The wheat stem rust fungus Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. Resistance of host lines is often governed by recognition of fungal effector proteins (avirulence/virulence proteins) by plant resistance proteins (R proteins). We have taken a mutational genomics approach to identify Avr genes in Pgt. We isolated spontaneous mutants with virulence for Sr50, Sr5, Sr27, Sr21 or Sr45 by selection on resistant host lines. Sequence analysis of the Sr50 virulent mutant revealed that virulence resulted from the exchange of a whole chromosome between the two haploid nuclei of this dikaryotic organism, resulting in loss of the avirulence allele. This confirms the important role of somatic exchange events in virulence evolution in Pgt. The AvrSr50 gene was identified from the 25 candidate effector genes from this chromosome by transient co-expression with the cloned Sr50 gene in N. benthamiana. AvrSr50 recognition was confirmed in wheat by viral expression. Recognition of the AvrSr50 protein by the host Sr50 immune receptor is based on direct interaction and we have identified critical amino acid polymorphisms contributing to the escape from recognition in virulent isolates. Identification of AvrSr50 has enabled development of tools for testing effector function in wheat including viral overexpression and wheat protoplast transient expression assays. Spontaneous mutants for several other Avr loci have also been sequenced and a new Pacbio-based genome assembly for the Australian parental Pgt isolate has facilitated the delineation of these loci. Three mutants with virulence for Sr27 contain overlapping deletions and a single candidate gene for AvrSr27 has been identified. Likewise, AvrSr5 mutants contain large deletions spanning several candidate effector genes
