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Protective role of the Arabidopsis leaf microbiota against a bacterial pathogen

An Author Correction to this article was published on 06 January 2022

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Abstract

The aerial parts of plants are host to taxonomically structured bacterial communities. Members of the core phyllosphere microbiota can protect Arabidopsis thaliana against foliar pathogens. However, whether plant protection is widespread and to what extent the modes of protection differ among phyllosphere microorganisms are not clear. Here, we present a systematic analysis of plant protection capabilities of the At-LSPHERE, which is a collection of >200 bacterial isolates from A. thaliana, against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. In total, 224 bacterial leaf isolates were individually assessed for plant protection in a gnotobiotic system. Protection against the pathogen varied, with ~10% of leaf microbiota strains providing full protection, ~10% showing intermediate levels of protection and the remaining ~80% not markedly reducing disease phenotypes upon infection. The most protective strains were distributed across different taxonomic groups. Synthetic community experiments revealed additive effects of strains but also that a single strain can confer full protection in a community context. We also identify different mechanisms that contribute to plant protection. Although pattern-triggered immunity coreceptor signalling is involved in protection by a subset of strains, other strains protected in the absence of functional plant immunity receptors BAK1 and BKK1. Using a comparative genomics approach combined with mutagenesis, we reveal that direct bacteria–pathogen interactions contribute to plant protection by Rhizobium Leaf202. This shows that a computational approach based on the data provided can be used to identify genes of the microbiota that are important for plant protection.

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Fig. 1: Overview of experimental screening system.
Fig. 2: Overview of plant protection in the At-LSPHERE.
Fig. 3: Protective strains of the At-LSPHERE colonize the phyllosphere at high density.
Fig. 4: SynCom experiments reveal community-specific gain of protection.
Fig. 5: Plant protection in the bak1/bkk1 plant background is strongly reduced in a subset of strains based on luminescence analysis.
Fig. 6: The T6SS is associated with plant protection in Rhizobium spp. in the At-LSPHERE.

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Data availability

Sequencing data for this study have been deposited in the European Nucleotide Archive under accession PRJEB47672. Other genome data are available from NCBI under accessions PRJNA297956, PRJNA471493 and PRJNA84361. The eggNOG database is available from http://eggnog45.embl.de/#/app/home. Rhizobium genomes used for comparative genomics are available from https://img.jgi.doe.gov/m under IMG Genome IDs 2643221743, 2643221780, 2643221832, 2643221860, 2643221889, 2643221891, 2643221896, 2643221905, 2643221915, 2643221931 and 2643221933-36. Source data are provided with this paper.

Code availability

The scripts used for sequencing data processing, genome assembly and data analysis are available at https://gitlab.ethz.ch/chvogel1/vogel_natmicro_2021/.

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Acknowledgements

We thank M. Bortfeld-Miller for technical assistance and A. Levy for helpful discussions. A. thaliana bak1/bkk1 was kindly provided by C. Zipfel (University of Zurich, Switzerland). The study was supported by an ERC Advanced Research Grant (PhyMo - 668991 to J.A.V.), ETH Zurich, a grant from the German Research Foundation (DECRyPT, no. SPP2125 to J.A.V.) and the NCCR Microbiomes (to J.A.V.), funded by the Swiss National Science Foundation.

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Authors and Affiliations

Authors

Contributions

C.V., D.B.P. and J.A.V. conceived and designed the study. C.V., D.B.P. and M.S. performed the protection screen and validation. C.V., D.B.P., M.S. and N.B. performed experiments testing SynComs. C.V. tested in vitro and in planta effect of Leaf202 and its T6SS mutant with help from M.S. C.V. and M.S. extracted DNA for genome sequencing. C.V. analysed sequencing data and assembled genomes. C.V. and D.B.P. analysed screening and validation data. C.V. analysed SynCom data with help from M.S. and analysed T6SS data. C.V. and J.A.V. wrote the manuscript.

Corresponding author

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 the anonymous reviewers 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 Protection potential by protective At-LSPHERE strains scales with infection titre.

A. thaliana were inoculated with fully protective strains Leaf15, Leaf154 or Leaf21 (protection score >90), or with protective strains Leaf205 or Leaf233 (protection score >75) and infected with Pst at the regular infection titre (OD 0.00003), a 100x higher (OD 0.003) or a 10’000x higher (OD 0.3) infection titre. a) Luminescence indicative of pathogen colonization was measured at 6 d post infection (dpi). Shown are boxplots and individual data points. Letters indicate significant differences for each infection titre based on ANOVA followed by Tukey’s post-hoc test (P < 0.05, n = 16–24). Exact P values and number of biological replicates are provided in Supplementary Table 3. Boxplots depict the median and interquartile range with whiskers extending to maximum 1.5x the interquartile range. b) Plants were scored for disease at 21 dpi on a scale of 1 (healthy) to 5 (dead). c) Exemplary images of plants at 21 dpi showing protection of plants by fully protective strains at high infection titre and reduced protection by Leaf205 and Leaf233 at increasing infection titre.

Source data

Extended Data Fig. 2 Protection and luminescence reduction correlate.

Mean protection score and mean luminescence reduction (that is pathogen colonization reduction) correlate well for most strains (Pearson’s R = 0.927, t = 36.8, df = 221, P < 2.2 × 10−16). Colours refer to phylum/class. a.u., arbitrary units.

Source data

Extended Data Fig. 3 Random SynComs of 10 non-protective strains or all Methylobacterium do not protect Arabidopsis against Pst.

Plants were inoculated with random SynComs of 10 strains (M10.2-M10.6) containing only non-protective strains, all 32 non-protective Methylobacterium spp. (M10.1), Fr1 or the control SynCom M10.7, which contains one protective strain before infection with lux-tagged P. syringae DC3000 (Pst). a) Distribution of disease scores on a scale of 1 (healthy) to 5 (dead) at 21 d post infection (dpi). b) Luminescence of the pathogen at 6 dpi. Boxplots depict the median and interquartile range with whiskers extending to maximum 1.5x the interquartile range. P values for the comparison to axenic infected controls are indicated (two-sided Welch’s t-test, corrected for multiple testing using Holm’s method, n = 15–23). Exact number of replicates are provided in Supplementary Table 4.

Source data

Extended Data Fig. 4 Random SynComs of a mixture of 10 non-protective and intermediate protective strains improve plant phenotype.

Plants were inoculated with random SynComs of 10 strains or Fr1 before infection with lux-tagged P. syringae DC3000 (Pst). a) Distribution of disease scores at 13 d post infection (dpi) on a scale of 1 (healthy) to 5 (dead). b) Luminescence as proxy of pathogen colonization at 6 dpi. Boxplots depict the median and interquartile range with whiskers extending to maximum 1.5x the interquartile range. P values for the comparison to axenic infected controls are indicated (two-sided Welch’s t-test, corrected for multiple testing using Holm’s method, n = 14–24). Exact number of replicates are provided in Supplementary Table 5. c) Colonization by individual SynComs on non-infected plants at 12 d post inoculation. Shown are the mean and individual data points of 3 replicates consisting of two plants each. d) Correlation of mean luminescence and mean disease score (R = 0.92, t = 14.76, d.f. = 39, P < 2.2 × 10−16).

Source data

Extended Data Fig. 5 Dropout of one or two strains from a random SynCom of 10 strains can affect plant protection by SynComs.

Plants were inoculated with random SynComs of 10 strains, dropout communities thereof or individual strains and infected with lux-tagged P. syringae DC3000 (Pst). a) Distribution of disease scores at 13 d post infection (dpi) on a scale of 1 (healthy) to 5 (dead). b) Luminescence as proxy of pathogen colonization at 6 dpi. Boxplots depict the median and interquartile range with whiskers extending to maximum 1.5x the interquartile range. P-values for indicated comparisons are shown (two-sided Welch’s t-test, n = 21-24 plants per condition). Exact number of replicates are provided in Supplementary Table 6. c) Colonization by the individual SynComs on non-infected plants at 12 d post inoculation. Shown are the mean and individual data points of 3 replicates consisting of 2 plants each.

Source data

Extended Data Fig. 6 SynComs of three strains can improve protection phenotypes relative to individual strains.

Plants were inoculated with SynComs of three strains (M3.1, M3.2 and M3.3; comprised of non-protective and intermediate protective strains) or the strains individually and infected with lux-tagged P. syringae DC3000 (Pst). a) Distribution of disease scores on a scale of 1 (healthy) to 5 (dead) at 13 d post infection (dpi). b) Pathogen luminescence at 6 dpi. Boxplots depict the median and interquartile range with whiskers extending to maximum 1.5x the interquartile range. Letters indicate statistical significance within each SynCom (one-way ANOVA with Tukey’s post-hoc test, n = 16–24). Exact P values and number of replicates are provided in Supplementary Table 8.

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Extended Data Fig. 7 T6SS gene cluster presence in At-LSPHERE strains and Sphingomonas melonis Fr1.

The outer rings reflect mean protection scores against lux-tagged P. syringae DC3000 on Col-0 plants and the presence of predicted T6SS gene clusters, respectively. a.u., arbitrary units.

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Supplementary Information

Supplementary Note and Figs. 1–7.

Reporting Summary.

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Supplementary Tables 1–12, 14.

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Supplementary Table 13.

Supplementary Data 1

Results for plant protection by individual strains against lux-tagged P. syringae DC3000 (Pst) by individual batches indicated with Experiment IDs on top. Shown are the distribution of disease scores at 13 d post infection (dpi) on a scale of 1 (healthy) to 5 (dead) (top panel), the luminescence as proxy of pathogen colonization at 6 dpi and the colonization by the individual strains on non-infected plants at 12 d post inoculation. Asterisks in the luminescence panel indicate significantly different luminescence relative to axenic infected controls (two-sided Welch’s t-test, corrected for multiple testing using Holm’s method, n = 11–24). The correlation of mean luminescence (log10(p/s)) at 6 dpi and mean disease score at 13 dpi is shown in the bottom panel. Colours of the three bottom panels correspond to phylum/class. Triangles in panel colonization indicate samples with colonization below detection limit, with a value just below the detection limit shown. For a list of the different strains tested in each batch (that is, Round and Part), exact P values and number of replicates within each experiment, we refer the reader to Supplementary Table 2.

Supplementary Data 2

Plant protection and colonization in the bak1/bkk1 plant background. Col-0 and bak1/bkk1 plants were inoculated with individual bacterial strains and infected with lux-tagged P. syringae DC3000 (Pst). Top panel: distribution of disease scores at 16 d post infection on a scale of 1 (healthy) to 5 (dead). Bottom panel: colonization level (log10(c.f.u. mg–1)) of the tested strains in non-infected plants 12 d after inoculation. Shown are the median and individual data points. P values of significant differences between wild-type and bak1/bkk1 plants are indicated (two-sided Welch’s test, Benjamini–Hochberg adjusted P < 0.05, n = 6). For exact P values and number of replicates we refer the reader to Supplementary Table 10.

Supplementary Data 3

Source data for Supplementary Data 1.

Supplementary Data 4

Source data for Supplementary Data 2.

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Vogel, C.M., Potthoff, D.B., Schäfer, M. et al. Protective role of the Arabidopsis leaf microbiota against a bacterial pathogen. Nat Microbiol 6, 1537–1548 (2021). https://doi.org/10.1038/s41564-021-00997-7

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