Abstract
We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum, indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage’s 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes (pilT, egl, pehC, hrPB, and phcA), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and its carrier plant pathogenic bacterial strain by determining the effect of the prophage Rs551 and its repressor on the virulence and competitive fitness of its carrier strain UW551 of R. solanacearum.
Highlights
Bacterial wilt, a soil borne vascular disease caused by Ralstonia solanacearum, is one of the most devastating bacterial diseases in tropical, subtropical, and temperate regions of the world
Wild-type, race 3 biovar 2, phylotype IIB sequevar 1, φRs551 carrier UW551 with a 342-bp prophage region containing orf14 replaced with a 616-bp Gm cassette, GmR UW551 with a 3,321-bp prophage region including orf14 and orf1 to orf8 replaced with a 616-bp Gm cassette, GmR Wild-type, biovar 1, phylotype IIB sequevar 4, φRs551S
R. solanacearum mutants were confirmed by their ability to grow on triphenyltetrazolium chloride (TZC) plates containing gentamicin, and by PCR for the absence of the 342-bp prophage region in the UW551 orf14 mutant and the 3,321-bp region in the UW551 prophage mutant
Summary
A soil borne vascular disease caused by Ralstonia solanacearum, is one of the most devastating bacterial diseases in tropical, subtropical, and temperate regions of the world. R. solanacearum normally invades host plants from soil through wounds in roots, reproduces in the xylem vessels, and spreads rapidly through the plant’s vascular system resulting in wilting and death of the plant (Vasse et al, 2000). Ralstonia solanacearum is historically classified into five races and five biovars based on host range and biochemical properties, respectively. The race 3 biovar 2 (phylotype IIB sequevars 1 and 2) of R. solanacearum causes devastating brown rot of potato, and is a quarantine pathogen in many countries and listed as a select agent in the United States (Agricultural Bioterrorism Protection Act of 2002, 2002)
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