Abstract:
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.
