Gene editing of the E3 ligase fine-tunes ROS production for enhanced bacterial disease resistance in tomato.

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis NADPH oxidase RBOHD is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase PIRE. Arabidopsis knockouts exhibit enhanced ROS production and resistance to the foliar pathogen . Here, we identified 170 homologs, which emerged in Tracheophytes and expanded in Angiosperms. We investigated the role of (tomato) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a dependent manner. Using CRISPR-cas9, we generated mutants in two homologs (). edited lines () in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens and . However, exhibited similar levels of colonization as wild type upon inoculation with diverse soilborne pathogens. These results indicate that phosphorylation and ubiquitination crosstalk regulate RBOHs in multiple plant species, and is a promising target for foliar disease control. This study also highlights the pathogen-specific role of , indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated interactions, positioning as a promising target for improving overall plant health.