CRISPR/Cas9-mediated generation of tomato RAD23 knockouts for studying phytoplasma-induced symptoms

Phytoplasma infection often induces abnormal flower development, such as phyllody and cauliflower-like inflorescence, leading to compromised fruit development and significant crop yield reduction. In Arabidopsis, the phytoplasma effector SAP54 interacts with RAD23 proteins to induce floral abnormalities, partially alleviated in rad23 mutants. To investigate whether a similar mechanism may occur in tomatoes, in this study, CRISPR/Cas9 technology was employed to generate knockout tomato mutants of SlRAD23C and SlRAD23D. Target-specific guide RNAs (sgRNAs) were designed to disrupt the coding sequences of SlRAD23Cand SlRAD23D. PCR screening and sequencing confirmed successful deletions in the targeted regions, leading to truncated protein products in mutant lines. These mutants will be further analyzed for their response to potato purple top phytoplasma-induced flower deformation to develop tomato lines less susceptible to phytoplasma-related reproductive reduction.