Characterization of tol-pal gene cluster mutants and identification of determinants in phage infection in Ralstonia solanacearum
Date Issued
2008
Date
2008
Author(s)
Chu, Yu-Ju
Abstract
Ralstonia solanacearum is a soil-borne bacterium infecting the vascular system and causing deadly wilting symptoms in many economically important crops. Gaining insight into pathogenesis mechanism of this devastating pathogen may provide information for future disease control. This study contains two main themes. First, the tol-pal gene cluster is widely conserved in many Gram-negative bacteria, involving in membrane integrity and filamentous phage DNA transport. Interestingly, R. solanacearum mutants of strain Pss190 possessing transposon insertions in this gene cluster displayed normal pathogenesis on tomato plants but reduced virulence on Arabidopsis, suggesting the plausible involvement of this gene cluster in host adaptation. Allelic mutants displayed consistent differential pathogenesis on the two host plants, confirming the transopon insertions are responsible for the altered phenotype. Proliferation and colonization of these mutants in solanaceous host Nicotiana benthamiana and tomato plants were not significantly different from the wild-type strain, while their colonization in Arabidopsis was significantly reduced. The tol-pal mutants also conferred certain phenotypic alterations which may possibly correlate to the differential pathogenesis, including reduced motility, altered capability in uptake and oxidation of carbon sources, lability of membrane structure and altered cell morphology (blebs-formation). Complementation test have further confirmed that tol-pal gene cluster involves in membrane stability. These results together suggest a role of tol-pal gene cluster in the adaptation to different host environments. Furthermore, promoter assays revealed that R. solanacearum tol-pal gene cluster could be organized as three transcription units and their activity is moderated throughout the growth phase. This is the first report investigating the function and organization of the tol-pal gene cluster in a vascular phytopathogenic bacterium. Secondly, because the tol-pal gene cluster has been reported to involve in phage infection, and R. solanacearum-infecting bacteriophages can be a putative agent for control of bacterial wilt, we are interested in studying phage-R. solanacearum interaction. To systematically identify R. solanacearum determinants for phage infection, a transposon–insertional mutant screening approach was employed to isolate phage-resistant mutants of R. solanacearum strain Pss4. Twelve mutants were identified to be resistant to a locally isolated phage ΦRS29 from a preliminary screen of approximately 2,000 mutants, and seven loci were identified. These loci are predicted to be involved two main functional groups: (a) Biogenesis of cell-wall and related components (ampG, wecA, hldD, rfaD, RSc2201); (b) Transporters (RSc2958, RSc2962). This is the first study reporting determinants of R. solanacearum involved in interaction with phages. Current results suggest that key steps for successful phage infection include recognition and adsorption of R. solanacearum cell wall and related components by the phages and injection of phage genomic DNA into the host cell via host transporters.
Subjects
Ralstonia solanacearum
tol-pal gene cluster
host adaptation
phage infection
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