The miR166a-MpATHB8 module regulates anthocyanin accumulation to increase rust resistance in Malus ‘Profusion’ leaves

Apple rust, caused by the fungal pathogen Gymnosporangium yamadae, leads to substantial yield losses and significant economic damage. In the rust-resistant cultivar Malus ‘Profusion’, rust infection triggers anthocyanin synthesis at infection sites as a defense mechanism to restrict fungal proliferation. Although small noncoding RNAs (miRNAs) play important roles in regulating anthocyanin biosynthesis, their specific functions under rust stress remain poorly characterized. In this study, small RNA sequencing revealed that miR166a is a key rust-responsive regulator. Its direct targeting and negative regulation of MpATHB8 were confirmed through luciferase assays, GUS staining, and gene expression analyses. Functional validation via transient and stable transformation in Malus demonstrated that suppressing miR166a expression using short tandem target mimics or overexpressing MpATHB8 promoted anthocyanin accumulation and enhanced resistance to rust. In contrast, overexpressing miR166a or silencing MpATHB8 suppressed anthocyanin synthesis and increased susceptibility to the pathogen. Further evidence indicates that the MpATHB8 protein activates anthocyanin biosynthesis by binding to and inducing the promoter of MpMYB10b. These findings reveal a miR166a-MpATHB8-MpMYB10b regulatory module that enhances rust resistance through anthocyanin metabolism in M. ‘Profusion’. Our findings provide novel insights into the miRNA-mediated regulation of anthocyanin metabolism and facilitate the breeding of rust-resistant and anthocyanin-enriched Malus cultivars.