Plant root tips are covered by a protective sleeve of loosely attached border cells that can release a matrix containing proteins, polysaccharides, and DNA. In animal immune systems, extracellular DNA forms the backbone of neutrophil extracellular traps (NETs) deployed by immune cells to immobilize and kill invading microbes. Some animal pathogens can secrete DNases to degrade NETs and facilitate infection. Plant border cells release DNA-containing extracellular traps in response to the high-impact plant pathogenic bacterium Ralstonia solanacearum. R. solanacearum secretes two DNases that free the pathogen from these extracellular traps. The bacterium needs these DNases for full virulence and normal colonization of its host plants. This work reveals that, like animal pathogens, the plant pathogen R. solanacearum can overcome a DNA-based host defense system with secreted enzymes.
Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum. (2016) PLoS Pathog 12(6): e1005686. doi: 10.1371/journal.ppat.1005686
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.