MoCul4 assists MoKmt1 to catalyze H3K9 methylation and regulates the growth, development, and pathogenicity in Magnaporthe oryzae

Rice blast, caused by the filamentous fungus Magnaporthe oryzae, is a serious disease of rice. In this study, by using homologous recombination, gene knockout mutants of ΔMokmt1 (a histone methyltransferase) and ΔMocul4 (Cullin4 subunit of an E3 ubiquitin ligase) were generated. Phenotypic analysis revealed that both MoKmt1 and MoCul4 are essential for fungal growth, development, and pathogenicity. Compared to the wild-type strain, the ΔMokmt1 mutant exhibited a slower growth rate (11.6% reduction) and significantly decreased virulence. Western blot analysis confirmed a significant decrease of H3K9 trimethylation (H3K9me3) levels in ΔMokmt1. These results illustrate MoKmt1 is the key enzyme catalyzing H3K9me3 in M. oryzae, which is critical for fungal growth, development, and pathogenicity. Notably, the ΔMocul4 mutant displayed similar but more severe developmental defects than ΔMokmt1, including a 32.8% reduction in growth rate, 90.4% decrease in conidiation, and a significant reduction in lesion area. Both MoCul4 and MoKmt1 were localized in the nucleus. Like ΔMokmt1, H3K9me3 levels in ΔMocul4 were nearly abolished, suggesting that MoCul4 modulates the methyltransferase activity of MoKmt1. RNA-seq analysis further revealed that 1,033 out of 2,910 differentially expressed genes (35.5%) were co-regulated by both MoKmt1 and MoCul4. The expression level of MoMAT1-1 and MoMAT A1-1, located in heterochromatin, was upregulated in both mutants, indicating compromised integrity of heterochromatin. This study demonstrates that MoCul4, functioning in the same pathway as MoKmt1 in M. oryzae, orchestrates the MoKmt1-mediated H3K9me3 epigenetic regulatory network, thereby providing a theoretical foundation for developing novel fungicides targeting epigenetic regulation.