<i>Ralstonia solanacearum</i> needs exDNases for full virulence on tomato.

<p>(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 <i>R</i>. <i>solanacearum</i> wildtype strain GMI1000, <i>ΔnucA</i>, <i>ΔnucB</i>, and the <i>ΔnucA/B</i> 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, <i>P</i><0.05). (D) The <i>R</i>. <i>solanacearum ΔnucA/B</i> 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 <i>ΔnucA/B</i> 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, <i>P</i><0.05). (E) The <i>R</i>. <i>solanacearum ΔnucA/B</i> 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 <i>ΔnucA/B</i>. 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 <i>ΔnucA/B</i>/ WT = 0.88 (Wilcoxon signed-ranked test, <i>P</i><0.05).</p

Plant root border cells release DNA-containing extracellular traps in response to <i>R</i>. <i>solanacearum</i>.

<p>(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 <i>R</i>. <i>solanacearum</i> cells. (B) A pea root border cell (yellow arrow) treated with GFP-expressing <i>R</i>. <i>solanacearum</i> (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 <i>R</i>. <i>solanacearum</i>, 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). <i>R</i>. <i>solanacearum</i> cells (green arrow) were captured by traps originating from a collapsed pea root border cell (yellow arrow).</p

Comparative Transcriptome Analysis Reveals Cool Virulence Factors of <i>Ralstonia solanacearum</i> Race 3 Biovar 2

<div><p>While most strains of the plant pathogenic bacterium <i>Ralstonia solanacearum</i> are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of <i>R</i>. <i>solanacearum</i> R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature <i>in planta</i>. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking <i>lecM</i>, <i>aidA</i>, or <i>aidC</i> were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The <i>lecM</i> and <i>aidC</i> mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the <i>lecM</i> mutant was defective in biofilm formation <i>in vitro</i>. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.</p></div

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

<p>(A) Histone H4, a component of the root cap secretome, is bactericidal to <i>R</i>. <i>solanacearum</i>. 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 <i>R</i>. <i>solanacearum</i>. 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.</p

Mutation of <i>R</i>. <i>solanacearum</i> strain UW551 <i>lecM</i>, <i>aidA</i>, or <i>aidC</i> but not <i>solI</i> differentially reduced bacterial virulence at 20°C.

<p>Virulence was measured on wilt-susceptible tomato plants at 20°C and 28°C via soil soak inoculation (A-E) or at 20°C via cut petiole inoculation (F). Each point represents the mean of three biological replicates, each containing 16 plants per strain per temperature. The area under disease progress curve (AUDPC) was measured for each strain in A-C and each mutant’s AUDPC relative to wild type is shown (E). Asterisks indicate that virulence of wild type and mutant strains were significantly different (* <i>P</i>< 0.05, ** <i>P</i>< 0.01, ANOVA). Each mutant was also significantly reduced in virulence (<i>P</i>< 0.01, repeated measures ANOVA) compared to the wild type following cut petiole inoculation (F).</p

UW551ΔlecM and UW551ΔaidC had reduced survival in potato tubers at 4°C.

<p>Potato tubers were injected with different <i>R</i>. <i>solanacearum</i> strains, and bacterial cell numbers were counted by grinding and dilution plating tubers at different times after inoculation. The experiment was repeated three times, with three tubers per strain per time point. At 6, 9 and 12 weeks after inoculation, the population sizes of UW551ΔlecM and UW551ΔaidC in tubers were significantly lower than those of the wild type parent strain (<i>P</i><0.05, ANOVA).</p

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

<p>(A) Kinetics of root border cell extracellular trap release. Pea root border cells (RBC) were inoculated with 10⁶ cells of <i>R</i>. <i>solanacearum</i> 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 <i>R</i>. <i>solanacearum</i> wild-type strain GMI1000 (WT), type III secretion system mutant <i>hrpB</i>, exopolysaccharide deficient mutant <i>epsB</i>, flagellin deficient mutant <i>fliC</i>, 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.</p

Expression of lectin genes in <i>R</i>. <i>solanacearum</i> strains GMI1000 and UW551 under various conditions.

<p>^aValues shown are scaled log2 signal intensities from strain-specific whole genome microarrays hybridized to labeled cDNA extracted from <i>R</i>. <i>solanacearum</i> cells grown in rich culture medium (CPG) or <i>in planta</i> (IP). These absolute expression values were used to allow comparisons across strains.</p><p>^b<i>np</i>: gene not present in this strain.</p><p>Expression of lectin genes in <i>R</i>. <i>solanacearum</i> strains GMI1000 and UW551 under various conditions.</p

A cluster of genes adjacent to those encoding the SolI/R quorum sensing system were up-regulated in <i>R</i>. <i>solanacearum</i> strain UW551 at 20°C, in culture.

<p>Some were also upregulated <i>in planta</i>. Arrows represent open reading frames. The numbers above the arrows indicate the expression fold-change for each gene at 20°C compared to 28°C, in culture/<i>in planta</i>, determined by whole-genome microarray analysis as described in the text. The arrangement and expression levels of the corresponding genes in strain GMI1000 are also shown.</p

<i>R</i>. <i>solanacearum</i> genes encode two secreted DNases.

<p>(A) DNase activity of cell-free bacterial culture supernatant from <i>R</i>. <i>solanacearum</i> 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: <i>R</i>. <i>solanacearum</i> 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 <i>R</i>. <i>solanacearum</i> 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, *<i>P</i><0.05, **<i>P</i><0.01, ****<i>P</i><0.0001). Abbreviations: WT, wild-type strain; <i>ΔnucA</i>, <i>R</i>. <i>solanacearum nucA</i> mutant; <i>ΔnucB</i>, <i>R</i>. <i>solanacearum nucB</i> mutant; <i>ΔnucA/B</i>, <i>R</i>. <i>solanacearum</i> double nuclease mutant; nucBcom or nucBcom; <i>R</i>. <i>solanacearum</i> mutant complemented with the corresponding wild-type gene; RBC, root border cells; Rs, <i>R</i>. <i>solanacearum</i> cells.</p