Recent loss of the Dim2 DNA methyltransferase decreases mutation rate in repeats and changes evolutionary trajectory in a fungal pathogen.

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.

Dissecting the Biology of the Fungal Wheat Pathogen Zymoseptoria tritici: A Laboratory Workflow.

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 crop-infecting 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. © 2020 The Authors. Basic Protocol 1: Purification of Z. tritici field isolates from leaf material Basic Protocol 2: Molecular identification of Z. tritici isolates Support Protocol 1: Rapid extraction of Z. tritici genomic DNA Support Protocol 2: Extraction of high-quality Z. tritici genomic DNA Basic Protocol 3: In vitro culture and long-term storage of Z. tritici isolates Basic Protocol 4: Analysis of Z. tritici virulence in wheat Support Protocol 3: Preparation of Z. tritici inoculum.