Set2 family regulates mycotoxin metabolism and virulence via H3K36 methylation in pathogenic fungus Aspergillus flavus

Aspergillus flavus infects various crops with aflatoxins, and leads to aspergillosis opportunistically. Though H3K36 methylation plays an important role in fungal toxin metabolism and virulence, no data about the biological function of H3K36 methylation in A. flavus virulence has been reported. Our study showed that the Set2 histone methyltransferase family, AshA and SetB, involves in morphogenesis and mycotoxin anabolism by regulating related transcriptional factors, and they are important for fungal virulence to crops and animals. Western-blotting and double deletion analysis revealed that AshA mainly regulates H3K36me2, whereas SetB is mainly responsible for H3K36me3 in the nucleus. By construction of domain deletion A. flavus strain and point mutation strains by homologous recombination, the study revealed that SET domain is indispensable in mycotoxin anabolism and virulence of A. flavus, and N455 and V457 in it are the key amino acid residues. ChIP analysis inferred that the methyltransferase family controls fungal reproduction and regulates the production of AFB1 by directly regulating the production of the transcriptional factor genes, including wetA, steA, aflR and amylase, through H3K36 trimethylation in their chromatin fragments, based on which this study proposed that, by H3K36 trimethylation, this methyltransferase family controls AFB1 anabolism through transcriptional level and substrate utilization level. This study illuminates the epigenetic mechanism of the Set2 family in regulating fungal virulence and mycotoxin production, and provides new targets for controlling the virulence of the fungus A. flavus. AUTHOR SUMMARY The methylation of H3K36 plays an important role in the fungal secondary metabolism and virulence, but no data about the regulatory mechanism of H3K36 methylation in the virulence of A. flavus have been reported. Our study revealed that, in the histone methyltransferase Set2 family, AshA mainly catalyzes H3K36me2, and involves in the methylation of H3K36me1, and SetB mainly catalyzes H3K36me3 and H3K36me1. Through domain deletion and point mutation analysis, this study also revealed that the SET domain was critical for the normal biological function of the Set2 family and that N455 and V457 in the domain were critical for AshA. By ChIP-seq and ChIP-qPCR analysis, H3K36 was found to be trimethylation modified in the promotors and ORF positions of wetA, steA, aflR and the amylase gene (AFLA_084340), and further qRT-PCR results showed that these methylation modifications regulate the expression levels of these genes. According to the results of ChIP-seq analysis, we proposed that, by H3K36 trimethylation, this methyltransferase family controls the metabolism of mycotoxin through transcriptional level and substrate utilization level. All the results from this study showed that Set2 family is essential for fungal secondary metabolism and virulence, which lays a theoretical groundwork in the early prevention and treatment of A. flavus pollution, and also provides an effective strategy to fight against other pathogenic fungi.