Leaf microbiome dysbiosis triggered by T2SS-dependent enzyme secretion from opportunistic Xanthomonas pathogens

Abstract

In healthy plants, the innate immune system contributes to maintenance of microbiota homoeostasis, while disease can be associated with microbiome perturbation or dysbiosis, and enrichment of opportunistic plant pathogens like Xanthomonas. It is currently unclear whether the microbiota change occurs independently of the opportunistic pathogens or is caused by the latter. Here we tested if protein export through the type-2 secretion system (T2SS) by Xanthomonas causes microbiome dysbiosis in Arabidopsis thaliana in immunocompromised plants. We found that Xanthomonas strains secrete a cocktail of plant cell wall-degrading enzymes that promote Xanthomonas growth during infection. Disease severity and leaf tissue degradation were increased in A. thaliana mutants lacking the NADPH oxidase RBOHD. Experiments with gnotobiotic plants, synthetic bacterial communities and wild-type or T2SS-mutant Xanthomonas revealed that virulence and leaf microbiome composition are controlled by the T2SS. Overall, a compromised immune system in plants can enrich opportunistic pathogens, which damage leaf tissues and ultimately cause microbiome dysbiosis by facilitating growth of specific commensal bacteria.

Fig. 1. Microbiota shift and plant disease driven by Xanthomonas Leaf131 in rbohD knockout plants.

a, Composition of synthetic bacterial communities SynCom-137 + Xanthomonas Leaf131 or SynCom-137 in rbohD or rbohD/RBOHD plants was compared with Col-0 wild-type plants. Effect size represents percentage of total variance explained by genotype (shown by dot size and absolute value) and statistical significance is expressed with P values determined by PERMANOVA (Benjamini–Hochberg adjusted, n = 16). Number of differentially abundant strains (as shown in b) is shown by dot colour. b, Heatmap shows subset of strains in SynCom-137 with significant log₂ fold changes (log₂FC, P < 0.05) in rbohD or rbohD/RBOHD compared with Col-0 wild-type plants in the presence (+) or absence (−) of Xanthomonas Leaf131. Black rectangles show significant changes, P < 0.05 (n = 16, two-sided Wald test, Benjamini–Hochberg adjusted). Complete heatmap of all strains in SynCom-137 is shown in Extended Data Fig. 1c. c, Fresh weight of aboveground plant tissue of Col-0, rbohD and rbohD/RBOHD mock inoculated, with SynCom-137 or SynCom-137 + Xanthomonas Leaf131. Box plots show the median with upper and lower quartiles and whiskers present 1.5× interquartile range (n = 16, two-sided Mann–Whitney U test, P values indicated above box plots). Corresponding plant phenotypes are shown in Extended Data Fig. 1d. d, CFU counts of Pseudomonas Leaf434 per gram plant fresh weight after inoculation of germ-free Col-0, rbohD and rbohD/RBOHD plants with Pseudomonas Leaf434 as single inoculation or in binary inoculation with Xanthomonas Leaf131 Tn7::Gm-lux. Box plots show the median with upper and lower quartiles and whiskers present 1.5× interquartile range (n = 12, two-sided Mann–Whitney U test, P values indicated above box plots). Source data

Fig. 2. Xanthomonas Leaf131 and Leaf148 degrade plant tissue.

a, Leaf discs of Col-0 and rbohD plants (6 weeks old) were mock inoculated (10 mM MgCl₂) or inoculated with Xanthomonas Leaf131 or Leaf148 (OD of 0.02) and incubated for 20 h. b, Time-course measurement and quantification of leaf disc brightness (arbitrary unit, AU) from experiment described in a. Statistical differences between Col-0 and rbohD at varying timepoints are indicated above box plots, with P value on the right or left of horizontal line indicating comparison (two-sided Mann–Whitney U test, n = 8). Box plots show the median with upper and lower quartiles and whiskers present 1.5× interquartile range. c, Leaf disc of 5-week-old rbohD plants mock (10 mM MgCl₂) inoculated or with Xanthomonas Leaf131 or Leaf148 (OD of 0.02) and incubated for 48 h. Scale bar, 1 mm. Source data

Fig. 3. T2SS Xps requirement for leaf tissue degradation and secretion of plant polymer-degradative enzymes.

a, Leaf discs of Col-0 and rbohD plants (5 weeks old) were mock treated (0.5× LB) or treated with cell-free supernatant (0.22 µm filter sterilized) of Xanthomonas Leaf131 or Leaf148 liquid cultures and incubated for 48 h. b, Genomic region of the T2SS operons xps and xcs in Xanthomonas Leaf131 and Leaf148. Letters indicate gene names and black line shows region of gene deletion. c,d, Leaf disc brightness was measured 24 h after inoculation with mock solution or with Xanthomonas wild-type or mutant strains of Leaf131 (c) or Leaf148 (d). Leaf discs were generated from Col-0 or rbohD plants (6 weeks old). Box plots show the median with upper and lower quartiles and whiskers present 1.5× interquartile range. Significant differences were calculated with ANOVA and two-sided Tukey’s honest significant difference post hoc test (n = 8, letters indicate significance groups, α = 0.05). e, Agar plates containing either skimmed milk, PGA, CMC, azo-xyloglucan or RBB-Xylan. Drops of 4 µl Xanthomonas Leaf131 wild-type or mutant suspension were pipetted onto agar plate. Pictures were taken 24 h after incubation at 22 °C. Quantification of halo diameter is shown in Supplementary Fig. 4. f, Leaf discs were treated with 0.22 µm filter-sterilized supernatant of liquid cultures from Xanthomonas Leaf131 or Leaf148 wild-type and xpsxcs mutants or mock solution (0.5× LB). Leaf discs were incubated for 48 h at 22 °C. AU, arbitrary unit; SN, supernatant; WT, wild type. Source data

Fig. 4. T2SS Xps requirement for full virulence and fitness of Xanthomonas Leaf131 in planta.

a, Phenotype of 5-week-old Col-0 plants (blue arrow) and rbohD plants (green arrow) mock inoculated or with Xanthomonas Leaf131 wild type (WT) or T2SS mutants xps, xcs and xpsxcs. Scale bars, 1 cm. b, Measurement of fresh weight from plants shown in a. c, CFU counts of Xanthomonas Leaf131 per gram plant fresh weight from samples in b. Box plots show the median with upper and lower quartiles and whiskers present 1.5× interquartile range. Significant differences in b (n = 20) and c (n = 12) were calculated with ANOVA and two-sided Tukey’s honest significant difference post hoc test (letters indicate significance groups, α = 0.05). Log reduction of bacterial abundance shown in Source Data Fig. 4. Source data

Fig. 5. Additional virulence factors contribute to leaf degradation and virulence of Xanthomonas Leaf131.

a, Leaf discs of 5-week-old rbohD plants were mock (10 mM MgCl₂) inoculated or inoculated with Xanthomonas Leaf131 wild type (WT) or gene deletion mutants (OD of 0.02) and incubated for 24 h. b, Fresh weight of aboveground plant tissue of 5-week-old gnotobiotic rbohD plants, either mock inoculated or inoculated with Xanthomonas Leaf131 wild type or gene deletion mutants. c, Leaf discs of 5-week-old rbohD plants were mock treated (0.5× LB) or treated with cell-free supernatant (0.22 µm filter sterilized) of liquid cultures from Xanthomonas Leaf131 wild type and gene deletion mutants. Leaf discs were incubated for 24 h at 22 °C. Black circles, rectangles and squares indicate data from three bacterial cultures. d, CFU counts of Xanthomonas Leaf131 per gram plant fresh weight from samples in b. Box plots show the median with upper and lower quartiles and whiskers present 1.5× interquartile range. Significant difference in a (n = 8), b (n = 20), c (n = 24) and d (n = 12) of gene deletion mutants compared with Leaf131 wild type was determined by two-sided Mann–Whitney U test, and P values are indicated above box plots. Source data

Fig. 6. Microbiota shift in rbohD depends on T2SS-related virulence of Xanthomonas Leaf131.

a, Composition of synthetic bacterial community SynCom-137 containing Xanthomonas Leaf131 wild type or mutants xps, xpsxcs and dsbB was compared with SynCom-137 alone in Col-0 and rbohD plants. Effect size represents percentage of total variance explained by genotype (shown by dot size and absolute value) and statistical significance is expressed with P values determined by PERMANOVA (Benjamini–Hochberg adjusted, n = 16). Number of differentially abundant strains (as shown in b) is represented by dot colour. b, Heatmap shows subset of strains of SynCom-137 with significant log₂ fold changes (log₂FC, P < 0.05) in rbohD plants inoculated either with only SynCom-137 or with SynCom-137 containing Xanthomonas Leaf131 wild type or the mutants xps, xpsxcs and dsbB. Black rectangles show significant changes, P < 0.05 (n = 16, two-sided Wald test, Benjamini–Hochberg adjusted). The heatmap of all strains in SynCom-137 is shown in Extended Data Fig. 5b. c, Relative abundance of Xanthomonas Leaf131 wild type or the mutants xps, xpsxcs and dsbB within SynCom-137 in Col-0 and rbohD plants. Ratios below violin plots represent frequency of samples where Xanthomonas Leaf131 was not detected. Violin plots show the median with upper and lower quartiles (n = 16, two-sided Mann–Whitney U test; P values are indicated above violin plots). d, CFU counts of Pseudomonas Leaf434 per gram plant fresh weight after inoculation of germ-free Col-0 and rbohD plants with Pseudomonas Leaf434 as single inoculation (−) or as binary inoculation with either Xanthomonas Leaf131 wild type or the mutants xps and xpsxcs. Box plots show the median with upper and lower quartiles and whiskers present 1.5× interquartile range. Significant differences were calculated with ANOVA and two-sided Tukey’s honest significant difference post hoc test (n = 12, letters indicate significance groups, α = 0.05). Source data

Extended Data Fig. 1. Microbiota composition of SynCom-137 and plant phenotype in presence and absence of Xanthomonas Leaf131.

a) Principal component analysis of SynCom-137+Xanthomonas Leaf131 and of b) SynCom-137 in Col-0 (blue), rbohD (green) and rbohD/RBOHD (light blue). Axes show principal components PC1 and PC2 with their explained variance (%). Statistical analysis (PERMANOVA) analysis is represented by effect size shown in Fig. 1Ac) Heatmap shows log₂ fold changes (log2FC) of strains in SynCom-137 in rbohD or rbohD/RBOHD compared to Col-0 wild-type plants in the presence (+) or absence (-) of Xanthomonas Leaf131. Black rectangles show significant changes, p-value < 0.05 (n = 16, two-sided Wald test, Benjamini–Hochberg adjusted). Subset of same data is shown in Fig. 1b. d) Plant phenotype of Col-0 (blue arrow), rbohD (green arrow) and rbohD/RBOHD (light blue arrow) mock inoculated or with SynCom-137 or SynCom-137+Xanthomonas Leaf131. Two representative replica growth boxes are shown for each treatment. Source data

Extended Data Fig. 2. Xanthomonas disrupts leaf tissue cohesion and requires plant wound.

a) Time-course of leaf discs from five-week-old rbohD plants inoculated with Xanthomonas Leaf131 (OD=0.02). Scale bar represents 1 mm. b) Leaf discs of rbohD plants in 96-well plate after 48 hours incubation with Xanthomonas Leaf131 were vortexed for two seconds. Scale bar represents 1 mm in left and middle panel, and 0.5 mm in right panel. c) Leaf discs of five-week-old Col-0 plants after 48 hours incubation with Xanthomonas Leaf131. Scale bar represents 1 mm in left and middle panel, and 0.5 mm in right panel. Experiment shown in panel A-C was repeated at least ten times. d) Leaves of five-week-old rbohD plants floating in water with undamaged leaf edge (left panels) or wounded leaf edge (right panels) were mock inoculated (10 mM MgCl₂) or with Xanthomonas Leaf131 or Leaf148 (OD=0.02).

Extended Data Fig. 3. Plant genotype influences leaf disc degradation and susceptibility to Xanthomonas.

a) Time-course of leaf discs brightness from six-week-old Col-0, rbohD, fls2/efr/cerk1 (fec) and bak1/bkk1/cerk1 (bbc) plants inoculated with Xanthomonas Leaf131 or b) Leaf148. Statistical differences of leaf disc brightness between plant genotype at varying time points is indicated with p-value above box plots (two-sided Mann–Whitney U-test, n = 4). Box plots show the median with upper and lower quartiles and whiskers present 1.5x interquartile range. c) Plant phenotype of gnotobiotic Col-0, rbohD and bbc plants mock inoculated (blue arrow) or with Xanthomonas Leaf131 (yellow arrow) or Xanthomonas Leaf148 (orange arrow) at 24 days post inoculation. Source data

Extended Data Fig. 4. Proteomic analysis of supernatant from Xanthomonas Leaf131 and Leaf148 liquid culture identified T2SS-specific proteins.

a) Coomassie stained SDS-PAGE of cell-free supernatant from Xanthomonas Leaf131 and Leaf148 wildtypes and xpsxcs mutants liquid cultures (n=3). Black arrows with numbers indicate protein bands excised from gel and analysed by LC-MS/MS with results of different fractions shown in Supplementary Table 2. b) Genomic region encoding T2SS-dependent secreted proteins pectate lyase (ASF73_20170) and lysyl endopeptidase (ASF73_20190). Orange line indicates in-frame deletion of gene cluster. c) Leaf discs of Col-0 or rbohD plants (six weeks old) were mock treated or with Xanthomonas Leaf131 wildtype or mutant strains with gene deletions of ASF73_13775, ASF73_18370, ASF73_04230, ASF73_20170-ASF73_20190. Box plots show the median with upper and lower quartiles and whiskers present 1.5x interquartile range. Sample size, n = 8. d) Table shows candidate genes of T2SS-dependent secreted proteins identified by LC-MS/MS (Supplementary Table 1) and gene identifiers of homologues in other Xanthomonas species as described in the literature. Source data

Extended Data Fig. 5. Microbiota composition in presence of virulent or attenuated Xanthomonas Leaf131.

a) Heatmap shows log₂ fold changes (log2FC) of strains in SynCom-137 in rbohD compared to Col-0 wild-type plants in the presence (+) or absence (−) of Xanthomonas Leaf131. b) Heatmap shows log₂ fold changes (log2FC) of strains in the presence of either Xanthomonas Leaf131 wildtype or the mutants xps, xpsxcs, or dsbB compared to SynCom-137 without Leaf131. Black rectangles show significant changes, p-value < 0.05 (n = 16, two-sided Wald test, Benjamini–Hochberg adjusted). Subset of same data is shown in Fig. 6a. c) Principal component analysis of community in rbohD plants (upper panel) and Col-0 (lower panel) inoculated only with SynCom-137 or SynCom-137 containing either Xanthomonas Leaf131, xps, xpsxcs, or dsbB. Axes show principal components PC1 and PC2 with their explained variance (%). Source data