N6-Methyladenine DNA modification regulates pathogen virulence in nematodes

Understanding the global regulatory mechanisms that control pathogen virulence gene expression is essential for elucidating the molecular basis of pathogenicity. N6-methyladenine (6mA) plays a crucial role in regulating gene expression in response to various environmental stresses; however, its role in pathogen virulence remains largely unexplored. Here, we report the widespread occurrence of 6mA across 17 nematode isolates and map its genomic landscape in six notorious agriculturally important pathogen root-knot nematodes (RKNs). We demonstrated that 6mA is characterized by a conserved GAG motif across nematodes, but exhibits species-specific distribution patterns and distinct effects on gene expression. In particular, its enrichment in transposable elements differs between polyploid and diploid nematodes, suggesting lineage-specific epigenetic regulation potentially associated with polyploidy. We further identified two functional 6mA demethylases, MiNMAD-1 and MiNMAD-2, and confirmed their catalytic activity and active sites. Host-induced gene silencing of minmad-1 significantly increased plant resistance to three polyploid RKN species. A detailed functional analysis revealed that minmad-1 knockdown disrupted virulence gene expression during the parasitic stage, thereby reducing nematode infectivity. Together, our findings suggest 6mA demethylase as a key epigenetic regulator of RKNs virulence, providing new insights into nematode biology and offering promising targets for the development of sustainable control strategies. Significance Statement6mA methyltransferase regulates the expression of virulence genes in certain pathogenic bacteria and plays a critical role in their infectivity. However, the regulation of virulence gene expression in eukaryotic pathogens, particularly plant pathogens, remains poorly understood. Most studies on pathogen virulence have focused on individual effectors, with little insight into global regulatory mechanisms. Here, we demonstrate that 6mA broadly shapes virulence gene expression patterns in polyploid RKNs. Moreover, transgenic tobacco, tomato, and rice plants expressing dsRNA against nematode demethylases showed significantly enhanced resistance to RKNs. These findings establish 6mA demethylase as a promising epigenetic target for controlling plant-parasitic nematodes.