog encodes a protein that confers resistance to Pm.
  • the isolated nucleic acid molecule comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 16 or a fragment, homolog or analog thereof and encodes a protein with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 17 or a fragment, homolog or analog thereof, wherein the protein confers resistance to Pm.
  • Each possibility represents a separate embodiment of the invention.
  • the nucleic acid molecule comprises a nucleic acid sequence that encodes the Rx_N domain of Pm69 or a functional fragment, homolog or analog thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that encodes the LRR domain of Pm69 or a functional fragment, homolog or analog thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that encodes the NB-ARC domain of Pm69 or a functional fragment, homolog or analog thereof.
  • the Rx_N domain comprises VLKPVLEKLAALLGDEYKRFKGVRKDIKSIARELAAMEAFLLKMSEEEDPDVQDK FWMNEVRELSYDM (SEQ ID NO: 34).
  • the Rx_N domain consists of SEQ ID NO: 34.
  • the nucleic acid sequence comprises a sequence that encodes the Rx_N domain.
  • a sequence that encodes the Rx_N domain comprises gtcctgaagcccgtcctggagaagctggcagctctgcttggtgacgagtacaagcggttcaagggagtgcgcaaggatatcaagt ccatcgctcgtgagctcgctgccatggaggcttttctctcaagatgtccgaggaggaggatccagatgtttgga tgtgga tgtgga tgtg (SEQ ID NO: 37).
  • a sequence that encodes the Rx_N domain consists of SEQ ID NO: 37.
  • the LRR domain comprises LSHMRSLTVSGNWVDEMSPLSFSHQLRVLDLEGCYLAQRHDLLAHLGSLCQLRY LSLGNAYLGDLLLAIQIGQLKSLICLRVGGRFKIKFRPGVVRELECLQELSMINLSK SPHVAKEERHETKERVEGISFEDMPDESEKGWEEESEGHERNEQSEIVS (SEQ ID NO: 35).
  • the ERR domain consists of SEQ ID NO: 35.
  • the nucleic acid sequence comprises a sequence that encodes the LRR domain.
  • a sequence that encodes the LRR domain comprises ttgtcccacatgaggtcacttactgtgtcgggcaactgggttgacgaaatgtcgcccctttcttttcccatcaattacgtgtattggattt ggagggctgctatcttgcgcaacgccatgatttacttgcgcatctcgggagtttatgccagttgagatatctttcgttgggaaatgcat acctcggtgacctcttattggcgatccaaattgggcagctaaaaagtctgatatgctttaagagttggcggtcgcttcaaaatcaaattc aggccaggtgtggttagggaactggagtgtctgcaatgatcaatttattattattatggatggtg
  • the NB-ARC domain comprises IDETKAEIIKLLGEENGQVPRQQQLKILSIVGFGGMGKTTLANQVYQDLKGEFQYR AFISVSQNPDLMKILRTILSEITGISYPGTEAGCIEQLIDKIKDFLADKRYLIVIDDIWD IKHWEVIRCALANNHYENRVITTTRDRDVARKVGGAYELKPLPDETSKILFFGRIF GINNDCPDDLVEVSETIMKKCGGVPLAIITIAGLLASRERNKREWNKLCDSIGSGLD NSPDVKTMRNILALSY (SEQ ID NO: 36).
  • the NB-ARC domain consists of SEQ ID NO: 36.
  • the nucleic acid sequence comprises a sequence that encodes the NB-ARC domain.
  • a sequence that encodes the NB-ARC domain comprises atccttagtgaaattactggtataagctatcctggcaccgaagcagggtgcatagaacaactcatcgacaagatcaaagatttcctag cagacaaaaggtatcttattgtcatagatgatatatgggacataaaacattgggaagtgatccgatgtgctctagctaataatcattatg agaatagagtaatcacaacaacccgtgatcgcgacgttgcacgtaaagttggtggcgcctatgagcttaaacccctccctgatgag acatccaaaatattattctttggaagaatttttggtattaataatgact
  • the Rx_N domain comprises at least 85% homology to SEQ ID NO: 34 and retains the domain function.
  • the LRR domain comprises at least 85% homology to SEQ ID NO: 35 and retains domain function.
  • the NB-ARC domain comprises at least 85% homology to SEQ ID NO: 36 and retains domain function.
  • domain function is conferring resistance to Pm.
  • the sequence comprises at least 85% homology to SEQ ID NO: 37.
  • the sequence comprises at least 85% homology to SEQ ID NO: 38.
  • the sequence comprises at least 85% homology to SEQ ID NO: 39.
  • the nucleic acid molecule is a vector.
  • the vector is an artificial vector.
  • the vector is an expression vector.
  • the expression vector is a plant expression vector.
  • a vector is a plasmid.
  • a vector is a viral vector.
  • a vector is a lentiviral vector.
  • the vector is for use in expressing Pm69.
  • the vector is for use in expressing Pm69 in a plant or cell thereof.
  • the vector is for use in conferring resistance to Pm.
  • the vector is for use in conferring resistance to Pm to a plant or cell thereof.
  • a vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • expression control element e.g., a promoter, enhancer
  • selectable marker e.g., antibiotic resistance
  • the vector may be a DNA plasmid delivered via non-viral methods or via viral methods.
  • the viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector, a virgaviridae viral vector, or a poxviral vector.
  • the barley stripe mosaic virus (BSMV), the tobacco ratle virus and the cabbage leaf curl geminivirus (CbLCV) may also be used.
  • the promoters may be active in plant cells.
  • the promoters may be a viral promoter.
  • the nucleic acid molecule further comprises at least one transcriptional regulatory element.
  • the vector comprises at least one transcriptional regulatory element.
  • the transcription regulatory element is a promoter.
  • the promoter is a plant promoter.
  • the promoter is active in plants or a plant cell.
  • the promoter is a viral promoter.
  • the promoter is a constitutive promoter.
  • the promoter is the endogenous Pm69 promoter or a fragment thereof capable of driving transcription.
  • driving transcription is driving transcription in a plant or plant cell.
  • the endogenous promoter comprises at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 kb upstream of the transcriptional start site of Pm69. Each possibility represents a separate embodiment of the invention.
  • the endogenous promoter comprises the first 2000 nucleotides of SEQ ID NO: 14.
  • the promoter is a heterologous promoter.
  • the nucleic acid sequence is operatively linked to a transcriptional regulatory element.
  • the nucleic acid molecule is operatively linked to a transcriptional regulatory element.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • the promoter is operably linked to an isolated DNA of the invention.
  • the transcriptional regulatory element is a heterologous transcriptional regulatory element.
  • the transcriptional regulatory element is an endogenous transcriptional regulatory element.
  • the vector is an expression vector that drives expression of Pm69.
  • expression refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product.
  • expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide).
  • transcription of the nucleic acid fragment e.g., transcription resulting in mRNA or other functional RNA
  • polypeptide e.g., transcription resulting in mRNA or other functional RNA
  • Expressing of a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome.
  • the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), such as biolistic use of coated particles, and needle-like particles, Agrobacterium Ti plasmids and/or the like.
  • electroporation e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • heat shock e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • infection by viral vectors e.g., as described in From et al., Pro
  • promoter refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. The promoter may extend upstream or downstream of the transcriptional start site, and may be any size ranging from a few base pairs to several kilo-bases.
  • nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II).
  • RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
  • plant expression vectors are used.
  • the expression of a polypeptide coding sequence is driven by a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 3:17-311 (1987)] are used.
  • plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J.
  • constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)].
  • Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
  • expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention.
  • SV40 vectors include pSVT7 and pMT2.
  • vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallo thionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • recombinant viral vectors which offer advantages such as systemic infection and targeting specificity, are used for in vivo expression.
  • systemic infection is inherent in the life cycle of, for example, the retrovirus and is the process by which a single infected cell produces many progeny virions that infect neighboring cells.
  • the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles.
  • viral vectors are produced that are unable to spread systemically. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • plant viral vectors are used.
  • a wildtype virus is used.
  • a deconstructed virus such as are known in the art is used.
  • Agrobacterium is used to introduce the vector of the invention into a virus.
  • the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield, or activity of the expressed polypeptide.
  • the vector further comprises at least one nucleic acid sequence of a tandem kinase-pseudokinase (TKP)-containing gene. In some embodiments, the vector further comprises at least one nucleic acid sequence of a pathogen resistance gene. In some embodiments, the vector comprises a sequence encoding a TKP-containing gene. In some embodiments, the vector comprises a sequence encoding at least one pathogen resistance gene.
  • TKP tandem kinase-pseudokinase
  • the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a tandem kinase-pseudokinase (TKP)-containing gene.
  • TKP tandem kinase-pseudokinase
  • a TKP-containing gene is a gene with an active kinase and inactive pseudokinse domain in tandem.
  • the TKP-containing gene comprises no other protein motifs.
  • the TKP-domain is homologous to the TKP domain of WTK1.
  • the TKP-domain containing protein is Wtkl (Yrl5).
  • the TKP-containing gene is selected from wheat tandem kinase 1 (WTK1), leucine-rich -repeat receptor kinases subfamily 6B (LRR-6B), receptorlike cytoplasmic kinases subfamily 7 (RLCK_7), leucine -rick-repeat receptor kinases subfamily 3 (LFF_3), receptor-like cytoplasmic kinases subfamily 8 (RLCK_8), cell wall- associated kinase (WAK), concanavalin A-like lectin protein kinase (L-LPK), other kinases with no published family (RK_1), leucine -rich-repeat receptor kinases subfamily 12
  • WTK1 wheat tandem kinase 1
  • LRR-6B leucine-rich -repeat receptor kinases subfamily 6B
  • RLCK_7 receptorlike cytoplasmic kinases subfamily 7
  • LFF_3 leucine -rick-repeat
  • the TKP-containing gene is a WTK1, LRR-6B, RLCK_7, LFF_3, RLCK_8, WAK, L-LPK, RK_1, RLCK_7, LRR_12 or LRR_6B subfamily gene. Each possibility represents a separate embodiment of the invention.
  • the TKP-containing gene is selected from TraesCS 1 A01G061500.1, TraesCSlA01G197000.2, TraesCSlA01G432400.1
  • the TKP containing gene is selected from TraesCSlA01G061500.1, TraesCSlA01G197000.2
  • BnaA07gl4690D BnaA09g41440D, BnaC04g38500D, BnaA03gl5120D,
  • BnaA02g06510D Potri.017G055000, Potri.001G315000, SOBIC.010G171600.1.P, SOBIC.005G096400.1.P, SOBIC.008G022300.2.P, SOBIC.001G353800.1.P,
  • the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a pathogen-resistance gene.
  • pathogen-resistance gene refers to a gene that provides a plant or plant cell with resistance to a pathogen.
  • the pathogen is a bacterial pathogen.
  • the pathogen is a fungal pathogen.
  • pathogen is yellow (stripe) rust (Pst).
  • yellow stripe rust is the fungus Puccinia striiformis f. sp.
  • Pathogen-resistance genes are well known in the art, and include, but are not limited to Yrl5, Yrl8, Yr5, Yr36, Yr46, Yrl7, Yr29, LrlO, Lrl3, Yr58, Srl3 and Sr 21.
  • Yrl8 is also known as Lr34 and Sr57.
  • Yr29 is also known as Lr46.
  • Yr46 is also known as Lr67.
  • Yrl8 is also known as Lr34 and Sr57.
  • the pathogen resistance gene is selected from Yrl5, Yrl8, Yr5, Yr36, Yr46, Yrl7, Yr29, LrlO, Lrl3, Yr58, Srl3 and Sr 21. In some embodiments, the pathogen resistance gene is selected from Yrl5, Yrl8, Yr5, Yr36, and Yr46. In some embodiments, the pathogen resistance gene is selected from Yrl5, Yrl8, Yr5, and Yr36. In some embodiments, the pathogen resistance gene is selected from Yrl5, Yrl8, and Yr5. In some embodiments, the pathogen resistance gene is selected from Yrl5 and Yr5. In some embodiments, the pathogen resistance gene is Yrl5.
  • the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yrl5 gene. Each possibility represents a separate embodiment of the invention.
  • the artificial vector further comprises a nucleic acid sequence with at least 85% homology to a Yrl5 gene.
  • the artificial vector further comprises a Yrl5 gene or fragment, homolog or analog thereof that confers resistance to Pst.
  • the Yrl5 gene comprises the nucleotide sequence atggattaccaaggaaacaattttaatgatttctttcaaactaatgggcattttgtacttaaaagagtggacaacaactataaactgcgg tcattcactgaaaaggagatagagcacattacagacagatatagcacttcgcttggtaatggctcgttcggtgatgtctacaaaggaggaa gattagacgatcaacgtccagtcgcagtaaagagatacaaaatggaaccaagaaagaggagtttgccaaggaggtgatagtgc attcccagataaaccataagaacgttgtcagattgttaggctgctgcacagaggaaatgcttttatggagtttatctgtataggaggttatct
  • the Yrl5 gene consists of SEQ ID NO: 29. In some embodiments, the Yrl5 gene is operably linked to a plant promoter. In some embodiments, the artificial vector further comprises a nucleic acid sequence that encodes for a Yrl5 protein.
  • the Yrl5 protein comprising the sequence MDYQGNNFNDFFQTNGHFVLKRVDNNYKLRSFTEKEIEHITDRYSTSLGNGSFGD VYKGREDDQRPVAVKRYKNGTKKEEFAKEVIVHSQINHKNVVREEGCCTEENAE MIVMEFICNGNLYNILHCGNADGPIPFPLDKRLDIAIESAEALSCMHSMYSPVLHGD IKPANILLDEKYLPKLSDFGIARLLSTDEAQRTKTVIGCIGYVDPLFCQSGILTTKSD VYSFGVVLLEMITRKKATDGATSLTQCFAEALGGKKVRQLFDVEIANDKKKVKLI EDIAKLAATCLKLEDKMRPTMVEVADRLRRIRKALPQRKGESSTGINNGLIRTGKA EDLPTISLDEMKKLTRNFSDGALIGESSQGRVLFEELSYGKRYAFKSSQEIDLKIEAI SRLKHKNVVQLLGNWVEGNKYV
  • the Yrl5 protein (WTK1) consists of SEQ ID NO: 30.
  • the Yrl5 homolog or analog encodes a protein with at least 85% homology to SEQ ID NO: 30 and confers resistance to Pst. In some embodiments, homology is identity.
  • the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yr5 gene. Each possibility represents a separate embodiment of the invention.
  • the artificial vector further comprises a Yr5 gene.
  • the Yr5 gene is operably linked to a plant promoter.
  • the artificial vector further comprises a nucleic acid sequence that encodes for a Yr5 protein.
  • the Yr5 genes encodes a protein comprising the sequence
  • the sequence of Yr5 comprises a sequence selected from the sequences provided in Accession numbers JQ318576.1, JQ318577.1, JQ318578.1, JQ318579.1, JQ318580.1, JQ318581.1, JQ318582.1, JQ318583.1, JQ318584.1, JQ318585.1, JQ318586.1, JQ318587.1, and JQ318588.1.
  • the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yrl8 gene.
  • the artificial vector further comprises a Yr 18 gene.
  • the Yrl8 gene is operably linked to a plant promoter.
  • the artificial vector further comprises a nucleic acid sequence that encodes for a Yr 18 protein.
  • the Yr 18 gene encodes a protein with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to the sequence MDIALASAAATWLINKLLDRLSDYAIKKLLGSEGLDAEASSLRDALRRATLVLGA VPAGAAAGVRIGNDQLLPQIDLVQRLATDLARHLDELEYYDVKKKVKKNQKSSN PLSKMNLPLTQAGQSKPKYNRTDIKQIRDTVGYLHSICDDVHKALLLDKLDAIKQ AAQDASTDKRETVDNFTENPRNKVFPREEMKDIIELINSAASSDQELLVVPIVGAG GVGKTTLARLVYHDPEVKDKFDIMLWIYVSANFDEVKLTQGILEQIPECEFKSAKN LTVLQRGINKYLTKRFLLVLDDMWEESEGRWDKLLAPLRSAQAKGNVLLVTTRK LSVARITSNTEAHIDLDGMKKDDFWLFFKRCIFGDENYQGQ
  • the Yr 18 gene comprises a nucleotide sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to the sequence provided in accession number XM_015795636.1.
  • the sequence of Yr5 comprises a sequence selected from the sequences provided in Accession numbers EU423905.1, EF489022.1, and EU423903.1.
  • the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yr46 gene.
  • the artificial vector further comprises a Yr46 gene.
  • Yr46 is the Lr67 gene.
  • the Yr46 gene is operably linked to a plant promoter.
  • the artificial vector further comprises a nucleic acid sequence that encodes for a Yr46 protein.
  • Yr46 comprises or consists of the sequence atgccgggcggggggttcgccgtgtcggcgccgtccggcgtggagttcgaggccaagatcacgcccatcgtcatcatctcctgc atcatggcggccaccggcggcctcatgttcggctacgacgtcggcatctcaggcggagtgacatcgatggacgatttcctgcgtg agttctcccggcggtgctgcgcggaagaaccaggacaaggagagcaactactgcaagtacgacaaccagggcctgcagctc tcacctcgccggctcacctcgcaccttcttcgctctacaccacccgccaccttcttcgctctacaccacccgccacct
  • the artificial vectors of the invention comprise at least one promoter for transcription in a plant cell. In some embodiments, the artificial vectors of the invention comprise at least one promoter for expression in a plant cell. In some embodiments, the at least one promoter is operably linked to a Yrl5 gene. In some embodiments, the vector comprises a nucleic acid molecule of the invention and the Yrl5 gene. In some embodiments, the nucleic acid molecule of the invention and the Yr 15 gene are operably linked to the same promoter. In some embodiments, the nucleic acid molecule of the invention and the Yr 15 gene are operably linked to different promoters.
  • the at least one promoter is operably linked to a Yr5 gene.
  • the vector comprises a nucleic acid molecule of the invention and the Yr5 gene.
  • the nucleic acid molecule of the invention and the Yr5 gene are operably linked to the same promoter.
  • the nucleic acid molecule of the invention and the Yr5 gene are operably linked to different promoters.
  • a plant cell comprising a nucleic acid molecule of the invention.
  • a plant cell comprising a vector of the invention.
  • the plant cell is a transgenic cell.
  • a “transgenic cell” refers to a cell that has undergone human manipulation on the genomic or gene level.
  • the transgenic cell has had exogenous nucleic acid molecule introduced into it.
  • exogenous molecule is an exogenous DNA.
  • the exogenous molecule is an exogenous RNA.
  • the exogenous molecule is an exogenous vector.
  • a transgenic cell comprises a cell that has a vector introduced into it.
  • a transgenic cell is a cell which has undergone genome mutation or modification.
  • a transgenic cell is a cell that has undergone genome editing. In some embodiments, genome editing is CRISPR genome editing. In some embodiments, a transgenic cell is a cell that has undergone targeted mutation of at least one base pair of its genome. In some embodiments, the nucleic acid molecule or vector is stably integrated into the cell. In some embodiments, the transgenic cell expresses a nucleic acid molecule of the invention. In some embodiments, the transgenic cell expresses a vector of the invention. In some embodiments, the transgenic cell expresses a protein of the invention.
  • the transgenic cell is a cell that comprises a pm69 non-functional allele and/or pseudogene that has been mutated or modified into a functional Pm69 gene.
  • the pm69 non-functional allele and/or pseudogene has been modified to comprise SEQ ID NO: 14.
  • the pm69 non-functional allele and/or pseudogene has been modified to comprise SEQ ID NO: 15.
  • the pm69 non-functional allele and/or pseudogene has been modified to comprise SEQ ID NO: 16.
  • a pm69 non-functional allele has been modified to encode a protein comprising the amino acid sequence provided in SEQ ID NO: 17.
  • a pm69 non-functional allele has been modified to encode a protein comprising an amino acid sequence with at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 17, and which confers resistance to Pm.
  • CRISPR technology is used to modify a pm69 non-functional allele.
  • a plant comprising a plant cell of the invention.
  • the plant is a transgenic plant.
  • the plant is a grain/cereal plant.
  • the plant is any plant that without addition of the vectors or nucleic acid molecules of the invention can be infected by Pm.
  • the plant is selected from barley, rye, triticale, oat, and wheat.
  • the plant is selected from barley, rye, triticale, oat, wheat, rice and maize.
  • the plant is wheat.
  • the transgenic plant cell is resistant to Pm.
  • the transgenic plant cell cannot be infected by Pm.
  • Pm does not grow on the transgenic plant cell.
  • Pm grows poorly on the transgenic plant cell. In some embodiments, Pm grows worse on the transgenic plant cell than on a plant cell that does not comprise a vector or nucleic acid molecule of the invention.
  • a transgenic plant produces a post- haustorial immune response to Pm.
  • a transgenic plant cell produces intracellular ROS in response to Pm.
  • in response to Pm is in response to exposure to Pm. In some embodiments, exposure is contact.
  • produces is increases. In some embodiments, increases is as compared to the cell not exposed to Pm. In some embodiments, increases is as compared to a non-transgenic cell exposed to Pm.
  • increase is by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500%.
  • Each possibility represents a separate embodiment of the invention.
  • a plant is a part of a plant. In some embodiments, a part of a plant is a seed. In some embodiments, a plant is any portion, see, tissue or organ thereof comprising at least one transgenic plant cell of the invention. In some embodiments, the transgenic plant or portion thereof consists of transgenic plant cells of the invention. In some embodiments, the plant or portion thereof comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% transgenic cells of the invention. Each possibility represents a separate embodiment of the invention. In some embodiments, the percentage of transgenic cells is a percentage high enough to confer resistance to Pm.
  • a protein comprising Pm69 (NLR6) or a fragment analog, homolog or derivative thereof that confers resistance to Pm.
  • the protein is an isolated protein. In some embodiments, isolated is from any other proteins. In some embodiments, the protein comprises at least 85% sequence homology to SEQ ID NO: 17 and confers resistance to Pm. In some embodiments, homology is identity. In some embodiments, the protein comprises SEQ ID NO: 17. In some embodiments, the protein consists of SEQ ID NO: 17. In some embodiments, the protein is NLR6. In some embodiments, the protein is Pm69. In all such embodiments, it will be understood that the protein will retain the ability to confer resistance to Pm to a cell or plant to which it is introduced.
  • derivative refers to any polypeptide that is based off the polypeptide of the invention and still confers resistance to Pm.
  • a derivative is not merely a fragment of the polypeptide, nor does it have amino acids replaced or removed (an analog), rather it may have additional modification made to the polypeptide, such as post-translational modification.
  • a derivative may be a derivative of a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 100 consecutive amino acids of the polypeptide of the invention.
  • the protein comprises an Rx_N domain. In some embodiments, the protein comprises a LRR domain. In some embodiments, the protein comprises a NB-ARC domain. In some embodiments, the domain are the domains of Pm69. In some embodiments, the domains are functional.
  • a method of conferring resistance to Pm to a plant cell comprising expressing in the cell at least one of a nucleic acid molecule of the invention, a vector of the invention and a protein of the invention, thereby conferring resistance to Pm to a plant cell.
  • a method of conferring resistance to Pm to a plant comprising expressing in the cell of the plant at least one of a nucleic acid molecule of the invention, a vector of the invention and a protein of the invention, thereby conferring resistance to Pm to a plant.
  • the expressing is in a sufficient number of cells such as to confer resistance to the plant.
  • a sufficient number of cells is at least 20, 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99 or 100% of the cells of the plant.
  • Each possibility represents a separate embodiment of the invention.
  • a sufficient number of cells is at least 25% of the cells of the plant.
  • a sufficient number of cells is at least 50% of the cells of the plant.
  • a method of conferring resistance to Pm to a plant cell comprising converting at least one pm69 non-functional allele of the cell into a functional Pm69 gene, thereby conferring resistance to Pm to a plant cell.
  • the plant cell is not resistant to Pm. In some embodiments, the plant cell does not comprise a functional Pm69 gene. In some embodiments, the cell comprises a pm69 non-functional allele. In some embodiments, the cell comprises a pm69 pseudogene.
  • said converting comprises genome editing.
  • the genome editing is CRISPR genome editing.
  • the converting comprises targeted mutation of the pm69 non-functional allele and/or pseudogene.
  • the functional Pm69 gene comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO:16. Each possibility represents a separate embodiment of the invention.
  • the functional Pm69 gene comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention.
  • the functional Pm69 gene comprises a nucleic acid sequence that encodes a protein with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 17. Each possibility represents a separate embodiment of the invention.
  • the functional Pm69 comprises a functional Rx_N domain, LRR domain and NB-ARC domain.
  • the function Pm69 comprises methionine 55, serine 507, glycine 606, serine 200 and at least one of glycine 11 and arginine 553 within SEQ ID NO: 17.
  • the method further comprises conferring resistance to Pst. In some embodiments, resistance to Pst is conferred to the plant cell. In some embodiments, resistance to Pst is conferred to the plant. In some embodiments, the method further comprises expressing in the plant cell any one of Yrl5, Yr5, Yr36, Yrl8 and Yr46. In some embodiments, the method further comprises expressing in the plant cell Yrl5.
  • a method for detecting a functional Pm69 gene in a sample comprising: a. providing nucleic acid molecules from the sample; and b. detecting a nucleic acid molecule comprising SEQ ID NO: 16 in the provided nucleic acid molecules; thereby, detecting a functional Pm69 gene in a sample.
  • a method for detecting a functional Pm69 gene in a sample comprising: a. providing nucleic acid molecules from the sample; and b. detecting a DNA molecule comprising a sequence that encodes an mRNA with at least 80% homology to SEQ ID NO: 16 or an mRNA comprising a sequence with at least 80% homology to SEQ ID NO: 16; and c.
  • steps b-c comprise detecting a nucleic acid molecule comprising SEQ ID NO: 16 in the provided nucleic acid molecule.
  • the determining is in the mRNA encoded by the detected DNA molecule.
  • the determining is in the detected mRNA.
  • the detecting comprises amplifying nucleic acid molecules in the sample.
  • the detecting comprises reverse transcribing nucleic acid molecule in the sample.
  • the detecting comprises sequencing nucleic acid molecules in the sample.
  • the amplifying comprises PCR.
  • the sequencing is Sanger sequencing.
  • the sequencing is deep sequencing.
  • the sequencing is next-generation sequencing.
  • Detecting a product of amplification is well known in the art and includes but is not limited to gel electrophoreses and column purification.
  • the one or more primers comprise a tag and the detecting a product of the amplifying comprises detecting the tag.
  • the tag is a fluorescent tag.
  • the tag is a FRET tag.
  • Fluorescently-tagged PCR is well known in the art, and detection of the amplification product may be performed with any fluorometer.
  • Methods of sequencing and of preparing a sequencing library are well known in the art. Any sequencing method including the use of any sequencer is also contemplated.
  • the sample is from a plant.
  • the plant is a cereal.
  • the cereal is selected from wheat, barley, oats, triticale and rye.
  • the cereal is selected from wheat, barley, oats, triticale, rye, rice and maize.
  • the cereal is wheat.
  • the plant is any plant that can be infected by Pm.
  • the sample is from a leaf of the plant. In some embodiments, the sample is from a mature plant. In some embodiments, the sample is from any one of cultivated plant germplasm, pre-breeding materials, and elite plant cultivars.
  • germplasm refers to any living tissue from which a new plant can be grown. In some embodiments, the germplasm is a seed. In some embodiments, the germplasm is any one of a seed, a leaf, stem, tissue culture cells, embryoids and pollen. In some embodiments, the germplasm comprises only a few cells. In some embodiments, the germplasm comprises enough material to perform PCR.
  • pre-breeding materials refers to materials that are not generally directly be used for breeding, but which contain genetic information that can be transferred to breeding materials.
  • cultivars refers to a plant or group of plants selected for desirable characteristics and maintained by propagation. It will be understood by one skilled in the art, that it is advantageous to those growing cereal plants, to integrate a functional Pm69 gene into the genomes of their crops. Further, it will be advantageous to integrate it into all of the crop and not just a portion. As such, the grower will need to confirm the presence of the functional gene, potentially at every stage of the transfer of the gene to the crop.
  • the molecules and methods of the invention can be used for this purpose, and thus the methods can be performed at any step of the process of integrating Pm69 into their crops, and with any material that might be used in this process.
  • the provided nucleic acids comprise at least one of genomic DNA, RNA and cDNA reverse-transcribed from RNA from the sample.
  • the hybridizing comprises at least one of, PCR, southern blotting and northern blotting.
  • PCR may be selected when the nucleic acids are cDNA
  • southern blotting may be selected when the nucleic acids are genomic DNA
  • northern blotting may be selected when the nucleic acids are RNA.
  • the PCR is any one of RT-PCR, qPCR, real-time PCR, or conventional end-point PCR.
  • detecting the hybridizing comprises detection of a PCR product.
  • the detecting comprises gel electrophoreses.
  • the detecting comprises sequencing, deep sequencing or next-generation sequencing.
  • a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
  • Plant materials Wild emmer wheat accession G305-3M (Accession number: CGN19852, genesys-pgr.org/10.18730/lNA3M), the Pm69 (PmG3M) gene donor line, was collected from Upper Galilee, Israel. G305-3M was crossed with the susceptible T. durum wheat line Langdon (LDN) to generate segregating mapping populations. RIL population was used for the construction of a sub-centiMorgan (cM) genetic linkage map. Differential wheat lines carrying different Pm genes (Pm21, TdPm60 and Pm30) were used for testing the virulence of Bgt isolate #70.
  • Bgt and Pst inoculation and disease assessment showed high virulence on many cultivated T. durum, T. aestivum and T. monococcum accessions, as well as on several Pm genes (e.g., Pmla-b, Pm2, Pm3a-d, Pm5a-b, Pm6, Pm7, Pml7, Pm22, Pm30 and Pml + Pm2 + Pm9), but it is avirulent on Pm69. Therefore, Bgt #70 was used in the current study for phenotyping of the Pm69 mapping populations and EMS mutants.
  • Pmla-b e.g., Pm2, Pm3a-d, Pm5a-b, Pm6, Pm7, Pml7, Pm22, Pm30 and Pml + Pm2 + Pm9
  • Plant genomic DNA was extracted using the CTAB method from leaves of 2-3 -week-old wheat seedlings.
  • CAPS and Sequence-Tagged Site (STS) markers were developed based on the local synteny of wheat with major cereals like rice, Brachypodium, Sorghum and barley genomes.
  • the primers for SNP-based Kompetitive Allele Specific PCR (KASP) markers were developed using PolyMarker (polymarker.tgac.ac.uk/) based on the specific SNPs identified by the wheat 90K iSelect SNP array and the wheat 15K SNP array (Trait Genetics®, Germany).
  • the International Wheat Genome Sequencing Consortium (IWGSC) RefSeq assembly vl.O, Wild Emmer Genome Assembly (Zavitan WEWSeq v.1.0) and the Durum Wheat (cv. Svevo) RefSeq Release 1.0 were used to design SSR markers by the BatchPrimer3 website tools (probes.pw.usda.gov/batchprimer3/). Those reference genomes also contributed the SNP information used for designing of KASP markers after verifying them by Sanger sequencing of PCR products. PCR reactions were performed by using 2xTaq PCR Master Mix (TIANGEN, Cat#KT201). KASP marker analysis was performed via the StepOnePlus Real-Time PCR system (Applied Biosystems, USA).
  • Pm69-flanking markers (uhwk386 and uhwk399) were used to select 147 RILs that carry critical recombination events in the Pm69 gene region after screening of 5500 F2 plants (G305-3M xLDN). These RILs were used for further dissection of the target locus by using the graphical genotyping approach. The genetic distances between the detected markers and Pm69 were calculated based on genotypic and phenotypic data using JoinMap 5.0. The physical location of the primers of each marker was identified by BLAST + search against the sequences of the chromosome arm 6BL of wheat reference genomes. Genetic and physical maps of Pm69 were constructed using MapChart v2.2.
  • the G305 ONT contigs were sliced into 1Mb segments with pyfasta and mapped to the the wild emmer assembly WEW_v2.0 using bwa-mem software (Li 2013).
  • the G305-3M ONT contigs anchored to the Pm69 region in Zavitan were compared to the Zavitan and Svevo genomes using Minimap (github.com/lh3/minimap2). Minimap results were visualized using ggplot geom_segment.
  • Susceptible M3-M4 independent mutants one from RIL 169B and four from G305-3M EMS-treated populations, were selected and confirmed with 30 Pm69 flanking markers that showed the same haplotype as G305-3M, ruling out the possibility of cross-pollination from other susceptible lines.
  • Transcriptome sequencing Leaf samples from the four susceptible mutants and the G305-3M wild type were collected 24 hours after 2, 1,3 -Benzo thiadiazole treatment (Sigma- Aldrich, USA). These leaves were submerged in RNAlater (Sigma-Aldrich, UK) and sent for RNA extraction and sequencing in Novogene-UK. Samples were sequenced on Illumina NovaSeq instrument. About 40 million 150 bp paired-end (PE) reads were obtained for each sample. Those reads were aligned to the G305-3M ONT genomic DNA contigs using GSNAP with default settings followed by sorting, duplicate removal, and indexing using SAMtools.
  • PE 150 bp paired-end
  • Mutation detection was done by visualizing the obtained bam files with IGV genome browser focusing on the contgis that were anchored to the PmG305 region. Regions on the contigs that had >4 read depth along at least Ikb and had mutations in all susceptible mutant samples relative to the resistant G305-3M wild-type RNA reads and to the reference G305-3M genome were looked for. The mutations had to be in all reads covering the site and to have read quality >30. Moreover, de-novo assembly of the wild type RNAseq reads was obtained using TRANSABYSS software.
  • Chromosome sorting Mitotic metaphase chromosome suspensions were prepared from tetrapioid wheat lines G305-3M and LDN, and from hexapioid introgression wheat line SC28RRR-26. Briefly, cell cycle of meristematic root tip cells was synchronized using hydroxyurea, and mitotic cells were accumulated in metaphase using amiprohos -methyl. Suspensions of intact chromosomes were prepared by mechanical homogenization of 100 formaldehyde-fixed root tips in 600 pl LB01 buffer.
  • GAA microsatellites and/or GAA and ACG microsatellites were labelled on chromosomes by fluorescence in situ hybridization in suspension (FISHIS) using FITC-labelled oligonucleotides (Sigma, Saint Louis, USA) and chromosomal DNA was stained by DAPI (4’,6-diamidino 2-phenylindole) at 2 pg/ml.
  • FISHIS fluorescence in situ hybridization in suspension
  • DAPI 4’,6-diamidino 2-phenylindole
  • Bivariate flow karyotypes FITC vs. DAPI fluorescence were acquired using a FACS Aria II SORP flow cytometer and sorter (Becton Dickinson Immunocytometry Systems, USA).
  • the samples were analyzed at rates of 1,500-2,000 particles and different positions of sorting windows were tested on bivariate flow karyotype FITC vs. DAPI to achieve the highest purity in the sorted 6B fractions.
  • the content of flow-sorted fractions was estimated using microscopy analysis of slides, containing 1,500-2,000 chromosomes, sorted into a 10-pl drop of PRINS buffer. Sorted chromosomes were identified by FISH with probes for DNA repeats pScl 19.2, Afa family and 45S rDNA. At least 100 chromosomes were classified for each sample using a standard karyotype.
  • Nanopore and Illumina sequencing DNA extraction and QC: High molecular weight (HMW) DNA was extracted from isolated nuclei and purified following a modified salting out DNA extraction protocol (10X Genomics). Stock HMW DNA was size selected on a Blue Pippin instrument (Sage Science) with the high pass protocol and electrophoretic conditions to retain fragments > 30 kb. Eluate was bead cleaned and concentrated. Size selected DNAs were quantified by fluorometry (Qubit 2.0) and DNA integrity was evaluated using a Tapestation 2200 instrument (Agilent). HMW DNAs were stored at 4oC until library preparation.
  • HMW High molecular weight
  • Library preparation Long molecule libraries (ID: Oxford Nanopore Technologies) were prepared following the standard ID ligation protocol (LSK109) with minor modifications to retain and enrich for HMW molecules. Briefly 1.2pg of size selected, end-repaired HMW DNAs were used as input into each library preparation reaction. Libraries were sequenced with R9 flow cell on a PromethlON instrument with high accuracy base-calling enabled. Raw read data were filtered for size and quality score, and approximately 305 Gb filtered data ( ⁇ 23X coverage) was used for assembly. Illumina DNA prep libraries were prepared, indexed, and sequenced to ⁇ 25X coverage on the NovaSeq 6000 S4 flow cell for polishing (PE reads: 150 bp).
  • Genome assembly, polishing and scaffolding Raw fast5 files generated by ONT sequencing of G305-3M were base-called using Guppy version 3.6 (Oxford Nanopore Technology) to produce fastq files. Fastq files from multiple flow-cells were concatenated to form a consolidated fastq file, which was then used for genome assembly using Smartdenovo with the default parameters80. The raw assembly from Smartdenovo was then subjected to a single round of long read polishing using Medaka version 144 (github.com/nanoporetech/medaka) followed by two rounds of short read polishing using Pilon. Assembly stats were calculated using QUAST version 5.0.2 (academic.oup.com/bioinformatics/article/29/8/1072/228832).
  • the G305 ONT contigs were sliced into 1Mb segments with pyfasta and mapped to the wild emmer assembly WEW_v2.0 using BWA-MEM software.
  • the G305-3M ONT contigs anchored to the Pm69 region were compared to the Zavitan (WEW_v2.0) and Svevo RefSeq Rel. 1.0 genomes using Minimap (github.com/lh3/minimap2), and visualized using ggplot geom_segment in R platform.
  • Transcriptome sequencing Wheat leaf samples from the four susceptible mutants and the G305-3M resistant wild type were collected 24 hours after 0.1 mM 2,1,3- Benzo thiadiazole (BTH) treatment with 0.05% Tween-20. Treated leaves were preserved in RNAlater (Sigma-Aldrich, UK) and sent for RNA extraction and sequencing in Novogene- UK. Samples were sequenced on Illumina NovaSeq 6000 instrument. About 40 million 150 bp paired-end (PE) reads were obtained for each sample. Those RNAseq reads were aligned to the G305-3M ONT genomic DNA contigs using GSNAP with default settings followed by sorting, duplicate removal, and indexing using SAMtools.
  • BTH 2,1,3- Benzo thiadiazole
  • Mutation detection was done by visualizing the obtained bam files with IGV genome browser focusing on the contigs that were anchored to the Pm69 region. We looked for the regions on the contigs that had >4 read depth along at least Ikb and had mutations in all susceptible mutant samples relative to the resistant G305-3M wild-type RNA reads and to the reference G305-3M genome. The mutations had to be in all reads covering the site and to have read quality >30. Moreover, de novo assembly of the wild-type RNAseq reads were obtained using TRANS ABYSS software.
  • Pm69 was amplified from cDNA library of G305-3M using the VeriFiTM Polymerase (PCRBIO, UK), then inserted into a cloning plasmid using 5 minTM TA/Blunt-Zero Cloning Kit (Vazyme, China), and transformed into E. coli strain DH5a.
  • the plasmid was extracted by Hybrid-QTM Plasmid Rapidprep Kit (Gene All, Korea) and sequenced using BigDye Terminator vl.l Cycle Sequencing Kit (Applied Biosystems, USA) on ABI 3130 instrument (Applied Biosystems, USA).
  • RNA extraction and quantitative Real-Time-PCR (qRT-PCR): Total RNA was extracted from Bgt #70-inoculated, and non-inoculated G305-3M leaf segments collected along 10 different time points (0, 3, 6, 9, 12, 16, 24, 36, 48, 72 hpi) using the RNeasy Plant Mini Kit (Qiagen, Germany). The cDNA was synthesized from total RNA using a qScriptTM cDNA Synthesis Kit (Quantabio, USA).
  • the gene-specific primers of the Pm69 and the housekeeping gene Ubiquitin were used for qRT-PCR amplification performed on a StepOne thermal cycler (ABI, USA) in a volume of 10 pl containing 5 pl of SYBR Green FastMix (Quantabio, USA), 250 nM primers and optimized dilution of cDNA template.
  • the program included an initial step at 95 °C for 30 s followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 10 s. Relative expression of the target genes was calculated by 2 A (ubiquitin CT-Target CT) ⁇ standard error of the mean (SEM). All of the qRT-PCRs were performed in triplicate, each with at least three (up to five) independent biological repetitions. Primers used are summarized in Table 1.
  • VIGS Virus-induced gene silencing
  • Equimolar amount of Agrobacterium tumefaciens strain GV3101 with pCa-BSMV-a, pCa- BSMV-[3 and pCa-BSMV-y vectors carrying target or control genes were used to inoculate Nicotiana benthamiana leaves to produce virus transcripts. Infiltrated A. benthamiana leaves were then used to extract the sap and further inoculate the second leaves of two-weeks old wheat plantlets. When the pCa-BSMV-y-PDS silenced plants showed chlorosis in the leaves, those plants were inoculated with Bgt #70 in a growth chamber. Two weeks post inoculation, the reaction of wheat plants to Bgt inoculation was recorded.
  • Example 1 The Challenges of chromosome walking in a complex structurally variable region
  • a recombinant inbred line (RIL) mapping population segregating for Pm69 was generated by crossing the susceptible durum wheat (Triticum turgidum ssp. durum) Langdon (LDN) with the resistant WEW accession G305-3M.
  • G305-3M and the Fi generation (G305- 3M x LDN) showed post-haustorial immune responses to Bgt #70 accompanied by intracellular ROS production and host cell death, while LDN showed a highly susceptible response resulting in the development of massive pathogen colonies (Fig. 1A).
  • G305-3M showed a wide-spectrum resistance against 55 tested Bgt isolates originating from four continents Asia, Europe, North America, and South America (Table 2).
  • Table 2 The reactions of wheat differential lines to a set of 54 powdery mildew isolates collected in Israel, Switzerland, USA, Netherland, Chile and Paraguay.
  • GF7 A homozygous resistant F3 line containing Pm21
  • GF8 A homozygous susceptible F3 line that doesn’t contain Pm21
  • Jing 411 and 781 are susceptible parents in the pedigree of producing this Pm21 mapping population.
  • Wild emmer wheat accessions G18-16, C20, and G-305-3M are the donors of PmG16, Pm30, and Pm69, respectively.
  • Example 2 The challenges of using MutChromSeq based on chromosome sorting in tetrapioid wheat.
  • chromosomes 6B, IB, 7B, 4B and 5B formed a composite and poorly resolved population on bivariate flow karyotype. Chromosomes were sorted from the composites in hopes of further enriching chromosome 6B, but only a maximum purity of 47% and 51% in the case of G305-3M and LDN, respectively, was achievable. The sorted fractions were contaminated by other chromosomes (IB, 7B, 4B and 5B in case of G305-3M, and chromosomes IB, 7B and 5B in case of LDN).
  • Example 3 Dissection of the structural variation complexity by ONT sequencing of G305-3M
  • ONT sequencing was used for whole-genome sequencing ( ⁇ 23x coverage) of WEW accession G305-3M. After assembling the reads, 2,489 Contigs were obtained, with N50 value of 11.2 Mb and N90 value of 2.6 Mb. The longest contig was 70.65 Mb, and the total length of the genome assembly was 10 GB, which is typical for the tetrapioid wheat genome.
  • Pm69 physical map was constructed by anchoring the ONT contigs to the Pm69 genetic map using the co-dominant PCR markers.
  • Two contigs utgl7163 (1.1 Mb) and utg5064 (1.09 Mb) were perfectly anchored to the genetic map by four Pm69 flanking markers.
  • An additional ONT contig utg4926 (156.5 kb) was identified by searching for gene sequences that reside in the Pm69 collinear region of the WEW_v2.0 genome. Based on the three identified ONT contigs, 15 markers were developed that were incorporated into the genetic map by graphical genotyping of the RIL population.
  • the Pm69 cosegregating markers spanned part of contig utg5064 (120400 bp to end), the whole contig utg4926 (0-156528 bp), and part of contigs utgl7163 (0-380148 bp) (Fig. IB).
  • RNAseq reads of four independent EMS-derived susceptible mutants were mapped onto the G305-3M ONT contigs (M3-5 generations, Fig. 3A, 10). This approach revealed eight expressed candidate genes in the Pm69 physical region, seven in utgl7163, one in utg5046, while no expressed genes were found in utg4926. Seven candidates were predicted as NLRs and one as a FAR1 Related Sequence (FRS) transcription factor (Fig. 3B).
  • FRS FAR1 Related Sequence
  • NLR-6 Only one gene spanning 12280 bp (SEQ ID NO: 14) on G305-3M contig utgl7163, named NLR-6, had five different point mutations (Table 3) in the four susceptible mutants. These five mutations in NLR-6 were all G/C to A/T transitions typical of EMS mutagenesis, while the remaining seven genes in this region did not have any mutations.
  • the predicted 2763 bp coding sequence (CDS) of NLR-6 (SEQ ID NO: 15) was validated by Sanger sequencing using G305-3M cDNA.
  • the -12.3 kb genomic region of the gene contains one 5’ UTR and two translated exons (SEQ ID NO: 16).
  • the region contains two introns, one of them located in the 5’ UTR region (Fig. 3C).
  • All the identified point mutations of NLR-6 were validated by Sanger sequencing and were confirmed as missense mutations.
  • their resistant sister lines, derived from the same Mo plants, harbored the Pm69 wild allele were validated by Sanger sequencing using G305-3M cDNA.
  • NLR6 NLR6
  • SEQ ID NO: 17 The predicted structure of the NLR-6 protein contains an N- terminal coil coil Rx_N domain with RanGAP interaction sites, an NB-ARC domain and an LRR domain (Fig. 3D).
  • the Rx_N-terminal domain predicted to have a coiled-coil structure with four helical bundle fold, is found in many plant resistance proteins.
  • Example 6 Functional validation of Pm69 through virus-induced gene silencing (VIGS) [0176]
  • Virus-induced gene silencing (VIGS) constructs were designed to target two genomic positions in Pm69 respectively (Table 4).
  • a phytoene desaturase (PDS) gene silencing construct was used to test the efficacy of the VIGS system in tetrapioid (G305-3M) and hexapioid (a bread wheat introgression line cv. Ruta + Pm69) backgrounds.
  • the inoculation with the Pm(59-silencing constructs resulted in susceptibility to Bgt #70 of the leaves in G305-3M and Pm69 introgression line, while the negative controls showed no visible Bgt symptoms on the leaves (Fig.
  • FIG. 4A-B Histopathological characterization showed that Pm69 control cells (BSMV:GFP) accumulated intracellular ROS and prevented the invasion of the Bgt germinating spores. Silencing of PDS is visualized as white streaks resulting from photobleached chlorophyll (Fig. 4A), while in the Pm69 VIGS silenced leaves, there was detected a mosaic pattern of germinating spores that invaded the cells successfully and developed colonies, alongside spores that activated HR cell death responses, as in the control resistant plants (Fig. 4C).
  • BSMV:GFP Pm69 control cells
  • Quantitative reverse transcription PCR showed a significant reduction of expression levels of Pm69 mRNA in the Pm69 VIGS silenced leaves compared with GFP silenced leaves (p ⁇ 0.05; Fig. 4A-B). Silencing of Pm69 in G305-3M and the introgression line (Ruta + Pm69) resulted in susceptible phenotypes, therefore, providing functional validation for the role of the Pm69 gene (NLR- 6) in conferring resistance against wheat powdery mildew.
  • Example 7 The Pm69 is a rare allele
  • the functional molecular marker uhw403 was used for a large-scale screening of the distribution of Pm69 among 538 wheat accessions of which 310 are WEW from across the Fertile Crescent and 228 represent other wheat species (Fig. 5A). Only G305-3M yielded positive PCR amplification. To further estimate the presence of the gene in the WEW gene pool, a return was made to the original G305-3M collection site south of Kadita, Northern Israel, and an additional 64 WEW accessions were collected in a radius of less than 1km from the original collection site (Fig. 5B). Even there, only three WEW accessions were found that gave amplification of uhw403 marker and showed high resistance to Bgt #70 (Fig. 5B-D).
  • Pm69 was transferred into elite Israeli bread wheat cultivar ‘Ruta’ using marker-assisted selection (MAS) following the “durum as a bridge” approach. Furthermore, Pm69 was also introgressed into the durum wheat cultivar ‘Svevo’, which contains Sri 3b. These homozygous ILs with different segments of G305-3M chromosome have been selected from the BC4F2 populations and showed high resistance to Bgt #70 (Fig. 7A).
  • MAS marker-assisted selection
  • G305-3M contains the yellow rust resistance gene Yrl5, which was confirmed by ONT sequencing data, present in 4.57 Mbp contig utgl l61 and showed high resistance to Pst #5006 (Fig. 7A).
  • These resistant ILs can be used in wheat resistance breeding programs (Fig. 7B).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Botany (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Isolated nucleic acid molecules comprising a nucleic acid sequence that encodes a functional Pm69 protein that confers resistance to powdery milder (Pm) are provided. Functional Pm69 protein as well as artificial vectors, and transgenic plants expressing the nucleic acid molecule or protein are also provided. Methods of conferring resistance to Pm and detecting a functional Pm69 gene are also provided.

Description

PM69 AND USE THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/307,820, filed February 8, 2022, and U.S. Provisional Patent Application No. 63/417,716, filed October 20, 2022, the contents of which are all incorporated herein by reference in their entirety.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[002] The contents of the electronic sequence listing (CRML-P-043-PCT.xml; Size: 63,350 bytes; and Date of Creation: February 5, 2023) is herein incorporated by reference in its entirety.
FIELD OF INVENTION
[003] The present invention is in the field of wheat genomics and disease resistance.
BACKGROUND OF THE INVENTION
[004] Powdery mildew (Pm) caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgf) is one of the most destructive wheat diseases worldwide. To date, more than 110 Pm resistance genes/alleles have been identified and mapped in wheat and its wild relatives1, of which only about 10% have been cloned. Pm3b/Pm8, Pm2a, Pm21, Pm60IMlIW172, Pm5e, Pm41 and Pmla encode nucleotide -binding leucine-rich repeat (NLR) protein. Pm24 encodes a tandem kinase protein and Pm4 encodes a putative chimeric protein of a serine/threonine kinase, multiple C2 domains, and transmembrane regions. Pm38 (Lr34IYrl8ISr57, ABC transporter) and Pm46 (Lr67IYr46ISr55, hexose transporter) show broad- spectrum adult plant resistance to powdery mildew and rust diseases. However, Pm resistance genes are frequently overcome due to the rapid evolution of Bgt isolates. Thus, the identification and cloning of novel disease resistance genes (R-genes) and deployment into cultivated wheat germplasm can enrich the repertoire of genes available for resistance breeding.
[005] Positional cloning has been widely used to clone genes. The published reference genomes of several wheat species serve as powerful tools for the dissection of the target genomic regions and provide a reliable source for candidate gene prediction. However, genes responsible for the phenotype of interest may be absent from the reference genotypes, especially when the target genes are derived from wild relatives. Frequently, robust disease resistance genes (R-gene) encode intracellular immune receptors, with nucleotide -binding leucine-rich repeat (NLR) domain architecture. Analysis of 16 hexapioid wheat genomes revealed that only 31-34% of the NLR signatures are shared across all the genomes, indicating that this gene family is subjected to rapid evolutionary processes. Analysis of the Chinese Spring (CS) bread wheat genome assembly revealed 2,151 NLR-like genes, of which 1,298 were arranged in 547 gene clusters, many of them showing more than 75% similarity within each cluster, likely formed by tandem duplications. Such clusters of highly similar genes make it particularly challenging to localize a causal R-gene, especially when relying heavily on published reference genomes. The construction of a Bacterial Artificial Chromosome (BAC) library with 100-200 kb insert size may be useful for assembling high- quality physical maps to support positional cloning, however, this technology is time and labor-intensive, especially when working with complex genomes, such as wheat.
[006] To overcome the limitations of positional cloning R-genes and reduce the genomic complexity of wheat, several methods based on next-generation sequencing (NGS) have been developed and their efficiency was demonstrated for cloning of novel R-genes. These methods include Mutagenesis and Resistance gene Enrichment and Sequencing (MutRenSeq), Association genetics with R gene enrichment Sequencing (AgRenSeq), Mutant Chromosome flow sorting and short-read Sequencing (MutChromSeq), and Targeted Chromosome-based Cloning via long-range Assembly (TACCA). However, these methods still have some limitations. For example, MutRenSeq and AgRenSeq only identify NLR genes that can be captured by hybridization, while TACCA and MutChromSeq rely on the purification of individual chromosomes that carry mutations in the target genes. Moreover, Bulked segregant RNA sequencing (BSR-Seq) and bulked segregant Core Genome Targeted sequencing (CGT-Seq) have been used successfully for identifying R-genes, but these methods rely on the selection scales of individuals for mixing genomic bulked pools.
[007] Wheat has a large and complex genome with more than 85% repetitive sequences making genome sequence assembly challenging, particularly when using short-read (100- 250 bp) sequencing technologies. With the rapid development of sequencing methodologies, the long-read sequencing platforms such as Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing and Oxford Nanopore Technologies (ONT) sequencing generate sufficiently long (> 10 kb) reads that result in more contiguous sequence assemblies. Long-read sequencing technologies have been successfully used for barley and bread wheat genome assemblies. These technologies can improve physical mapping certainty, detection of structural variants, and transcript isoform identification.
[008] Wild emmer wheat (Tritium turgidum ssp. dicoccoides, WEW, 2/7 = 4% = 28, AABB), the tetrapioid progenitor of hexapioid bread wheat (T. aestivum, 2n=6x=42, AABBDD), is a valuable genetic resource for resistance genes. More than 20 Pm resistance genes have been identified and mapped in WEW. Among them, only two were cloned, which were Pm41 localized on chromosome arm 3BL and TdPm60 localized on chromosome arm 7 AL. Pm.41 and TdPm60 showed abundance of 1.81% and 25.6% among the tested natural WEW accessions, respectively. Pm69 (PmG3M), identified from WEW accession G305- 3M, is a dominant gene conferring a wide-spectrum resistance to Bgt isolates from around the globe. Pm69 is the only gene derived from WEW that was mapped to the telomeric region of chromosome arm 6BL.The full sequence of PM69, which can be integrated into cereal strains that lack it and thus confer Pm resistance is greatly needed.
SUMMARY OF THE INVENTION
[009] The present invention provides isolated nucleic acid molecules comprising a nucleic acid sequence that encodes a functional Pm69 protein that confers resistance to powdery milder (Pm). Functional Pm69 protein as well as artificial vectors, and transgenic plants expressing the nucleic acid molecule or protein are also provided. Methods of conferring resistance to Pm and detecting a functional Pm69 gene are also provided.
[010] According to a first aspect, there is provided an isolated nucleic acid molecule comprising a nucleic acid sequence that encodes a protein with at least 85% homology to SEQ ID NO: 17 and that confers resistance to powdery mildew (Pm).
[Oi l] According to some embodiments, the nucleic acid sequence encodes a protein with at least 85% identity to SEQ ID NO: 17 and that confers resistance to Pm.
[012] According to some embodiments, the nucleic acid sequence comprising at least 85% homology to SEQ ID NO: 15 or 16. [013] According to some embodiments, the nucleic acid sequence comprises at least 85% identity to SEQ ID NO: 15 or 16.
[014] According to some embodiments, the protein that confers resistance comprises methionine 55, serine 507, glycine 606, serine 200 and at least one of glycine 11 and arginine 553 within SEQ ID NO: 17.
[015] According to some embodiments, the protein that confers resistance comprises a functional Rx_N domain, LRR domain and NB-ARC domain such that the protein confers resistance to PM.
[016] According to some embodiments, the functional Rx_N domain comprises SEQ ID NO: 34, the functional LRR domain comprises SEQ ID NO: 35 or the functional NB-ARC domain comprises SEQ ID NO: 36.
[017] According to some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule.
[018] According to some embodiments, conferring Pm resistance is conferring Pm resistance to cereal plant.
[019] According to some embodiments, the cereal plant is a wheat plant.
[020] According to some embodiments, the nucleic acid sequence comprises at least 85% identity to SEQ ID NO: 16.
[021] According to some embodiments, the nucleic acid sequence consists of SEQ ID NO: 16.
[022] According to some embodiments, the isolated nucleic acid molecule further comprises a transcription regulatory element operatively linked to the nucleic acid sequence.
[023] According to some embodiments, resistance comprises a post-haustorial immune responses to the Pm.
[024] According to some embodiments, the post-haustorial immune response comprises intracellular reactive oxidation species (ROS) production in response to Pm.
[025] According to another aspect, there is provided an artificial vector comprising the isolated nucleic acid molecule of the invention.
[026] According to some embodiments, the artificial vector further comprises at least one nucleic acid sequence of a pathogen-resistance gene. [027] According to some embodiments, the pathogen is Stripe Rust (Pst).
[028] According to some embodiments, the pathogen-resistance gene is selected from Yrl5, Yr5, Yr36, Yrl8 and Yr46.
[029] According to some embodiments, the pathogen-resistance gene is Yr 15.
[030] According to some embodiments, the artificial vector comprises at least one transcriptional regulatory element active in plant cells and operatively linked to the nucleic acid sequence.
[031] According to some embodiments, the transcriptional regulatory element is a promoter.
[032] According to some embodiments, the promoter is a heterologous promoter of the Pm69 endogenous promoter.
[033] According to some embodiments, the plant is a cereal plant.
[034] According to some embodiments, the cereal plant is selected from wheat, barley, rye, triticale, oat, rice and maize.
[035] According to some embodiments, the artificial vector is for use in conferring resistance to Pm to a cell of a plant.
[036] According to some embodiments, resistance comprises a post-haustorial immune responses to the Pm.
[037] According to some embodiments, the post-haustorial immune response comprises intracellular reactive oxidation species (ROS) production in response to Pm.
[038] According to another aspect, there is provided a transgenic plant or cell thereof comprising a nucleic acid molecule of the invention or an artificial vector of the invention.
[039] According to some embodiments, the plant is a cereal plant.
[040] According to some embodiments, the cereal plant is any one of barley, rye, triticale, oat, wheat, rice and maize.
[041] According to some embodiments, the cereal plant is wheat.
[042] According to another aspect, there is provided an isolated protein comprising at least 85% homology to SEQ ID NO: 17 and comprising the ability to confer resistance to Pm.
[043] According to some embodiments, the isolated protein comprises at least 85% identity to SEQ ID NO: 17. [044] According to some embodiments, the isolated protein consists of an amino acid sequence with at least 85% identity to SEQ ID NO: 17.
[045] According to some embodiments, the isolated protein consists of SEQ ID NO: 17.
[046] According to another aspect, there is provide a method of conferring resistance to Pm to a plant or a cell thereof, the method comprising at least one of: a. expressing in the cell of the plant at least one of an isolated nucleic acid molecule of the invention, an artificial vector of the invention, and an isolated protein of the invention; and b. converting at least one pm69 non-functional allele of the cell of the plant into a functional Pm69 gene, thereby conferring resistance to Pm to a plant or cell thereof.
[047] A method for detecting a functional Pm69 gene in a sample, which a functional Pm69 gene confers resistance to Pm, comprising: a. providing nucleic acid molecules from the sample; b. detecting a DNA molecule comprising a sequence that encodes an mRNA with at least 80% homology to SEQ ID NO: 16 or an mRNA comprising a sequence with at least 80% homology to SEQ ID NO: 16; and c. determining that the mRNA encodes a protein comprising at least 85% identity to SEQ ID NO: 17 and comprising methionine 55, serine 507, glycine 606, serine 200 and at least one of glycine 11 and arginine 553; thereby detecting a functional Pm69 gene in a sample.
[048] According to some embodiments, steps b-c comprise detecting a nucleic acid molecule comprising SEQ ID NO: 16 in the provided nucleic acid molecules.
[049] According to some embodiments, the sample is from a plant.
[050] According to some embodiments, the plant is a cereal plant.
[051] According to some embodiments, the cereal plant is selected from wheat, barley, oat, triticale, rye, rice and maize.
[052] According to some embodiments, the sample is from any one of cultivated plant germplasm, pre-breeding materials, and elite plant cultivars.
[053] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[054] Figures 1A-B: Fine mapping of Pm69 from WEW G305-3M. (1A) Macroscopic observation of the response of G305-3M, LDN and Fl leaves to Bgt # 70 infection at 7 days post-infection (dpi); DAB staining of leaves at 3 dpi to detect ROS accumulation visualized as Reddish-brown coloration; Trypan blue staining of leaves at 3 dpi to visualize fungal structures and plant cell death as blue coloration; MH: mature haustorium; HP: haustorial primordium; Hy: hyphae. Scale bars = 50 pm. (IB) Genomic region containing Pm69 genetic region on the long arm of wheat chromosome 6B (top row). The locations of Pm69 flanking genetic markers on the WEW _v2.0 reference genome (second row). The genetic map of Pm69. The green-colored markers were developed based on WEW _v2.0, the blue-colored markers were developed based on ONT contigs, (third row). The 6BL ONT contig assembly in the Pm69 genetic region of G305-3M. (fourth row). The physical map of the Pm69 region is marked in blue.
[055] Figures 2A-B: Comparison of the G305-3M ONT contigs with the (2A) 6B pseudomolecule of WEW_v2.0 and (2B) durum wheat Svevo RefSeq Rel. 1.0 around the Pm69 genetic region. X-axis: the cumulative length of G305-3M contigs; Y-axis: the physical location of 6B pseudomolecule in the reference genome. Contigs utg380-utgl7163 marked with different colors belong to the ONT assembly of G305-3M
[056] Figures 3A-D: The workflow of identification of Pm69 by ONT contigs by MutRNAseq. (3A) The phenotypes of EMS-derived mutants (M-40, M-22, M-12, and M- 8) and wild type (G305-3M) infected with Bgt #10. M-40, M-22, M-12 and M-8 and G305- 3M were analyzed by RNA-seq to identify SNPs among the three ONT contigs. (3B) The expressed genes in the Pm69 genetic region identified by aligning the RNAseq reads to G305-3M ONT contigs. Vertical red line: the locations of SNPs in the susceptible mutants identified after mapping the RNASeq reads to G305-3M ONT contigs. (3C) The distribution of the independent mutations. (3D) The predicted structure of Pm69 protein with Rx_N, NB- ARC and LRR domains, and the precise location of all the identified mutants. [057] Figures 4A-C: Functional validation of Pm69 candidate through VIGS. (4A) VIGS of Pm69 candidate gene is in G305-3M. (4B) VIGS of Pm69 candidate gene in the introgression line (Ruta+Pm69). BSMV:GFP was used as a negative control. BSMV:wild type, the wild type of pCa-BSMV- vector, was used as a negative control. BSMV: PDS is the PDS gene silencing construct used to check the efficiency of the VIGS system; BMSV:Pm69-l and Pm69-2 were the constructs that targeted 5’UTR- Rx_N domain and NB-ARC domain, respectively. Asterisks indicate the level of significance by t-test of the Pm69 expression levels in the target plants compared with the negative control of BSMV: wild type, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). (4C) Histopathology characterization of the Pm69 wild-type plants (BSMV:GFP) and Pm69- silencing plants (BSMV:Pm69-l and BSMV:Pm69-2). DAB and Coomassie brilliant blue staining of Pm69 or GFP silenced leaves in G305-3M at 7 dpi with Bgt #70. Scale bars = 100 pm.
[058] Figures 5A-D: The geographic distribution of the Pm69 allele in WEW populations. (5A) The geographic distribution of wild emmer wheat (WEW) natural populations (black circles) which were screened for the presence of Pm69 functional allele. (5B) The geographic distribution of WEW individual plants collected near the recorded G305-3M collection site (marked by a blue triangle, south of Kadita, Northern Israel). (5C) A submap of 5B showing the three accessions contain Pm69. The maps were obtained from Google Maps. (5D) The Phenotypes in response to infection with Bgt #70 and agarose gel electrophoresis of PCR products amplified by marker uhw403 (549 bp for Pm69) and M- Pm60-Sl (831 bp for TdPmOO) from representative WEW accessions.
[059] Figure 6: Micro-Collinearity analysis of the Pm69 genetic region among different Triticeae genomes. Lines indicate genes with high collinearity. The numbers on the yellow-colored NLRs present different NLR clades based on the evolutionary analysis, which means the same number of NLRs may have evolved by duplication events. The dark blue marks Pm69 and baby blue marks Pm69 homologs. The red triangle marks the locations of Pm69 or its homologs with highest similarity. The pink color marks OPR11 homologs which are more conserved among different genomes than the Pm69 homologs. The green color marks other non-NLR proteins. The NLRs show more genetic diversity than their flanking non-NLR proteins.
[060] Figures 7A-B: Introgression of the Pm69 into the cultivated wheat and pyramided with yellow rust resistance genes. (7A) The phenotypes of different wheat parental and introgression lines to Bgt #70 and Pst #5006. WEW: G305-3M contains Pm69 and G25 contains Yrl5. Durum wheat: LDN and Svevo. Common wheat: Ruta and AvocetTr75. (7B) The whole plant structure of the G305-3M, Ruta and a representative introgression line Ruta + Pm69.
[061] Figure 8: The genetic map of Pm69. The deletion bin map of wheat chromosome arm 6BL (left); Primary genetic map of the Pm69 by using a small population (2500 F2 individuals) (middle); High-resolution genetic map of Pm69 on wheat chromosome 6BL (right). The mapping population of 147 homozygous recombinant inbred lines (RILs, F6 generation) which were developed from 5500 F2 individuals (G305-3M * LDN) based on the graphical genotyping approach. The blue markers, codominant; Red markers, LDN dominant; Green markers, G305-3M dominant.
[062] Figure 9: The phenotypic response of different wheat accessions to Bgt #70. Bgt #70 showed a strong virulence to several wheat species. Wild emmer wheat (WEW) accession: Zavitan; Durum accessions: Kronos, LDN and Svevo; Bread wheat accessions: Fielder, Morocco, Ruta and Chinese Spring; T. monococcum accessions: NGB 10953, NGB 10965, NGB 10969 and NGB22782, are susceptible to Bgt #70. The WEW accession: G305-3M and G18-16 that contained functional Pm genes (Pm69 and TdPm60. respectively) showed high resistance to Bgt #70. The Zavitan, Svevo and CS were highly susceptible to Bgt #70, suggesting that the three reference genomes in Figure S2 did not contain the functional Pm69 allele.
[063] Figure 10: The phenotypic response of EMS-derived mutants to Bgt #70 and Bgt #15. The resistant plant is the sister line of each mutant, which was derived from the same Mo plant. Bgt #15 is another isolate used for Pm69 genetic mapping.
[064] Figure 11: The expression patterns of Pm69 in G305-3M. The expression levels of Pm69 (fold change as compared to ubiquitin levels) in the By/-inoculatcd plants compared with the negative control of non-inoculated plants at different time point. The time points ranged from 0-72 hpi in both non-inoculated (mock) and Bgt #70-inoculated plants. Asterisks indicate the level of significance by t-test, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), of the difference in Pm69 gene expression in non-inoculated and By/-inoculatcd plants.
[065] Figure 12: Sequence alignments of Pm69 protein and five representative susceptible Pm69 homologs. The red colored region presents the conserved part of the protein. The sequence of those homologs could be found on the website of WheatOmics 1.0 and EnsemblPlants by using their ID names. The alignments were constructed by the multiple alignment tool CLUSTALW from the NCBI. [066] Figure 13: Three Pm69-similar sequences in the whole genome of WEW_ v2.0. Blasting the Pm69 protein sequence against Zavitan reference genome WEW_v2.0 revealed three copies in 716-719 Mbp with different lengths of introns, 615, 15676 and 3390 bp, respectively. The blue color marks exon 1 and green color marks exon 2. Identity to Pm69 is provided.
DETAILED DESCRIPTION OF THE INVENTION
[067] The present invention, in some embodiments, provides isolated nucleic acid molecules comprising a nucleic acid sequence that encodes a functional Pm69 protein that confers resistance to powdery milder (Pm). Isolated nucleic acid molecule comprising a nucleic acid sequence that encodes a protein with at least 85% homology to SEQ ID NO: 17 and that confers resistance to Pm are also provided. The functional Pm69 protein as well as artificial vectors, and transgenic plants expressing the nucleic acid molecule or protein are provided as are methods of conferring resistance to Pm and detecting a functional Pm69 gene.
[068] The invention is based, at least in part, of the surprising discovery of the sequence of Pm69 which confers resistance to powdery mildew (Pm). Further, when Pm69 was deployed into cultivated wheat breeding lines in conferred resistance to Pm and pyramiding of Pm69 with a yellow rust gene, Yrl5, enriching the wheat disease resistance breeding resource. The Rx_N domain, LRR domain and NB-ARC domain were found to be required for this resistance and multiple pm69 non-functional alleles with mutations in the coding region lack a Pm-resistance phenotype and are found in many domesticated species that are susceptible to Pm. Furthermore, by comparing these non-functional alleles to the functional allele it is possible to distinguish between non-functional alleles and/or pseudogenes and the functional allele in the future.
[069] By a first aspect, there is provided a nucleic acid molecule comprising a nucleic acid sequence that encodes a protein that confers resistance to Pm.
[070] As used herein, “Pm” refers to powdery mildew, which is also called Blumeria graminis f. sp. tritici (Bgf). It is a biotrophic fungus that is one of the most destructive wheat diseases worldwide. In some embodiments, Pm comprises all strains of powdery mildew. In some embodiments, Pm is cereal plant Pm. In some embodiments, Pm is wheat Pm. [071] In some embodiments, the protein that confers resistance is Pm69. Pm69 is also known as nucleotide-binding leucine-rich repeat 6 (NLR6). The nucleotide and protein sequence for Pm(59/Pm69 is first disclosed herein. In some embodiments, Pm69 is a functional Pm69. In some embodiments, functional is functional in conferring Pm resistance. In some embodiments, functional is functional in inducing a post-haustorial immune response. In some embodiments, the immune response is in response to Pm infection. In some embodiments, a post-hausorial immune response comprises intracellular reactive oxidation species (ROS) production. In some embodiments, the production is in response to Pm infection. As used herein, “Pm69” refers to a functional Pm69 gene, which confers resistance to Pm. As used herein, “pm69” refers to a non-functional pm69 gene, which does not confer resistance to Pm. The term "gene" is used broadly to refer to any nucleic acid associated with a biological function. Thus, the term "gene" includes coding sequences (CDS) and/or regulatory sequences required for expression. The term "gene" can also apply to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
[072] As used herein, the term “confers resistance” refers to increasing the survival of a plant or cell when challenged with a pathogen i.e., Pm. In some embodiments, increasing survival is a decrease in the growth of the pathogen. In some embodiments, increasing survival is a decrease in the spread of the pathogen. In some embodiments, increasing survival comprises lack of infection by the pathogen. In some embodiments, resistance comprises not being able to be infected by the pathogen. In some embodiments resistance comprises increased survival as compared to survival without the vector, nucleic acid moleucle, or protein of the invention. In some embodiments, the increase refers to at least 10% increase, 20% increase, 30% increase, 40% increase, 50% increase, 60% increase, 70% increase, 80% increase, 90% increase, or 100% increase in survival. Each possibility represents a separate embodiment of the invention. In some embodiments, the decrease refers to at least 10% decrease, 20% decrease, 30% decrease, 40% decrease, 50% decrease, 60% decrease, 70% decrease, 80% decrease, 90% decrease, or 100% decrease. Each possibility represents a separate embodiment of the invention. In some embodiments, the increase is at least a 50% increase. In some embodiments, the decrease is at least a 50% decrease. In some embodiments, conferring resistance comprises conferring a post- haustorial immune response to the pathogen. In some embodiments, conferring resistance comprises conferring an intracellular ROS response to the pathogen. [073] In some embodiments, the functional Pm69 gene comprises the Pm69 CDS. In some embodiments, the Pm69 CDS comprises the nucleic acid sequence atggaggcggctctcgtgagtgtggccacgggggtcctgaagcccgtcctggagaagctggcagctctgcttggtgacgagtac aagcggttcaagggagtgcgcaaggatatcaagtccatcgctcgtgagctcgctgccatggaggcttttctcctcaagatgtccga ggaggaggatccagatgtacaagacaaattttggatgaatgaggtgcgggagctctcctatgatatggaggatgccattgacgact tcatgcaaagcgttggtgacaaagacgaaaagcctgatgacttcattgagaagatcaaaaacacactagaaaagttggggaagat gaaggctcgccgccggattggcaaggagattcaggatctaaagaaacagatcatacaggtgggtgacaggaatgcaaggtaca aaggtcgtcaaaccttctctagcaccaaaaatgaaattgtagaccctagaattcttgctagattcgagcatgcatcaaaactcgttgga attgatgaaaccaaagccgagataatcaagctattaggcgaagaaaatggacaagtgccaaggcaacaacaactgaagatactct ctattgttggatttggaggaatggggaagacaactcttgcaaaccaagtgtatcaagacctcaaaggcgaatttcagtatcgagcttt catatcggtgtcacaaaatccagacttgatgaaaatcctcagaactatccttagtgaaattactggtataagctatcctggcaccgaag cagggtgcatagaacaactcatcgacaagatcaaagatttcctagcagacaaaaggtatcttattgtcatagatgatatatgggacat aaaacattgggaagtgatccgatgtgctctagctaataatcattatgagaatagagtaatcacaacaacccgtgatcgcgacgttgc acgtaaagttggtggcgcctatgagcttaaacccctccctgatgagacatccaaaatattattctttggaagaatttttggtattaataat gactgtccagatgatttggtcgaagtatctgaaaccatcatgaagaaatgtggcggcgtgccattggctatcatcaccattgctggttt attggccagtcgggaaaggaataaaagggagtggaacaagttgtgtgattctatcggttcgggacttgacaatagtcctgatgtgaa gacgatgagaaatatattggcccttagctatcgccatctacctatccacctaaaaacttgcttgttgtatctaagtatatttcctgaagac tacattattcaaggagacagattgatatggaggtggatatgtgaaggttttcttaatggggtacaggatgaggacttatttgagcttggt gagagctacatcacggagctcataaacagaggattgatccaagcggtggactcctataggtatgtcaggacaacggattgccgtgt gcatgacttggttctagaatttatcagctccatttctattgaagaaaatttttgtactgtattgcatgataagaaggccaagtcagctgcg ataacaagcaaggtccgcaggttatttctgcaactccagcatgttgagatgcctcaggggagattgatattgtcccacatgaggtca cttactgtgtcgggcaactgggttgacgaaatgtcgcccctttctttttcccatcaattacgtgtattggatttggagggctgctatcttg cgcaacgccatgatttacttgcgcatctcgggagtttatgccagttgagatatctttcgttgggaaatgcatacctcggtgacctcttatt ggcgatccaaattgggcagctaaaaagtctgatatgcttaagagttggcggtcgcttcaaaatcaaattcaggccaggtgtggttag ggaactggagtgtctgcaagagttgtcaatgatcaatttatctaagtctccgcacgttgccaaagagctaaggcatttgactaaactg agggttcttggtatctctttcgaagatatgcctgatgagagcttgaagggttggcttttggagtctctaggtcacctgaggaatttgcag agcttaattgtgagcacttttggttgtgtatctttggatttcttttgggaaggctggacatcccctcctcgcaacctccgtagatttcattca tccttttcctacctgtcgttgtggatcagcccgtcaaatcttcgggagctctccatcatagagatcaagctggacaacctgcggcggg aggatcttgacatacttggttcctttccttctttgcaatccctccggctgaggggctacaagaatatctgggatgaagaaaggaaaca gtggccggtgatcagtgctgatactttccagtgcctgcgggagtgtgtgttggggatacttcccatgggaggaaatatgttcgcacc aggagcaatgcccaaggtacaaagccttacatttgattgttacatcgaggatgtatttagcttgggtttggcgaacctcccatctctcc gtgatagcttgggcttggagaatctcccttctctccaggagctccatgtttgcttagaagaattcacccatgggtccatcacccgaga agcatatgacaaagcgaaggctgcaattaggtgtgcagcagacaaccatcccaaccgccccacccttaaggtgggcatgtggttc tgtccatcatgggcgagcaactcggactccgcatag (SEQ ID NO: 16). In some embodiments, the Pm69 CDS consists of SEQ ID NO: 16. In some embodiments, SEQ ID NO: 16 is a cDNA sequence devoid of introns. In some embodiment SEQ ID NO: 16 is a DNA sequence not found in nature.
[074] In some embodiments, the functional Pm69 gene comprises the Pm69 genomic sequence which comprises the nucleic acid sequence cgctgcacaagtctcagttgctgcacttgcctcacgccgtcacgtgtcgccggagtctggtcgacggacgacacggctggacacc gtctacgctccagccacacaaaattaagcgagctagcacgtcctgcaacccatggaggcggctctcgtgagtgtggccacgggg gtcctgaagcccgtcctggagaagctggcagctctgcttggtgacgagtacaagcggttcaagggagtgcgcaaggatatcaagt ccatcgctcgtgagctcgctgccatggaggcttttctcctcaagatgtccgaggaggaggatccagatgtacaagacaaattttgga tgaatgaggtgcgggagctctcctatgatatggaggatgccattgacgacttcatgcaaagcgttggtgacaaagacgaaaagcct gatgacttcattgagaagatcaaaaacacactagaaaagttggggaagatgaaggctcgccgccggattggcaaggagattcag gatctaaagaaacagatcatacaggtgggtgacaggaatgcaaggtacaaaggtcgtcaaaccttctctagcaccaaaaatgaaat tgtagaccctagaattcttgctagattcgagcatgcatcaaaactcgttggaattgatgaaaccaaagccgagataatcaagctatta ggcgaagaaaatggacaagtgccaaggcaacaacaactgaagatactctctattgttggatttggaggaatggggaagacaactc ttgcaaaccaagtgtatcaagacctcaaaggcgaatttcagtatcgagctttcatatcggtgtcacaaaatccagacttgatgaaaatc ctcagaactatccttagtgaaattactggtataagctatcctggcaccgaagcagggtgcatagaacaactcatcgacaagatcaaa gatttcctagcagacaaaaggtatcttattgtcatagatgatatatgggacataaaacattgggaagtgatccgatgtgctctagctaat aatcattatgagaatagagtaatcacaacaacccgtgatcgcgacgttgcacgtaaagttggtggcgcctatgagcttaaacccctc cctgatgagacatccaaaatattattctttggaagaatttttggtattaataatgactgtccagatgatttggtcgaagtatctgaaaccat catgaagaaatgtggcggcgtgccattggctatcatcaccattgctggtttattggccagtcgggaaaggaataaaagggagtgga acaagttgtgtgattctatcggttcgggacttgacaatagtcctgatgtgaagacgatgagaaatatattggcccttagctatcgccat ctacctatccacctaaaaacttgcttgttgtatctaagtatatttcctgaagactacattattcaaggagacagattgatatggaggtgga tatgtgaaggttttcttaatggggtacaggatgaggacttatttgagcttggtgagagctacatcacggagctcataaacagaggatt gatccaagcggtggactcctataggtatgtcaggacaacggattgccgtgtgcatgacttggttctagaatttatcagctccatttctat tgaagaaaatttttgtactgtattgcatgataagaaggccaagtcagctgcgataacaagcaaggtccgcaggttatttctgcaactc cagcatgttgagatgcctcaggggagattgatattgtcccacatgaggtcacttactgtgtcgggcaactgggttgacgaaatgtcg cccctttctttttcccatcaattacgtgtattggatttggagggctgctatcttgcgcaacgccatgatttacttgcgcatctcgggagttt atgccagttgagatatctttcgttgggaaatgcatacctcggtgacctcttattggcgatccaaattgggcagctaaaaagtctgatat gcttaagagttggcggtcgcttcaaaatcaaattcaggccaggtgtggttagggaactggagtgtctgcaagagttgtcaatgatca atttatctaagtctccgcacgttgccaaagagctaaggcatttgactaaactgagggttcttggtatctctttcgaagatatgcctgatg agagcttgaagggttggcttttggagtctctaggtcacctgaggaatttgcagagcttaattgtgagcacttttggttgtgtatctttgga tttcttttgggaaggctggacatcccctcctcgcaacctccgtagatttcattcatccttttcctacctgtcgttgtggatcagcccgtca aatcttcgggagctctccatcatagagatcaagctggacaacctgcggcgggaggatcttgacatacttggttcctttccttctttgca atccctccggctgaggggctacaagaatatctgggatgaagaaaggaaacagtggccggtgatcagtgctgatactttccagtgc ctgcgggagtgtgtgttggggatacttcccatgggaggaaatatgttcgcaccaggagcaatgcccaaggtacaaagccttacattt gattgttacatcgaggatgtatttagcttgggtttggcgaacctcccatctctccgtgatagcttgggcttggagaatctcccttctctcc aggagctccatgtttgcttagaagaattcacccatgggtccatcacccgagaagcatatgacaaagcgaaggctgcaattaggtgt gcagcagacaaccatcccaaccgccccacccttaaggtgggcatgtggttctgtccatcatgggcgagcaactcggactccgcat agtttccttgttgtatgcatatcttaactctgaagtggaactaagtacctatgtagtgtgttgcgtgagcactgcttttcttctattgtaatac tacactttttgtacatttttgtatgttttgttctacaaattttgtacgttttgtacattgatgatgtatctttagccatttaatttggacgacaatca ttttgcttttttg (SEQ ID NO: 15). In some embodiments, the Pm69 genomic sequence consists of SEQ ID NO: 15. In some embodiments, SEQ ID NO: 15 is a DNA sequence of the full transcript sequence of the Pm69 mRNA. It will be understood by a skilled artisan that SEQ ID NO: 15 can also be an RNA sequence in which the thymine residues are uracil. In some embodiments, the Pm69 genomic sequence comprises the genomic sequence of Pm69 and 2 kilobases upstream and/or downstream from that sequence. In some embodiments, SEQ ID NO: 14 comprises the Pm69 genomic sequence with 2 kilobases upstream and two kilobases downstream from SEQ ID NO: 15.
[075] In some embodiments, the Pm69 CDS is a cDNA reverse transcribed from an RNA which codes for the amino acid sequence MEAALVSVATGVLKPVLEKLAALLGDEYKRFKGVRKDIKSIARELAAMEAFLLK MSEEEDPDVQDKFWMNEVRELSYDMEDAIDDFMQSVGDKDEKPDDFIEKIKNTL EKLGKMKARRRIGKEIQDLKKQIIQVGDRNARYKGRQTFSSTKNEIVDPRILARFE HASKLVGIDETKAEIIKLLGEENGQVPRQQQLKILSIVGFGGMGKTTLANQVYQDL KGEFQYRAFISVSQNPDLMKILRTILSEITGISYPGTEAGCIEQLIDKIKDFLADKRYL IVIDDIWDIKHWEVIRCALANNHYENRVITTTRDRDVARKVGGAYELKPLPDETSK ILFFGRIFGINNDCPDDLVEVSETIMKKCGGVPLAIITIAGLLASRERNKREWNKLC DSIGSGLDNSPDVKTMRNILALSYRHLPIHLKTCLLYLSIFPEDYIIQGDRLIWRWIC EGFLNGVQDEDLFELGESYITELINRGLIQAVDSYRYVRTTDCRVHDLVLEFISSISI EENFCTVLHDKKAKSAAITSKVRRLFLQLQHVEMPQGRLILSHMRSLTVSGNWVD EMSPLSFSHQLRVLDLEGCYLAQRHDLLAHLGSLCQLRYLSLGNAYLGDLLLAIQI GQLKSLICLRVGGRFKIKFRPGVVRELECLQELSMINLSKSPHVAKELRHLTKLRVL GISFEDMPDESLKGWLLESLGHLRNLQSLIVSTFGCVSLDFFWEGWTSPPRNLRRF HSSFSYLSLWISPSNLRELSIIEIKLDNLRREDLDILGSFPSLQSLRLRGYKNIWDEER KQWPVISADTFQCLRECVLGILPMGGNMFAPGAMPKVQSLTFDCYIEDVFSLGLA NLPSLRDSLGLENLPSLQELHVCLEEFTHGSITREAYDKAKAAIRCAADNHPNRPT LKVGMWFCPSWASNSDSA (SEQ ID NO: 17). In some embodiments, the Pm69 protein comprises SEQ ID NO: 17. In some embodiments, the Pm69 protein consists of SEQ ID NO: 17. [076] In some embodiments, Pm69 comprises a functional Rx_N domain. An Rx_N domain is also known as an Rx N-terminal domain. Rx_N domains are found in many plant resistance proteins at the protein’s N-terminus. It is predicted to be a coiled-coil, but some work has shown that it adopts a four helical bundle fold. In some embodiments, the Rx_N domain of Pm69 comprises or consists of amino acids 12-79 of SEQ ID NO: 17. In some embodiments, the Rx_N domain comprises a methionine at position 55 in SEQ ID NO: 17. In some embodiments, the Rx_N domain does not comprise an alanine at position 55 in SEQ ID NO: 17. In some embodiments, a functional Pm69 comprises a methionine at position 55 of SEQ ID NO: 17. In some embodiments, a functional Pm69 does not comprise an alanine at position 55 of SEQ ID NO: 17. In some embodiments, the Rx_N domain comprises a glycine at position 11 of SEQ ID NO: 17. In some embodiments, the Rx_N domain does not comprise an arginine at position 11 of SEQ ID NO: 17. In some embodiments, the functional Pm69 comprises a glycine at position 11 of SEQ ID NO: 17. In some embodiments, the functional Pm69 does not comprise an arginine at position 11 of SEQ ID NO: 17. In some embodiments, the Rx_N domain comprises an arginine at position 553 of SEQ ID NO: 17. In some embodiments, the Rx_N domain does not comprise a lysine at position 553 of SEQ ID NO: 17. In some embodiments, the functional Pm69 comprises an arginine at position 553 of SEQ ID NO: 17. In some embodiments, the functional Pm69 does not comprise a lysine at position 553 of SEQ ID NO: 17. In some embodiments, the Rx_N domain comprises a glycine at position 11 and an arginine at position 553 of SEQ ID NO: 17. In some embodiments, the Rx_N domain does not comprise an arginine at position 11 and a lysine at position 553 of SEQ ID NO: 17. In some embodiments, the functional Pm69 comprises a glycine at position 11 and an arginine at position 553 of SEQ ID NO: 17. In some embodiments, the functional Pm69 does not comprise an arginine at position 11 and a lysine at position 553 of SEQ ID NO: 17.
[077] In some embodiments, Pm69 comprises a functional leucine rich repeat (LRR) domain. LRR domains generally consists of 2-45 leucine-rich repeats with each repeat being about 20-30 residues long. Structurally these domains adopt an arc or horseshoe shape, with the concave face consisting of parallel P-strands and the convex face representing a more variable region of secondary structures including helices. In some embodiments, the LRR domain of Pm69 comprises or consists of amino acids 549-708 of SEQ ID NO: 17. In some embodiments, the LRR domain comprises a glycine at position 606 of SEQ ID NO: 17. In some embodiments, the LRR domain does not comprise a glutamic acid at position 606 of SEQ ID NO: 17. In some embodiments, the functional Pm69 comprises a glycine at position 606 of SEQ ID NO: 17. In some embodiments, the functional Pm69 does not comprise a glutamic acid at position 606 of SEQ ID NO: 17.
[078] In some embodiments, Pm69 comprises a functional NB-ARC domain. NB-ARC domains are functional ATPase domains that generally regulate signaling. In particular, its nucleotide-binding state is proposed to regulate activity of the R protein. In some embodiments, the NB-ARC domain of Pm69 comprises or consists of amino acids 172-416 of SEQ ID NO: 17. In some embodiments, the NB-ARC domain comprises a serine at position 200 of SEQ ID NO: 17. In some embodiments, the NB-ARC domain does not comprise a phenylalanine at position 200 of SEQ ID NO: 17.
[079] In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the DNA is cDNA. In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the DNA or RNA is double stranded. In some embodiments, the DNA or RNA is single stranded. In some embodiments, the DNA molecule is a vector.
[001] In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule. As used herein, the term "isolated nucleic acid molecule" refers to a nucleic acid molecule that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the nucleic acid in nature. Typically, a preparation of isolated DNA or RNA contains the nucleic acid in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. In some embodiments, an isolated nucleic acid molecule is at least 90% pure. In some embodiments, an isolated nucleic acid molecule is at least 95% pure. In some embodiments, pure is with respect to other nucleic acid molecule. In some embodiments, pure is with respect to other non-nucleic acid cellular components. In some embodiments, the isolated DNA is cDNA. In some embodiments, the isolated nucleic acid is any one of DNA, RNA, and cDNA. In some embodiments, the isolated nucleic acid molecule is a synthesized nucleic acid molecule. Synthesis of nucleic acid molecules is well known in the art and may be performed, for example, by ligating or covalently linking by primer linkers multiple nucleic acid molecules together or by in vitro amplification (e.g., PCR) or reversetranscription.
[002] The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising nucleotides. Nucleotides are comprised of nucleosides and phosphate groups. The nitrogenous bases of nucleosides include, for example, naturally occurring purine or pyrimidine nucleosides as found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). Sequences given herein are given as DNA sequences comprising thymine but it will be understood that they can also refer to an RNA with the same sequence but with the thymines replaced by uracils.
[003] The term “nucleic acid molecule” includes but is not limited to singlestranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNAs, circular nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, amplification products, modified nucleic acids, plasmid or organellar nucleic acids, and artificial nucleic acids such as oligonucleotides.
[004] In some embodiments, the nucleic acid sequence comprises at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to the Pm69 coding sequence. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleic acid sequence comprises at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 16. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleic acid sequence comprises at least 80% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 85% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 90% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 95% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 97% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 99% homology to SEQ ID NO: 16. In some embodiments, the nucleic acid sequence comprises at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention. In some embodiments, the nucleic acid sequence comprises at least 85% homology to SEQ ID NO: 15. In some embodiments, homology is identity.
[005] In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 14. In some embodiments, the nucleic acid sequence consists of SEQ ID NO: 14. In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 15. In some embodiments, the nucleic acid sequence consists of SEQ ID NO: 15. In some embodiments, the nucleic acid sequence comprises SEQ ID NO: 16. In some embodiments, the nucleic acid sequence consists of SEQ ID NO: 16. [006] In some embodiments, the nucleic acid sequence encodes a protein that confers resistance to Pm. In some embodiments, a protein that confers resistance comprises at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 17 or a fragment analog or homolog thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, a protein that confers resistance comprises at least 85% homology to SEQ ID NO: 17. In some embodiments, homology is identity. In some embodiments, a fragment is a fragment that confers resistance. In some embodiments, a fragment is a fragment comprising an Rx_N domain, a LRR domain or a NB-ARC domain. In some embodiments, a fragment is a fragment comprising an Rx_N domain, a LRR domain and a NB-ARC domain. In some embodiments, a fragment comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 amino acids of SEQ ID NO: 17. Each possibility represents a separate embodiment of the invention. In some embodiments, amino acids of SEQ ID NO: 17 are consecutive amino acids of SEQ ID NO: 17. In some embodiments, an analog or homolog is an analog or homolog that confers resistance.
[007] As used herein, the term "analog" or “homolog” includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities to confer resistance to Pm. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.
[080] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides, polypeptides and proteins described have modifications rendering them more stable while in the organism or more capable of penetrating into cells. In one embodiment, the terms “peptide”, "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.
[081] In some embodiments, conferring resistance is conferring resistance to a plant. In some embodiments, conferring resistance is conferring resistance to a plant cell. In some embodiments, a plant is a cell of a plant. In some embodiments, the plant is a cereal plant. In some embodiments, the plant is a grain plant. In some embodiments, the grain/cereal plant is wheat. In some embodiments, the grain plant is selected from barley, rye, oat, triticale, spelt and wheat. In some embodiments, the grain plant is selected from barley, rye, rice, maize, triticale, oat, spelt and wheat.
[082] As used herein, the term “wheat” refers to a plant of the genus Triticum. Wheat can be used for the production of grain such as is used for bread, cereal or pasta for non-limiting examples. In some embodiments, wheat is bread wheat or duram wheat. In some embodiments, wheat is duram wheat. In some embodiments, wheat comprises spelt. In some embodiments, wheat is Triticum turgidum.
[083] In some embodiments, the isolated nucleic acid molecule comprises a fragment, a homolog or an analog to the Pm69 gene, wherein the fragment, homoIgo or analog encodes a protein that confers resistance to Pm. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 16 or a fragment, homolog or analog thereof and encodes a protein with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology to SEQ ID NO: 17 or a fragment, homolog or analog thereof, wherein the protein confers resistance to Pm. Each possibility represents a separate embodiment of the invention.
[084] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that encodes the Rx_N domain of Pm69 or a functional fragment, homolog or analog thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that encodes the LRR domain of Pm69 or a functional fragment, homolog or analog thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that encodes the NB-ARC domain of Pm69 or a functional fragment, homolog or analog thereof.
[085] In some embodiments, the Rx_N domain comprises VLKPVLEKLAALLGDEYKRFKGVRKDIKSIARELAAMEAFLLKMSEEEDPDVQDK FWMNEVRELSYDM (SEQ ID NO: 34). In some embodiments, the Rx_N domain consists of SEQ ID NO: 34. In some embodiments, the nucleic acid sequence comprises a sequence that encodes the Rx_N domain. In some embodiments, a sequence that encodes the Rx_N domain comprises gtcctgaagcccgtcctggagaagctggcagctctgcttggtgacgagtacaagcggttcaagggagtgcgcaaggatatcaagt ccatcgctcgtgagctcgctgccatggaggcttttctcctcaagatgtccgaggaggaggatccagatgtacaagacaaattttgga tgaatgaggtgcgggagctctcctatgatatg (SEQ ID NO: 37). In some embodiments, a sequence that encodes the Rx_N domain consists of SEQ ID NO: 37.
[086] In some embodiments, the LRR domain comprises LSHMRSLTVSGNWVDEMSPLSFSHQLRVLDLEGCYLAQRHDLLAHLGSLCQLRY LSLGNAYLGDLLLAIQIGQLKSLICLRVGGRFKIKFRPGVVRELECLQELSMINLSK SPHVAKEERHETKERVEGISFEDMPDESEKGWEEESEGHERNEQSEIVS (SEQ ID NO: 35). In some embodiments, the ERR domain consists of SEQ ID NO: 35. In some embodiments, the nucleic acid sequence comprises a sequence that encodes the LRR domain. In some embodiments, a sequence that encodes the LRR domain comprises ttgtcccacatgaggtcacttactgtgtcgggcaactgggttgacgaaatgtcgcccctttctttttcccatcaattacgtgtattggattt ggagggctgctatcttgcgcaacgccatgatttacttgcgcatctcgggagtttatgccagttgagatatctttcgttgggaaatgcat acctcggtgacctcttattggcgatccaaattgggcagctaaaaagtctgatatgcttaagagttggcggtcgcttcaaaatcaaattc aggccaggtgtggttagggaactggagtgtctgcaagagttgtcaatgatcaatttatctaagtctccgcacgttgccaaagagcta aggcatttgactaaactgagggttcttggtatctctttcgaagatatgcctgatgagagcttgaagggttggcttttggagtctctaggt cacctgaggaatttgcagagcttaattgtgagc (SEQ ID NO: 38). In some embodiments, a sequence that encodes the LRR domain consists of SEQ ID NO: 38.
[087] In some embodiments, the NB-ARC domain comprises IDETKAEIIKLLGEENGQVPRQQQLKILSIVGFGGMGKTTLANQVYQDLKGEFQYR AFISVSQNPDLMKILRTILSEITGISYPGTEAGCIEQLIDKIKDFLADKRYLIVIDDIWD IKHWEVIRCALANNHYENRVITTTRDRDVARKVGGAYELKPLPDETSKILFFGRIF GINNDCPDDLVEVSETIMKKCGGVPLAIITIAGLLASRERNKREWNKLCDSIGSGLD NSPDVKTMRNILALSY (SEQ ID NO: 36). In some embodiments, the NB-ARC domain consists of SEQ ID NO: 36. In some embodiments, the nucleic acid sequence comprises a sequence that encodes the NB-ARC domain. In some embodiments, a sequence that encodes the NB-ARC domain comprises atccttagtgaaattactggtataagctatcctggcaccgaagcagggtgcatagaacaactcatcgacaagatcaaagatttcctag cagacaaaaggtatcttattgtcatagatgatatatgggacataaaacattgggaagtgatccgatgtgctctagctaataatcattatg agaatagagtaatcacaacaacccgtgatcgcgacgttgcacgtaaagttggtggcgcctatgagcttaaacccctccctgatgag acatccaaaatattattctttggaagaatttttggtattaataatgactgtccagatgatttggtcgaagtatctgaaaccatcatgaagaa atgtggcggcgtgccattggctatcatcaccattgctggtttattggccagtcgggaaaggaataaaagggagtggaacaagttgtg tgattctatcggttcgggacttgacaatagtcctgatgtgaagacgatgagaaatatattggcccttagctat (SEQ ID NO: 39). In some embodiments, a sequence that encodes the NB-ARC domain consists of SEQ ID NO: 39.
[088] In some embodiments, the Rx_N domain comprises at least 85% homology to SEQ ID NO: 34 and retains the domain function. In some embodiments, the LRR domain comprises at least 85% homology to SEQ ID NO: 35 and retains domain function. In some embodiments, the NB-ARC domain comprises at least 85% homology to SEQ ID NO: 36 and retains domain function. In some embodiments, domain function is conferring resistance to Pm. In some embodiments, the sequence comprises at least 85% homology to SEQ ID NO: 37. In some embodiments, the sequence comprises at least 85% homology to SEQ ID NO: 38. In some embodiments, the sequence comprises at least 85% homology to SEQ ID NO: 39.
[089] By another aspect, there is provided a vector comprising an isolated DNA of the invention.
[090] In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is an artificial vector. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector is a plant expression vector. In some embodiments, a vector is a plasmid. In some embodiments, a vector is a viral vector. In some embodiments, a vector is a lentiviral vector. In some embodiments, the vector is for use in expressing Pm69. In some embodiments, the vector is for use in expressing Pm69 in a plant or cell thereof. In some embodiments, the vector is for use in conferring resistance to Pm. In some embodiments, the vector is for use in conferring resistance to Pm to a plant or cell thereof.
[091] Expressing of a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. In some embodiments, the gene is in an expression vector such as plasmid or viral vector. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
[092] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector, a virgaviridae viral vector, or a poxviral vector. The barley stripe mosaic virus (BSMV), the tobacco ratle virus and the cabbage leaf curl geminivirus (CbLCV) may also be used. The promoters may be active in plant cells. The promoters may be a viral promoter.
[093] In some embodiments, the nucleic acid molecule further comprises at least one transcriptional regulatory element. In some embodiments, the vector comprises at least one transcriptional regulatory element. In some embodiments, the transcription regulatory element is a promoter. In some embodiments, the promoter is a plant promoter. In some embodiments, the promoter is active in plants or a plant cell. In some embodiments, the promoter is a viral promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is the endogenous Pm69 promoter or a fragment thereof capable of driving transcription. In some embodiments, driving transcription is driving transcription in a plant or plant cell. In some embodiments, the endogenous promoter comprises at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 kb upstream of the transcriptional start site of Pm69. Each possibility represents a separate embodiment of the invention. In some embodiments, the endogenous promoter comprises the first 2000 nucleotides of SEQ ID NO: 14. In some embodiments, the promoter is a heterologous promoter.
[094] In some embodiments, the nucleic acid sequence is operatively linked to a transcriptional regulatory element. In some embodiments, the nucleic acid molecule is operatively linked to a transcriptional regulatory element. The term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, the promoter is operably linked to an isolated DNA of the invention. In some embodiments, the transcriptional regulatory element is a heterologous transcriptional regulatory element. In some embodiments, the transcriptional regulatory element is an endogenous transcriptional regulatory element.
[095] In some embodiments, the vector is an expression vector that drives expression of Pm69. The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. Thus, expression of a nucleic acid molecule may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and/or translation of RNA into a precursor or mature protein (polypeptide). Expressing of a gene within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell’s genome. [096] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), such as biolistic use of coated particles, and needle-like particles, Agrobacterium Ti plasmids and/or the like.
[097] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. The promoter may extend upstream or downstream of the transcriptional start site, and may be any size ranging from a few base pairs to several kilo-bases.
[098] In some embodiments, nucleic acid sequences are transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.
[099] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.
[0100] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo- 5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallo thionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
[0101] In some embodiments, recombinant viral vectors, which offer advantages such as systemic infection and targeting specificity, are used for in vivo expression. In one embodiment, systemic infection is inherent in the life cycle of, for example, the retrovirus and is the process by which a single infected cell produces many progeny virions that infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread systemically. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
[0102] In some embodiments, plant viral vectors are used. In some embodiments, a wildtype virus is used. In some embodiments, a deconstructed virus such as are known in the art is used. In some embodiments, Agrobacterium is used to introduce the vector of the invention into a virus.
[0103] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, agrobacterium Ti plasmids and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
[0104] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield, or activity of the expressed polypeptide.
[0105] In some embodiments, the vector further comprises at least one nucleic acid sequence of a tandem kinase-pseudokinase (TKP)-containing gene. In some embodiments, the vector further comprises at least one nucleic acid sequence of a pathogen resistance gene. In some embodiments, the vector comprises a sequence encoding a TKP-containing gene. In some embodiments, the vector comprises a sequence encoding at least one pathogen resistance gene.
[0106] In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a tandem kinase-pseudokinase (TKP)-containing gene. Each possibility represents a separate embodiment of the invention.
As used herein a TKP-containing gene is a gene with an active kinase and inactive pseudokinse domain in tandem. In some embodiments, the TKP-containing gene comprises no other protein motifs. In some embodiments, the TKP-domain is homologous to the TKP domain of WTK1. In some embodiments, the TKP-domain containing protein is Wtkl (Yrl5). In some embodiments, the TKP-containing gene is selected from wheat tandem kinase 1 (WTK1), leucine-rich -repeat receptor kinases subfamily 6B (LRR-6B), receptorlike cytoplasmic kinases subfamily 7 (RLCK_7), leucine -rick-repeat receptor kinases subfamily 3 (LFF_3), receptor-like cytoplasmic kinases subfamily 8 (RLCK_8), cell wall- associated kinase (WAK), concanavalin A-like lectin protein kinase (L-LPK), other kinases with no published family (RK_1), leucine -rich-repeat receptor kinases subfamily 12
(LRR_12) and cysteine rich kinases (LRR_8B) subfamilies. In some embodiments, the TKP- containing gene is a WTK1, LRR-6B, RLCK_7, LFF_3, RLCK_8, WAK, L-LPK, RK_1, RLCK_7, LRR_12 or LRR_6B subfamily gene. Each possibility represents a separate embodiment of the invention. In some embodiments, the TKP-containing gene is selected from TraesCS 1 A01G061500.1, TraesCSlA01G197000.2, TraesCSlA01G432400.1
TraesCSlB01G079900.1, TraesCSlD01G033500.1, TraesCS2A01G510300.1
TraesCS2B01G538000.1, TraesCS2B01G538200.1, TraesCS2D01G123700.1
TraesCS2D01G124300.1, TraesCS2D01G511500.1, TraesCS2D01G579800.3
TraesCS3B01G579200.1, TraesCS4A01G334900.1, TraesCS4A01G335000.1
TraesCS5A01G161500.1, TraesCS5A01G241300.2, TraesCS5A01G449800.1
TraesCS5B01G005400.3, TraesCS5B01G159000.1, TraesCS5B01G239600.1
TraesCS5D01G166400.1, TraesCS5D01G241800.1, TraesCS5D01G247800.1
TraesCS5D01G459500.1, TraesCS5D01G459700.1, TraesCS5D01G537200.1 TraesCS6A01G020100.1, TraesCS6A01G036400.1, TraesCS6B01G029600.1,
TraesCS6B01G050800.1, TraesCS6B01G050900.1, TraesCS6B01G051000.1,
TraesCS6D01G025700.1, TraesCS6D01G042200.1, TraesCS6D01G042300.1,
TraesCS7B01G048900.1, TraesCS7D01G147900.1, HORVUlHrlGOl 1660.17,
HORVUlHrlG051220.15, HORVU5HrlG050470.1, HORVU5HrlG107460.3,
HGRVU6HrlG003940.7, HORVU6HrlG025940.2, HORVU7HrlG001450.11,
HGRVU7HrlG001600.12, SclLoc00250465.1, Sc5LocO 1920045.3, Sc2Loc00020948.6, GsOltO310500-01, 0s07t0493200-01, Os07t0493800-00, 0s07t0494300-00,
0sl0t0141200-00, GslOtO143866-OO, 0sl lt0173432-00, 0sl lt0445300-01,
Gsl ltO5535OO-OO, 0sl lt0556400-00, AQK57443.1, AQK57450.1, AQK57451.1, AQK57454.1, AQK58522.1, AQK90211.1, AQK92446.1, ONM26931.1, AT2G32800.1, Ppls31_26V6.1, PGSC0003DMP400002294, BnaA07gl4690D, BnaA09g41440D, BnaC04g38500D, BnaA03gl5120D, BnaA02g06510D, Potri.017G055000,
Potri.001G315000, SOB IC.010G171600. l.P, SOBIC.005G096400.1.P,
SOBIC.008G022300.2.P, SOBIC.001G353800.1.P, SOBIC.008G148200.2.P,
SOBIC.010G028950.1.P, SOBIC.005G154100.1.P, SOBIC.009G246800.1.P,
SOBIC.005G155100.1.P, SOBIC.005G154800.1.P, SOBIC.005G060700.2.P,
SOBIC.001G354100.2.P, WTK1, MLOC, and RPG1. In some embodiments, the TKP containing gene is selected from TraesCSlA01G061500.1, TraesCSlA01G197000.2
TraesCSlA01G432400.1, TraesCSlB01G079900.1, TraesCSlD01G033500.1
TraesCS2A01G510300.1, TraesCS2B01G538000.1, TraesCS2B01G538200.1
TraesCS2D01G123700.1, TraesCS2D01G124300.1, TraesCS2D01G511500.1
TraesCS2D01G579800.3, TraesCS3B01G579200.1, TraesCS4A01G334900.1
TraesCS4A01G335000.1, TraesCS5A01G161500.1, TraesCS5A01G241300.2
TraesCS5A01G449800.1, TraesCS5B01G005400.3, TraesCS5B01G159000.1
TraesCS5B01G239600.1, TraesCS5D01G166400.1, TraesCS5D01G241800.1.
TraesCS5D01G247800.1, TraesCS5D01G459500.1, TraesCS5D01G459700.1
TraesCS5D01G537200.1, TraesCS6A01G020100.1, TraesCS6A01G036400.1
TraesCS6B01G029600.1, TraesCS6B01G050800.1, TraesCS6B01G050900.1
TraesCS6B01G051000.1, TraesCS6D01G025700.1, TraesCS6D01G042200.1.
TraesCS6D01G042300.1, TraesCS7B01G048900.1, TraesCS7D01G147900.1
HORVUlHrlG011660.17, HORVUlHrlG051220.15, HGRVU5HrlG050470.1
HORVU5HrlG107460.3, HORVU6HrlG003940.7, HORVU6HrlG025940.2
HORVU7HrlG001450.11, HORVU7HrlG001600.12, SclLoc00250465.1.
Sc5Loc01920045.3, Sc2Loc00020948.6, GsOltO310500-01, 0s07t0493200-01 Os07t0493800-00, 0s07t0494300-00, Os lOtO 141200-00, 0sl0t0143866-00,
0sl lt0173432-00, 0sl lt0445300-01, 0sl lt0553500-00, 0sl lt0556400-00, AQK57443.1, AQK57450.1, AQK57451.1, AQK57454.1, AQK58522.1, AQK90211.1, AQK92446.1, ONM26931.1, AT2G32800.1, Ppls31_26V6.1, PGSC0003DMP400002294,
BnaA07gl4690D, BnaA09g41440D, BnaC04g38500D, BnaA03gl5120D,
BnaA02g06510D, Potri.017G055000, Potri.001G315000, SOBIC.010G171600.1.P, SOBIC.005G096400.1.P, SOBIC.008G022300.2.P, SOBIC.001G353800.1.P,
SOBIC.008G148200.2.P, SOBIC.010G028950.1.P, SOBIC.005G154100.1.P,
SOBIC.009G246800.1.P, SOBIC.005G155100.1.P, SOBIC.005G154800.1.P,
SOBIC.005G060700.2.P, SOBIC.001G354100.2.P and MLOC.
[0107] In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a pathogen-resistance gene. Each possibility represents a separate embodiment of the invention. As used herein, the term “pathogen-resistance gene” refers to a gene that provides a plant or plant cell with resistance to a pathogen. In some embodiments, the pathogen is a bacterial pathogen. In some embodiments, the pathogen is a fungal pathogen. In some embodiments, pathogen is yellow (stripe) rust (Pst). In some embodiments, yellow stripe rust is the fungus Puccinia striiformis f. sp. Tritici (Pst). Pathogen-resistance genes are well known in the art, and include, but are not limited to Yrl5, Yrl8, Yr5, Yr36, Yr46, Yrl7, Yr29, LrlO, Lrl3, Yr58, Srl3 and Sr 21. Yrl8 is also known as Lr34 and Sr57. Yr29 is also known as Lr46. Yr46 is also known as Lr67. Yrl8 is also known as Lr34 and Sr57. In some embodiments, the pathogen resistance gene is selected from Yrl5, Yrl8, Yr5, Yr36, Yr46, Yrl7, Yr29, LrlO, Lrl3, Yr58, Srl3 and Sr 21. In some embodiments, the pathogen resistance gene is selected from Yrl5, Yrl8, Yr5, Yr36, and Yr46. In some embodiments, the pathogen resistance gene is selected from Yrl5, Yrl8, Yr5, and Yr36. In some embodiments, the pathogen resistance gene is selected from Yrl5, Yrl8, and Yr5. In some embodiments, the pathogen resistance gene is selected from Yrl5 and Yr5. In some embodiments, the pathogen resistance gene is Yrl5.
[0108] In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yrl5 gene. Each possibility represents a separate embodiment of the invention. In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 85% homology to a Yrl5 gene. In some embodiments, the artificial vector further comprises a Yrl5 gene or fragment, homolog or analog thereof that confers resistance to Pst. In some embodiments, the Yrl5 gene comprises the nucleotide sequence atggattaccaaggaaacaattttaatgatttctttcaaactaatgggcattttgtacttaaaagagtggacaacaactataaactgcgg tcattcactgaaaaggagatagagcacattacagacagatatagcacttcgcttggtaatggctcgttcggtgatgtctacaaaggaa gattagacgatcaacgtccagtcgcagtaaagagatacaaaaatggaaccaagaaagaggagtttgccaaggaggtgatagtgc attcccagataaaccataagaacgttgtcagattgttaggctgctgcacagaggaaaatgctcttatgattgttatggagtttatctgta atggaaacctctacaacatccttcactgtggcaatgcagatggtcctatccccttccctttggacaaacgtttggacatcgccatcga gtcagcggaagcactatcatgcatgcattcaatgtacagtcctgtccttcatggtgacattaaacctgccaatatactgttggatgaaa agtacttgccaaagctatctgattttggaatagcaagactgctttctactgatgaggcccagcgtaccaaaactgttattggttgcatag gttatgtagaccctttgttttgtcagagcggtattctaactacaaagagtgatgtatacagttttggagttgttctgttggaaatgatcacc cgaaaaaaagcgacggatggggctactagtcttactcaatgtttcgccgaggccctgggagggaagaaggtgagacaattgtttg atgtggaaattgccaatgacaagaagaaggtgaaattgatcgaagatattgcgaagttagcagctacatgcttgaaactggaggat aaaatgcgtccgacaatggttgaggtagcagatagacttaggaggattagaaaagctctcccccagcgcaagggtgaaagctcta caggcatcaacaatgggctcataagaacaggaaaggcagaggatctaccaactatttcccttgatgaaatgaagaaactaacaag aaacttcagtgatggtgctctaataggagagagctcacaaggcagagttttgttcgaagagttaagttatggaaagagatatgcattc aagtcttctcaagaaattgatttgaagattgaagcaatttcaagactgaaacacaagaacgttgtccaacttctcggaaattgggtcga aggaaacaaatatgttcttgcttacgagtatgtatcggggggcacgttgcatgatattcttcacagagagggtgataagggtgtcagt ggagccaggccaggagcagctctatcatggatgcagagagtgaagattgccttaagtgcagcagaagggcttgagttcctccatc agaaggcagagcctcaggtcacccacggtaacatcatgtccagcaagatacttctctttgacaaagataatgcaaaggttggcggc gttggtatctccaatgtactggtgcgtgataacatggttcactgtcacagttttagacaggactgtgacatggatcgtatggatggtatt cgttatcacccagatgattactatgtcgatctatatgctgctactggacagtgtaacgcaaagagcgatgtatacgccttcggggttgt gctgctggagcttttaaccggtcgcgaggcagttgatcatgcactacccaaaggcaagcagagcctcgtgacatgggtatacaac catggcgaggagaaaagcccatggagatgggaaaacaatattatggaagacaatgtgttaacaaaaacaagttttagtgaagatat ggtgcagcgatgcgtggatccaaggcttaaaggatattaccatcgcagtgctgttaccaagatgggtgcgatcgcgtcgctatgcg tgaattacaatccagatctccgaccaaacatgagcactgtcgtcaagggtctgaggcaattgttgcaaaagtga (SEQ ID NO: 29). In some embodiments, the Yrl5 gene consists of SEQ ID NO: 29. In some embodiments, the Yrl5 gene is operably linked to a plant promoter. In some embodiments, the artificial vector further comprises a nucleic acid sequence that encodes for a Yrl5 protein. In some embodiments, the Yrl5 protein (WTK1) comprising the sequence MDYQGNNFNDFFQTNGHFVLKRVDNNYKLRSFTEKEIEHITDRYSTSLGNGSFGD VYKGREDDQRPVAVKRYKNGTKKEEFAKEVIVHSQINHKNVVREEGCCTEENAE MIVMEFICNGNLYNILHCGNADGPIPFPLDKRLDIAIESAEALSCMHSMYSPVLHGD IKPANILLDEKYLPKLSDFGIARLLSTDEAQRTKTVIGCIGYVDPLFCQSGILTTKSD VYSFGVVLLEMITRKKATDGATSLTQCFAEALGGKKVRQLFDVEIANDKKKVKLI EDIAKLAATCLKLEDKMRPTMVEVADRLRRIRKALPQRKGESSTGINNGLIRTGKA EDLPTISLDEMKKLTRNFSDGALIGESSQGRVLFEELSYGKRYAFKSSQEIDLKIEAI SRLKHKNVVQLLGNWVEGNKYVLAYEYVSGGTLHDILHREGDKGVSGARPGAA LSWMQRVKIALSAAEGLEFLHQKAEPQVTHGNIMSSKILLFDKDNAKVGGVGISN VLVRDNMVHCHSFRQDCDMDRMDGIRYHPDDYYVDLYAATGQCNAKSDVYAF GVVLLELLTGREAVDHALPKGKQSLVTWVYNHGEEKSPWRWENNIMEDNVLTK TSFSEDMVQRCVDPRLKGYYHRSAVTKMGAIASLCVNYNPDLRPNMSTVVKGLR QLLQK (SEQ ID NO: 30). In some embodiments, the Yrl5 protein (WTK1) consists of SEQ ID NO: 30. In some embodiments, the Yrl5 homolog or analog encodes a protein with at least 85% homology to SEQ ID NO: 30 and confers resistance to Pst. In some embodiments, homology is identity.
[0109] In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yr5 gene. Each possibility represents a separate embodiment of the invention. In some embodiments, the artificial vector further comprises a Yr5 gene. In some embodiments, the Yr5 gene is operably linked to a plant promoter. In some embodiments, the artificial vector further comprises a nucleic acid sequence that encodes for a Yr5 protein. In some embodiments, the Yr5 genes encodes a protein comprising the sequence
KEYFNEFAWVTVSQKFKGIDLLNDILKQITGASYESSKATDQIQENEIGKKIHDFLL QRRYLLVLDDVWEADTWEQINRAAKVSPDTNNGSRVLLTTRKKDVAHHIQMPTY VCDLKLMDEEKSWELFKSKALPSYRTYMICNPDKF (SEQ ID NO: 31). In some embodiments, the sequence of Yr5 comprises a sequence selected from the sequences provided in Accession numbers JQ318576.1, JQ318577.1, JQ318578.1, JQ318579.1, JQ318580.1, JQ318581.1, JQ318582.1, JQ318583.1, JQ318584.1, JQ318585.1, JQ318586.1, JQ318587.1, and JQ318588.1.
[0110] In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yrl8 gene. Each possibility represents a separate embodiment of the invention. In so embodiments, the artificial vector further comprises a Yr 18 gene. In some embodiments, the Yrl8 gene is operably linked to a plant promoter. In some embodiments, the artificial vector further comprises a nucleic acid sequence that encodes for a Yr 18 protein. In some embodiments, the Yr 18 gene encodes a protein with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to the sequence MDIALASAAATWLINKLLDRLSDYAIKKLLGSEGLDAEASSLRDALRRATLVLGA VPAGAAAGVRIGNDQLLPQIDLVQRLATDLARHLDELEYYDVKKKVKKNQKSSN PLSKMNLPLTQAGQSKPKYNRTDIKQIRDTVGYLHSICDDVHKALLLDKLDAIKQ AAQDASTDKRETVDNFTENPRNKVFPREEMKDIIELINSAASSDQELLVVPIVGAG GVGKTTLARLVYHDPEVKDKFDIMLWIYVSANFDEVKLTQGILEQIPECEFKSAKN LTVLQRGINKYLTKRFLLVLDDMWEESEGRWDKLLAPLRSAQAKGNVLLVTTRK LSVARITSNTEAHIDLDGMKKDDFWLFFKRCIFGDENYQGQRKLQNIAKKIATRLN GNPLAAKSVGTLLRRNINEDYWTRILDSNEWKLQESIDDIIPALKLSYNQLPYRLQL LFSYCAMFPKGYNFDKGQLICTWIALGFVMNERKKLEDEGSDCFDDLVDRSFFQK YGVSQYYTVHDLMHDVAQEVSINKCLIIDGSDLRTVPSSICHLSIWTEPVYNEQSIE RNDNFEEKLDAVQDNVLGSLECLILAGVYDENYSAKFVKTLERVRYVRMLQLTA MPFNSDILLSSIKKLIHLRYLELRCTSDKPKSLPEAICKLYHLQVLDVQHWSGLNDL PKDMSNLVNLRHLFVPGSGSLHSKISRVGELKFLQELKEFQVQEADGFEISQLGNIN EIRGSLSILGLETVKKKGDATRARLKDKKHLRTLSLTWGSASGSTTTVQKEVMEG LKPHENLSHLLVYNYSGATPSWLLGDSFSLGNLESLHLQDCAAVKILPPFEEMPFL KKLSLVCMPCLKSIRIDFNSADEEDELELSEIEISKCLALTSIRLHSCKALTMLSINDC EALGSLEGLSFSEKLKQCVVQGCPKLPSGFIAN (SEQ ID NO: 32). Each possibility represents a separate embodiment of the invention. In some embodiments, the Yr 18 gene comprises a nucleotide sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to the sequence provided in accession number XM_015795636.1. In some embodiments, the sequence of Yr5 comprises a sequence selected from the sequences provided in Accession numbers EU423905.1, EF489022.1, and EU423903.1.
[0111] In some embodiments, the artificial vector further comprises a nucleic acid sequence with at least 95%, 90%, 85%, 80%, 75%, or 70% homology to a Yr46 gene. Each possibility represents a separate embodiment of the invention. In some embodiments, the artificial vector further comprises a Yr46 gene. In some embodiments, Yr46 is the Lr67 gene. In some embodiments, the Yr46 gene is operably linked to a plant promoter. In some embodiments, the artificial vector further comprises a nucleic acid sequence that encodes for a Yr46 protein. In some embodiments, Yr46 comprises or consists of the sequence atgccgggcggggggttcgccgtgtcggcgccgtccggcgtggagttcgaggccaagatcacgcccatcgtcatcatctcctgc atcatggcggccaccggcggcctcatgttcggctacgacgtcggcatctcaggcggagtgacatcgatggacgatttcctgcgtg agttcttcccggcggtgctgcgccggaagaaccaggacaaggagagcaactactgcaagtacgacaaccagggcctgcagctc ttcacctcgtcgctctacctcgccggcctcaccgccaccttcttcgcctcctacaccacccgccgcctcggacgccgcctcaccatg ctcatcgccggcgtcttcttcatcatcggcgtcatcttcaacggggccgcccagaacctcgccatgctcatcatcggcaggatcctg cttcgttgcggcgtcggcttcgccaaccaggccgttcccctgttcctgtcggagatcgcgccgacgaggatccgcggcgggctca acatcctgttccagctgaacgtgaccatcggcatcctgttcgcgaacctggtgaactacggcacgagcaagatccacccgtgggg ctggcggctgtcgctgtcgctggccggcatcccggcggcgatgctcaccctgggcgcgctcttcgtcaccgacacccccaacag cctcatcgagcgcggccacctggaggagggcaaggcggtgctcaagcggatccgcggcaccgacaacgtggagccggagtt caacgagatcgtggaggcgagccgcatcgcgcaggaggtgaagcacccgttccggaacctgctccagcgccggaaccgccc gcagctggtcatcgccgtgctgctccagatcttccagcagttcacggggatcaacgccatcatgttctacgcccccgtgctgttcaa cacgctcgggttcaagagcgacgcgtcgctctactcggcggtgatcacgggcgccgtcaacgtgctggccacgctggtgtcggt gtacgccgtggaccgcgccgggcggcgcgcgctgctgctggaggctggcgtgcagatgttcctgtcgcaggtggtgatcgccg tggtgctgggcatcaaggtgacggacaagtcggacaacctgggccacgggtgggccatcctgttggtggtcatggtgtgcaccta cgtggcctccttcgcctggtcctggggcccgctggggtggctcatccccagcgagacgttcccgctggagacgcggtcggcgg ggcagagcgtgacggtgtgcgtcaacctgctcttcaccttcctcatcgcgcaggccttcctctccatgctctgccacctcaagttcgc catcttcatcttcttctcggcctgggtgctcgtcatgtccgtcttcgtgctcttcttcctcccggagaccaagaacgtgcccatcgagga gatgaccgacaaggtgtggaagcagcactggttctggaagagattcatggacgacgacgaccaccaccacaacatcgccaacg gcaagaacgccaccgtctga (SEQ ID NO: 33).
[0112] In some embodiments, the artificial vectors of the invention comprise at least one promoter for transcription in a plant cell. In some embodiments, the artificial vectors of the invention comprise at least one promoter for expression in a plant cell. In some embodiments, the at least one promoter is operably linked to a Yrl5 gene. In some embodiments, the vector comprises a nucleic acid molecule of the invention and the Yrl5 gene. In some embodiments, the nucleic acid molecule of the invention and the Yr 15 gene are operably linked to the same promoter. In some embodiments, the nucleic acid molecule of the invention and the Yr 15 gene are operably linked to different promoters. In some embodiments, the at least one promoter is operably linked to a Yr5 gene. In some embodiments, the vector comprises a nucleic acid molecule of the invention and the Yr5 gene. In some embodiments, the nucleic acid molecule of the invention and the Yr5 gene are operably linked to the same promoter. In some embodiments, the nucleic acid molecule of the invention and the Yr5 gene are operably linked to different promoters.
[0113] By another aspect, there is provided a plant cell comprising a nucleic acid molecule of the invention. By another aspect, there is provided a plant cell comprising a vector of the invention.
[0114] In some embodiments, the plant cell is a transgenic cell. As used herein, a “transgenic cell” refers to a cell that has undergone human manipulation on the genomic or gene level. In some embodiments, the transgenic cell has had exogenous nucleic acid molecule introduced into it. In some embodiments, exogenous molecule is an exogenous DNA. In some embodiments, the exogenous molecule is an exogenous RNA. In some embodiments, the exogenous molecule is an exogenous vector. In some embodiments, a transgenic cell comprises a cell that has a vector introduced into it. In some embodiments, a transgenic cell is a cell which has undergone genome mutation or modification. In some embodiments, a transgenic cell is a cell that has undergone genome editing. In some embodiments, genome editing is CRISPR genome editing. In some embodiments, a transgenic cell is a cell that has undergone targeted mutation of at least one base pair of its genome. In some embodiments, the nucleic acid molecule or vector is stably integrated into the cell. In some embodiments, the transgenic cell expresses a nucleic acid molecule of the invention. In some embodiments, the transgenic cell expresses a vector of the invention. In some embodiments, the transgenic cell expresses a protein of the invention. In some embodiments, the transgenic cell, is a cell that comprises a pm69 non-functional allele and/or pseudogene that has been mutated or modified into a functional Pm69 gene. In some embodiments, the pm69 non-functional allele and/or pseudogene has been modified to comprise SEQ ID NO: 14. In some embodiments, the pm69 non-functional allele and/or pseudogene has been modified to comprise SEQ ID NO: 15. In some embodiments, the pm69 non-functional allele and/or pseudogene has been modified to comprise SEQ ID NO: 16. In some embodiments, a pm69 non-functional allele has been modified to encode a protein comprising the amino acid sequence provided in SEQ ID NO: 17. In some embodiments, a pm69 non-functional allele has been modified to encode a protein comprising an amino acid sequence with at least 99%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 17, and which confers resistance to Pm. In some embodiments, CRISPR technology is used to modify a pm69 non-functional allele.
[0115] By another aspect, there is provided a plant comprising a plant cell of the invention.
[0116] In some embodiments, the plant is a transgenic plant. In some embodiments, the plant is a grain/cereal plant. In some embodiments, the plant is any plant that without addition of the vectors or nucleic acid molecules of the invention can be infected by Pm. In some embodiments, the plant is selected from barley, rye, triticale, oat, and wheat. In some embodiments, the plant is selected from barley, rye, triticale, oat, wheat, rice and maize. In some embodiments, the plant is wheat. In some embodiments, the transgenic plant cell is resistant to Pm. In some embodiments, the transgenic plant cell cannot be infected by Pm. In some embodiments, Pm does not grow on the transgenic plant cell. In some embodiments, Pm grows poorly on the transgenic plant cell. In some embodiments, Pm grows worse on the transgenic plant cell than on a plant cell that does not comprise a vector or nucleic acid molecule of the invention. In some embodiments, a transgenic plant produces a post- haustorial immune response to Pm. In some embodiments, a transgenic plant cell produces intracellular ROS in response to Pm. In some embodiments, in response to Pm is in response to exposure to Pm. In some embodiments, exposure is contact. In some embodiments, produces is increases. In some embodiments, increases is as compared to the cell not exposed to Pm. In some embodiments, increases is as compared to a non-transgenic cell exposed to Pm. In some embodiments, increase is by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500%. Each possibility represents a separate embodiment of the invention.
[0117] In some embodiments, a plant is a part of a plant. In some embodiments, a part of a plant is a seed. In some embodiments, a plant is any portion, see, tissue or organ thereof comprising at least one transgenic plant cell of the invention. In some embodiments, the transgenic plant or portion thereof consists of transgenic plant cells of the invention. In some embodiments, the plant or portion thereof comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% transgenic cells of the invention. Each possibility represents a separate embodiment of the invention. In some embodiments, the percentage of transgenic cells is a percentage high enough to confer resistance to Pm.
[0118] By another aspect, there is provide a protein encoded by a nucleic acid molecule of the invention.
[0119] By another aspect, there is provided a protein comprising Pm69 (NLR6) or a fragment analog, homolog or derivative thereof that confers resistance to Pm.
[008] In some embodiments, the protein is an isolated protein. In some embodiments, isolated is from any other proteins. In some embodiments, the protein comprises at least 85% sequence homology to SEQ ID NO: 17 and confers resistance to Pm. In some embodiments, homology is identity. In some embodiments, the protein comprises SEQ ID NO: 17. In some embodiments, the protein consists of SEQ ID NO: 17. In some embodiments, the protein is NLR6. In some embodiments, the protein is Pm69. In all such embodiments, it will be understood that the protein will retain the ability to confer resistance to Pm to a cell or plant to which it is introduced.
[009] The term “derivative” as used herein, refers to any polypeptide that is based off the polypeptide of the invention and still confers resistance to Pm. A derivative is not merely a fragment of the polypeptide, nor does it have amino acids replaced or removed (an analog), rather it may have additional modification made to the polypeptide, such as post-translational modification. Further, a derivative may be a derivative of a fragment of the polypeptide of the invention, however, in such a case the fragment must comprise at least 100 consecutive amino acids of the polypeptide of the invention.
[0120] In some embodiments, the protein comprises an Rx_N domain. In some embodiments, the protein comprises a LRR domain. In some embodiments, the protein comprises a NB-ARC domain. In some embodiments, the domain are the domains of Pm69. In some embodiments, the domains are functional.
[0121] By another aspect, there is provided a method of conferring resistance to Pm to a plant cell, the method comprising expressing in the cell at least one of a nucleic acid molecule of the invention, a vector of the invention and a protein of the invention, thereby conferring resistance to Pm to a plant cell.
[0122] By another aspect, there is provided a method of conferring resistance to Pm to a plant, the method comprising expressing in the cell of the plant at least one of a nucleic acid molecule of the invention, a vector of the invention and a protein of the invention, thereby conferring resistance to Pm to a plant.
[0123] In some embodiments, the expressing is in a sufficient number of cells such as to confer resistance to the plant. In some embodiments, a sufficient number of cells is at least 20, 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 99 or 100% of the cells of the plant. Each possibility represents a separate embodiment of the invention. In some embodiments, a sufficient number of cells is at least 25% of the cells of the plant. In some embodiments, a sufficient number of cells is at least 50% of the cells of the plant.
[0124] By another aspect, there is provided a method of conferring resistance to Pm to a plant cell, the method comprising converting at least one pm69 non-functional allele of the cell into a functional Pm69 gene, thereby conferring resistance to Pm to a plant cell.
[0125] In some embodiments, the plant cell is not resistant to Pm. In some embodiments, the plant cell does not comprise a functional Pm69 gene. In some embodiments, the cell comprises a pm69 non-functional allele. In some embodiments, the cell comprises a pm69 pseudogene.
[0126] In some embodiments, said converting comprises genome editing. In some embodiments, the genome editing is CRISPR genome editing. In some embodiments, the converting comprises targeted mutation of the pm69 non-functional allele and/or pseudogene. In some embodiments, the functional Pm69 gene comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO:16. Each possibility represents a separate embodiment of the invention. In some embodiments, the functional Pm69 gene comprises a nucleic acid sequence with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention. In some embodiments, the functional Pm69 gene comprises a nucleic acid sequence that encodes a protein with at least 99%, 97%, 95%, 90%, 85%, 80%, 75%, or 70% homology or identity to SEQ ID NO: 17. Each possibility represents a separate embodiment of the invention. In some embodiments, the functional Pm69 comprises a functional Rx_N domain, LRR domain and NB-ARC domain. In some embodiments, the function Pm69 comprises methionine 55, serine 507, glycine 606, serine 200 and at least one of glycine 11 and arginine 553 within SEQ ID NO: 17.
[0127] In some embodiments, the method further comprises conferring resistance to Pst. In some embodiments, resistance to Pst is conferred to the plant cell. In some embodiments, resistance to Pst is conferred to the plant. In some embodiments, the method further comprises expressing in the plant cell any one of Yrl5, Yr5, Yr36, Yrl8 and Yr46. In some embodiments, the method further comprises expressing in the plant cell Yrl5.
[0128] By another aspect, there is provided a method for detecting a functional Pm69 gene in a sample, the method comprising: a. providing nucleic acid molecules from the sample; and b. detecting a nucleic acid molecule comprising SEQ ID NO: 16 in the provided nucleic acid molecules; thereby, detecting a functional Pm69 gene in a sample.
[0129] By another aspect, there is provided a method for detecting a functional Pm69 gene in a sample, the method comprising: a. providing nucleic acid molecules from the sample; and b. detecting a DNA molecule comprising a sequence that encodes an mRNA with at least 80% homology to SEQ ID NO: 16 or an mRNA comprising a sequence with at least 80% homology to SEQ ID NO: 16; and c. determining that the mRNA encodes a protein comprising at least 85% identity to SEQ ID NO: 17 and comprising methionine 55, serine 507, glycine 606, serine 200 and at least one of glycine 11 and arginine 553; thereby detecting a functional Pm69 gene in a sample.
[0130] In some embodiments, steps b-c comprise detecting a nucleic acid molecule comprising SEQ ID NO: 16 in the provided nucleic acid molecule. In some embodiments, the determining is in the mRNA encoded by the detected DNA molecule. In some embodiments, the determining is in the detected mRNA. In some embodiments, the detecting comprises amplifying nucleic acid molecules in the sample. In some embodiments, the detecting comprises reverse transcribing nucleic acid molecule in the sample. In some embodiments, the detecting comprises sequencing nucleic acid molecules in the sample. In some embodiments, the amplifying comprises PCR. In some embodiments, the sequencing is Sanger sequencing. In some embodiments, the sequencing is deep sequencing. In some embodiments, the sequencing is next-generation sequencing.
[0131] Detecting a product of amplification is well known in the art and includes but is not limited to gel electrophoreses and column purification. In some embodiments, the one or more primers comprise a tag and the detecting a product of the amplifying comprises detecting the tag. In some embodiments, the tag is a fluorescent tag. In some embodiments, the tag is a FRET tag. Fluorescently-tagged PCR is well known in the art, and detection of the amplification product may be performed with any fluorometer. Methods of sequencing and of preparing a sequencing library are well known in the art. Any sequencing method including the use of any sequencer is also contemplated.
[0132] In some embodiments, the sample is from a plant. In some embodiments, the plant is a cereal. In some embodiments, the cereal is selected from wheat, barley, oats, triticale and rye. In some embodiments, the cereal is selected from wheat, barley, oats, triticale, rye, rice and maize. In some embodiments, the cereal is wheat. In some embodiments, the plant is any plant that can be infected by Pm.
[0133] In some embodiments, the sample is from a leaf of the plant. In some embodiments, the sample is from a mature plant. In some embodiments, the sample is from any one of cultivated plant germplasm, pre-breeding materials, and elite plant cultivars. As used herein, “germplasm” refers to any living tissue from which a new plant can be grown. In some embodiments, the germplasm is a seed. In some embodiments, the germplasm is any one of a seed, a leaf, stem, tissue culture cells, embryoids and pollen. In some embodiments, the germplasm comprises only a few cells. In some embodiments, the germplasm comprises enough material to perform PCR. As used herein, “pre-breeding materials” refers to materials that are not generally directly be used for breeding, but which contain genetic information that can be transferred to breeding materials. As used herein, “cultivars” refers to a plant or group of plants selected for desirable characteristics and maintained by propagation. It will be understood by one skilled in the art, that it is advantageous to those growing cereal plants, to integrate a functional Pm69 gene into the genomes of their crops. Further, it will be advantageous to integrate it into all of the crop and not just a portion. As such, the grower will need to confirm the presence of the functional gene, potentially at every stage of the transfer of the gene to the crop. The molecules and methods of the invention can be used for this purpose, and thus the methods can be performed at any step of the process of integrating Pm69 into their crops, and with any material that might be used in this process.
[0134] In some embodiments, the provided nucleic acids comprise at least one of genomic DNA, RNA and cDNA reverse-transcribed from RNA from the sample. In some embodiments, the hybridizing comprises at least one of, PCR, southern blotting and northern blotting. One skilled in the art will appreciate that the method of hybridization will be selected to match the source of the provided nucleic acids. As non-limiting examples, PCR may be selected when the nucleic acids are cDNA, southern blotting may be selected when the nucleic acids are genomic DNA and northern blotting may be selected when the nucleic acids are RNA. In some embodiments, the PCR is any one of RT-PCR, qPCR, real-time PCR, or conventional end-point PCR. In some embodiments, detecting the hybridizing comprises detection of a PCR product. In some embodiments, the detecting comprises gel electrophoreses. In some embodiments, the detecting comprises sequencing, deep sequencing or next-generation sequencing.
[0135] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.
[0136] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.
[0137] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."
[0138] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
[0139] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
[0140] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
EXAMPLES
[0141] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I- III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes LIII Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
Materials and Methods
[0142] Plant materials: Wild emmer wheat accession G305-3M (Accession number: CGN19852, genesys-pgr.org/10.18730/lNA3M), the Pm69 (PmG3M) gene donor line, was collected from Upper Galilee, Israel. G305-3M was crossed with the susceptible T. durum wheat line Langdon (LDN) to generate segregating mapping populations. RIL population was used for the construction of a sub-centiMorgan (cM) genetic linkage map. Differential wheat lines carrying different Pm genes (Pm21, TdPm60 and Pm30) were used for testing the virulence of Bgt isolate #70. A core collection of WEW accessions, originating from the Fertile Crescent and obtained from the Wild Cereals Gene Bank (ICGB) at the University of Haifa, Israel, was used to screen the Pm69 alleles. Modern Israeli bread wheat cultivar ‘Ruta’ and durum wheat cultivar 'Svevo', highly susceptible to Bgt #70, were used as recurrent parents for the development of hexapioid and tetrapioid introgression lines. Hexapioid wheat lines Avocet+Yrl5 that harbors Yrl5 were used as Yr gene donor lines or recurrent parents for pyramiding Pm69 with Yr genes.
[0143] Bgt and Pst inoculation and disease assessment: Bgt #70 showed high virulence on many cultivated T. durum, T. aestivum and T. monococcum accessions, as well as on several Pm genes (e.g., Pmla-b, Pm2, Pm3a-d, Pm5a-b, Pm6, Pm7, Pml7, Pm22, Pm30 and Pml + Pm2 + Pm9), but it is avirulent on Pm69. Therefore, Bgt #70 was used in the current study for phenotyping of the Pm69 mapping populations and EMS mutants. The reactions to Bgt inoculation were examined visually, with the infection type (IT) recorded on a 0-4 scale at 7 days post-inoculation (dpi). Plants with an IT 0-2 (no disease colonies or less than 1 mm) were considered resistant and those with IT 3-4 (scattered disease spots of greater than 1 mm) are susceptible. Israeli Pst isolate #5006 was used for the yellow rust resistance test as described in previous study50. It was scored at 14 dpi using a 0-9 scale: 0-3 = resistant, none to trace sporulation; 4-6 = intermediate, light to moderate sporulation; 7-9 = susceptible, abundant sporulation. ROS accumulation and cell death were evaluated in inoculated wheat leaves (3 dpi).
[0144] Development of molecular markers, genetic and physical maps: Plant genomic DNA was extracted using the CTAB method from leaves of 2-3 -week-old wheat seedlings. For mapping Pm69, CAPS and Sequence-Tagged Site (STS) markers were developed based on the local synteny of wheat with major cereals like rice, Brachypodium, Sorghum and barley genomes. The primers for SNP-based Kompetitive Allele Specific PCR (KASP) markers were developed using PolyMarker (polymarker.tgac.ac.uk/) based on the specific SNPs identified by the wheat 90K iSelect SNP array and the wheat 15K SNP array (Trait Genetics®, Germany). The International Wheat Genome Sequencing Consortium (IWGSC) RefSeq assembly vl.O, Wild Emmer Genome Assembly (Zavitan WEWSeq v.1.0) and the Durum Wheat (cv. Svevo) RefSeq Release 1.0 were used to design SSR markers by the BatchPrimer3 website tools (probes.pw.usda.gov/batchprimer3/). Those reference genomes also contributed the SNP information used for designing of KASP markers after verifying them by Sanger sequencing of PCR products. PCR reactions were performed by using 2xTaq PCR Master Mix (TIANGEN, Cat#KT201). KASP marker analysis was performed via the StepOnePlus Real-Time PCR system (Applied Biosystems, USA).
[0145] Pm69-flanking markers (uhwk386 and uhwk399) were used to select 147 RILs that carry critical recombination events in the Pm69 gene region after screening of 5500 F2 plants (G305-3M xLDN). These RILs were used for further dissection of the target locus by using the graphical genotyping approach. The genetic distances between the detected markers and Pm69 were calculated based on genotypic and phenotypic data using JoinMap 5.0. The physical location of the primers of each marker was identified by BLAST + search against the sequences of the chromosome arm 6BL of wheat reference genomes. Genetic and physical maps of Pm69 were constructed using MapChart v2.2.
[0146] Construction of Pm69 physical map and comparison with other reference genomes: The co-dominant markers in the Pm69 genetic region and candidate genes from Zavitan V2 were blasted against the contigs of G305-3M ONT assembly. The contigs that showed perfect match with these markers were selected for construction of the Pm69 physical map and development of new genetic markers. The new markers were dominant markers that amplified only G305-3M alleles. Those markers were used to genotype the 72 RILs to characterize the size of the chromosome region that is co-segregating with Pm69. The G305 ONT contigs were sliced into 1Mb segments with pyfasta and mapped to the the wild emmer assembly WEW_v2.0 using bwa-mem software (Li 2013). The G305-3M ONT contigs anchored to the Pm69 region in Zavitan were compared to the Zavitan and Svevo genomes using Minimap (github.com/lh3/minimap2). Minimap results were visualized using ggplot geom_segment.
[0147] Mutant development: To produce loss-of-function mutants of the Pm69 gene, we mutagenized the wild emmer accession G305-3M and homozygous resistant F6 RIL-(169B) by using 0.3%-0.5% Ethyl methane sulfonate (EMS) treatments. We generated 1530 G305- 3M and 162 169B MO plants which were developed to M2 families. We inoculated 10-20 plants of each M2 family with Bgt #70 for screening for susceptible mutants under controlled greenhouse conditions. Susceptible mutants and their resistant sister lines were selected and grown to M3-M4 generation. Resistant sister lines were kept as controls for verification of the Pm69 candidate gene. Susceptible M3-M4 independent mutants, one from RIL 169B and four from G305-3M EMS-treated populations, were selected and confirmed with 30 Pm69 flanking markers that showed the same haplotype as G305-3M, ruling out the possibility of cross-pollination from other susceptible lines.
[0148] Transcriptome sequencing: Leaf samples from the four susceptible mutants and the G305-3M wild type were collected 24 hours after 2, 1,3 -Benzo thiadiazole treatment (Sigma- Aldrich, USA). These leaves were submerged in RNAlater (Sigma-Aldrich, UK) and sent for RNA extraction and sequencing in Novogene-UK. Samples were sequenced on Illumina NovaSeq instrument. About 40 million 150 bp paired-end (PE) reads were obtained for each sample. Those reads were aligned to the G305-3M ONT genomic DNA contigs using GSNAP with default settings followed by sorting, duplicate removal, and indexing using SAMtools. Mutation detection was done by visualizing the obtained bam files with IGV genome browser focusing on the contgis that were anchored to the PmG305 region. Regions on the contigs that had >4 read depth along at least Ikb and had mutations in all susceptible mutant samples relative to the resistant G305-3M wild-type RNA reads and to the reference G305-3M genome were looked for. The mutations had to be in all reads covering the site and to have read quality >30. Moreover, de-novo assembly of the wild type RNAseq reads was obtained using TRANSABYSS software.
[0149] Chromosome sorting: Mitotic metaphase chromosome suspensions were prepared from tetrapioid wheat lines G305-3M and LDN, and from hexapioid introgression wheat line SC28RRR-26. Briefly, cell cycle of meristematic root tip cells was synchronized using hydroxyurea, and mitotic cells were accumulated in metaphase using amiprohos -methyl. Suspensions of intact chromosomes were prepared by mechanical homogenization of 100 formaldehyde-fixed root tips in 600 pl LB01 buffer. GAA microsatellites and/or GAA and ACG microsatellites were labelled on chromosomes by fluorescence in situ hybridization in suspension (FISHIS) using FITC-labelled oligonucleotides (Sigma, Saint Louis, USA) and chromosomal DNA was stained by DAPI (4’,6-diamidino 2-phenylindole) at 2 pg/ml. Bivariate flow karyotypes FITC vs. DAPI fluorescence were acquired using a FACS Aria II SORP flow cytometer and sorter (Becton Dickinson Immunocytometry Systems, USA). The samples were analyzed at rates of 1,500-2,000 particles and different positions of sorting windows were tested on bivariate flow karyotype FITC vs. DAPI to achieve the highest purity in the sorted 6B fractions. The content of flow-sorted fractions was estimated using microscopy analysis of slides, containing 1,500-2,000 chromosomes, sorted into a 10-pl drop of PRINS buffer. Sorted chromosomes were identified by FISH with probes for DNA repeats pScl 19.2, Afa family and 45S rDNA. At least 100 chromosomes were classified for each sample using a standard karyotype.
[0150] Nanopore and Illumina sequencing: DNA extraction and QC: High molecular weight (HMW) DNA was extracted from isolated nuclei and purified following a modified salting out DNA extraction protocol (10X Genomics). Stock HMW DNA was size selected on a Blue Pippin instrument (Sage Science) with the high pass protocol and electrophoretic conditions to retain fragments > 30 kb. Eluate was bead cleaned and concentrated. Size selected DNAs were quantified by fluorometry (Qubit 2.0) and DNA integrity was evaluated using a Tapestation 2200 instrument (Agilent). HMW DNAs were stored at 4oC until library preparation. Library preparation: Long molecule libraries (ID: Oxford Nanopore Technologies) were prepared following the standard ID ligation protocol (LSK109) with minor modifications to retain and enrich for HMW molecules. Briefly 1.2pg of size selected, end-repaired HMW DNAs were used as input into each library preparation reaction. Libraries were sequenced with R9 flow cell on a PromethlON instrument with high accuracy base-calling enabled. Raw read data were filtered for size and quality score, and approximately 305 Gb filtered data (~23X coverage) was used for assembly. Illumina DNA prep libraries were prepared, indexed, and sequenced to ~25X coverage on the NovaSeq 6000 S4 flow cell for polishing (PE reads: 150 bp).
[0151] Genome assembly, polishing and scaffolding: Raw fast5 files generated by ONT sequencing of G305-3M were base-called using Guppy version 3.6 (Oxford Nanopore Technology) to produce fastq files. Fastq files from multiple flow-cells were concatenated to form a consolidated fastq file, which was then used for genome assembly using Smartdenovo with the default parameters80. The raw assembly from Smartdenovo was then subjected to a single round of long read polishing using Medaka version 144 (github.com/nanoporetech/medaka) followed by two rounds of short read polishing using Pilon. Assembly stats were calculated using QUAST version 5.0.2 (academic.oup.com/bioinformatics/article/29/8/1072/228832).
[0152] Construction of Pm69 physical map and comparison with other reference genomes: The co-dominant markers in the Pm69 genetic region and candidate genes from WEW_v2.0 were used for searching the contigs of G305-3M ONT assembly. The contigs that showed a perfect match with these markers were selected for the construction of the Pm69 physical map. The new mapping markers were developed based on the polymorphism between the ONT contigs and wheat reference genomes. Those markers were used to genotype the 31 representative RILs to characterize the size of the chromosome region that is co-segregating with Pm69. The G305 ONT contigs were sliced into 1Mb segments with pyfasta and mapped to the wild emmer assembly WEW_v2.0 using BWA-MEM software. The G305-3M ONT contigs anchored to the Pm69 region were compared to the Zavitan (WEW_v2.0) and Svevo RefSeq Rel. 1.0 genomes using Minimap (github.com/lh3/minimap2), and visualized using ggplot geom_segment in R platform.
[0153] Transcriptome sequencing: Wheat leaf samples from the four susceptible mutants and the G305-3M resistant wild type were collected 24 hours after 0.1 mM 2,1,3- Benzo thiadiazole (BTH) treatment with 0.05% Tween-20. Treated leaves were preserved in RNAlater (Sigma-Aldrich, UK) and sent for RNA extraction and sequencing in Novogene- UK. Samples were sequenced on Illumina NovaSeq 6000 instrument. About 40 million 150 bp paired-end (PE) reads were obtained for each sample. Those RNAseq reads were aligned to the G305-3M ONT genomic DNA contigs using GSNAP with default settings followed by sorting, duplicate removal, and indexing using SAMtools. Mutation detection was done by visualizing the obtained bam files with IGV genome browser focusing on the contigs that were anchored to the Pm69 region. We looked for the regions on the contigs that had >4 read depth along at least Ikb and had mutations in all susceptible mutant samples relative to the resistant G305-3M wild-type RNA reads and to the reference G305-3M genome. The mutations had to be in all reads covering the site and to have read quality >30. Moreover, de novo assembly of the wild-type RNAseq reads were obtained using TRANS ABYSS software.
[0154] Gene annotations, cloning of Pm69 and Sanger sequencing: Gene annotation of the ONT contigs mapped to Pm69 flanking region was carried on GeneSAS 6.0 server (www.gensas.org/gensas) using BLAST for EST matching and Augustus for structural prediction. Annotation of resistance genes was done using NLR annotator, PfamScan (ftp.ebi.ac.uk/pub/databases/Pfam/Tools/), Fgenesh gene-finder
(www.softberry.com/berry.phtml) and NCBI BLASTP. The primers for cloning and sequencing of Pm69 were designed based on ONT contig utgl7163 sequences and the RNA- seq assembly using BatchPrimer3 website tools. Pm69 was amplified from cDNA library of G305-3M using the VeriFi™ Polymerase (PCRBIO, UK), then inserted into a cloning plasmid using 5 minTM TA/Blunt-Zero Cloning Kit (Vazyme, China), and transformed into E. coli strain DH5a. The plasmid was extracted by Hybrid-QTM Plasmid Rapidprep Kit (Gene All, Korea) and sequenced using BigDye Terminator vl.l Cycle Sequencing Kit (Applied Biosystems, USA) on ABI 3130 instrument (Applied Biosystems, USA).
[0155] RNA extraction and quantitative Real-Time-PCR (qRT-PCR): Total RNA was extracted from Bgt #70-inoculated, and non-inoculated G305-3M leaf segments collected along 10 different time points (0, 3, 6, 9, 12, 16, 24, 36, 48, 72 hpi) using the RNeasy Plant Mini Kit (Qiagen, Germany). The cDNA was synthesized from total RNA using a qScript™ cDNA Synthesis Kit (Quantabio, USA). The gene-specific primers of the Pm69 and the housekeeping gene Ubiquitin were used for qRT-PCR amplification performed on a StepOne thermal cycler (ABI, USA) in a volume of 10 pl containing 5 pl of SYBR Green FastMix (Quantabio, USA), 250 nM primers and optimized dilution of cDNA template. The program included an initial step at 95 °C for 30 s followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 10 s. Relative expression of the target genes was calculated by 2A(ubiquitin CT-Target CT) ± standard error of the mean (SEM). All of the qRT-PCRs were performed in triplicate, each with at least three (up to five) independent biological repetitions. Primers used are summarized in Table 1.
[0156] Table 1: Primers used.
Figure imgf000046_0001
GCTATCGCCATCTACCTATC
Pm69-qPCR GCAATCCGTTGTCCTGAC (13)
(12)
[0157] Virus-induced gene silencing (VIGS): The construction of VIGS vectors and inoculation were carried out as is known in the art. To specifically silence the Pm69 gene without off-targets in the G305-3M transcriptome, we used si-Fi software to predict 150-350 nt gene regions with the efficient siRNAs. Two selected regions were amplified and inserted in the pCa-BSMV-y vector, as well as the control insertions of GFP and PDS genes. Equimolar amount of Agrobacterium tumefaciens strain GV3101 with pCa-BSMV-a, pCa- BSMV-[3 and pCa-BSMV-y vectors carrying target or control genes were used to inoculate Nicotiana benthamiana leaves to produce virus transcripts. Infiltrated A. benthamiana leaves were then used to extract the sap and further inoculate the second leaves of two-weeks old wheat plantlets. When the pCa-BSMV-y-PDS silenced plants showed chlorosis in the leaves, those plants were inoculated with Bgt #70 in a growth chamber. Two weeks post inoculation, the reaction of wheat plants to Bgt inoculation was recorded.
[0158] Wheat transformation: The full-length CDS of PmG69 is amplified from cDNA of G305-3M, cloned into the pDONR207 by BP Clonase™ II Enzyme mix (Invitrogen, USA), and constructed into the destination vector pYPQ210 by LR Clonase™ II Enzyme mix (Invitrogen, USA), under Zea mays Ubil promoter with bar gene for plant selection. Agrobacterium tumefaciens strain EHA105 is used for plasmid transformation in the wheat callus from the immature embryos. The 1-5 mg/L glufosinate-ammonium (SIGMA, Switzerland) is used for the transformant selection.
[0159] Phylogeny and synteny analysis: The evolutionary tree was built using MEGA-X (aligning by MUSCLE, Neighbor-Joining algorithm, bootstrap: 500 times) and the tree was draw using iTOL v6 (itol.embl.de/). The Neighbor-Joining tree is built based on protein sequence of genes in the NLR cluster by MAFFT88 and Fastree89. Synteny was analyzed by TGT90 http://wheat.cau.edu.cn/TGT/. Pm69 homologs were identified by blasting the Pm69 protein sequence to the database of WheatOmics 1.0, EnsemblPlants, and NCBI. The alignment of Pm69 homologs was done by the multiple alignment tool CLUSTALW from NCBI and displayed using ESPript (espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).
[0160] Statistical analysis: Statistical analysis was performed using JMP® version 15.1 statistical packages (SAS Institute, Cary, NC, USA). Comparison between the treatments were performed by Student's t-test. Asterisks indicate the level of significance at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). [0161] Data availability: The datasets generated during and/or analyzed during the current study are publicly available as follows. The ONT sequencing contigs were deposited. Raw reads of the MutRNAseq were deposited to the NCBI Sequence Read Archive under BioProject ID PRJNA795708. The Pm69 gene and transcript sequence was deposited in NCBI Genbank. The scripts used in this analysis have been published on the GitHub repository. Seeds of WEW G305-3M, mutants and introgression lines of Pm69 are available from the Wild Cereals Gene Bank (ICGB) at the University of Haifa, Israel. The data provided herein is also available on bioRxiv at biorxiv.org/content/ 10.1101/2022.10.14.512294vl, herein incorporated by reference in its entirety. Relevant supplemental material is also available on bioRxiv at biorxiv.org/content/ 10.1101/2022.10.14.512294v 1.supplementary-material, herein incorporated by reference in its entirety.
Example 1: The Challenges of chromosome walking in a complex structurally variable region
[0162] A recombinant inbred line (RIL) mapping population segregating for Pm69 was generated by crossing the susceptible durum wheat (Triticum turgidum ssp. durum) Langdon (LDN) with the resistant WEW accession G305-3M. G305-3M and the Fi generation (G305- 3M x LDN) showed post-haustorial immune responses to Bgt #70 accompanied by intracellular ROS production and host cell death, while LDN showed a highly susceptible response resulting in the development of massive pathogen colonies (Fig. 1A). G305-3M showed a wide-spectrum resistance against 55 tested Bgt isolates originating from four continents Asia, Europe, North America, and South America (Table 2). For fine mapping of Pm69, 5500 F2 plants (11,000 gametes) were screened with DNA markers uhwk386 and uhwk399 to develop 147 F4-7 RILs that carried informative recombination events within the target region. In total, an additional 33 DNA markers were developed and mapped within this genetic region (Fig. 8), enabling to map Pm69 within a 0.21 cM interval between markers uhw367 and uhwk389 (Fig. IB).
[0163] Table 2: The reactions of wheat differential lines to a set of 54 powdery mildew isolates collected in Israel, Switzerland, USA, Netherland, Chile and Paraguay.
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
[0164] 1) The first 42 powdery mildew isolates (from #1 to #113) were collected from different wheat species (T. dicoccoides, T. durum and T. aestivum) from different wheat plots in Israel. In order to obtain a set of Swiss representative random mildew samples, a car was driven with the spore trap mounted on the roof in the main wheat growing areas of Switzerland (5 isolates; 96229-96275). Assessment of the G-305-3M accession to Bgt isolates from the other eight locations worldwide (USA-CM1 and CM2; 2 isolates from Netherlands, 2 isolates from Chile and 1 isolate from Paraguay the last 5 rows in the table) were reported in Gerechter-Amitai et al., 1989, “Resistance to powdery mildew in wild emmer (Triticum dicoccoides Korn.)”, Euphytica, 33(2), pp.273-280, herein incorporated by reference in its entirety. Those areas are dominated by bread wheat cultivars. 2) GF7: A homozygous resistant F3 line containing Pm21; GF8: A homozygous susceptible F3 line that doesn’t contain Pm21; Jing 411 and 781 are susceptible parents in the pedigree of producing this Pm21 mapping population. Wild emmer wheat accessions G18-16, C20, and G-305-3M are the donors of PmG16, Pm30, and Pm69, respectively. R: resistant (IT=0-l); M-R: mild resistant (IT=2); S: susceptible (IT=3-4).
[0165] These closely linked genetic markers were anchored to three wheat reference genomes and it was found that Pm69 genetic map showed a higher collinearity level with the WEW_v2.0 assembly than with that of durum wheat Svevo (RefSeq Rel. 1.0) and bread wheat Chinese Spring (IWGSC RefSeq vl.O). As Zavitan, Svevo and Chinese Spring are susceptible to Bgt #70 (Fig. 9), they were not expected to contain functional pm69 alleles. In Zavitan, this region is spanning 1.02 Mb comprising 23 genes, of which 18 are NERs. However, 10 markers physically mapped in this region showed co-segregation with the Pm69 phenotype, suggesting suppressed recombination. In addition, 12 markers developed based on these candidate genes amplified PCR products in the susceptible parent Langdon, but not in the Pm69 donor line G305-3M. Taken together, these results indicated that structural variation between G305-3M and LDN probably caused the suppression of recombination around the Pm69 genetic region and thus prevented further chromosome walking towards Pm69 based on the published reference genomes.
Example 2: The challenges of using MutChromSeq based on chromosome sorting in tetrapioid wheat.
[0166] As an alternative approach, an attempt was made to use MutChromSeq for cloning of Pm69. To produce pm69 loss-of-function mutants, ethyl methane sulfonate (EMS) treatment was used to mutagenize the WEW accession G305-3M and resistant F7-8 RILs. A total of five independent susceptible mutant lines (four in the G305-3M background and one in the resistant RIL background) were detected and grown to M4 generation (Fig. 10).
[0167] Next, an attempt was made to isolate 6B chromosomes from G305-3M and LDN by flow cytometry-based chromosome sorting. Because of similar size and GAA microsatellite content, chromosomes 6B, IB, 7B, 4B and 5B formed a composite and poorly resolved population on bivariate flow karyotype. Chromosomes were sorted from the composites in hopes of further enriching chromosome 6B, but only a maximum purity of 47% and 51% in the case of G305-3M and LDN, respectively, was achievable. The sorted fractions were contaminated by other chromosomes (IB, 7B, 4B and 5B in case of G305-3M, and chromosomes IB, 7B and 5B in case of LDN).
[0168] An additional attempt was made to flow sort chromosome 6B from the hexapioid introgression line SC28RRR-26 (a bread wheat introgression line cv. Ruta + Pm69). The position of chromosome 6B population on the flow karyotype indicated its higher DNA content relative to other B-genome chromosomes. Improved discrimination of chromosome 6B resulted in a higher purity (73.1%) of the fraction flow sorted from the hexapioid wheat background. At that moment EMS-derived mutants of the introgression line were not yet available and it would take at least a couple of years to create these mutants, thus, alternative approaches were considered.
Example 3: Dissection of the structural variation complexity by ONT sequencing of G305-3M
[0169] The ONT long-read sequencing technology offered an attractive alternative to rapidly generate long contigs that span the Pm69 genomic interval, thus overcoming difficulties conferred by the structural variations in the target gene region. ONT sequencing was used for whole-genome sequencing (~23x coverage) of WEW accession G305-3M. After assembling the reads, 2,489 Contigs were obtained, with N50 value of 11.2 Mb and N90 value of 2.6 Mb. The longest contig was 70.65 Mb, and the total length of the genome assembly was 10 GB, which is typical for the tetrapioid wheat genome.
Example 4: Construction of Pm69 physical map using ONT contigs
[0170] Pm69 physical map was constructed by anchoring the ONT contigs to the Pm69 genetic map using the co-dominant PCR markers. Two contigs utgl7163 (1.1 Mb) and utg5064 (1.09 Mb) were perfectly anchored to the genetic map by four Pm69 flanking markers. An additional ONT contig utg4926 (156.5 kb) was identified by searching for gene sequences that reside in the Pm69 collinear region of the WEW_v2.0 genome. Based on the three identified ONT contigs, 15 markers were developed that were incorporated into the genetic map by graphical genotyping of the RIL population. Finally, the Pm69 cosegregating markers spanned part of contig utg5064 (120400 bp to end), the whole contig utg4926 (0-156528 bp), and part of contigs utgl7163 (0-380148 bp) (Fig. IB).
[0171] A comparison of G305-3M ONT contigs with the 6B pseudomolecules of WEW_ v2.0 and the Svevo reference genomes showed a very low level of collinearity around the Pm69 genetic region, indicating massive structural rearrangements between them (Fig. 2A and 2B, respectively). In contrast, more distant regions flanking Pm69 showed a high collinearity level between the G305-3M contigs and the two reference genomes (Fig. 2A- B).
Example 5: Identification of Pm69 candidate gene by using ONT-MutRNAseq
[0172] To identify a candidate for Pm69 in the final physical interval, the RNAseq reads of four independent EMS-derived susceptible mutants were mapped onto the G305-3M ONT contigs (M3-5 generations, Fig. 3A, 10). This approach revealed eight expressed candidate genes in the Pm69 physical region, seven in utgl7163, one in utg5046, while no expressed genes were found in utg4926. Seven candidates were predicted as NLRs and one as a FAR1 Related Sequence (FRS) transcription factor (Fig. 3B). Only one gene spanning 12280 bp (SEQ ID NO: 14) on G305-3M contig utgl7163, named NLR-6, had five different point mutations (Table 3) in the four susceptible mutants. These five mutations in NLR-6 were all G/C to A/T transitions typical of EMS mutagenesis, while the remaining seven genes in this region did not have any mutations.
[0173] The predicted 2763 bp coding sequence (CDS) of NLR-6 (SEQ ID NO: 15) was validated by Sanger sequencing using G305-3M cDNA. The -12.3 kb genomic region of the gene contains one 5’ UTR and two translated exons (SEQ ID NO: 16). The region contains two introns, one of them located in the 5’ UTR region (Fig. 3C). All the identified point mutations of NLR-6 were validated by Sanger sequencing and were confirmed as missense mutations. Moreover, their resistant sister lines, derived from the same Mo plants, harbored the Pm69 wild allele. An additional susceptible mutant line M-3, identified by Sanger sequencing, also contained a point mutation (C599T, protein S200F) in NLR6 (Fig. 3C, Table 3). The predicted structure of the NLR-6 protein (SEQ ID NO: 17) contains an N- terminal coil coil Rx_N domain with RanGAP interaction sites, an NB-ARC domain and an LRR domain (Fig. 3D). The Rx_N-terminal domain, predicted to have a coiled-coil structure with four helical bundle fold, is found in many plant resistance proteins.
[0174] Table 3: Molecular characterization of the Pm69 EMS mutants
Figure imgf000053_0001
[0175] The expression pattern of Pm69 NLR-6) was further examined at the different time points from zero to 72 hours post-infection (hpi) in both non-inoculated (mock) and inoculated G305-3M plants. The expression level of Pm69 did not significantly change in the Bgt #70-infected plants during 0-16 hpi, compared with non-inoculated control. However, Pm69 expression was significantly decreased (p<0.05) in the inoculated plants at 16-72 hpi (Fig. 11).
Example 6: Functional validation of Pm69 through virus-induced gene silencing (VIGS) [0176] Virus-induced gene silencing (VIGS) constructs were designed to target two genomic positions in Pm69 respectively (Table 4). A phytoene desaturase (PDS) gene silencing construct was used to test the efficacy of the VIGS system in tetrapioid (G305-3M) and hexapioid (a bread wheat introgression line cv. Ruta + Pm69) backgrounds. The inoculation with the Pm(59-silencing constructs resulted in susceptibility to Bgt #70 of the leaves in G305-3M and Pm69 introgression line, while the negative controls showed no visible Bgt symptoms on the leaves (Fig. 4A-B). Histopathological characterization showed that Pm69 control cells (BSMV:GFP) accumulated intracellular ROS and prevented the invasion of the Bgt germinating spores. Silencing of PDS is visualized as white streaks resulting from photobleached chlorophyll (Fig. 4A), while in the Pm69 VIGS silenced leaves, there was detected a mosaic pattern of germinating spores that invaded the cells successfully and developed colonies, alongside spores that activated HR cell death responses, as in the control resistant plants (Fig. 4C). Quantitative reverse transcription PCR (qRT-PCR) showed a significant reduction of expression levels of Pm69 mRNA in the Pm69 VIGS silenced leaves compared with GFP silenced leaves (p < 0.05; Fig. 4A-B). Silencing of Pm69 in G305-3M and the introgression line (Ruta + Pm69) resulted in susceptible phenotypes, therefore, providing functional validation for the role of the Pm69 gene (NLR- 6) in conferring resistance against wheat powdery mildew.
[0177] Table 4: Markers used in the VIGS of Pm69
Figure imgf000054_0001
Example 7: The Pm69 is a rare allele
[0178] A search in 37 published Triticeae genome sequences identified 51 Pm69 homolog s/orthologs from 19 hexapioid wheat, 3 tetrapioid, and 6 Aegilops accessions, which showed 82-89% protein sequence similarity with Pm69 (Table 5). Most of the closest Pm69 homologs were from 6B and 6D chromosomes or the chromosome 6 of Aegilops. Protein sequence analysis of pm69 homologs showed that their variation part was mainly present in the LRR domain compared with the Pm69 allele (Fig. 12). Phylogenetic analysis of Pm69 homologs showed that Pm69 from G305-3M was a unique allele, and other homologs were showing high diversity in the Rx_N and LRR domains compared with Pm69. Phenotyping of 11 representative accessions that cover the most alleles among these homologs showed that probably all of them were susceptible to Bgt #70 (Table 5).
[0179] Table 5: Sequence alignment of Pm69 with the similar proteins from different wheat reference genomes. Susceptibility score (IT) to Bgt #70; 0= not susceptible, 4= most susceptible.
Figure imgf000054_0002
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
[0180] The functional molecular marker uhw403 was used for a large-scale screening of the distribution of Pm69 among 538 wheat accessions of which 310 are WEW from across the Fertile Crescent and 228 represent other wheat species (Fig. 5A). Only G305-3M yielded positive PCR amplification. To further estimate the presence of the gene in the WEW gene pool, a return was made to the original G305-3M collection site south of Kadita, Northern Israel, and an additional 64 WEW accessions were collected in a radius of less than 1km from the original collection site (Fig. 5B). Even there, only three WEW accessions were found that gave amplification of uhw403 marker and showed high resistance to Bgt #70 (Fig. 5B-D). Sanger sequencing confirmed that these accessions contain a functional Pm69 allele identical to that of G305-3M. Moreover, eight resistant accessions were identified to contain TdPm60 by marker M-Pm60-Sl , and an additional five accessions contained unknown Pm genes. Therefore, it can be concluded that Pm69 is a rare NLR allele among natural WEW populations, even within its population of origin near the collection site of its donor accession G305-3M. Example 8: Pm69 is located within a rapidly evolving NLR cluster
[0181] Micro-collinearity analysis among different wheat genomes revealed high copy number variation of NLRs in Pm69 genetic regions, as well as multiple putative structural rearrangements (Fig. 6). The WEW G305-3M harbors 47 NLRs and Zavitan contains 42 NLRs, while all other domesticated wheat reference assemblies contain only 8-24 NLRs, within 0.5 - 3.1 Mb physical intervals comprising this cluster. These collinearity NLR clusters were also identified in chromosomes 6A and 6D from different wheat genome references within 6-23 and 7-19 NLRs, respectively. Interestingly, the stem rust resistance protein Sri 3 alleles, originating from chromosome 6A of T. turgidum subsp. dicoccum, belong to this homoeologous NLR cluster (Fig. 6). Although the Pm69 was close to Sri 3 in the evolutionary tree comprising all cloned functional R-proteins from the Triticeae, their sequence similarity is low (49%), suggesting they are not true orthologs. Evolutionary analysis of 743 clustered NLRs around Pm69 loci from 23 wheat reference genomes, separates 8 different clades, Pm69 belong to clade 7 containing 42 genes/alleles which could be identified as Pm69 homologs, while Sri 3 was present in clade 2 containing 95 genes. Altogether, this result showed that Pm69 is present in a NLR cluster containing abundant genetic variation and complex evolution events, which increased the difficulty of cloning this gene.
[0182] A highly similar Pm69 homolog (NLR9, 92% similarity) was identified on contig utg5064 in this NLR cluster when searching for Pm69 homologs in the ONT genome assembly of G305-3M, suggesting a duplication event. Blasting the cDNA sequence of Pm69 against WEWseq_v2.0 reference genome revealed three copies on chromosome 6B in the range of 717-718Mbp, with DNA identity >92% in exon 1 and >86% in exon 2 (Fig. 13). Moreover, 1-2 duplication copies of pm69 homologs were found in several wheat references (Table 5). Probably, the duplication events might contribute to the Pm69 evolution.
[0183] Inside the NLR cluster, 12-oxophytodienoate reductase 11 (OPR11), known to be involved in the biosynthesis of jasmonic acid (JA), showed a highly conserved protein sequence with 1-2 copies among different Triticeae genomes, suggesting that opposing evolutionary forces are acting in this small genetic interval. Moreover, more distant regions flanking the Pm69 clusters showed a high collinearity level, with similar gene content and order. This might also imply that different evolutionary pressures may act on NLRs relative to other genes, probably imposed by natural selection (Fig. 6). Example 9: Introgression of the Pm69 into cultivated wheat and pyramiding with yellow rust (Er) resistant genes
[0184] As part of the aim to develop resistant pre -breeding genetic resources, Pm69 was transferred into elite Israeli bread wheat cultivar ‘Ruta’ using marker-assisted selection (MAS) following the “durum as a bridge” approach. Furthermore, Pm69 was also introgressed into the durum wheat cultivar ‘Svevo’, which contains Sri 3b. These homozygous ILs with different segments of G305-3M chromosome have been selected from the BC4F2 populations and showed high resistance to Bgt #70 (Fig. 7A).
[0185] Moreover, it was found that G305-3M contains the yellow rust resistance gene Yrl5, which was confirmed by ONT sequencing data, present in 4.57 Mbp contig utgl l61 and showed high resistance to Pst #5006 (Fig. 7A). Pm69 with Yr 15 were pyramided in the hexapioid cultivar 'Avocet' background, and homozygous plants (AvocctYr 15+Pm69) were selected by MAS, which showed high resistance to both Pst #5006 (IT = 1-3) and Bgt #70 (IT = 0;-l) (Fig. 7A). These resistant ILs can be used in wheat resistance breeding programs (Fig. 7B).
[0186] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:
1. An isolated nucleic acid molecule comprising a nucleic acid sequence that encodes a protein with at least 85% homology to SEQ ID NO: 17 and that confers resistance to powdery mildew (Pm).
2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid sequence encodes a protein with at least 85% identity to SEQ ID NO: 17 and that confers resistance to Pm.
3. The isolated nucleic acid molecule of claim 1 or 2, wherein said nucleic acid sequence comprising at least 85% homology to SEQ ID NO: 15 or 16.
4. The isolated nucleic acid molecule of claim 3, wherein said nucleic acid sequence comprises at least 85% identity to SEQ ID NO: 15 or 16.
5. The isolated nucleic acid molecule of any one of claims 1 to 4, wherein said protein that confers resistance comprises methionine 55, serine 507, glycine 606, serine 200 and at least one of glycine 11 and arginine 553 within SEQ ID NO: 17.
6. The isolated nucleic acid molecule of any one of claims 1 to 5, wherein said protein that confers resistance comprises a functional Rx_N domain, LRR domain and NB- ARC domain such that said protein confers resistance to PM.
7. The isolated nucleic acid molecule of claim 6, wherein said functional Rx_N domain comprises SEQ ID NO: 34, said functional LRR domain comprises SEQ ID NO: 35 or said functional NB-ARC domain comprises SEQ ID NO: 36.
8. The isolated nucleic acid molecule of any one of claims 1 to 7, wherein said nucleic acid molecule is a DNA molecule or an RNA molecule.
9. The isolated nucleic acid molecule of any one of claims 1 to 8, wherein conferring Pm resistance is conferring Pm resistance to cereal plant.
10. The isolated nucleic acid molecule of claim 9, wherein said cereal plant is a wheat plant.
11. The isolated nucleic acid molecule of any one of claims 1 to 10, wherein said nucleic acid sequence comprises at least 85% identity to SEQ ID NO: 16.
12. The isolated nucleic acid molecule of any one of claims 1 to 11, wherein said nucleic acid sequence consists of SEQ ID NO: 16.
13. The isolated nucleic acid molecule of any one of claims 1 to 12, further comprising a transcription regulatory element operatively linked to said nucleic acid sequence.
14. The isolated nucleic acid molecule of any one of claims 1 to 13, wherein resistance comprises a post-haustorial immune responses to said Pm. The isolated nucleic acid molecule of claim 14, wherein said post-haustorial immune response comprises intracellular reactive oxidation species (ROS) production in response to Pm. An artificial vector comprising the isolated nucleic acid molecule of any one of claims 1 to 15. The artificial vector of claim 16, further comprising at least one nucleic acid sequence of a pathogen-resistance gene. The artificial vector of claim 17, wherein said pathogen is Stripe Rust (Pst). The artificial vector of claim 18, wherein said pathogen-resistance gene is selected from Yrl5, Yr5, Yr36, Yrl8 and Yr46. The artificial vector of claim 19, wherein said pathogen-resistance gene is Yrl5. The artificial vector of any one of claims 16 to 19, comprising at least one transcriptional regulatory element active in plant cells and operatively linked to said nucleic acid sequence. The artificial vector of claim 21, wherein said transcriptional regulatory element is a promoter. The artificial vector of claim 22, wherein said promoter is a heterologous promoter of the Pm69 endogenous promoter. The artificial vector of any one of claims 21 to 23, wherein said plant is a cereal plant. The artificial vector of claim 24, wherein said cereal plant is selected from wheat, barley, rye, triticale, oat, rice and maize. The artificial vector of any one of claims 16 to 25, for use in conferring resistance to Pm to a cell of a plant. The artificial vector of claim 26, wherein resistance comprises a post-haustorial immune responses to said Pm. The artificial vector of claim 27, wherein said post-haustorial immune response comprises intracellular reactive oxidation species (ROS) production in response to Pm. A transgenic plant or cell thereof comprising a nucleic acid molecule of any one of claims 1 to 15 or an artificial vector of any one of claims 16 to 28. The transgenic plant or cell thereof of claim 29, wherein the plant is a cereal plant. The transgenic plant or cell thereof of claim 30, wherein said cereal plant is any one of barley, rye, triticale, oat, wheat, rice and maize. The transgenic plant or cell thereof of claim 31, wherein the cereal plant is wheat. An isolated protein comprising at least 85% homology to SEQ ID NO: 17 and comprising the ability to confer resistance to Pm. The isolated protein of claim 33, comprising at least 85% identity to SEQ ID NO: 17. The isolated protein of claim 33 or 34, consisting of an amino acid sequence with at least 85% identity to SEQ ID NO: 17. The isolated protein of any one of claims 33 to 35, consisting of SEQ ID NO: 17. A method of conferring resistance to Pm to a plant or a cell thereof, the method comprising at least one of: a. expressing in said cell of said plant at least one of an isolated nucleic acid molecule of any one of claims 1 to 15, an artificial vector of any one of claims 16 to 28, and an isolated protein of any one of claims 33 to 36; and b. converting at least one pm69 non-functional allele of said cell of said plant into a functional Pm69 gene, thereby conferring resistance to Pm to a plant or cell thereof. A method for detecting a functional Pm69 gene in a sample, which a functional
Pm69 gene confers resistance to Pm, comprising: a. providing nucleic acid molecules from said sample; b. detecting a DNA molecule comprising a sequence that encodes an mRNA with at least 80% homology to SEQ ID NO: 16 or an mRNA comprising a sequence with at least 80% homology to SEQ ID NO: 16; and c. determining that said mRNA encodes a protein comprising at least 85% identity to SEQ ID NO: 17 and comprising methionine 55, serine 507, glycine 606, serine 200 and at least one of glycine 11 and arginine 553; thereby detecting a functional Pm69 gene in a sample. The method of claim 38, wherein steps b-c comprise detecting a nucleic acid molecule comprising SEQ ID NO: 16 in the provided nucleic acid molecules. The method of claim 38 or 39, wherein said sample is from a plant. The method of claim 40, wherein said plant is a cereal plant. The method of claim 41, wherein said cereal plant is selected from wheat, barley, oat, triticale, rye, rice and maize. The method of any one of claims 40 to 42, wherein said sample is from any one of cultivated plant germplasm, pre -breeding materials, and elite plant cultivars.
PCT/IL2023/050137 2022-02-08 2023-02-08 Pm69 and use thereof WO2023152742A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202263307820P 2022-02-08 2022-02-08
US63/307,820 2022-02-08
US202263417716P 2022-10-20 2022-10-20
US63/417,716 2022-10-20

Publications (1)

Publication Number Publication Date
WO2023152742A1 true WO2023152742A1 (en) 2023-08-17

Family

ID=87563853

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2023/050137 WO2023152742A1 (en) 2022-02-08 2023-02-08 Pm69 and use thereof

Country Status (1)

Country Link
WO (1) WO2023152742A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111732644A (en) * 2019-03-20 2020-10-02 中国科学院遗传与发育生物学研究所 Powdery mildew resistance related protein Pm41, and coding gene and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111732644A (en) * 2019-03-20 2020-10-02 中国科学院遗传与发育生物学研究所 Powdery mildew resistance related protein Pm41, and coding gene and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 13 November 2020 (2020-11-13), ANONYMOUS : "PREDICTED: Triticum dicoccoides disease resistance protein RGA5-like (LOC119325579), mRNA", XP093084188, retrieved from NCBI Database accession no. XM_037599308.1 *
DATABASE Protein 13 November 2020 (2020-11-13), ANONYMOUS : "disease resistance protein RGA5-like [Triticum dicoccoides]", XP093084187, retrieved from NCBI Database accession no. XP_037455205.1 *
LI YINGHUI; WEI ZHEN-ZHEN; FATIUKHA ANDRII; JAIWAR SAMIDHA; WANG HANCHAO; HASAN SAMIHA; LIU ZHIYONG; SELA HANAN; KRUGMAN TAMAR; FA: "TdPm60 identified in wild emmer wheat is an ortholog of Pm60 and constitutes a strong candidate for PmG16 powdery mildew resistance", THEORETICAL AND APPLIED GENETICS, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 134, no. 9, 8 June 2021 (2021-06-08), Berlin/Heidelberg, pages 2777 - 2793, XP037534513, ISSN: 0040-5752, DOI: 10.1007/s00122-021-03858-3 *
WEI ZHEN-ZHEN, KLYMIUK VALENTYNA, BOCHAROVA VALERIA, POZNIAK CURTIS, FAHIMA TZION: "A Post-Haustorial Defense Mechanism is Mediated by the Powdery Mildew Resistance Gene, PmG3M, Derived from Wild Emmer Wheat", PATHOGENS, vol. 9, no. 6, 28 May 2020 (2020-05-28), pages 418, XP093084192, DOI: 10.3390/pathogens9060418 *

Similar Documents

Publication Publication Date Title
Li et al. A domestication-associated gene GmPRR3b regulates the circadian clock and flowering time in soybean
Sato et al. Comprehensive structural analysis of the genome of red clover (Trifolium pratense L.)
Kofuji et al. Evolution and divergence of the MADS-box gene family based on genome-wide expression analyses
US11473100B2 (en) Stripe rust resistance gene Wtk1 (Yr 15) and use thereof
US7642403B2 (en) Marker mapping and resistance gene associations in soybean
Cole et al. Diversity in receptor‐like kinase genes is a major determinant of quantitative resistance to Fusarium oxysporum f. sp. matthioli
Livaja et al. BSTA: a targeted approach combines bulked segregant analysis with next-generation sequencing and de novo transcriptome assembly for SNP discovery in sunflower
AU2020202265B2 (en) Genetic markers for myb28
Kaufmann et al. Isolation, molecular characterization, and mapping of four rose MLO orthologs
US20210105961A1 (en) Mads-box domain alleles for controlling shell phenotype in palm
Brotman et al. Molecular markers linked to papaya ring spot virus resistance and Fusarium race 2 resistance in melon
CN111868075A (en) Wheat comprising male fertility restorer allele
Hewitt et al. Wheat leaf rust resistance gene Lr13 is a specific Ne2 allele for hybrid necrosis
Li et al. Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69
Li et al. Long-read genome sequencing accelerated the cloning of Pm69 by resolving the complexity of a rapidly evolving resistance gene cluster in wheat
KR100984736B1 (en) Marker for selecting brown planthopper-resistant rice cultivar
Cao et al. Pepper variome reveals the history and key loci associated with fruit domestication and diversification
BR112014031416B1 (en) Method of identifying a soybean plant or soybean germplasm with marked resistance to cyst nematode
US10519463B2 (en) Androecious cucurbit plants, methods of obtaining and uses of said cucurbit plants
Wang et al. Fighting wheat powdery mildew: from genes to fields
Liu et al. Pm57 from Aegilops searsii encodes a novel tandem kinase protein conferring powdery mildew resistance in bread wheat
Yu et al. Reference genome-assisted identification of stem rust resistance gene Sr62 encoding a tandem kinase
Donia et al. Identification, evolutionary patterns and intragenic recombination of the gametophytic self incompatibility pollen gene (SFB) in Tunisian Prunus Species (Rosaceae)
Huang et al. Identification and transfer of a new Pm21 haplotype with high genetic diversity and a special molecular resistance mechanism
WO2023152742A1 (en) Pm69 and use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23752561

Country of ref document: EP

Kind code of ref document: A1