Phylogenetic and functional analyses of -methyladenosine RNA methylation factors in the wheat scab fungus .

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