In eukaryotes, N⁶-methyladenosine (m⁶A) RNA modification plays a crucial role in governing the fate of RNA molecules and has been linked to various developmental processes. However, the phyletic distribution and functions of genetic factors responsible for m⁶A modification remain largely unexplored in fungi. To get insights into the evolution of m⁶A machineries, we reconstructed global phylogenies of potential m⁶A writers, readers, and erasers in fungi. Substantial copy number variations were observed, ranging from up to five m⁶A writers in early-diverging fungi to a single copy in the subphylum Pezizomycotina, which primarily comprises filamentous fungi. To characterize m⁶A factors in a phytopathogenic fungus Fusarium graminearum, we generated knockout mutants lacking potential m⁶A factors including the sole m⁶A writer MTA1. However, the resulting knockouts did not exhibit any noticeable phenotypic changes during vegetative and sexual growth stages. As obtaining a homozygous knockout lacking MTA1 was likely hindered by its essential role, we generated MTA1-overexpressing strains (MTA1-OE). The MTA1-OE5 strain showed delayed conidial germination and reduced hyphal branching, suggesting its involvement during vegetative growth. Consistent with these findings, the expression levels of MTA1 and a potential m⁶A reader YTH1 were dramatically induced in germinating conidia, followed by the expression of potential m⁶A erasers at later vegetative stages. Several genes including transcription factors, transporters, and various enzymes were found to be significantly upregulated and downregulated in the MTA1-OE5 strain. Overall, our study highlights the functional importance of the m⁶A methylation during conidial germination in F. graminearum and provides a foundation for future investigations into m⁶A modification sites in filamentous fungi.IMPORTANCEN⁶-methyladenosine (m⁶A) RNA methylation is a reversible posttranscriptional modification that regulates RNA function and plays a crucial role in diverse developmental processes. This study addresses the knowledge gap regarding phyletic distribution and functions of m⁶A factors in fungi. The identification of copy number variations among fungal groups enriches our knowledge regarding the evolution of m⁶A machinery in fungi. Functional characterization of m⁶A factors in a phytopathogenic filamentous fungus Fusarium graminearum provides insights into the essential role of the m⁶A writer MTA1 in conidial germination and hyphal branching. The observed effects of overexpressing MTA1 on fungal growth and gene expression patterns of m⁶A factors throughout the life cycle of F. graminearum further underscore the importance of m⁶A modification in conidial germination. Overall, this study significantly advances our understanding of m⁶A modification in fungi, paving the way for future research into its roles in filamentous growth and potential applications in disease control.

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