Method for modifying the resistance profile of spinacia oleracea to downy mildew

Abstract

The present invention relates to a method for modifying the resistance profile of a spinach plant to Peronospora farinosa f. sp. spinaciae, comprising introducing a WOLF allele or a resistance-conferring part thereof into the genome of said spinach plant, or modifying an endogenous WOLF allele in the genome of said spinach plant. The invention further provides a method for selecting a spinach plant comprising a novel WOLF gene that confers resistance to Peronospora farinosa f. sp. spinaciae in a spinach plant and a method for identifying a WOLF allele that confers resistance to one or more pathogenic races of Peronospora farinosa f. sp. spinaciae in a spinach plant and to primers for use in these methods.

What is claimed is: 
     
         1 . A method for modifying the resistance profile of a spinach plant to  Peronospora farinosa  f. sp.  spinaciae,  comprising introducing a WOLF allele or a resistance-conferring part thereof into the genome of said spinach plant, or modifying an endogenous WOLF allele in the genome of said spinach plant, wherein the WOLF allele encodes a CC-NB S-LRR protein that comprises in its amino acid sequence:
 a) the motif “MAEIGYSVC” at its N-terminus, and   b) the motif “KWMCLR” or “HVGCVVDR”.   
     
     
         2 . The method as claimed in  claim 1 , wherein the WOLF allele encodes a WOLF protein according to any one of SEQ ID NOs referred to in Table 3. 
     
     
         3 . The method as claimed in  claim 1 , wherein introducing a WOLF allele into the genome of a spinach plant comprises the step of expressing in a spinach cell a recombinant nucleic acid construct comprising a coding sequence encoding one or more WOLF polypeptides. 
     
     
         4 . The method as claimed in  claim 3 , wherein the nucleic acid construct is designed for stable incorporation into the genome of a plant cell. 
     
     
         5 . The method as claimed in  claim 1 , wherein modifying the endogenous WOLF allele is achieved by means of genome editing techniques or mutagenesis techniques. 
     
     
         6 . The method as claimed in  claim 5 , wherein the endogenous WOLF allele is replaced by a modified WOLF allele by means of genome editing techniques. 
     
     
         7 . A spinach plant comprising a WOLF allele as defined in  claim 1 . 
     
     
         8 . The spinach plant as claimed in  claim 7 , which is obtainable by the method of  claim 1 . 
     
     
         9 . The spinach plant according to  claim 7 , having a modified resistance profile to  Peronospora farinosa  f. sp.  spinaciae  as compared to an isogenic spinach plant that has not been modified by the method of  claim 1 . 
     
     
         10 . A spinach plant comprising one or more WOLF alleles, wherein said WOLF allele is selected from the nucleotide sequences referred to in Table 3 or is a nucleotide sequence encoding a polypeptide having an amino acid sequence referred to in Table 3 or is a nucleotide sequence encoding a polypeptide having a sequence similarity of 95%, 96%, 97%, 98%, or 99% with any one of the amino acid sequences referred to in Table 3, or a having a sequence similarity of 95%, 96%, 97%, 98%, or 99% with any one of the nucleotide sequences referred to in Table 3. 
     
     
         11 . Propagation material of a plant as claimed in claim7, wherein a plant grown or regenerated from the material comprises a resistance-conferring WOLF allele as defined in  claim 1 . 
     
     
         12 . A cell of a spinach plant as claimed in  claim 7 , which cell comprises a WOLF allele as defined in  claim 1 . 
     
     
         13 . A seed capable of growing into a spinach plant as claimed in  claim 7 . 
     
     
         14 . Harvested leaves of a spinach plant as claimed in  claim 7 . 
     
     
         15 . A food product comprising the harvested leaves of  claim 14 . 
     
     
         16 . A WOLF allele having a nucleotide sequence selected from SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:96, SEQ ID NO:102, SEQ ID NO:145, SEQ ID NO:155. 
     
     
         17 . A vector comprising a WOLF allele according to  claim 16 . 
     
     
         18 . A spinach plant comprising the vector of  claim 17 . 
     
     
         19 . A method for selecting a spinach plant comprising a novel WOLF allele that confers resistance to  Peronospora farinosa  f. sp.  spinaciae  in a spinach plant, comprising:
 a) determining the sequence of the LRR domain or part thereof of a WOLF allele in the genome of a spinach plant;   b) comparing said sequence to the sequences referred to in Table 3;   c) if the sequence of the LRR domain or part thereof is substantially different from the sequences referred to in Table 3, select the spinach plant that harbours said sequence in its genome as a spinach plant that comprises a novel WOLF allele.   
     
     
         20 . The method as claimed in  claim 19 , wherein the selected plants are subsequently tested for their resistance to different pathogenic races of  Peronospora farinosa  f. sp.  spinaciae.    
     
     
         21 . A method for identifying a WOLF allele that confers resistance to one or more pathogenic races of  Peronospora farinosa  f. sp.  spinaciae  in a spinach plant, comprising:
 a) phenotypically selecting a spinach plant that is resistant to one or more pathogenic races of  Peronospora farinosa  f. sp.  spinaciae;      b) determining the sequence of the LRR domain or part thereof of a WOLF allele that is present in the genome of said spinach plant, and   c) optionally comparing the sequence to a reference sequence representing the WOLF gene to be identified.   
     
     
         22 . The method as claimed in  claim 19  or  21 , wherein part of a WOLF allele is amplified from the plant's genome by means of PCR. 
     
     
         23 . A primer pair for amplifying the LRR domain or part thereof of a WOLF allele from the genome of a spinach plant, wherein the forward primer is a nucleic acid molecule comprising the sequence of SEQ ID NO:1 or SEQ ID NO:3, and the reverse primer is a nucleic acid molecule comprising the sequence of SEQ ID NO:2. 
     
     
         24 . An allele of an alpha-WOLF gene, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif “MAEIGYSVC” at its N-terminus; and b) the motif “KWMCLR”; and wherein the LRR domain of the protein has in order of increased preference 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence similarity to any one of the amino acid sequences having SEQ ID NO:111. SEQ ID NO:113, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:139, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170; and wherein the allele when present in a spinach plant confers resistant to one or more  Peronospora farinosa  f. sp.  spinaciae  races or isolates. 
     
     
         25 . An allele of a beta-WOLF gene, wherein the protein encoded by said allele is a CC-NBS-LRR protein that comprises in its amino acid sequence: a) the motif “MAEIGYSVC” at its N-terminus; and b) the motif “HVGCVVDR”; and wherein the LRR domain of the protein has in order of increased preference 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence similarity to any one of the amino acid sequences having SEQ ID NO:109, SEQ ID NO:115. SEQ ID NO:137; and wherein the allele when present in a spinach plant confers resistant to one or more  Peronospora farinosa  f. sp.  spinaciae  races or isolates. 
     
     
         26 . A spinach plant comprising a WOLF allele as defined in  claim 1 , wherein the LRR domain of the WOLF allele has an amino acid sequence selected from the group consisting of SEQ ID NO:109, SEQ ID NO:111. SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO: 152, SEQ ID NO:154, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, and SEQ ID NO:170. 
     
     
         27 . A hybrid spinach plant comprising two WOLF alleles as defined in  claim 1 , wherein the LRR domain of the first WOLF allele and second WOLF allele each have an amino acid sequence selected from the group consisting of SEQ ID NO:109, SEQ ID NO:111. SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO: 152, SEQ ID NO:154, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, and SEQ ID NO:170. 
     
     
         28 . A hybrid spinach plant as claimed in  claim 27 , wherein the first and second allele are selected such that the combination of the two alleles provides resistance to races Pfs1, Pfs2, Pfs3, Pfs4, Pfs5, Pfs6, Pfs7, Pfs8, Pfs9, Pfs10, Pfs11, Pfs12, Pfs13, Pfs14, Pfs15 and Pfs16 and optionally isolate US1508.