Botrytis cinerea Loss and Restoration of Virulence during in vitro Culture Follows Flux in Global DNA Methylation

There is compelling evidence highlighting the importance of DNA methylation in controlling gene expression of higher animals and plants during development and in response to challenges. However, less is known of the extent to which DNA methylation is involved in determining the virulence of pathogenic fungi. Here we confirm previous reports that the virulence of plant pathogenic fungi (in this case B. cinerea) decreases during prolonged in vitro culture, and that virulence levels recover after one passage on A. thaliana. We next observed correlative associations between progressive loss and restoration of virulence of these samples and flux in global DNA methylation patterns measured using Methylation Sensitive Amplification Polymorphisms, suggesting a link between DNA methylation and virulence. To test this hypothesis we used Next Generation Sequencing to re-sequence the genomes of B. cinerea cultures of different ages (i.e, 1 and 8 months) and to generate the first single base resolution of the B. cinerea methylome by Whole Genome Bisulfite Sequencing of three culture times (1, 8 month and 8 months plus one passage on A. thaliana). This work showed that while genetic mutations could not explain the observed changes in virulence, modification to the methylation levels of 25% of all B. cinerea genes known to be implicated in virulence were consistent with a functional link existing between methylation status and virulence in this important pathogen.