The landscape of cotton DNA methylation and its epigenetic regulation in Verticillium wilt resistance

Verticillium wilt (VW) is a devastating disease that causes severe losses in cotton yield and fiber quality. While DNA methylation is known to regulate various processes in plant development and stress responses, its specific role in conferring host resistance remains unclear. To elucidate the regulatory mechanism of DNA methylation in VW resistance, we compared the genome-wide DNA methylation profiles of a resistant cotton cultivar (NDM8) and a susceptible cultivar (TM-1) under both normal and V. dahliae-infected conditions using whole-genome bisulfite sequencing. Our results revealed that a lower overall level of DNA methylation was associated with enhanced VW resistance. We also found that the A-subgenome exhibited a higher methylation level than the D-subgenome, indicating an asymmetric methylation distribution between subgenomes. Furthermore, except for chromosomes A09, D07, D09, and D10, the chromosomal arms consistently showed significantly lower methylation levels than the centromeric regions. Upon V. dahliae infection, altered DNA methylation patterns predominantly led to reduced gene expression. Analyses of differentially methylated regions (DMRs) and genes (DMGs), combined with gene silencing assays, demonstrated that the RNA-directed DNA methylation (RdDM) pathway plays a central regulatory role in cotton resistance to VW. Key components of this pathway, including Pol IV, Pol V, AGO4, RDR2, and DCL3, were implicated in this process. Moreover, we confirmed that AGO4 interacts with four proteins, including ADH1, to collectively regulate VW resistance. In summary, our study unveils an epigenetic mechanism underlying disease resistance in cotton, providing new insights into the defense response against Verticillium wilt.