Precision recreation and multiplex stacking of disease-resistance alleles in tomato using optimized prime editing

Prime editing enables precise modification of plant genomes and provides a means to recreate beneficial alleles underlying agronomically important traits. However, efficient and multiplexable prime editing systems remain limited in dicot crops. Here, we developed optimized prime editing systems for tomato (Solanum lycopersicum) and applied them to engineer and stack disease-resistance alleles. Using three tomato prime editors, TomPE2, TomPE6c, and TomPE7, we recreated a naturally occurring resistance-associated deletion identified in pepper by precisely deleting two leucine residues from the CYSTM-domain proteins SlBs5 and SlBs5L. The resulting tomato lines exhibited enhanced resistance to Xanthomonas, demonstrating that a resistance allele can be functionally recreated across related crop species. In parallel, introduction of premature stop codons into the susceptibility gene SlDMR6 conferred resistance to both Xanthomonas and Pseudomonas syringae. Transcriptome profiling revealed distinct defense states: Slbs5/Slbs5L plants predominantly exhibited enhanced pathogen-induced defense responses, whereas Sldmr6 plants showed elevated basal defense. Comparison of prime editor architectures and culture temperatures revealed differences in editing activity and precision, with TomPE6c producing the highest proportion of precise edits. We further developed a modular multiplex prime editing system capable of simultaneously modifying multiple loci, enabling resistance alleles and regulatory variants to be combined within a single generation. The engineered alleles were stably inherited, and resistance phenotypes were maintained under elevated temperature conditions. Together, these results establish prime editing as an effective platform for precise recreation, engineering, and multiplex stacking of disease-resistance alleles and provide a framework for accelerating resistance breeding in crops.