SlWRKY33 and SlPUB23 Negatively Regulate Rx4-Mediated Field Resistance to Bacterial Spot Race T3 in Tomato

Bacterial spot, caused by Xanthomonas species, is a destructive tomato disease that reduces yield and fruit quality worldwide. The tomato resistance gene Rx4 confers hypersensitive response and field resistance to race T3 of Xanthomonas euvesicatoria pv. perforans, but downstream components associated with Rx4-mediated field resistance remain unclear. Here, we compared the susceptible processing tomato line OH 88119 with its near-isogenic line Rx4-1806 after spray inoculation with the race T3 strain Xv829. Transcriptome profiling at 1, 6, and 72 h post-inoculation identified limited transcriptional differences at 1 and 6 h, but 2247 differentially expressed genes at 72 h, including 1712 genes downregulated in Rx4-1806. Enrichment analyses highlighted plant–pathogen interaction, plant hormone signal transduction, and MAPK signaling pathways. Among candidate defense-related genes, SlWRKY33 was downregulated in Rx4-1806 and selected for functional validation. CRISPR/Cas9-mediated knockout of SlWRKY33 enhanced resistance in both OH 88119 and Rx4-1806, whereas SlWRKY33 overexpression increased infected leaf area and bacterial population. Knockout of SlPUB23 enhanced resistance in Rx4-1806 but not in OH 88119. These results suggest that SlWRKY33 and SlPUB23 negatively regulate tomato field resistance to bacterial spot race T3, with SlPUB23 functioning in an Rx4-dependent manner.