Three putative DNA methyltransferases of Verticillium dahliae differentially contribute to DNA methylation that is dispensable for growth, development and virulence

Background: DNA methylation is an important epigenetic control mechanism that in many fungi is restricted to genomic regions containing transposable elements (TEs). Two DNA methyltransferases, Dim2 and Dnmt5, are known to perform methylation at cytosines in fungi. While most ascomycete fungi encode both Dim2 and Dnmt5, only few functional studies have been performed in species containing both. Methods: In this study, we report functional analysis of both Dim2 and Dnmt5 in the plant pathogenic fungus Verticillium dahliae. Results: Our results show that Dim2, but not Dnmt5 or the putative sexual-cycle-related DNA methyltransferase Rid, is responsible for the majority of DNA methylation under the tested conditions. Single or double DNA methyltransferase mutants did not show altered development, virulence, or transcription of genes or TEs. In contrast, Hp1 and Dim5 mutants that are impacted in chromatin-associated processes upstream of DNA methylation are severely affected in development and virulence and display transcriptional reprogramming in specific hypervariable genomic regions (so-called adaptive genomic regions) that contain genes associated with host colonization. As these adaptive genomic regions are largely devoid of DNA methylation and of Hp1- and Dim5-associated heterochromatin, the differential transcription is likely caused by pleiotropic effects rather than by differential DNA methylation. Conclusion: Overall, our study suggests that Dim2 is the main DNA methyltransferase in V. dahliae and, in conjunction with work on other fungi, is likely the main active DNMT in ascomycetes, irrespective of Dnmt5 presence. We speculate that Dnmt5 and Rid act under specific, presently enigmatic, conditions or, alternatively, act in DNA-associated processes other than DNA methylation.