Promoter methylation of <scp>MdRNS3a</scp> contributes to <i>Alternaria</i> leaf spot resistance in apple ( <i>Malus × domestica</i> )

Apple leaf spot severely threatens global apple production, and with chemical control posing environmental and health risks, resistance breeding necessitates exploration of a previously unknown regulatory mechanism that distinguishes ALT1-susceptible from -resistant apple cultivars. Small RNA sequencing of Alternaria alternata f. sp. mali (ALT1)-infected susceptible apple 'Golden Delicious' revealed a novel tRNA-Val-derived tsRNA (tsRVal) that negatively regulates MdTIR-1 expression to modulate ALT1 resistance. Our findings revealed that tsRVal biosynthesis required cleavage by the ribonuclease T2 family member MdRNS3a. Interestingly, we found that DNA methylation of the MdRNS3a promoter negatively regulates its expression in the resistant apple cultivar Han Fu, which in turn abrogates tsRVal biogenesis and maintains resistance to ALT1. Conversely, ALT1 inoculation induced promoter hypomethylation in GD, leading to robust MdRNS3a activation, significant tsRVal accumulation, and the subsequent development of pathogenic phenotypes. Our findings further support a regulatory model wherein MdbHLH3 functions as a methylation-sensitive transcriptional activator of MdRNS3a via promoter binding, thereby determining tsRVal biosynthesis and the resultant leaf spot sensitivity. Our study demonstrates that the methylation status of the MdRNS3a promoter can serve as a reliable epigenetic marker for distinguishing resistant and susceptible apple varieties, thus providing a valuable tool to facilitate disease-resistant apple breeding programs.