Unusual evolutionary mechanisms to escape Effector-Triggered-Immunity in the fungal phytopathogen Leptosphaeria maculans

Leptosphaeria maculans is the fungus responsible for the stem canker disease of oilseed rape (Brassica napus). AvrLm3 and AvrLm7, two avirulence effector genes of L. maculans, are involved in an unusual relationship: the presence of AvrLm7 suppresses the Rlm3-mediated resistance of AvrLm3. Following the large-scale cropping of Rlm7 cultivars in Europe, the on-going breakdown of the Rlm7 resistance and the concomitant inactivation of AvrLm7 was accompanied by the resurgence of isolates expressing the AvrLm3 phenotype. Here, we evaluated adaptation mechanisms to escape Rlm3-mediated ETI by assessing AvrLm3 polymorphism in a collection of 236 L. maculans isolates. In isolates virulent towards both Rlm3 and Rlm7 (a3a7), the loss of the Rlm3-mediated resistance response was due to two distinct mechanisms. First, when AvrLm7 was inactivated (deletion or inactivating mutations), amino acid substitutions in AvrLm3 generated virulent isoforms of the protein. Secondly, point mutations in AvrLm7 could maintain the masking of the AvrLm3 phenotype while also allowing escape from the Rlm7 recognition. This virulence mechanism was found in 56.4% of the a3a7 isolates, and these contained an avirulent allele of AvrLm3. Signatures of positive selection were observed in AvrLm3 and, in spite of the telomeric location of AvrLm3, no a3a7 isolates exhibited deletion events or inactivating mutations in AvrLm3 as commonly observed for other avirulence genes of L. maculans. The complex evolutionary mechanisms enabling L. maculans to escape Rlm3-mediated resistance while preserving AvrLm3 sequence integrity, along with observed reduced aggressiveness of isolates silenced for AvrLm3, serves to emphasize importance of this effector in pathogenicity towards B. napus.