Phylogenetic and functional analyses ofN6-methyladenosine RNA methylation factors in the wheat scab fungusFusarium graminearum

Abstract

AbstractIn eukaryotes,N6-methyladenosine (m6A) RNA modification plays crucial roles 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 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 fungusFusarium graminearum, we generated knockout mutants lacking potential m6A factors including the sole m6A writerMTA1. However, the resulting knockouts did not exhibit any noticeable phenotypic changes during vegetative and sexual growth stages. As obtaining a homozygous knockout lackingMTA1was likely hindered by its essential role, we generatedMTA1-overexpressing strains (MTA1-OE). TheMTA1-OE5 strain showed delayed conidial germination and reduced hyphal branching, suggesting its involvement during vegetative growth. Consistent with these findings, the expression levels ofMTA1and a potential m6A readerYTH1were 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 up- and down-regulated in theMTA1-OE5 strain. Overall, our study highlights the functional importance of the m6A methylation during conidial germination inF. graminearumand 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 fungusFusarium graminearumprovides insights into the essential role of the m6A writerMTA1in conidial germination and hyphal branching. The observed effects of overexpressingMTA1on fungal growth and gene expression patterns of m6A factors throughout the life cycle ofF. graminearumfurther 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.