A Truncated WRKY Protein Enhances Drought Resistance in Wild Tomatoes Through the SlWRKY16‐CIP2b‐ <i>SlSYP121</i> Module

Drought stress is a major abiotic factor that severely affects plant growth and food production. Identifying drought-resistant genes and their regulatory mechanisms is essential for mitigating the negative impacts of drought on plants. In this study, we identified a natural single nucleotide polymorphism (SNP) mutation in SlWRKY16 that is closely linked to drought tolerance in tomato. This SNP leads to the expression of a truncated SlWRKY16 protein. The CRISPR/Cas9 knockout of SlWRKY16, which produces this truncated SlWRKY16 protein, exhibits enhanced drought tolerance, whereas the overexpression lines demonstrate the opposite effect. Yeast two-hybrid screening demonstrated that SlWRKY16 physically interacted with CONSTANS Interacting Protein 2b (CIP2b). The CIP2b knockout mutants displayed increased sensitivity to drought stress. Importantly, this drought-sensitive phenotype was rescued in double mutants (cip2b/slwrky16). RNA-seq analysis revealed that a syntaxin gene (SlSYP121) co-expressed with both SlWRKY16 and CIP2b. Electrophoretic mobility shift assays confirmed that SlWRKY16 directly binds to the promoter of SlSYP121 and represses its expression, while the truncated SlWRKY16 protein failed to bind. Moreover, SlSYP121 acts as a positive regulator of drought tolerance. Our findings further demonstrate that the interaction between CIP2b and SlWRKY16 reduces the binding affinity of SlWRKY16 to the SlSYP121 promoter. This study identified a key SNP associated with differences in drought tolerance between wild and cultivated tomato, elucidated the regulatory function of the SlWRKY16-CIP2b-SlSYP121 module in the tomato drought response, and enhanced our understanding of the molecular mechanisms underlying plant drought resistance.