Cell Density-Regulated Adhesins Contribute to Early Disease Development and Adhesion in Ralstonia solanacearum

Abstract

Adhesins (adhesive proteins) help bacteria stick to and colonize diverse surfaces and often contribute to virulence. The genome of the bacterial wilt pathogen Ralstonia solanacearum (Rs) encodes dozens of putative adhesins, some of which are upregulated during plant pathogenesis. Little is known about the role of these proteins in bacterial wilt disease. During tomato colonization, three putative Rs adhesin genes were upregulated in a ΔphcA quorum-sensing mutant that cannot respond to high cell densities: radA (Ralstonia adhesin A), rcpA (Ralstonia collagen-like protein A), and rcpB. Based on this differential gene expression, we hypothesized that adhesins repressed by PhcA contribute to early disease stages when Rs experiences a low cell density. During root colonization, Rs upregulated rcpA and rcpB, but not radA, relative to bacteria in the stem at mid-disease. Root attachment assays and confocal microscopy with ΔrcpA/B and ΔradA revealed that all three adhesins help Rs attach to tomato seedling roots. Biofilm assays on abiotic surfaces found that Rs does not require RadA, RcpA, or RcpB for interbacterial attachment (cohesion), but these proteins are essential for anchoring aggregates to a surface (adhesion). However, Rs did not require the adhesins for later disease stages in planta, including colonization of the root endosphere and stems. Interestingly, all three adhesins were essential for full competitive fitness in planta. Together, these infection stage-specific assays identified three proteins that contribute to adhesion and the critical first host-pathogen interaction in bacterial wilt disease. IMPORTANCE Every microbe must balance its need to attach to surfaces with the biological imperative to move and spread. The high-impact plant-pathogenic bacterium Ralstonia solanacearum can stick to biotic and abiotic substrates, presumably using some of the dozens of putative adhesins encoded in its genome. We confirmed the functions and identified the biological roles of multiple afimbrial adhesins. By assaying the competitive fitness and the success of adhesin mutants in three different plant compartments, we identified the specific disease stages and host tissues where three previously cryptic adhesins contribute to success in plants. Combined with tissue-specific regulatory data, this work indicates that R. solanacearum deploys distinct adhesins that help it succeed at different stages of plant pathogenesis.

Keywords: Ralstonia solanacearum; adhesin; adhesion; bacterial wilt; biofilm; cohesion; plant pathogens; soil-borne pathogen; tomato.