m 6 A methyltransferase AflIme4 orchestrates mycelial growth, development and aflatoxin B 1 biosynthesis in Aspergillus flavus.

Aflatoxin B1 (AFB1 ), a highly toxic secondary metabolite produced by Aspergillus flavus, poses a severe threat to agricultural production, food safety and human health. The methylation of mRNA m6 A has been identified as a regulator of both the growth and AFB1 production of A. flavus. However, its intracellular occurrence and function needs to be elucidated. Here, we identified and characterized a m6 A methyltransferase, AflIme4, in A. flavus. The enzyme was localized in the cytoplasm, and knockout of AflIme4 significantly reduced the methylation modification level of mRNA. Compared with the control strains, ΔAflIme4 exhibited diminished growth, conidial formation, mycelial hydrophobicity, sclerotium yield, pathogenicity and increased sensitivity to CR, SDS, NaCl and H2 O2 . Notably, AFB1 production was markedly inhibited in the A. flavus ΔAflIme4 strain. RNA-Seq coupled with RT-qPCR validation showed that the transcriptional levels of genes involved in the AFB1 biosynthesis pathway including aflA, aflG, aflH, aflK, aflL, aflO, aflS, aflV and aflY were significantly upregulated. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) analysis demonstrated a significant increase in m6 A methylation modification levels of these pathway-specific genes, concomitant with a decrease in mRNA stability. These results suggest that AflIme4 attenuates the mRNA stability of genes in AFB1 biosynthesis by enhancing their mRNA m6 A methylation modification, leading to impaired AFB1 biosynthesis. Our study identifies a novel m6 A methyltransferase AflIme4 and highlights it as a potential target to control A. flavus growth, development and aflatoxin pollution.