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Mapping phyllosphere microbiota interactions in planta to establish genotype–phenotype relationships

Abstract

Host-associated microbiomes harbour hundreds of bacterial species that co-occur, creating the opportunity for manifold bacteria–bacteria interactions, which in turn contribute to the overall community structure. The mechanisms that underlie this self-organization among bacteria remain largely elusive. Here, we studied bacterial interactions in the phyllosphere microbiota. We screened for microbial interactions in planta by adding 200 endogenous strains individually to a 15-member synthetic community and tracking changes in community composition upon colonization of the model plant Arabidopsis. Ninety percent of the identified interactions in planta were negative, and phylogenetically closely related strains elicited consistent effects on the synthetic community, providing support for trait conservation. Community changes could be largely explained by binary interactions; however, we also identified a higher-order interaction of more than two interacting strains. We further focused on a prominent interaction between two members of the Actinobacteria. In the presence of Aeromicrobium Leaf245, the population of Nocardioides Leaf374 was reduced by almost two orders of magnitude. We identified a potent antimicrobial peptidase in Aeromicrobium Leaf245, which resulted in Nocardioides Leaf374 lysis. A respective Leaf245 mutant strain was necessary and sufficient to restore Nocardioides colonization in planta, demonstrating that direct bacteria–bacteria interactions were responsible for the population shift originally observed. Our study highlights the power of synthetic community screening and uncovers a strong microbial interaction that occurs despite a spatially heterogeneous environment.

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Fig. 1: A 15-strain focal SynCom to map bacteria–bacteria interactions.
Fig. 2: Interactions of the At-LSPHERE collection with the focal SynCom in planta.
Fig. 3: Biochemical characterization of the interaction between Nocardioides Leaf374 and Aeromicrobium Leaf245.
Fig. 4: Leaf374 phyllosphere colonization is less affected when co-inoculated with Leaf245 EMS mutants that no longer secrete ASF05_00205 compared with the wild type.
Fig. 5: Producer and target range of the NlaP.

Data availability

The 16S rRNA gene amplicon sequencing reads and the reads obtained from Leaf245 EMS mutant genome sequencing are available online at the European nucleotide archive (https://www.ebi.ac.uk/ena/) under the accession numbers PRJEB50894 and PRJEB47688, respectively. The SILVA SSU Ref NR database (release 132) can be found in www.arb-silva.de/download/archive. Source data are provided with this paper.

Code availability

The code used for analysis of 16S rRNA gene amplicon data can be found in the package ‘phylloR’ available on GitHub (https://github.com/MicrobiologyETHZ/phylloR/). No unpublished algorithms were used.

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Acknowledgements

We thank A. Minder and S. Kobel at the Genetic Diversity Center Zurich (GDC, ETH Zurich) for MiSeq amplicon sequencing service, C. Field (Institute of Microbiology, ETH Zurich) for amplicon raw read processing, the Functional Genomics Center Zurich (FGCZ) for protein identification service and P. Schulze-Lefert (Max Planck Institute for Plant Breeding Research, Cologne, Germany) for sharing At-RSPHERE isolates. This work was supported by the Swiss National Science Foundation through NRP72 (no. 407240_167051) and as part of NCCR Microbiomes, a National Centre of Competence in Research (no. 51NF40_180575) and a European Research Council Advanced Grant (PhyMo; no. 668991).

Author information

Authors and Affiliations

Authors

Contributions

M.S. and J.A.V. designed the research. M.S., M.B.-M. and C.M.V. conducted the in planta interaction screen and binary competition experiments. M.S. prepared the amplicon sequencing libraries and analysed amplicon sequencing data, performed biochemical analyses of Leaf245 supernatants and heterologous expression of proteins. M.M. conducted microscopy time course experiment to monitor Leaf374 lysis. M.S. prepared and M.S. and C.M.V screened the Leaf245 EMS library. C.M.V. closed the Leaf245 genome and mapped point mutations of genome re-sequenced EMS clones. M.S. screened the inhibition spectrum of NlaP producer strains, performed statistical analyses and visualized the data. M.S. and J.A.V. wrote the manuscript with contributions from all authors.

Corresponding author

Correspondence to Julia A. Vorholt.

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Nature Microbiology thanks Gwyn Beattie, Steven Lindow, Linda Thomashow 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 Changes in focal community strain abundances upon strain drop-in.

Heatmap showing log2-transformed fold changes (treatment vs control) for each focal community strain (top) upon strain drop-in (left). Significant changes (DESeq2-normalized counts, Wald test, Benjamini-Hochberg corrected, padj ≤ 0.01, nfocal community = 16, ndrop-in = 3) are indicated with a black frame. Drop-in strains are colored by phylum or Proteobacteria class. Exact p-values are provided in Supplementary Table 2.

Source data

Extended Data Fig. 2 Phylogenetic distance of interacting strains.

Focal strains that showed ≥ 1 interaction are shown. The phylogenetic distance of the focal strain to all added strains is indicated on the x-axis. Log2-fold changes (log2FC) are shown on the y-axis. Significant fold-changes are highlighted with blue (negative interactions) or red (positive interactions) fill color. Strains that belong to the same ASV as a focal strain have a light grey fill color.

Source data

Extended Data Fig. 3 Phyllosphere colonization of Leaf220 and Leaf408.

Phyllosphere colonization of a) Leaf220 or b) Leaf408 in mono-association or in combination with other strains (indicated below). Shown are the median and individual data points of log10-transformed CFU per gram fresh weight across two independent experiments. Colors/shapes refer to experiment. Exact p-values (Krusksal-Wallis test with post-hoc Dunn test, Bonferroni adjusted p) and log2 fold-changes (log2FC) for comparisons to the mono-association condition are indicated above or below the graph, respectively. For corresponding Leaf88 colonization levels see Fig. 2c.

Source data

Extended Data Fig. 4 Validation of Leaf374-Leaf245 interaction by sequential inoculation of A. thaliana.

Phyllosphere colonization of a) Nocardioides Leaf374 and b) Aeromicrobium Leaf245 in mono-association or in combination with the other strain. Shown are the median and individual data points of log10-transformed CFU per gram fresh weight recovered after plant colonization (n = 12). CFU were enumerated on MM maltose agar plates and R2A + M for Nocardioides Leaf374 and Aeromicrobium Leaf245, respectively. The timepoint of inoculation with Leaf245 or Leaf374 is indicated below the graph as days (d) after planting. For mono-association controls half of the replicates were inoculated on day7 (black) or day8 (grey) and mock-treated with 10 mM MgCl2 on the other day. Exact p-values (two-sided Wilcoxon rank sum test) and log2 fold changes (log2FC) compared to the mono-association control are indicated above or below the graph, respectively.

Source data

Extended Data Fig. 5 Characterization of selected proteins identified in the supernatant of Leaf245.

a) List of proteins identified in the supernatant of Leaf245 and selected for heterologous expression in E. coli. For each gel band in Fig. 3d as well as the most active fractions from two independent experiments the rank abundance of the proteins based on total ion count as identified by mass spectrometry are shown. b, c, d) Leaf374 inhibition and lysis by heterologously produced candidate proteins. b) Activity of cleared cell lysate after expression of proteins (top label) in E. coli BL21 DE3 gold. Lysate was applied on Leaf374 overlay plates directly after preparation (left panel) or on Leaf374 overlay plates that were pre-incubated for 24 h to assess lysis (right panel). Lysate concentration was normalized to the final OD600 that each expression culture reached. Up to 50-fold dilutions in the buffer used for lysis (see methods) were prepared as indicated on the left. c) Activity of purified proteins against 24 h pre-grown Leaf374. d) Activity of ASF05_00205 native and mutant (S70L) protein against Leaf374. Concentrations of both purified proteins were normalized (0.1 mg mL−1) and a 10- and 100- fold dilution was prepared and assayed on a Leaf374 overlay plate directly after preparation (left panel) or after the overlay was pre-incubated for 24 h (right panel) to test for growth inhibition and cell lysis, respectively. Data shown for ASF05_00205 native protein is the same as shown in Fig. 3e and panel c) of this figure.

Extended Data Fig. 6 Aeromicrobium Leaf245 wild type and EMS mutant colonization level.

Phyllosphere colonization of Leaf245 in mono-association or in combination with Leaf374. Shown are the median and individual data points of log10-transformed CFU per g fresh weight across 2–3 independent experiments. Log2 fold changes and exact p-values (two-sided Wilcoxon rank sum test) between Leaf245 mono-association and co-colonization treatments with Leaf374 are shown above the graph. For the corresponding Leaf374 colonization see Fig. 4d.

Source data

Extended Data Fig. 7 In vitro inhibition assay with different producer and target cell densities.

The concentrations of the NlaP producer strains (Leaf245 and Root85) spotted on top (2 µL) and target strains (Leaf374 and Root122) within the agar are indicated on top and on the left of the graph, respectively. Pictures were taken 36 hours after overlay preparation and spotting. The standard concentrations used for all other assays performed in this study are highlighted in red.

Supplementary information

Supplementary Information

Supplementary Note and Figs. 1–13.

Reporting Summary

Peer Review File

Supplementary Video

Time-lapse video of Leaf374 cell lysis upon addition of Leaf245 supernatant. Imaging was started 1 min after supernatant addition and a picture was recorded every 20 s over the course of 20 min.

Supplementary Table 1

Supplementary Tables 1–7.

Supplementary Data 1

Demultiplexed 16S RNA gene amplicon sequencing read counts and corresponding metadata.

Supplementary Data 2

Source data for Supplementary Figs. 3, 4, 6 and 9–13.

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SDS–PAGE gel.

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Schäfer, M., Vogel, C.M., Bortfeld-Miller, M. et al. Mapping phyllosphere microbiota interactions in planta to establish genotype–phenotype relationships. Nat Microbiol 7, 856–867 (2022). https://doi.org/10.1038/s41564-022-01132-w

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