Specific Reconstruction on pRC Plasmid to Facilitate Its Universal Chromosomal Integration in Different Ralstonia...

The pRC system is an efficient tool for genetic studies in Ralstonia solanacearum, ensuring direct insertion of foreign gene elements into Ralstonia chromosome downstream of glms. This system is designed for double recombination across glms and the downstream region, which considerably simplifies genetic studies of complementation, overexpression, pathogenicity, and in-vivo promoter activity assays with monocopy in R. solanacearum, one of the most destructive plant-pathogenic bacteria worldwide. R. solanacearum is extremely heterogeneous and is currently referred to as a Ralstonia solanacearum species complex (RSSC). The glms gene is greatly conserved, but its downstream regions are mostly different in the RSSC, which limits the application of the current pRC plasmid in the RSSC. We compared all existing 132 genome sequences in a precise genomic glms downstream region and confirmed that the pRC system is appropriate for application of chromosomal integration in all RSSC strains but needs respective reconstruction on current pRC plasmids, since glms downstream regions are greatly variable in the RSSC. RSSC strains can be grouped according to identical glms downstream regions. This grouping provides valuable information for gene insertion in this chromosomal region, as it facilitates universal application of the pRC system in RSSC strains.