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

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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.