Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

PLoS Pathog. 2016 Jun 23;12(6):e1005686. doi: 10.1371/journal.ppat.1005686. eCollection 2016 Jun.

Abstract

Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / immunology
  • Bacterial Proteins / metabolism
  • Deoxyribonucleases / immunology
  • Deoxyribonucleases / metabolism*
  • Extracellular Traps / microbiology*
  • Pisum sativum / immunology
  • Pisum sativum / microbiology
  • Plant Diseases / immunology
  • Plant Immunity / immunology*
  • Plant Roots / immunology
  • Plant Roots / microbiology
  • Ralstonia solanacearum / metabolism*
  • Solanum lycopersicum / immunology
  • Solanum lycopersicum / microbiology
  • Virulence / physiology*
  • Virulence Factors / immunology
  • Virulence Factors / metabolism*

Substances

  • Bacterial Proteins
  • Virulence Factors
  • Deoxyribonucleases

Grants and funding

This work was supported by the USDA Hatch Program funding, administered by the University of Wisconsin-Madison College of Agricultural and Life Sciences, Project # WIS01502 (CA); National Science Foundation Symbiosis, Defense, and Self-Recognition Program Grant # IOS1456636 (CA); and Vietnam Education Foundation graduate fellowship (TMT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.