Evolutionary analyses of the avirulence effector AvrStb6 in global populations of Zymoseptoria tritici identify candidate amino acids involved in recognition

We analysed the population genetic diversity of AvrStb6, the first avirulence gene cloned from the wheat pathogen Zymoseptoria tritici, using 142 Z.tritici strains sampled from four wheat fields growing on three continents. Although AvrStb6 was located in a recombination hotspot, it was found in every strain, with 71 polymorphic sites that produced 41 distinct DNA haplotypes encoding 30 AvrStb6 protein isoforms. An AvrStb6 homologue was found in the closest known relative, Z.pseudotritici, but not in three other closely related Zymoseptoria species, indicating that this gene has emerged in Zymoseptoria quite recently. Two AvrStb6 homologues with nucleotide similarities greater than 70% were identified on chromosome 10 in all Z.tritici isolates, suggesting that AvrStb6 belongs to a multigene family of candidate effectors that has expanded recently through gene duplication. The AvrStb6 sequences exhibited strong evidence for non-neutral evolution, including a large number of non-synonymous mutations, with significant positive diversifying selection operating on nine of the 82 codons. It appears that balancing selection is operating across the entire gene in natural field populations. There was also evidence for co-evolving codons within the gene that may reflect compensatory mutations associated with the evasion of recognition by Stb6. Intragenic recombination also appears to have affected the diversity of AvrStb6.