Fig 1.
In planta expression and phenotypic contributions of Ralstonia lectin genes lecF and lecX. A) Expression of lectin genes lecF and lecX was upregulated in tomato root endosphere and stem.
Five-day-old axenic tomato seedlings were flood-inoculated with a low cell density of Rps GMI1000. Rhizoplane and root endosphere samples were harvested 6 hpi and 48 hpi, respectively. Mid-stem samples were harvested from 24-day-old tomato plants three days after petiole inoculation. Total RNA was extracted from plant tissue samples and qRT-PCR was used to quantify expression of lecF, lecX, and cell density control genes, epsB and iolG. Gene expression in the root interior and stem is shown on a base-2 logarithmic scale relative to expression in GMI1000 cells on the rhizoplane. Asterisks indicate a difference in gene expression between the stated condition and the rhizoplane (Student’s t-test; *P≤0.05, **P≤0.01). B and C) LexF and LecX negatively modulate Rps attachment to the root surface. Roots of 4-day-old seedlings were inoculated with 104 CFU of wild-type (gray), ΔlecF (light blue), ΔlecF+lecF (complemented mutant, striped blue), ΔlecX (light green), or ΔlecX+lecX (complemented mutant, striped green). At 2 hpi roots were washed, homogenized, and dilution plated to quantify the rhizoplane population. Each symbol represents four pooled roots. Data shown reflect three experiments, each with 6–10 technical replicates per treatment. Asterisks indicate a difference between wild-type and the lectin mutants and complemented strains (B, Kruskal-Wallis test, ΔlecF: P = 0.0089, ΔlecX: 0.0089; C, Mann-Whitney, P = 0.0008). D, E, and F) Mutating both lecF and lecX increased Rps root attachment and virulence but did not affect colonization of tomato roots or stems. Roots of 4-day-old tomato seedlings were inoculated with 104 CFU of Rps GMI1000 wild-type or the ΔlecF/X double mutant. After 48 h, roots were surface sterilized, homogenized, and dilution plated. Experiments were repeated three times with 9 to 12 technical replicates per treatment (Mann-Whitney test, P = 0.60). Horizontal yellow bars indicate the geometric mean. E) 21-day-old tomato plants were inoculated through a cut petiole with 2000 CFU of wild-type or ΔlecF/X Rps. At 3 dpi, mid-stem samples above the point of inoculation were harvested, homogenized, and dilution plated. Data shown are from three experiments, each with 11–15 plants per treatment (Mann-Whitney test, P = 0.95). The numbers above the x-axis denote the total number of uncolonized plants. F) 21-day-old tomato plants were soil soak inoculated with 50 mL of 108 CFU/mL wild-type GMI1000 or ΔlecF/X. Disease severity was rated over 14 days on a scale from 0 (no wilting) to 4 (76–100% of plant wilting). Each point indicates the area under the disease progress curve for one plant. Data shown are from three experiments, each with 11–15 plants per treatment (Student’s t-test, P = 0.0201). This figure was created in part using BioRender.
Fig 2.
Effects of deleting Rps lectins on biofilm formation in vitro.
A and B) LexF and LecX constrain Rps biofilm formation in static conditions. A) 107 CFU/mL of wild-type GMI1000, ΔlecF, ΔlecF+lecF (complemented mutant), ΔlecX, ΔlecX+lecX (complemented mutant), and ΔlecF/X in CPG broth were aliquoted into a 96-well PVC plate. After 24 h, biofilms were stained with 1% w/v crystal violet and measured at A590nm. Data are from three to five independent experiments, each with 12–36 technical replicates per treatment. Different letters over bars indicate differences among strains as determined by ANOVA (P<0.0001). B-E) 107 CFU/mL wild-type GMI1000, ΔlecF, ΔlecX, or ΔlecF/X suspended in CPG broth were aliquoted into 8-well chambered cover glass slides. Cultures were grown statically for 3 days at 28°C with fresh media added daily and cells were stained with SYTO9 before confocal imaging. Representative images show the orthogonal view at the middle of biofilm Z-stacks. Experiments were repeated twice with 3–4 technical replicates per treatment. The white scale bar indicates 100 μm. F) LecF and LecX are essential for biofilm formation in xylem sap under flow. 50x50 μm-cross-section microfluidic channels were coated with carboxymethyl cellulose-dopamine (CDC-DOPA). Rps wild-type GMI1000, ΔlecF, ΔlecX, and ΔlecF/X were suspended at 109 CFU/mL in ex vivo tomato xylem sap, seeded into channels for 6 h and then incubated for 3 days at a flow rate of 38 μL/h. Biofilms were stained with 1% crystal violet and imaged with a light microscope. The experiment was repeated twice and representative images are shown. White bar indicates 100 μm.
Fig 3.
Heterologous expression of GMI1000 lecF and lecX in other plant pathogenic Ralstonia.
A) Phylogenetic tree of the RSSC was constructed using 49 conserved genes and the RSSC Phylogenomics narrative on KBase [77,78]. Complete genomes of 187 RSSC strains and 3 other Ralstonia spp. were included in this analysis. Protein BLAST was used to determine lectin conservation and the outermost circles indicate the presence (filled icon) or absence (open icon) of lecF (blue), lecM (red) or lecX (green). Strains marked with yellow arrows were used for heterologous expression experiments. B) GMI1000 lecF and lecX were expressed in four Rs and Rps strains that naturally lack them. Strain characteristics and identity or similarity with GMI1000 lectin proteins are shown. Dark gray boxes indicate the gene is absent from that strain. C-F) Heterologous expression of lecF and lecX in Rs Phylotype II and Rps Phylotype III strains reduced static biofilm formation. C and D) Ralstonia strains CMR15, CMR15+lecX, UW386, UW386+lecX, UW163, UW163+lecF, UW551, and UW551+lecF were suspended at 107 CFU/mL in CPG broth, incubated in 96-well PVC plates for 24 h, stained with crystal violet, and biofilm was quantified as A590nm. For UW386, data reflect four independent experiments, each with 12–48 technical replicates. For CMR15, UW163, and UW551 strains, data represent three independent experiments with 12–48 technical replicates. Asterisks indicate a difference between the wild-type and heterologous-expression strains (Student’s t-test; C, P<0.0001, P = 0.0027; D, P<0.0001). E and F) 107 CFU/mL wild-type UW551 and UW551+lecF suspended in CPG broth were aliquoted into 8-well chambered cover glass slides and cultured. statically for 3 days at 28°C and then stained with SYTO9 for confocal imaging. Representative images show the orthogonal view at the middle of biofilm Z-stacks. Experiments were repeated twice with 2–4 technical replicates per treatment. The white scale bar indicates 100 μm.
Fig 4.
Effect of Rps lectins on the bacterial extracellular matrix.
A-B) LecF and LecX are required for normal colony coherence. 30 CFU of wild-type Rps GMI1000, ΔlecF, ΔlecX, ΔlecF/X, or ΔepsB were spread onto CPG media supplemented with tetrazolium chloride. Plates were photographed after 4 days’ incubation at 28°C. Representative colony images are shown in A and the white scale bar indicates 2 mm. B) Colony area was measured using ImageJ. Data shown reflect two independent experiments containing 14–31 technical replicates. Different letters above bars indicate differences among the strains (ANOVA, P<0.0001). C) Rps lectin mutants produce wild-type levels of EPS I. 108 CFU of wild-type GMI1000, ΔlecF, ΔlecX, ΔlecF/X, and ΔepsB were spread onto CPG plates. Following a 4-day incubation at 28°C, bacteria were scraped from plates and resuspended in water. 100 μL of this bacterial suspension was aliquoted into 96-well plates coated with Agdia anti-EPS-I monoclonal antibodies and DAS-ELISA was performed. Data shown reflect two independent experiments, each containing 6–8 technical replicates. Different letters above bars indicate differences among the strains (ANOVA, P<0.0001). D and E) Lectins contribute to wild-type viscosity of the Ralstonia extracellular matrix (ECM). 1 mL of colony biomass was scraped from 2% w/v agar plates following a three day incubation at 28°C. The biofilm colony viscosity of wild-type GMI1000, ΔlecF, ΔlecX, ΔlecF/X was measured at strain rates ranging from 0.01 to 1000 s-1 and the mean viscosity at a shear rate of 1.12 s-1 is displayed in panel D (D, ANOVA, P = 0.006; E, Repeated measures Two-way ANOVA, P<0.0001). F) Crude ECM from a ΔlecF/X double mutant was more adhesive than wild-type ECM, and LecF and LecX contribute to Rps attachment to ECM. Mixed cellulose ester (MCE) membranes were incubated in crude ECM extract from wild-type GMI1000 (solid box) or ΔlecF/X (striped box). Membranes were washed and incubated with a suspension of wild-type GMI1000 (gray) or ΔlecF/X (dark blue). After 1 h, membranes were gently washed, homogenized, and dilution plated to quantify the adhering bacteria. Data shown reflect three experiments with 5 technical replicates each. Different letters indicate significant differences (ANOVA, P<0.0001).
Fig 5.
Effect of Rps EPS I on bacterial attachment.
A-D) EPS I is not required for attachment to tomato roots, PVC, or glass under static conditions. A) Roots of 4-day-old seedlings were inoculated with 104 CFU of wild-type or ΔepsB for 2 h, then washed, homogenized, and dilution plated to quantify the rhizoplane population. Each dot represents four pooled roots. Data are shown from three experiments each containing 10 technical replicates (Mann-Whitney test, P = 0.2979). B) 107 CFU/mL GMI1000 and ΔepsB in CPG broth were incubated in a 96-well PVC plate for 24 h without shaking. Biofilms were stained with 1% crystal violet and measured at A590nm. Data shown reflects five independent experiments with 16–32 technical replicates (Student’s t-test; P = 0.92). C and D) 107 CFU/mL suspensions of wild-type GMI1000 (C) and ΔepsB (D) were incubated without shaking in 8-well chambered cover glass slides for 3 days at 28°C and then biofilms were stained with SYTO9 for confocal imaging. Representative images show the orthogonal view at the middle of biofilm Z-stacks. Experiments were repeated twice with 3–4 technical replicates each. The white scale bar indicates 100 μm. E) EPS I is essential for Rps biofilm formation under flow. 50x50 μm-cross-section microfluidic channels coated with carboxymethyl cellulose-dopamine (CDC-DOPA) were seeded for 6 h with Rps wild-type GMI1000 or ΔepsB suspended at 109 CFU/mL in ex vivo Bonny Best xylem sap. Xylem sap was then pumped through devices for 3 days at a flow rate of 38 μL/h. Biofilms were stained with 1% crystal violet and imaged with a light microscope. The experiment was repeated twice and representative images are shown. The white bar indicates 100 μm.
Fig 6.
Proposed roles of EPS I, LecF, and LecX in bacterial wilt disease.
Data presented here suggest that in the relatively static environment of the host rhizoplane, the lectins constrain Rps adhesion, thereby promoting pathogen dispersal in the rhizosphere and root invasion. Once inside the plant, Rps colonizes the water transporting xylem tissue. When Rps cells enter a healthy, flowing xylem vessel, LecF, LecX and EPS I mediate bacterial attachment to the vessel wall and to other bacteria. The lectins enable biofilm development by binding ECM polysaccharides. Under shear forces created by transpirational flow, the lectins increase the biofilm viscosity, supporting biofilm development and expansion. As the pathogen occludes vascular flow, creating a static environment, LecF and LecX once again negatively modulate attachment to facilitate Ralstonia dispersal to new vessels or escape from the host. The carbohydrate-binding lectins LecF and LecX thus have a novel environment-specific role in Rps attachment behaviors. This figure was created in part using BioRender.