A filamentous phage-based versatile toolkit for molecular studies in Ralstonia pseudosolanacearum

Abstract The Ralstonia solanacearum species complex (RSSC), a devastating soil-borne phytopathogen, urgently requires advanced genetic tools to unravel its pathogenicity mechanisms. Here, we present the pRSCq toolkit, a versatile plasmid system derived from the filamentous phage RSCq through genome minimization and engineering. By systematically deleting non-essential genes while retaining replication-critical elements, we developed stable plasmids (pRSCq1-4) with diverse antibiotic resistance markers and Golden Gate cloning compatibility. These plasmids exhibited exceptional stability in the absence of antibiotic selection and imposed a negligible burden on bacterial growth, motility, biofilm formation, or virulence. The toolkit’s utility was demonstrated through gene complementation of key regulators (PhcA and HrpG) and the protease ClpP, as well as through gene expression profiling and real-time monitoring of pathogen dynamics via a LuxCDABE reporter system ex planta and in planta . Furthermore, the pRSCq toolkit demonstrates compatibility with both pBBR1 plasmids and genomic integration systems, providing a versatile solution for coordinated multi-gene expression in RSSC. Thus, the pRSCq toolkit will bridge critical gaps in RSSC genetic engineering and promote the study of host–pathogen interactions.