Developing mutants of negative regulator of immune response gene (CPL-3) in tomato using CRISPR/Cas9

Bacterial infections pose a substantial threat to tomato production, impacting the biochemical and molecular attributes of the crop. This study elucidates the identification and characterization of RNA Polymerase II (RNAP) C-Terminal Domain Phosphatase-like 3 (SlCPL-3) genes in prominent commercial tomato cultivars (Rio-grande, Sazlica, and Falcon). Comprising 11 exons, the SlCPL-3 gene encodes two protein domains, CPDCs, and BRCT. Employing various bioinformatic tools, a predictive 3-D structure and an exploration of phosphorylation potential were conducted. Mutant development in tomatoes allowed an exploration of CPL-3 gene expression under biotic stress, utilizing Pst DC-3000 as the infectious pathogen. Results unveiled the activation of the SlCPL-3 gene during bacterial stress, with upregulated expression observed across various time intervals. Mutant plants exhibited diminished CPL-3 expression compared to wild-type plants, complemented by a subsequent reduction in PR-1 gene expression a marker linked with biotic stress. These findings support the hypothesis that targeting the CPL-3 gene through CRISPR/Cas9 holds promise for enhancing crop resilience against bacterial pathogens.