miR3398-VqMYB15 Regulates the Synthesis of Stilbene in Vitis quinquangularis.

Grapevine (Vitis vinifera L.) is an economically important fruit crop grown worldwide. Grapevine is cultivated extensively in China, and certain wild grapes exhibit excellent resistance to pathogens and stress. MicroRNAs (miRNAs) act as key regulators of plant growth, development, and immunity; however, their functions in grape stilbene synthesis are poorly understood. We identified an miRNA (miR3398) that negatively regulates the transcription factor MYB15 and participates in the synthesis of stilbene from Vitis quinquangularis (V. quinquangularis). MiR3398 and VqMYB15 showed completely opposite expression patterns after AlCl₃ treatment, and the interaction between miR3398 and VqMYB15 was confirmed using 5'-RACE ligase-mediated rapid amplification of cDNA ends, dual-luciferase reporter gene system, and western blot analysis. VqMYB15 could bind to the VqSTS48 promoter by using yeast one-hybrid and electrophoretic mobility shift assay, and overexpression of VqMYB15 promoted stilbene accumulation in grape leaves. Using an overexpression and silencing system, we found that miR3398 negatively targets VqMYB15 to synthesis of stilbenes. We used Al³⁺ as an elicitor, indicating that miR3398 plays an important role in the plant immunity of V. quinquangularis. We also found that miR3398 is involved in plant immunity by detecting its promoter activity in grape protoplasts, luciferase imaging, and transgenic Arabidopsis thaliana. More importantly, we found that an ethylene transcription factor, ERF057, can bind to the promoter of miR3398 using Y1H and EMSA assays and inhibit its transcription using DLR, luciferase imaging, and β-glucuronidase transcript assays. Overexpression of VqERF057 reduced miR3398 transcript in V. quinquangularis and transgenic grapevine calli, but increased the stilbene content. These findings contribute to the understanding of the biological functions of miR3398 regulates stilbene synthesis in grapevines and clarify the molecular mechanism underlying the interaction between miR3398 and VqMYB15.