The RsWRKY18-RsHSFA2 module confers thermotolerance by activating RsHSP22 during taproot thickening in radish

Radish (Raphanus sativus L.) is a cool-season root vegetable crop grown worldwide. Heat stress (HS) severely restricts radish taproot formation, resulting in the loss of yield and quality. However, the regulatory mechanism underlying radish taproot thickness under HS conditions has been largely unexplored. Here, we identified a heat-induced HSP protein, HEAT SHOCK PROTEIN 22 (RsHSP22), via comparative proteome analysis. Yeast one-hybrid (Y1H) assays, dual-luciferase reporter assays, and electrophoretic mobility shift assays demonstrated that WRKY DNA-BINDING PROTEIN 18 (RsWRKY18) and HEAT STRESS TRANSCRIPTION FACTOR A2 (RsHSFA2) bind to the W-box and HSE element of the promoter of RsHSP22 to activate its expression, respectively. RsWRKY18 and RsHSFA2 are heat-inducible and nucleus-localized transcription activators, and their expression levels in the heat-tolerant genotype "NAU-XBC" were higher compared to the heat-susceptible genotype "NAU-YB" after a short- or long-term HS treatment at the taproot thickening stage. Overexpression of RsWRKY18 and RsHSFA2 alleviated oxidative damage under HS and conferred heat tolerance in Arabidopsis (Arabidopsis thaliana) and radish, whereas silencing RsWRKY18 resulted in heat susceptibility and limited taproot thickening in radish. Furthermore, RsWRKY18 bound to the promoters of several taproot thickening-related genes, including Xyloglucan endotransglucosylase/hydrolase 32 (RsXTH32), Alpha-expansin 9 (RsEXPA9), KNOTTED-like from Arabidopsis thaliana 1 (RsKNAT1), and WUSCHEL homeobox-containing 14 (RsWOX14). Interestingly, RsWRKY18 interacted with RsHSFA2 coordinately to enhance the transactivation activity of both RsWRKY18 and RsHSFA2 on their target genes, especially under HS conditions. Overall, these results show that the module RsWRKY18-RsHSFA2 regulates the HS response and taproot thickening in radish and helps facilitate the development of heat-tolerant cultivars with superior yield and quality in radish and other root vegetables.