Characterization of Ralstonia solanacearum extracellular nucleases and their roles in the interaction with root border cell extracellular traps and biofilm modulation.

摘要

The bacterium Ralstonia solanacearum is a widespread plant pathogen of great agricultural and economic importance. The core genome of the Ralstonia species complex encodes two highly conserved predicted nucleases, namely NucA and NucB. Little is known about the role of these nucleases in the R. solanacearum life cycle or about their contribution to bacterial virulence and bacterial wilt disease development. The plant root cap secretome is rich in proteins and, surprisingly, extracellular DNA. Root border cells released extracellular DNA traps in response to R. solanacearum in a flagellin-dependent manner. These traps immobilized and killed the bacteria much as neutrophil extracellular traps capture animal pathogens. An R. solanacearum strain GMI1000 mutant lacking extracellular nucleases could not escape from trapps as well as wild-type. Purified NucA and NucB protein studies found they are both functional DNases that can degrade pea extracellular traps. These enzymes were required for full virulence of R. solanacearum on pea and tomato, specifically for bacterial attachment to root surfaces, colonization and symptom development.;Nuclease mutants of R. solanacearum were less virulent than the parental strain even when the bacteria were injected directly into tomato stems to bypass the natural root infection route. This suggests that extracellular nucleases play other roles beside assisting bacterial invasion of plant roots. Characterization of R. solanacearum biofilm confirmed that DNA is an important component of the biofilm, and extracellular DNases facilitated bacterial dispersal from established biofilms. Nuclease mutants formed thicker biofilms than wild-type in vitro and in planta. A translocation study with GFP-tagged bacteria indicated that a mutant lacking both nucleases reached wild-type population sizes but was impaired in systemic spread in tomato stems, possibly due to an abnormal in planta biofilm structure that included a dense mat of thick chromatin-like fibers. Together, these results indicate that R. solanacearum extracellular DNases serve multiple purposes in the life of this pathogen. They are an effective counterdefense against border cell extracellular traps at the early stage of root infection and they also modulate bacterial biofilm after R. solanacearum is established inside hosts.