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

  • ️Fri Jan 01 2016

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

Tuan Minh Tran et al. PLoS Pathog. 2016.

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Plant root border cells release DNA-containing extracellular traps in response to R. solanacearum.

(A) Fluorescence microscopy images of pea root border cells (yellow arrow) releasing extracellular DNA (white arrow, stained green or white with SYTOX Green) 30 min after exposure to R. solanacearum cells. (B) A pea root border cell (yellow arrow) treated with GFP-expressing R. solanacearum (green arrows), which are immobilized on traps containing extracellular DNA (stained blue with DAPI, white arrow). In the adjacent water control, the root border cell nuclei are stained blue but no extracellular DNA is visible. Untrapped bacteria were able to move freely in the suspension and appear blurred in the image. (C) Scanning electron microscopy showed that following treatment with R. solanacearum, pea border cells released web-like structures similar to neutrophil extracellular traps. (D) Root extracellular traps contained both small threads and thicker cables (left and right white arrows, respectively). R. solanacearum cells (green arrow) were captured by traps originating from a collapsed pea root border cell (yellow arrow).

Fig 2
Fig 2. Flagella and Flg22 triggered the release of exDNA by pea border cells.

(A) Kinetics of root border cell extracellular trap release. Pea root border cells (RBC) were inoculated with 106 cells of R. solanacearum strain GMI1000 (Rs), the preparation was stained with SYTOX Green, and imaged over time with an epifluorescence microscope. Images are representative of two experiments and at least four images were taken for each time point. (B) Flagellin-triggered formation of border cell extracellular traps. A suspension of 10,000 pea border cells were inoculated with a 1000-fold excess (107 cells) of either R. solanacearum wild-type strain GMI1000 (WT), type III secretion system mutant hrpB, exopolysaccharide deficient mutant epsB, flagellin deficient mutant fliC, or 20 μg/ml of flagellin-derived peptide Flg22 and stained with SYTOX Green to visualize DNA (white arrows on merged images). Live imaging was performed with a Zeiss Elyra 780 CLSM and SYTOX Green fluorescent, differential interference contrast microscopy (DIC) and merged images are shown. At least 5 images per treatment were taken from 30 min to 1 h post inoculation. The experiment was repeated three times and representative images are shown (bar = 50 μm). White arrows in (A) and (B) indicate exDNA.

Fig 3
Fig 3. Histones and DNA contribute to the bactericidal activity of pea root border cells.

(A) Histone H4, a component of the root cap secretome, is bactericidal to R. solanacearum. Percent live bacteria in the presence of histone H4 was determined by the BacLight LIVE/DEAD staining kit. A standard curve of percentage of live cells relative to SYTO9/PI fluorescence intensity was constructed using known ratios of live/dead bacteria (R2 = 0.99). The experiment was repeated twice, each with three technical replicates. (B) Bactericidal activity of root border cells on R. solanacearum. This effect was blocked by addition of either anti-Histone H4 antibody or DNase I. Asterisks indicate treatment significantly different from the bacteria-only control (one-way ANOVA, **** p<0.0001). Bars represent the mean of at least three independent experiments, each comprised of six technical replicates.

Fig 4
Fig 4. R. solanacearum genes encode two secreted DNases.

(A) DNase activity of cell-free bacterial culture supernatant from R. solanacearum strains grown in minimal medium, measured by DNase Alert assay on a fluorescence plate reader at 37°C over 3 h. Bars represent mean relative fluorescence units normalized to A600 of overnight culture. (B) Activity of purified nucleases on different DNA substrates. 1 μg of purified NucA or NucB were incubated with 1 μg of each DNA substrate: R. solanacearum genomic DNA, pea DNA, supercoiled plasmid DNA (pUCGM) and salmon sperm DNA) for 30 min at 37°C. Results were analyzed by electrophoresis in a 1% agarose gel. (C) NucA and NucB degrade DNA from root border cell traps. Pea border cells were incubated with R. solanacearum in the presence of NucA, NucB or DNase I as control. Relative DNA amount was measured by SYTOX Green fluorescence after 6 h of incubation. Asterisks indicate differences from the wild-type (one-way ANOVA, *P<0.05, **P<0.01, ****P<0.0001). Abbreviations: WT, wild-type strain; ΔnucA, R. solanacearum nucA mutant; ΔnucB, R. solanacearum nucB mutant; ΔnucA/B, R. solanacearum double nuclease mutant; nucBcom or nucBcom; R. solanacearum mutant complemented with the corresponding wild-type gene; RBC, root border cells; Rs, R. solanacearum cells.

Fig 5
Fig 5. Ralstonia solanacearum needs exDNases for full virulence on tomato.

(A-C) Disease progress on tomato. Twenty-one-day-old wilt-susceptible tomato plants (cv. Bonny Best) were inoculated by soil-drenching with 1x107 CFU/g soil of R. solanacearum wildtype strain GMI1000, ΔnucA, ΔnucB, and the ΔnucA/B double nuclease mutant. Plants were rated daily using a 0–4 disease index scale (0: no leaves wilted, 4: all leaves wilted). Each point represents the average disease index of 42 plants from three independent experiments. The virulence of all three nuclease mutants was different from wild-type strain (repeated-measures ANOVA, P<0.05). (D) The R. solanacearum ΔnucA/B double nuclease mutant was impaired in plant colonization, measured as bacterial population size in mid-stems of susceptible tomato (cv. Bonny Best) inoculated by soil-drenching with 107 CFU/g soil of either GMI1000 WT or the ΔnucA/B mutant. Data were obtained by grinding and serial dilution plating a mid-stem section at first sight of symptoms. Results are representative of two independent experiments with at least 10 plants per treatment (Student’s t-test, P<0.05). (E) The R. solanacearum ΔnucA/B nuclease double mutant had lower competitive fitness in tomato plants than the wildtype strain. Tomato plants were soil-drenched with a 1:1 mixture containing 1x108 CFU/ml each of GMI1000-gfp and ΔnucA/B. When wilt symptoms first appeared on a plant, the population size of each strain was determined by grinding a mid-stem slice and serially dilution plating on both tetracycline and kanamycin+gentamycin CPG plates. Data are representative of two independent experiments each including at least 30 plants. Competitive Index of ΔnucA/B/ WT = 0.88 (Wilcoxon signed-ranked test, P<0.05).

Fig 6
Fig 6. R. solanacearum exDNases contribute to tomato and pea root stunting and bacterial attachment to pea roots.

Seedlings of (A) pea (cv. Little Marvel) and (B) tomato (cv. Bonny Best) were grown on water agar for 2–3 days, inoculated with 107 cells (pea) or 106 cells (tomato) of R. solanacearum wild-type strain GMI1000 or exDNase mutant strains as indicated and grown in germination pouches at 24°C (pea) or 28°C (tomato) for 10 days. Red bars indicate approximate root tip length. Representative results of two independent experiments are shown. Bar graphs show average length of tomato root determined using measurements with ImageJ. Bars that have the same letter are not significantly different (Tukey’s HSD test, P<0.01). (C) and (D) ExDNase mutants are defective in attachment to pea roots, but not to tomato roots. Three-day old axenic tomato or pea seedlings were incubated for 2 h with 2.5x103 CFU of either wild-type strain GMI1000 or the ΔnucA/B mutant. Excised roots were washed in sterile water and blotted dry. Four roots were pooled as a technical replicate, ground, and serially dilution plated to quantify attached bacteria. Data shown are from three independent experiments; each contained 5 technical replicates. Bars represent proportion of wild-type root attachment (one-way ANOVA, *P< 0.05, ** P< 0.01).

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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.

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