High salinity can severely inhibit the growth and development of watermelon (Citrullus lanatus L.). WRKY proteins are believed to mediate the adaptation of plants to abiotic stresses. Here, we identified the ClWRKY61 gene, which positively regulates the tolerance of watermelon to salt stress. Knockout of the ClWRKY61 reduced salt tolerance, while overexpression of the ClWRKY61 enhanced salt tolerance in watermelon according to phenotypic and physiological analyses. Yeast two-hybrid assays revealed that ClWRKY61 interacts with the ClLEA55 protein, and this interaction was further confirmed by luciferase complementation imaging, transient bimolecular fluorescence complementation, and GST pull-down assays. Knockout of the ClLEA55 resulted in lower salt tolerance compared to the wild-type plants. RNA-seq analysis indicated 421 up-regulated and 133 down-regulated genes in the ClWRKY61 knockout line under salt stress, containing 293 differentially expressed genes with W-box in their promoters. Thirteen genes encoding phytoene synthase, MYB transcription factor, sucrose synthase, alpha/beta-hydrolases superfamily protein, glutathione reductase, sugar transporter, LEA protein, WRKY transcription factor, ERF transcription factor, alpha-glucan water dikinase, and calcium-dependent protein kinase showed transcriptional changes in ClWRKY61 knockout line, ClWRKY61 overexpression line, and ClLEA55 knockout line under salt stress. These results provide an opportunity to mediate the regulation of salt stress in watermelon with WRKY proteins.

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