RNA demethylase CsALKBH8 enhances postharvest disease resistance of citrus fruit against penicillium digitatum via...

N6-methyladenosine (m⁶A) is a widespread internal modification of eukaryotic mRNA and is increasingly recognized as an important regulator of plant growth, development, and stress adaptation. However, its involvement in postharvest disease resistance of fruit is still poorly defined. In the present study, we identified an RNA demethylase, CsALKBH8, and investigated its function in citrus defense against green mold. We found that Penicillium digitatum infection significantly reduced global m⁶A levels in citrus peel, accompanied by rapid induction of CsALKBH8 expression. In vitro assays confirmed that CsALKBH8 possesses m⁶A demethylase activity. Transient overexpression of CsALKBH8 in citrus peel markedly alleviated disease development, as reflected by lower infection severity and smaller lesions after pathogen challenge. Transcriptome analysis revealed that CsALKBH8 overexpression induced extensive reprogramming of gene expression, with significant enrichment of defense-related pathways, including reactive oxygen species (ROS) metabolism, phenylpropanoid biosynthesis, and plant-pathogen interaction. Furthermore, CsALKBH8 reduced m⁶A levels on transcripts of multiple defense-related genes, including CsPR-1, CsPODs, CsRBOHF, CsGSTs, and lignin biosynthesis-associated genes, thereby enhancing their expression. This epitranscriptomic regulation promoted the activation of ROS metabolism and lignin accumulation, leading to increased enzyme activities (NOX, SOD, POD, APX, GST, and LAC) and elevated levels of H₂O₂, O₂^-, and lignin, ultimately strengthening disease resistance in citrus fruit. Collectively, our findings demonstrate that CsALKBH8 enhances citrus resistance to P. digitatum through m⁶A-dependent post-transcriptional regulation of defense pathways, highlighting its potential as a molecular target for improving fruit storage quality and postharvest disease control.