DNA demethylation induced by self-DNA activates pattern-triggered immunity in postharvest citrus

Growing evidence indicates that extracellular self-DNA (sDNA) acts as a damage-associated molecular pattern (DAMP) that elicits protective responses in conspecific plants. However, the mechanism by which plant senses or recognizes sDNA to initiate pattern-triggered immunity (PTI) remains unclear. In citrus fruit, sDNA treatment induced PTI responses, accompanied by jasmonic acid (JA)-dependent defense and reduced susceptibility to the fungus Penicillium digitatum. DNA methylation profiling showed that P. digitatum infection increased methylation genome-wide, whereas sDNA treatment reduced it extensively. Corresponding differentially methylated regions (DMRs) were significantly enriched in genes involved in the plant-pathogen interaction pathway. Notably, sDNA reduced the DNA methylation level of a critical LRR-RLK gene, CsSOBIR1, maintaining its high expression during P. digitatum infection. The subsequent interaction between CsSOBIR1 and CsRLP7 facilitated sDNA recognition, triggering PTI responses and activating the downstream CsMAPKKK1-CsMAPKK2-CsMAPK4 cascade along with JA signaling. The sDNA-induced DNA demethylation was associated with the upregulation of several demethylase-encoding genes, including CsDML1.1, CsDML1.2, and CsDME. Consistently, treatment with the DNA methylation inhibitor 5-azacytidine (5'-Aza) enhanced citrus resistance to fungal infection, supporting the positive correlation between sDNA-mediated demethylation and immune response. These findings suggest that DNA demethylation is an important epigenetic component of sDNA-mediated signaling that potentiates PTI-associated defenses in citrus fruit.