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 Citrus remain largely unclear. Citrus reticulata cv. Sanhu hongju (Sanhu) is a drought-tolerant variety from Jiangxi Province, China. Here, we report a differentially expressed CrWRKY57 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 CrWRKY57 in lemon (Citrus limon) and tobacco (Nicotiana tabacum) 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 CrWRKY57 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 ClABF3, 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 CrCYCD6;1 by binding to W-box and ABRE elements, respectively. Furthermore, silencing CrCYCD6;1 in Sanhu also severely reduces primary root length and lateral root number. Collectively, our findings provide a new perspective of CrWRKY57 as a positive player in drought response and highlight the role of the CrWRKY57-CrABF-CrCYCD6;1 module in enhancing drought tolerance by modulating root development.

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