| Summary: | <i>Botrytis cinerea</i> is one of the most destructive fungal pathogens that cause gray mold rot in horticultural products, including fresh fruits, vegetables, and flowers, leading to serious economic losses. <i>B. cinerea </i>is difficult to control because it has strong stress resistance and complex infection modes. The pathogenic mechanisms of <i>B. cinerea</i> have been revealed at multiple levels, but little is known at the epigenetic level. In this study, we first revealed the important role of DNA methyltransferases in regulating the development and pathogenicity of <i>B. cinerea</i>. We showed that two DNA methyltransferases, BcDIM2 and BcRID2, showed a strong synergistic effect in regulating the pathogenicity of<i> B. cinerea</i>. The double knockout mutant <i>△Bcdim2rid2</i> showed slower mycelial growth, lower spore germination, attenuated oxidative tolerance, and complete pathogenicity loss on various hosts, which is related to the reduced expression of virulence-related genes in <i>△Bcdim2rid2 </i>and the induced resistance of the host. Although<i> B. cinerea</i> has multiple DNA methyltransferases, the global methylation level is very low, and few 5mC sites can be detected by BS-seq. These results first revealed the important role and the action mode of DNA methyltransferases in <i>B. cinerea</i>.
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