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The plant NADPH oxidase RBOHD is required for microbiota homeostasis in leaves

Nature Microbiology volume 6, pages 852–864 (2021)Cite this article

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Abstract

The plant microbiota consists of a multitude of microorganisms that can affect plant health and fitness. However, it is currently unclear how the plant shapes its leaf microbiota and what role the plant immune system plays in this process. Here, we evaluated Arabidopsis thaliana mutants with defects in different parts of the immune system for an altered bacterial community assembly using a gnotobiotic system. While higher-order mutants in receptors that recognize microbial features and in defence hormone signalling showed substantial microbial community alterations, the absence of the plant NADPH oxidase RBOHD caused the most pronounced change in the composition of the leaf microbiota. The rbohD knockout resulted in an enrichment of specific bacteria. Among these, we identified Xanthomonas strains as opportunistic pathogens that colonized wild-type plants asymptomatically but caused disease in rbohD knockout plants. Strain dropout experiments revealed that the lack of RBOHD unlocks the pathogenicity of individual microbiota members driving dysbiosis in rbohD knockout plants. For full protection, healthy plants require both a functional immune system and a microbial community. Our results show that the NADPH oxidase RBOHD is essential for microbiota homeostasis and emphasizes the importance of the plant immune system in controlling the leaf microbiota.

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Fig. 1: Characterization of the At-LSPHERE model community in the phyllosphere and endosphere of A. thaliana.
Fig. 2: Effect of plant genotype on the leaf endosphere community.
Fig. 3: The plant mutant rbohD shows a dysbiosis phenotype and assembles a microbiota enriched in Gammaproteobacteria.
Fig. 4: Xanthomonas Leaf131 and Leaf148 are opportunistic pathogens in immunocompromised rbohD.
Fig. 5: Xanthomonas Leaf131 causes dysbiosis in rbohD.

Data availability

Raw reads of demultiplexed 16S rDNA amplicon samples can be found in the European Nucleotide Archive under accession number PRJEB44158. Source data are provided with this paper.

Code availability

The code used to analyse all data and generate figures can be found at https://github.com/MicrobiologyETHZ/phylloR/releases/tag/v1.1. No unpublished algorithms or methods were used.

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Acknowledgements

We thank B. Maier, A. Imboden, B. Emmenegger and P. Kirner for technical assistance, and C. Vogel, M. Schäfer and L. Hemmerle for discussion and critical reading of the manuscript. DNA sequencing was done at the Genetic Diversity Centre Zurich with assistance from S. Kobel. We are grateful to the following laboratories for generous sharing of seeds: C. Zipfel (University of Zurich, Switzerland) for fls2 efr cerk1 (fec), bak1-5 bkk1-1 cerk1-2 (bbc), rbohD, rbohDrbohF, rbohD/pRbohD:3xFLAG–RBOHD, rbohD/pRBOHD:3xFLAG–RBOHD-S39AS339AS343A; J.-M. Zhou (Chinese Academy of Sciences, Beijing, China) for rbohD/pRBOHD:3xFLAG–RBOHD-S343AS347A; K. Shirasu and Y. Kadota (Riken Center for Sustainable Resource Science, Yokohama, Japan) for rbohDrbohF/pRBOHD:3xFLAG‐RBOHD and rbohDrbohF/pRBOHD:3xFLAG‐RBOHD-S343A-S347A; S.Y. He (Michigan State University, USA) for min7 fls2 efr cerk1-2 (mfec) and min7 bak1-5 bkk1-1 cerk1-2 (mbbc); T. Hamann (Norwegian University of Science and Technology, Norway) for pepr1-1 pepr2-1; T. Nürnberger (University of Tubingen, Germany) for lym3lym1-5-28; X. Dong (Duke University, USA) for cpr5-1 and cpr6-1. We acknowledge funding from ETH Zurich, a grant from the German Research Foundation (DECRyPT, no. SPP2125), the NCCR Microbiomes, funded by the Swiss National Science Foundation, and a European Research Council Advanced grant (no. PhyMo—668991).

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  1. Institute of Microbiology, ETH Zurich, Zurich, Switzerland

    Sebastian Pfeilmeier, Gabriella C. Petti, Miriam Bortfeld-Miller, Benjamin Daniel, Christopher M. Field, Shinichi Sunagawa & Julia A. Vorholt

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  1. Sebastian Pfeilmeier
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Contributions

The conceptualization came from S.P. and J.A.V. The investigation was carried out by S.P., G.C.P., M.B.-M., B.D. and C.M.F. Software and data curation were done by C.M.F. Visualization was done by S.P., G.C.P., C.M.F. and S.S. The original draft was written by S.P. and J.A.V. Review and editing of the paper was carried out by S.P., G.C.P., B.D., S.S. and J.A.V. Funding was acquired by S.P. and J.A.V. Supervision of the work was responsibility of S.P. and J.A.V. All authors read and approved the submitted version.

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Correspondence to Julia A. Vorholt.

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The authors declare no competing interests.

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Peer review information Nature Microbiology thanks Gwyn Beattie, Joshua Herr and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 SynCom-222 composition of inoculum, phyllosphere and endosphere.

Relative abundance of strains (or ASVs indicated by superscript circle) determined by 16 S rDNA amplicon sequencing of samples from SynCom-222 inoculum mix, phyllosphere and endosphere samples of A. thaliana Col-0. Box plots show the median with upper and lower quartiles and whiskers present 1.5x interquartile range. ‘Undetected’ and dot size indicate the number of plant replicates where a given strain was not detected (n = 12). Colors represent strain phylogeny.

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Extended Data Fig. 2 A. thaliana Col-0 leaves after infiltration with bacterial strains.

a, Bacterial load measured as colony forming units (CFU)/cm2 at 0 and 5 days post infiltration (dpi). Xanthomonas Leaf131, Pseudomonas Leaf59, Pseudorhodoferax Leaf265, Pseudomonas syringae pv. tomato DC3000 (Pst) hrcC- and wild-type Pst were infiltrated at OD = 0.002 (~106 CFU/ml) into leaves of soil-grown, four-week-old Col-0 plants. Infiltrated leaves were harvested, surface sterilized in 70% ethanol and homogenized before serial dilution plating. Box plots show the median with upper and lower quartiles and whiskers present 1.5x interquartile range. Statistical differences were calculated with two-tailed Mann–Whitney U-test (0 dpi, n = 4; 5 dpi, n = 8; ns, not significant; * p < 0.05, ** p < 0.01). b, Photographs of leaves from Col-0 five days after treatments as described above. White bar indicates one cm.

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Extended Data Fig. 3 Genotype effect on bacterial community in phyllosphere.

a, Effect of plant genotype on phyllosphere community. The bacterial community of each genotype was compared to Col-0 wild-type in principal component analysis (PCA, n = 12) followed by PERMANOVA (permutations = 10,000), and the effect size of the genotype was plotted in decreasing order. Effect size represents variance explained by genotype (p-value<0.05, Benjamini–Hochberg adjusted; n = 12). b, Relative abundance of phyllosphere bacteria on the indicated plant genotypes. Asterisks (or hash tags for Firmicutes) denote significant differences in taxa on genotypes compared to Col-0 in a two-sided t-test (p < 0.05, Benjamini-Hochberg adjusted, n = 12).

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Extended Data Fig. 4 Overview and clustering of community profiles on genotypes versus Col-0.

a, Strain changes in endosphere communities are displayed in a heatmap as log2 fold-change of strains in the endosphere of the individual genotypes versus Col-0 (columns). Strains or ASVs (indicated by superscript circle) of SynCom-222 are ordered and coloured by phylogeny. Hierarchical clustering (R command hclust, method ‘single’) of genotypes was performed on an Euclidean distance matrix of log2 fold changes between test conditions and controls. b, Strain changes in phyllosphere communities shown in the heatmap with genotype clustering as described above. Differential strain abundance was calculated using DESeq2, and statistical significance was expressed with p-values (two-sided Wald test, Benjamini-Hochberg adjusted): the black cell rectangle highlights significant changes p < 0.05.

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Extended Data Fig. 5 Microbiota-induced disease in rbohD is linked to the enrichment of Xanthomonadaceae.

a, Screening of plant phenotypes and disease symptoms in rbohD after colonization with individual bacterial strains. Germ-free, 10-day-old rbohD seedlings were inoculated with a bacterial suspension (OD600 ranging from 0.02 to 0.08), and the plant phenotype was examined after 3.5 weeks (n = 8). Inoculated plants showed a variety of phenotypes, for example, stunted plants, necrotic lesions, curled leaves or dead plants. Phenotype-inducing strains were selected based on symptoms and are highlighted by red rectangles in the phylogeny. b, Disease index was assessed in rbohD plants: 1, healthy; 2, mild symptoms on individual leaves; 3, stronger symptoms on multiple leaves; 4, strong symptoms on whole plant; 5, severe symptoms or dead plant. The graph displays the plant fresh weight (mg) of SynCom-137-inoculated rbohD plants (n = 20) per disease category. Box plots show the median with upper and lower quartiles and whiskers present 1.5x interquartile range. c, Bacterial load in SynCom-137-inoculated Col-0 and rbohD plants measured as colony forming units (CFU) per gram of plant fresh weight isolated from the endosphere and phyllosphere (n = 16; two-tailed Mann-Whitney U-test; ns, not significant; **, p < 0.01). Bacterial load in Col-0 and rbohD plants inoculated with the indicated SynCom represented by qPCR of the bacterial 16 S rDNA gene relative to plant gene. Box plots show the median with upper and lower quartiles and whiskers present 1.5x interquartile range. Statistical significance was calculated by two-tailed Mann-Whitney U-test (n = 3, each pool of four DNA samples; ns, not significant). qPCR data for SynCom-222 in Col-0 are the same as in Fig. 1c. d, Correlation analysis between the relative abundance of Xanthomonadaceae in phyllosphere samples of rbohD inoculated with the indicated SynCom and plant fresh weight. The Spearman coefficient ρ (p-value<0.01) was calculated using ggscatter command (ggpubr, R), grey area shows 95% confidence interval of regression line in green. Correlation data on endosphere samples was not possible due to bulk surface sterilization of plants. e, ROS accumulation was measured in leaf discs with a luminol-based assay after treatment with extracts from heat-killed bacteria. ROS production was recorded for 45 min, and luminescence counts were integrated over time. ROS triggered by individual treatments were normalized to ROS production by 10 nM flg22. Normalized ROS accumulation is shown for each bacterial strain. Barplots show mean and error bars show standard deviation (n = 16; combined data from two independent experiments). Red dots indicate rbohD-enriched strains.

Source data

Extended Data Fig. 6 The community assembly of rbohD and rbohDrbohF substantially differs from that of other PTI genotypes.

The heatmap shows log2 fold-changes in strain abundance on different genotypes compared to Col-0 wild-type. Columns show endosphere and phyllosphere samples from plants inoculated with either a, SynCom-222 or b, SynCom-223 or SynCom-137. Strains (or ASVs indicated by superscript circles) are ordered and colored by phylogeny. Statistical significance of differential strain abundances was calculated with the two-sided Wald test (DESeq2 package, R) and highlighted with a black cell rectangle for p < 0.05 (n = 12; Benjamini-Hochberg adjusted).

Source data

Extended Data Fig. 7 PTI-associated phosphorylation sites of RBOHD are involved in resistance to opportunistic pathogens.

a, ROS production of A. thaliana Col-0, rbohD, rbohF, rbohD/RBOHD, rbohD/RBOHD-S39AS339AS343A, rbohD/RBOHD-S343A-S347A, rbohD/RBOHD-S343A-S347A, rbohDrbohF/RBOHD, rbohDrbohF/RBOHD-S343A-S347A and rbohDrbohF, after treatment with 100 nM flg22. ROS production was measured in leaf discs from soil-grown plants with a luminol-based assay and expressed as integrated luminescence over 45 min (AU, arbitrary units). Box plots show the median with upper and lower quartiles and whiskers present 1.5x interquartile range. b, Fresh weight of Col-0, rbohD, rbohD/RBOHD, and rbohD/RBOHD-S343A-S347A inoculated with individual strains of Xanthomonas spp. Leaf131, Leaf148, Pst hrcC-, and Pst wild-type or mock-inoculated with 10 mM MgCl2. Germ-free 10-day-old seedlings were inoculated with OD = 0.02 of the respective strains. Box plots show the median with upper and lower quartiles and whiskers present 1.5x interquartile range. Significant differences were calculated between the mutants and their respective controls (n = 20; two-tailed Mann-Whitney U-test). Brackets above bar plots indicate comparison groups with p-values displayed above and fold-change below. Data from two independent experiments are shown in separate graphs.

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Extended Data Fig. 8 Leaf131 is required and sufficient for disease in rbohD.

Two independent replicate experiments with dropout synthetic communities as presented in Fig. 5. In addition, SynCom-REPI without (w/o) Leaf131 was tested and compared to SynCom-REPI. Significant differences were calculated with ANOVA and Tukey’s HSD post-hoc test (n = 20, letters indicate significance groups, α = 0.05).

Source data

Supplementary information

Reporting Summary

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Supplementary Table

At-LSPHERE strains and SynCom inoculum composition, A. thaliana genotypes and ASV count table and metadata.

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Source Data Fig. 1

Data tables for graphs and statistical analysis.

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Data tables for graphs and statistical analysis.

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Data tables for graphs and statistical analysis.

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Data tables for graphs and statistical analysis.

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Data tables for graphs and statistical analysis.

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Data tables for graphs and statistical analysis.

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Data tables for graphs and statistical analysis.

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Data tables for graphs and statistical analysis.

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Data tables for graphs and statistical analysis.

Source Data Extended Data Fig. 8

Data tables for graphs and statistical analysis.

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Pfeilmeier, S., Petti, G.C., Bortfeld-Miller, M. et al. The plant NADPH oxidase RBOHD is required for microbiota homeostasis in leaves. Nat Microbiol 6, 852–864 (2021). https://doi.org/10.1038/s41564-021-00929-5

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