DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ between eukaryotes. Many strains of the plant pathogenic fungus Zymoseptoria tritici appeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of the Ztdim2 DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation of Ztdim2 occurred recently as some Z. tritici isolates carry a functional Ztdim2 gene. Moreover, we show that Ztdim2 inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with and without functional Ztdim2. Presence of functional Ztdim2 correlated with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense.We identified low levels of 5mC in strains carrying inactive Ztdim2 alleles, suggesting that 5mC is maintained over time, presumably by an active Ztdnmt5 gene. Integration of a functional Ztdim2 allele in strains with mutated Ztdim2 restored normal 5mC levels, demonstrating de novo cytosine methylation activity of Ztdim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lacking Ztdim2. We found that the presence of Ztdim2 alters nucleotide composition by promoting C to T transitions (C→T) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that dense 5mC at TEs is a polymorphic trait in Z. tritici populations that can impact genome evolution. SignificanceCytosine DNA methylation (5mC) is known to silence transposable elements in fungi and thereby appears to contribute to genome stability. The genomes of plant pathogenic fungi are highly diverse, differing substantially in transposon content and distribution. Here, we show extensive differences of 5mC levels within a single species of an important wheat pathogen that were caused by inactivation of the DNA methyltransferase ZtDim2 in the majority of studied isolates. Presence of widespread 5mC increased point mutation rates in regions with active or mutated transposable elements during mitosis. The mutation pattern is dependent on the presence of ZtDim2 and resembles a mitotic version of Repeat-Induced Point mutation (RIP). Thus, loss of 5mC may represent an evolutionary trade-off offering adaptive potential at the cost of transposon control. occurred recently as two closely related sister species of Z. tritici, Zymoseptoria ardabiliae and Zymoseptoria pseudotritici were shown to carry a single intact dim2 gene and have 5mC (25).By genome analyses of multiple Z. tritici isolates from the center of origin of the pathogen, the Middle East, we discovered several Z. tritici isolates with an intact Ztdim2 gene. This finding suggests that the loss of 5mC not only occurred very recently but is a polymorphic trait in Z.triti...

DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ among eukaryotes. Strains of the plant pathogenic fungus Zymoseptoria tritici appeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of the dim2 DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation of dim2 occurred recently as some Z. tritici isolates carry a functional dim2 gene. Moreover, we show that dim2 inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with or without functional dim2 alleles. Presence of functional dim2 correlates with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense. We identified low levels of 5mC in strains carrying non-functional dim2 alleles, suggesting that 5mC is maintained over time, presumably by an active Dnmt5 DNA methyltransferase. Integration of a functional dim2 allele in strains with mutated dim2 restored normal 5mC levels, demonstrating de novo cytosine methylation activity of Dim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lacking functional dim2. We found that presence of a functional dim2 allele alters nucleotide composition by promoting C to T transitions (C→T) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that 5mC density at TEs is a polymorphic trait in Z. tritici populations that can impact genome evolution.

The fungus Zymoseptoria tritici is one of the most devastating pathogens of wheat. Aside from its importance as a disease-causing agent, this species has emerged as a powerful model system for evolutionary genetic studies of cropinfecting fungal pathogens. Z. tritici exhibits exceptionally high levels of genetic and phenotypic diversity as well as morphological plasticity, which can make experimental studies and comparability of results obtained in different laboratories, e.g., from infection assays, challenging. Therefore, standardized experimental methods are crucial for research on Z. tritici biology and the interaction of this fungus with its wheat host. Here, we describe a suite of well-tested and optimized protocols ranging from isolation of Z. tritici field specimens to analyses of virulence assays under controlled conditions. Several biological and technical aspects of working with Z. tritici under laboratory conditions are considered and carefully described in each protocol.