CRISPR RNA-Guided Integrase Enables Targeted Genome Engineering and Prophage Functional Genomics in Plant-Pathogenic Pseudomonas

Pseudomonas species include important plant pathogens responsible for substantial agricultural losses. Although CRISPR-INTEGRATE has been applied for genome engineering in medically relevant Pseudomonas, its utility in plant-pathogenic Pseudomonas has not been established. Here, we demonstrate CRISPR-INTEGRATE, an RNA-guided Tn7-like transposition system that enables targeted chromosomal integration without double-strand breaks or host-dependent homologous recombination, across three plant-pathogenic Pseudomonas backgrounds. Targeted disruption of hrpJ, hrcC, and gacA was confirmed by PCR and Sanger sequencing and resulted in distinct phenotypes, including impaired type III secretion-associated virulence and hypersensitive response, altered colony morphology and biofilm formation, and reduced motility. We further coupled CRISPR-INTEGRATE with Cre-lox recombination to excise a ~40-kb prophage-associated region from the P. syringae pv. tomato DC3000 chromosome. Genomic characterization identified a 33.45-kb predicted prophage within this region, putative attL/attR sites sharing an identical 14-bp core sequence, an orthologous empty locus containing a candidate attB site, and conserved prophage-associated gene organization across P. syringae genomes. Deletion of the region altered bacterial growth and increased susceptibility to phage infection, with the strongest effect observed for phage Plaza, while having no detectable effect on the tested plant virulence phenotypes. Together, these findings establish CRISPR-INTEGRATE as a portable genome-engineering platform for plant-pathogenic Pseudomonas and demonstrate its utility for functional interrogation of individual genes and large accessory genomic elements.