Circular RNA Vv‐circCOR27 modulates thermotolerance through attenuating VvHSP90 .2b‐ VvHsfA7a interaction in grapevine

Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules that arise from exon back-splicing. The identification and function investigation of circRNAs have been comprehensively explored in plants, however, the regulatory mechanisms are largely unknown. This study employed genetic transformation, circRNA pull-down assay, ribonucleoprotein immunoprecipitation (RIP), and the circRNA trimolecular fluorescence complementation (cTriFC) system to investigate the function and regulatory mechanisms of a circRNA Vv-circCOR27 in grapevine. Vv-circCOR27 expression was suppressed under heat stress and was markedly lower in thermotolerant cultivars. In addition, overexpression of Vv-circCOR27, using an expression cassette with endogenous introns inserted into circRNA-producing exons to minimize the background level of cognate linear RNA, was shown to exacerbate thermotolerance in grapevine embryogenic calli and seedlings. Biochemical assays demonstrate that Vv-circCOR27 binds directly to heat shock protein 90.2b (VvHSP90.2b). Further evidence indicated that Vv-circCOR27 inhibits the interaction between VvHsfA7a and VvHSP90.2b in vivo, which resulted in the downregulation of small heat shock protein (sHSP) genes under heat stress in Vv-circCOR27 overexpression calli. Collectively, this finding suggests a model in which thermotolerance is fine-tuned by Vv-circCOR27 via competitively binding to VvHSP90.2b, thereby attenuating its interaction with VvHsfA7a and subsequent transactivation. This work advances understanding of circRNA regulatory mechanisms in plants.