Characterization of a novel pilus system in Ralstonia solanacearum and its contribution to virulence on potato

• Main Author: Wairuri, Charles K.
• Other Authors: Van der Waals, Jacquie E. (Jacqueline Elise)
• Published: University of Pretoria 2013
• Subjects: UCTD
• Online Access: http://hdl.handle.net/2263/31299; Wairuri, C 2011, Characterization of a novel pilus system in Ralstonia solanacearum and its contribution to virulence on potato, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-09232011-145315/ >; http://upetd.up.ac.za/thesis/available/etd-09232011-145315/

Ralstonia solanacearum, a widely distributed soil-borne pathogen belonging to the β- subdivision of Proteobacteria, causes a lethal wilting disease of more than 450 plants species, including economically important crops such as tomato and potato. Although bacterial wilt pathogenesis is incompletely understood, genetic and molecular studies have implicated type IVa pili-mediated twitching motility, amongst other, as a factor contributing to disease development. Research performed over the last decade has led to the identification of the Tad (tight adherence) macromolecular transport system that is present in many bacterial and archaeal species. The tad genes encode the machinery required for the biogenesis and secretion of a novel type IVb pilin protein, designated Flp, which is required for colonization and virulence in several human pathogenic bacteria. Therefore, the aims of this investigation were essentially to mine the genome of R. solanacearum in order to identify homologues of the tad genes and to determine the importance of the Tad system in R. solanacearum virulence on potato. During the course of this study, two distinct tad gene clusters were identified in the genome of R. solanacearum. These tad loci are present in the megaplasmid and chromosome, respectively, and both gene clusters encode predicted protein products with homology to those encoded by the tad loci in human pathogenic bacteria such as Aggregatibacter actinomycetemcomitans and Pseudomonas aeruginosa. Towards determining the importance of the tad loci in R. solanacearum virulence, a mutation was introduced into the cpaF2 (RSP1085) gene of the megaplasmid tad locus, which encodes a putative NTPase and is highly conserved in different bacterial Tad systems. Comparative analysis of the R. solanacearum wild-type NB336 and mutant NB336 cpaF2 strains indicated that the mutant strain was not growth impaired in vitro or in planta, produced near wild-type levels of EPS, and exhibited swimming and twitching motility that was comparable to that of the wild-type strain. However, the mutant NB336 cpaF2 strain, in contrast to the wild-type NB366 strain, was significantly impaired in its ability to adhere to and colonize potato roots. Moreover, in a biologically representative soil soak inoculation virulence assay, the mutant NB336 cpaF2 strain was not able to cause disease on potato plants, and similar results were obtained when the mutant strain was inoculated directly onto cut potato leaf petioles. Notably, both the adherence and virulence phenotypes were restored when a wild-type copy of the cpaF2 gene was provided in trans. These results suggest that the Tad system located on the megaplasmid, makes an important contribution to R. solanacearum virulence on potato. It is likely that this contribution is of importance during the early stages in host plant colonization. === Dissertation (MSc)--University of Pretoria, 2011. === Microbiology and Plant Pathology === Unrestricted