CrWRKY57 and CrABF3 cooperatively activate <i>CrCYCD6;1</i> to modulate drought tolerance and root development

Drought is a major abiotic stress. WRKYs are one of the largest families of transcription factors (TFs) in plants. The effects of most WRKYs on developmental regulation and drought adaptation in <i>Citrus</i> remain largely unclear. <i>Citrus reticulata</i> cv. Sanhu hongju (Sanhu) is a drought-tolerant variety from Jiangxi Province, China. Here, we report a differentially expressed <i>CrWRKY57</i> gene in drought-treated Sanhu leaves through transcriptome analysis. Its transcriptional expression could be induced by abscisic acid (ABA) treatment and water deficit. Overexpression of <i>CrWRKY57</i> in lemon (<i>Citrus limon</i>) and tobacco (<i>Nicotiana tabacum</i>) confers enhanced drought tolerance, while RNA interference (RNAi)-mediated silencing in Sanhu increases dehydration susceptibility and reduces root volume. Moreover, virus-induced gene silencing-mediated knockdown of <i>CrWRKY57</i> in Sanhu reduces primary root length and lateral root number by nearly 50% compared to the control. The results of yeast two-hybrid, co-immunoprecipitation assays and bimolecular fluorescence complementation demonstrate that CrWRKY57 interacts with CrABF3, a key TF in ABA signaling. Silencing <i>ClABF3</i>, its homolog in lemon, also increases drought sensitivity and disrupts root system development. Together, CrWRKY57 and CrABF3 directly activate the promoter of the cell cycle gene <i>CrCYCD6;1</i> by binding to W-box and ABRE elements, respectively. Furthermore, silencing <i>CrCYCD6;1</i> in Sanhu also severely reduces primary root length and lateral root number. Collectively, our findings provide a new perspective of <i>CrWRKY57</i> as a positive player in drought response and highlight the role of the CrWRKY57-CrABF-<i>CrCYCD6;1</i> module in enhancing drought tolerance by modulating root development.