Distinct targeting of 5mC and 6mA DNA methylation during the onset of Flg22‐induced immunity between Arabidopsis and rice

Cytosine methylation drives transcriptional reprogramming during plant innate and acquired immunity. Beyond RNA adenosine methylation, DNA adenine methylation (6mA) has recently emerged as a candidate regulator of gene expression, though its role in plant immunity is unknown. Using the bacterial elicitor Flg22, we examined genome-wide DNA methylation responses of Arabidopsis thaliana and Oryza sativa to identify shared epigenetic responses across divergent genomes. High-resolution Oxford Nanopore sequencing enabled simultaneous detection of 5-methylcytosine (5mC) and 6mA DNA modifications. In both species, early immune activation was marked by non-CG 5mC hypomethylation at short euchromatic transposable elements. By 72 hpt, both species showed elevated 5mC levels at (peri)centromeric regions, targeted to pericentromeric non-CG sites in Arabidopsis and accompanied by lasting chromosomal arm hypomethylation in rice. Despite species-specific global 6mA responses, both species exhibited a conserved bimodal 6mA distribution flanking transcription start and termination sites of genes, intensified at defence-related genes following Flg22 elicitation. In Arabidopsis, these 6mA signatures correlated with Flg22-primed gene activation, while mutation of 6mA demethylase ALKBH1D increased susceptibility to Pseudomonas syringae and Meloidogyne incognita. Together, our findings reveal conserved 5mC dynamics during immune activation and highlight a role for 6mA in modulating defence-related transcriptional reprogramming.