miR395m/n negatively regulate resistance to Colletotrichum gloeosporioides in Malus domestica

Glomerella leaf spot (GLS), incited by Colletotrichum gloeosporioides, poses a significant threat to apple production. While microRNAs (miRNAs) are known to be critical regulators of plant-pathogen interactions, their specific roles in the apple-C. gloeosporioides interaction are not fully understood. In this study, we identified two novel miR395 family members, designated miR395m and miR395n, which are significantly upregulated in susceptible apple varieties upon C. gloeosporioides infection. Both miRNAs were found to target and cleave MdTHESEUS1 (MdTHE1) and MdLaccase12 (MdLAC12). Overexpression of miR395m and miR395n in resistant variety 'Fuji' ('FJ') compromised resistance to C. gloeosporioides, while silencing these miRNAs in 'Royal Gala' ('GL-3') resulted in attenuated disease symptoms compared to control plants. Consistently, silencing of MdTHE1 and MdLAC12 in 'FJ' increased susceptibility to C. gloeosporioides, whereas their overexpression in 'GL-3' diminished disease severity. In addition, overexpression of miR395m/n suppressed the expression of pathogenesis-related (PR) proteins, the reactive oxygen species (ROS) scavenging system, and reduced lignin content. Taken together, miR395m/n modulate resistance to C. gloeosporioides by regulating MdLAC12 and MdTHE1. This study reveals a conserved miRNA-mediated regulatory module that fine-tunes defense responses in apple and provides new insights into GLS resistance breeding.