Functional overlap of the Arabidopsis leaf and root microbiota

Abstract

Roots and leaves of healthy plants host taxonomically structured bacterial assemblies, and members of these communities contribute to plant growth and health. We established Arabidopsis leaf- and root-derived microbiota culture collections representing the majority of bacterial species that are reproducibly detectable by culture-independent community sequencing. We found an extensive taxonomic overlap between the leaf and root microbiota. Genome drafts of 400 isolates revealed a large overlap of genome-encoded functional capabilities between leaf- and root-derived bacteria with few significant differences at the level of individual functional categories. Using defined bacterial communities and a gnotobiotic Arabidopsis plant system we show that the isolates form assemblies resembling natural microbiota on their cognate host organs, but are also capable of ectopic leaf or root colonization. While this raises the possibility of reciprocal relocation between root and leaf microbiota members, genome information and recolonization experiments also provide evidence for microbiota specialization to their respective niche.

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Figure 1: Taxonomic overlap between At-RSPHERE and At-LSPHERE isolates and their representation in culture-independent microbiota profiling studies.
Figure 2: Analysis of functional diversity between sequenced isolates.
Figure 3: Functional analysis of sequenced isolates.
Figure 4: SynCom colonization of germ-free A. thaliana plants.
Figure 5: SynCom competition supports host-organ-specific community assemblies.

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Primary accessions

BioProject

European Nucleotide Archive

Data deposits

Sequencing reads (454 16S rRNA, MiSeq 16S rRNA and WGS HiSeq reads) have been deposited in the European Nucleotide Archive (ENA) under accession numbers PRJEB11545, PRJEB11583 and PRJEB11584, and genome assemblies and annotations corresponding to the leaf, root and soil culture collections have been deposited in the BioProject database under accession numbers PRJNA297956, PRJNA297942 and PRJNA298127. Isolates have been deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (https://www.dsmz.de/).

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Acknowledgements

We thank D. Lundberg, S. Lebeis, S. Herrera-Paredes, S. Biswas and J. Dangl for sharing the calcined clay utilization protocol before publication; M. Kisielow of the ETH Zurich Flow Cytometry Core Facility for help with bacterial cell sorting as well as M. Baltisberger, D. Jolic and D. Weigel for their help in finding natural Arabidopsis populations; E. Kemen and M. Agler for sharing the Illumina Mi-Seq protocol for profiling of defined communities before publication and A. Sczyrba for his advice with the genome assembly. This work was supported by funds to P.S.-L. from the Max Planck Society, a European Research Council advanced grant (ROOTMICROBIOTA), the ‘Cluster of Excellence on Plant Sciences’ program funded by the Deutsche Forschungsgemeinschaft, the German Center for Infection Research (DZIF), by funds to J.A.V. from ETH Zurich (ETH Research Grant ETH-41 14-2), a grant from the Swiss National Research Foundation (310030B_152835), and a European Research Council advanced grant (PhyMo).

Author information

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Authors

Contributions

J.A.V. and P.S.-L. initiated, coordinated and supervised the project. Y.B., M.R., N.D. and S.S. isolated root and soil bacteria strains. Y.B. collected root material and performed culture-independent community profiling. D.B.M., E.P. and M.R.-E. collected environmental leaf material, D.B.M. and E.P. isolated leaf strains and performed culture-independent community profiling. G.S. and R.G.-O. analysed culture-independent 16S rRNA amplicon sequencing data. Y.B., D.B.M. isolated DNA and prepared samples for genome sequencing. R.G.-O., P.C.M, B.H. and A.C.M. organized the genome sequencing data. R.G.-O. assembled and annotated draft genomes and performed comparative genome analyses. Y.B. and D.B.M. performed recolonization experiments; G.S. and R.G.-O. analysed the recolonization data. Y.B., D.B.M., R.G.-O., J.A.V. and P.S.-L. wrote the manuscript.

Corresponding authors

Correspondence to Julia A. Vorholt or Paul Schulze-Lefert.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Culture-dependent coverage of A. thaliana root- and leaf-associated OTUs identified in several cultivation-independent studies.

ad, The inner circle depicts taxonomic assignments of top 100 root-associated OTUs (filled dots) for the indicated phyla and families that were identified in the current (a), ref. 6 (b) and ref. 12 (c) studies with Cologne-soil-grown plants, and current leaf (d) study at locations around Tübingen and Zurich. Black squares of the outer ring highlight OTUs sharing ≥ 97% 16S rRNA gene similarity to Arabidopsis root or leaf bacterial culture collection.

Extended Data Figure 2 16S rRNA gene community profiling of phyllosphere samples from different locations.

ad, The indicated Beta-diversity indices were calculated from leaf samples (n = 60) collected from natural A. thaliana populations growing in the areas around Tübingen and Zurich. The indicated colour code refers to sampling locations, sampling sites, sampling season, and combined or individual leaves of respective plants.

Extended Data Figure 3 At-RSPHERE, At-LSPHERE and soil bacterial culture collections.

a, At-RSPHERE (n = 206 isolates), a culture collection of the A. thaliana root microbiota. b, At-LSPHERE (n = 224 isolates), a culture collection of the A. thaliana leaf microbiota. c, Bacteria isolated from Cologne soil (n = 33 isolates). Numbers inside white circles indicate the number of bacterial isolates sharing ≥ 97% sequence identity, but isolated from independent roots, leaves and soil batches.

Extended Data Figure 4 Taxonomy overlap between A. thaliana root- and leaf-associated bacterial community from plants grown in natural soils.

a, b, Rank abundance plots of top 20 genera (a) and OTUs (b) in root bacterial communities (n = 8) from Cologne with corresponding genera detected in leaf bacterial communities (n = 60) from Zurich and Tübingen. c, d, Rank abundance plots of top 20 genera (c) and OTUs (d) in leaf bacterial communities from Zurich and Tübingen with corresponding genera detected in root bacterial communities from Cologne. Boxplot whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the upper or lower quartiles.

Extended Data Figure 5 Phylogenetic distribution of ‘carbohydrate metabolism’ genes across sequenced isolates.

a, Phylogeny of sequenced leaf (n = 206), root (n = 194) and soil (n = 32) isolates based on the concatenated alignment of the 31 conserved AMPHORA phylogenetic marker genes. The origin of each genome (leaf, root or soil) is shown by different shapes and their taxonomic affiliation (phylum level) is depicted using various colours. Shaded areas correspond to the different clusters of genomes and are annotated with their consensus taxonomy (family level). b, Relative abundance of protein coding genes classified as belonging to the KEGG general term ‘carbohydrate metabolism’, measured as percentage of annotated proteins per genome.

Extended Data Figure 6 Phylogenetic distribution of ‘xenobiotic biodegradation and metabolism’ genes across sequenced isolates.

a, Phylogeny of sequenced leaf (n = 206), root (n = 194) and soil (n = 32) isolates based on the concatenated alignment of the 31 conserved AMPHORA phylogenetic marker genes. The origin of each genome (leaf, root or soil) is shown by different shapes and their taxonomic affiliation (phylum level; class level for Proteobacteria) is depicted using various colours. Shaded areas correspond to the different clusters of genomes and are annotated with their consensus taxonomy (family level). b, Relative abundance of protein coding genes classified as belonging to the KEGG general term ‘xenobiotics biodegradation and metabolism’, measured as percentage of annotated proteins per genome.

Extended Data Figure 7 V. vinifera metagenome comparison.

a, b, Functional enrichment analysis of V. vinifera root and soil shotgun metagenomes (a; n = 47) compared to A. thaliana culture collection root and soil genomes (b; n = 432). Functional category abundances correspond to the percentage of annotated genes in each genome or metagenome sample. Boxplot whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the upper or lower quartiles.

Extended Data Figure 8 Cluster analysis of Bray–Curtis distances between groups of samples in the SynCom colonization of germ-free A. thaliana experiments.

a, Comparison of pairwise distances within input samples and between input and output samples of the RS in clay experiments. b, Comparison of pairwise distances between samples within the same cluster and between different clusters of the RS in clay experiments. c, Comparison of pairwise distances between input samples and between input and output samples of the L spray experiments. d, Comparison of pairwise distances within samples within the same cluster and between different clusters of the L spray experiments. e, Comparison of pairwise distances between samples within the same cluster and between different clusters of the leaf output across experiments. f, Comparison of pairwise distances between leaf output samples in the RSL in clay experiments and leaf output samples in the L in clay and RS in clay experiments. g, Comparison of pairwise distances between root output samples in the RSL in clay experiments and root output samples in the L in clay and RS in clay experiments. All comparisons marked with asterisks were subjected to a Student’s t-test (P < 0.001 in each case). L in clay was tested with 6 independently prepared SynComs (n = 6); RSL in clay experiment was tested with 3 independently prepared SynComs, each used for 3 independent inoculations (n = 9). All other experiments were tested with 6 independently prepared SynComs and each preparation was used for 3 independent inoculations (n = 18). L, leaf-derived strains; RS, root- and soil-derived strains.

Extended Data Figure 9 Similarity of rank abundances of SynCom outputs with corresponding root- and leaf-associated OTUs of plants grown in natural environments.

ac, Rank abundance plots of SynCom root outputs (n = 69) with corresponding root-associated OTUs in natural communities (n = 8) from plants grown in the present study in Cologne soil at the taxonomic ranks of phylum (a), order (b) and family (c). df, Rank abundance plots of SynCom leaf outputs (n = 69) with corresponding leaf-associated OTUs in natural communities (n = 60) from plants grown in the present study around Tuebingen or Zurich at the taxonomic ranks of phylum (d), order (e) and family (f). Boxplot whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the upper or lower quartiles.

Extended Data Figure 10 Fractional contribution of At-LSPHERE and At-RPSHERE-specific OTUs and SynCom competition supports host organ-specific community assemblies.

a, Fractional contribution of At-LSPHERE and At-RPSHERE specific OTUs in the input, leaf and the root output communities in the ‘RSL in clay’ experiment (n = 9). b, c, PCoA of Bray–Curtis distances of root (b; n = 21) and leaf (c; n = 21) outputs of the ‘R in clay’, ‘RS in clay’, and ‘R spray’ SynCom experiments. R, root-derived isolates; S, soil-derived isolates; L, leaf-derived isolates. RSL in clay experiment was tested with 3 independently prepared SynComs, each used for 3 independent inoculations. All other experiments were tested with 3 independently prepared SynComs and each preparation was used for 3 independent inoculations. Boxplot whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range from the upper or lower quartiles.

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Bai, Y., Müller, D., Srinivas, G. et al. Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528, 364–369 (2015). https://doi.org/10.1038/nature16192

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