RsVQ4-RsWRKY26 module positively regulates thermotolerance by activating RsHSP70-20 transcription in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) is an important cool-season root vegetable crop worldwide. Heat stress (HS) severely restricts radish taproot formation and diminishes its yield and quality. Although WRKY transcription factors (TFs) play pivotal roles in several biotic and/or abiotic stress responses, how WRKY TFs mediate HS regulatory network remains elusive in radish. Herein, a WRKY-Ia TF gene, RsWRKY26, exhibited dramatically up-regulated expression under heat stress, especially at 8 h and 24 h. RsWRKY26 was localized to the nucleus and displayed transactivation activity in both the yeast and plant cells. Interestingly, the RsWRKY26-overexpressing radish plants exhibited significantly enhanced thermotolerance compared to the control plants, whereas its knock-down plants showed increased thermosensitivity. Both in vitro and in vivo assays showed that RsWRKY26 activated RsHSP70-20 transcription by directly binding to the specific W-box element in its promoter. Moreover, several biochemical assays revealed that a VQ motif-containing protein RsVQ4 interacted with RsWRKY26, which promoted DNA-binding activity of RsWRKY26 to regulate RsHSP70-20 expression. Notably, RsWRKY26 in turn binds to RsVQ4 promoter to activate its transcription, thereby forming a positive feedback loop to cooperatively regulate heat stress response in radish. Together, we revealed a novel RsVQ4-RsWRKY26-RsHSP70-20 module to promote the thermotolerance, which would facilitate dissecting the molecular regulatory network of RsWRKYmediated thermal morphogenesis in radish.