Loss of function of OsWRKY53-OsARF18-OsRR22 significantly enhances rice salt tolerance

Salt stress is a key abiotic stress factor limiting rice growth and development. Previous studies have shown that OsWRKY53, OsARF18, and OsRR22 not only serve as important negative regulators of salt tolerance in rice but are also crucial for growth and development. However, the relative strengths of salt tolerance among these three genes and their combined effects within the same rice variety have not yet been reported.

In this study, we employed CRISPR/Cas9-mediated genome editing to simultaneously disrupt OsWRKY53, OsARF18, and OsRR22 in the rice cultivar Shuanghui 459. Salt tolerance increases sequentially in single-gene, double-gene, and triple-gene mutants. Under 1.0% NaCl stress, the triple mutant exhibited approximately 80% survival versus complete lethality in the wild type (WT). Physiologically, ROS accumulation progressively declined, while key antioxidant enzyme activities (CAT, SOD, POD) significantly increased. This suggests that the enhanced salt tolerance of the mutants may be associated with increased antioxidant enzyme activity. More importantly, molecular analysis revealed that the WRKY domain of OsWRKY53 binds to the W-box elements in the promoters of OsARF18 and OsRR22, thereby downregulating their expression. Reciprocally, OsARF18 and OsRR22 knockout downregulated OsWRKY53. Furthermore, OsWRKY53 and OsRR22 directly interact at the protein level.

Taken together, our results reveal that OsWRKY53, OsARF18, and OsRR22 constitute a reciprocal negative-feedback loop, wherein these three transcriptional regulators mutually antagonize each other. The significant enhancement of salt tolerance in the triple mutant may be related to the relief of this antagonistic interaction. Importantly, the polygenic aggregation of OsWRKY53-OsARF18-OsRR22 not only significantly enhances rice salt tolerance but also does not affect normal plant growth and development. These findings provide new strategies for polygenic aggregation-based genetic improvement of salt-tolerant rice varieties.