Identification of Avirulence Genes in the Wheat-Puccinia graminis (Pgt) Pathosystem by EMS Mutagenesis and Diversity Analyses

The identification of avirulence (Avr) genes in biotrophic fungi, such as Pgt, represents a substantial challenge due to the lack of adequate functional validation tools. To overcome these limitations, we have performed the whole genome comparative analyses of multiple Sr35-virulent and –avirulent Pgt strains including chemically mutagenized strains that acquired virulence on the Sr35 resistance gene, and natural field isolates. The Sr35­-virulent strains were developed by treating the spores Sr35-avirulent RKQQC Pgt race with EMS. Except for the Sr35-carrying wheat line, all mutagenized strains showed similar virulence on the panel of lines carrying different stem rust resistance genes. The whole genome shotgun sequencing of 15 EMS mutants of Pgt identified a single gene carrying strong effect mutations in all mutants. A gene had a signal peptide suggesting that its protein could be directed towards the secretion pathway making it a strong Avr candidate. Resequencing of the candidate gene in a panel of Pgt strains revealed the presence of a DNA insertion creating premature termination codon in the coding sequence of all virulent isolates, and the lack of this insertion in all avirulent isolates. Preliminary experiments with tobacco agro-infiltration suggest that the combination (but not individual genes) of the effector candidate and Sr35 gene constructs can trigger hypersensitive response. Our results demonstrate that by applying whole genome next-generation sequencing to the populations of mutagenized and natural pathogen strains differing in the virulence to a specific resistance gene, it is feasible to identify and validate avirulence factors in biotrophic fungi.