Maximizing Plant Prime Editing Efficiency to Precisively Introduce Pepper Resistance Mutations into Tomato for the Construction of Multidisease Resistance
Background
Bacterial diseases are a major factor in drastically reducing yields in crop cultivation fields. As cultivation environments become unstable due to climate change, the importance of breeding technologies to secure resistance against complex pathogens has increased. Traditional breeding methods require multiple backcrosses to introduce desired resistance alleles, taking several years or more to develop a variety. The linkage drag problem, where unwanted wild-type genes are introduced along with the target gene due to chromosomal linkage, was also a major obstacle.
While targeted DNA cleavage technology using CRISPR-Cas9 was introduced, methods relying on double-strand breaks (DSB) made it difficult to induce intended precise mutations due to random insertions and deletions. Prime Editing (PE), which precisely performs base substitutions, insertions, and deletions at target sites without DNA cleavage, emerged as an alternative.
The problem was that, unlike monocot crops such as rice, the efficiency of prime editor operation in dicot crops like tomato was extremely low. Establishing a multiplex editing system to edit multiple genes simultaneously was also challenging. There was an urgent need to develop a high-efficiency genome editing platform capable of rapidly and precisely reproducing useful natural variations from wild or related species into commercial varieties to combat multiple pathogens.
Key Findings
Researchers newly designed an optimized prime editor system for tomato (Solanum lycopersicum), a representative dicot crop. Three types of prime editors for tomatoes with improved structures (TomPE2, TomPE6c, and TomPE7) were developed, and their activity and precision were systematically evaluated according to culture temperature and editor structure. Among the three editors, TomPE6c achieved the highest precise editing efficiency while minimizing off-target mutations.
Based on the established editing platform, the researchers undertook experiments to precisely reproduce natural disease-resistance alleles found in pepper into the tomato genome. Two leucine amino acid residues were precisely deleted from the SlBs5 and SlBs5L genes, which encode CYSTM-domain proteins, a class of cysteine-rich membrane proteins. The edited tomato lines with these two amino acid deletions showed significantly improved resistance to Xanthomonas, the causal agent of bacterial speck. This demonstrates that beneficial variants identified in other crop species can be precisely introgressed into related crops to confer the same defense functions.
In parallel, the researchers suppressed gene function by introducing premature stop codons into the SlDMR6 gene, which is involved in pathogen susceptibility. As a result, the resulting editing line exhibited strong complex resistance against not only Xanthomonas but also the canker pathogen Pseudomonas syringae.
Transcriptome profiling analysis revealed that the two resistance genes activated different immune mechanisms. The Slbs5 and Slbs5L variants exhibited a pathogen-induced defense state, suppressing immune responses under normal conditions but rapidly inducing defense responses upon pathogen invasion. In contrast, the Sldmr6 variant maintained a basal defense state with elevated expression levels of defense-related genes even before pathogen infection.
Researchers also completed a multiplex prime editing system capable of simultaneously editing multiple loci by modularizing multiple guide RNAs. They successfully accumulated resistance alleles with distinct defense pathways and expression regulatory variants in a single generation through a single transformation process. The introduced mutations were stably inherited in the next generation, and the resistance phenotype remained stable even under high-temperature conditions.
Significance and Outlook
This study successfully expanded prime editing technology, which was centered on monocot crops, to tomato, a major dicot vegetable crop, and demonstrated that useful variations from nature can be directly reproduced in the laboratory. By demonstrating that superior traits from other species can be realized through precise editing at the level of a few base pairs without interspecific hybridization, it laid the foundation for transforming the paradigm of new variety development.
In particular, the strategy of combining distinct immune mechanisms—pathogen-induced defense and basal defense—within a single generation serves as a powerful tool to overcome the vulnerability of resistance collapsing due to the emergence of pathogen variants. The fact that stable disease resistance was maintained even under abnormal high-temperature conditions caused by climate warming is a positive sign for the future breeding of crops with resistance to complex environmental stresses.
Several follow-up challenges remain for commercial field implementation. Technologies that efficiently select and mass-propagate transgene-free pure-line plants, in which foreign vector genes are completely eliminated, must be supported. Optimization research to overcome variations in tissue culture and regeneration efficiency among varieties must also continue when expanding the application range to other Solanaceous crops such as pepper, potato, and eggplant.
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.
This technology provides a solution for the horticultural sector, where new pathogen variants are rapidly spreading due to climate change, by enabling the immediate supply of customized resistant varieties. The development period for multi-disease resistant varieties, which previously took 5 to 7 years or more using traditional breeding methods, can be drastically reduced to 1–2 generations, or approximately one year. From a farmer's perspective, this enables the early introduction of highly resistant varieties to prevent yield loss while reducing chemical pesticide use, even in complex infection environments where Xanthomonas and Pseudomonas co-occur. Seed companies can also strengthen their competitiveness in the global seed market by establishing a modular breeding pipeline that rapidly benchmarks and accumulates superior natural genes from other crops into their lines.