Studies on the Role and Function of miR3630‐5p‐VqNAC92‐VqMYB14/VqRAP2.12‐VqSTSs in Vitis quinquangularis Plant Immunity

The key enzyme responsible for the synthesis of stilbenes, important phytoalexins, is stilbene synthase (STS), whose activity has been characterised in several plant species. However, the mechanisms through which upstream transcription factors (TFs) regulate stilbene synthesis remain unclear. In this study, the transcription factor (TF) VqNAC92 was shown to interact with VqMYB14 through yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), coimmunoprecipitation (Co-IP), and pull-down assays. By suppressing MYB14 via virus-induced gene silencing (VIGS) and subsequently overexpressing VqNAC92, performing a dual-luciferase reporter assay (DLRA) and an electrophoretic mobility shift assay (EMSA), as well as overexpressing VqMYB14/VqNAC92 in Vitis quinquangularis leaves, we found that VqNAC92 indirectly regulates VqSTS48 and promotes stilbene accumulation by regulating VqMYB14 expression at the protein and transcription levels. We also reported that VqNAC92 binds to the promoter of VqRAP2.12. Silencing or overexpression of VqRAP2.12 resulted in reduced or enhanced disease resistance, respectively. When VqRAP2.12 was introduced into the European grape cultivar 'Thompson Seedless', disease resistance significantly improved, and the expression levels of genes related to stilbene synthesis markedly increased. In addition, a known miRNA, miR3630-5p, was found to negatively regulate VqNAC92 and affect stilbene accumulation. In all, miR3630-5p negatively regulates VqNAC92, and VqNAC92 indirectly regulates VqSTS48 through VqMYB14/VqRAP2.12, revealing key mechanisms governing stilbene biosynthesis.