Genomic features of bacterial adaptation to plants

This article has been updated

Abstract

Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe–microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant–microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.

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Fig. 1: The genome dataset used in analysis, and differences in gene category abundances.
Fig. 2: Validation of predicted plant-associated genes by multiple approaches.
Fig. 3: Proteins and protein domains that were reproducibly enriched as plant-associated or root-associated in multiple taxa.
Fig. 4: A protein family shared by plant-associated bacteria, fungi, and oomycetes that resemble plant proteins.
Fig. 5: Co-occurring plant-associated and soil-associated flagellum-like gene clusters are sporadically distributed across Burkholderiales.
Fig. 6: Rapidly diversifying, high-copy-number Jekyll and Hyde plant-associated genes.
Fig. 7: Hyde1 proteins of Acidovorax citrulli AAC00-1 are toxic to E. coli and various plant-associated bacterial strains.

Change history

  • 05 April 2018

    In the version of this article initially published, owing to technical errors during production Supplementary Tables 2–26 were linked to the incorrect legends, and replacement files posted were corrupted. The errors have been corrected in the HTML version of the paper.

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Acknowledgements

The work conducted by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under contract no. DE-AC02-05CH11231. J.L.D. and S.G.T. were supported by NSF INSPIRE grant IOS-1343020, and J.L.D. was also supported by DOE–USDA Feedstock Award DE-SC001043 and by the Office of Science (BER), US Department of Energy, grant no. DE-SC0014395. S.H.P. was supported by NIH Training Grant T32 GM067553-06 and was a Howard Hughes Medical Institute (HHMI) International Student Research Fellow. D.S.L. was supported by NIH Training Grant T32 GM07092-34. J.L.D. is an Investigator of the HHMI, supported by the HHMI and the Gordon and Betty Moore Foundation (GBMF3030). M.E.F. was supported by NIH Dr. Ruth L. Kirschstein NRSA Fellowship F32-GM112345. D.A.P. and T.-Y.L. were supported by the Genomic Science Program, US Department of Energy, Office of Science, Biological and Environmental Research as part of the Oak Ridge National Laboratory Plant Microbe Interfaces Scientific Focus Area (http://pmi.ornl.gov) and Plant Feedstock Genomics Award DE-SC001043. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the US Department of Energy under contract DE-AC05-00OR22725. J.A.V. was supported by a SystemsX.ch grant (Micro2X) and a European Research Council (ERC) advanced grant (PhyMo). We thank I. Bertani, C. Bez, R. Bowers, D. Burstein, A. Chun Chen, D. Chiniquy, B. Cole, O. Cohen, A. Copeland, J. Eisen, E. Eloe-Fadrosh, M. Hadjithomas, O. Finkel, H. Schnitzel Meule Fux, N. Ivanova, J. Knelman, R. Malmstrom, R. Perez-Torres, D. Salomon, R. Sorek, T. Mucyn, R. Seshadri, T.K. Reddy, L. Ryan, and H. Sberro Livnat for general help, text editing, and ideas for this work. We thank R. Walcott (University of Georgia, Athens, GA, USA) for providing the Acidovorax citrulli VasD mutant strain.

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A.L. performed most data analysis and wrote the paper. I.S.G. performed phylogenetic inference, performed phylogenetically aware analyses, analyzed the data, provided the supporting website, and contributed to manuscript writing. M. Mittelviefhaus and J.A.V. designed and performed experiments related to Hyde1 gene function and contributed to manuscript writing. S.C. isolated single bacterial cells and prepared metadata for data analysis. F.M. analyzed data. S.H.P. analyzed data and contributed to manuscript writing. J.M. produced a mutant strain for Hyde1. K.W. tested Hyde1 toxicity inE. coli. G.D. and V.V. produced deletion mutants and designed and performed rice root colonization experiments. K.S. helped in data analysis. B.R.A. prepared metadata for data analysis. D.S.L., T.-Y.L., S.L., Z.J., M. McDonald, A.P.K., M.E.F., and S.L.D. isolated bacteria from different plants or managed this process. T.G.d.R. managed the sequencing project. S.R.G., D.A.P., and R.E.L. managed bacterial isolation efforts and contributed to manuscript writing. B.Z. managed Hyde1 deletion and toxicity testing. S.G.T. contributed to manuscript writing. T.W. managed single-cell isolation efforts and contributed to manuscript writing. J.L.D. directed the overall project and contributed to manuscript writing.

Corresponding authors

Correspondence to Susannah G. Tringe or Tanja Woyke or Jeffery L. Dangl.

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Competing interests

J.L.D. is a cofounder of and shareholder in, and S.H.P. collaborates with, AgBiome LLC, a corporation that aims to use plant-associated microbes to improve plant productivity.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–29 and Supplementary Note 1.

Life Sciences Reporting Summary

Supplementary Table 1

All genomes used. Lists of all genomes used from nine taxa (pre-filtration). Cells filled with yellow are Brassicaceae root isolates from the USA, cells filled with green are single cells isolated from Arabidopsis thaliana, cells filled with pink are poplar isolates, cells filled with blue are recently published leaf and root Arabidopsis and soil isolates from Europe, cells filled with purple are maize root isolates. “Filtered out?” column is ‘N’ if genome is retained for usage in analysis after QA process. “Representative genome taxid” – taxon id of another genome (different row in the same tab) representing at least two redundant genomes. Completeness and contamination values were calculated with CheckM. Full genome sequence, gene annotation, and metadata of each genome used can be found in the IMG website https://img.jgi.doe.gov/. For example the metadata of taxon id 2558860101 can be found in https://img.jgi.doe.gov/cgibin/mer/main.cgi?section=TaxonDetail&page=taxonDetail&ta xon_oid=2558860101.

Supplementary Table 2

Statistics of genomes in the taxa used

Supplementary Table 3

Sequencing and assembly information of new genomes

Supplementary Table 4

Abundance of the nine taxa in 16S marker gene surveys. The relative abundances of taxa composing a specific taxon were taken from the different publications and were added to yield the relative abundance of that taxon. In those cases with biological replicates, e.g. in Lundberg et al. Nature 2012 we used the median value.

Supplementary Table 5

Genome size comparison. Genome size comparison between the different isolation sites done by t-test and PhyloGLM. Each cell denotes the group with the largest genomes, if the difference is significant (P < 0.05). N.S. - not significant. PhyloGLM test takes into account the phylogenetic structure of the taxon.

Supplementary Table 6

COG-to-COG category mapping

Supplementary Table 7

Acinetobacter PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster inpfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used qvalue< 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn, we used Bonferroni-corrected P < 0.1.To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 8

Actinobacteria1 PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster in pfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used qvalue < 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn, we used Bonferroni-corrected P < 0.1. To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 9

Actinobacteria2 PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster in pfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used qvalue < 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn, we used Bonferroni-corrected P < 0.1. To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 10

Alphaproteobacteria PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster in pfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used q- value < 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn, we used Bonferroni-corrected P < 0.1. To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 11

Bacillales PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster in pfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn, we used Bonferroni-corrected P < 0.1. To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 12

Bacteroidetes PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster in pfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used qvalue < 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn, we used Bonferroni-corrected P < 0.1. To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 13

Burkholderiales PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster in pfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used qvalue < 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn, we used Bonferroni-corrected P < 0.1. To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 14

Pseudomonas PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster in pfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used qvalue < 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn, we used Bonferroni-corrected P < 0.1. To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 15

Xanthomonadaceae PA/NPA/RA/soil genes/domains. Phylogenetic diversity is the median pairwise distance between the genomes hosting the genes in the cluster. Values for each test are "Y", "N", or "Untested" (clusters were untested when there was insufficient phylogenetic signal, they were too small or were found in all genomes). To be considered as a significant cluster in pfam/COG/TIGRFAM/KO + hypergbin/hypergcn, we used qvalue < 0.05 (Benjamini Hochberg FDR corrected). To be considered as significant cluster in OrthoFinder + hypergbin/hypergcn we used Bonferroni-corrected P < 0.1. To be considered as a significant PA/RA cluster in phyloglmcn/phyloglmcn, we used q-value < 0.05 (Benjamini Hochberg FDR corrected) and an estimate > 0 (or estimate < 0 for significant NPA/soil). To be considered as a significant PA/RA cluster in Scoary, we used P < 0.05 for three tests: Fisher exact test (Benjamini Hochberg FDR corrected), worst pairing scenario test, and empirical test and odds ratio or Fisher exact test > 1 (odds ratio < 1 for NPA/soil).

Supplementary Table 16

Validation of PA/NPA/RA/soil genes through metagenomes. a. Samples used (n=38), b. Summary of results based on two sided t test.

Supplementary Table 17

Validation of PA genes in Paraburkholderia kururiensis M130. a. Mutant used and statistical tests results, b. Raw data: cfu/g root, 3. Primers used.

Supplementary Table 18

The number of operons predicted by different approaches.

Supplementary Table 19

Reproducible PA domains. a. Protein domains that are significantly PA in at least three taxa by at least two tests. NA – test results are not available (untested), NS – non-significant result. b. Fractions for LacI proteins within genomes, c. Fraction of pfam00248 domain within genomes.

Supplementary Table 20

DNA motifs predicted to be bound by LacI transcription factors. Predicted promoter sequences are intergenic sequences, at least 25 bp long, located upstream of carbohydrate metabolism and transport genes that are found directly adjacent to LacI genes. The most abundant kmers of different lengths were detected using wordcount (Emboss package). The most abundant motifs found in multiple taxa were compared against their distribution in random intergenic sequences using the Fisher exact test.

Supplementary Table 21

PREPARADOs. Pfam domains that are both significant PA/RA domains (reproducibly found as such in multiple taxa or by multiple approaches) and more abundant in plants than in bacteria according to Pfam (PREPARADOs). Pfams labeled in yellow are carbohydrate-related and are part of proteins found in eukaryotes and bacteria with full length sequence similarity, having an N-terminus signal peptide, and lacking a transmembrane domain. Cells marked in green are domains that are predicted to be secreted by Sec or T3SS (over >50% of the bacterial proteins having the domain are predicted to be secreted by these secretion systems).

Supplementary Table 22

Full-length proteins conserved between PA bacterial genes and eukaryotic genes. LAST alignment results of PREPARADO-containing proteins from bacteria (query) against plant, fungi, oomycetes, and protist proteins from Refseq (target). Only alignments that are over 40% identity and stretch across at least 90% of the query and target length are shown.

Supplementary Table 23

Jekyll and Hyde. Gene homologs of Jekyll and Hyde proteins based on protein homologs on IMG; To find all homologs and paralogs of Jekyll and Hyde genes (a-d) we used IMG blast search with e value threshold of 1e-5 against all IMG isolates, some of which were not included in the original comparartive analysis and hence their genes are not part of any cluster. Since Hyde1 proteins are rapidly evolving, they are scattered across multiple OrthoFinder orthogroups. Metadata in a-d was retrieved from IMG website. a. Jekyll protein homologs of Acidovorax gene Ga0102403_10160, b. Hyde1 protein homologs of Acidovorax protein Aave_1071, c. Hyde1-like protein homologs of Pseudomonas protein A243_06583, d. Hyde2 homologs of Ga0078621_123530, e. Hyde1-like-Hyde2 loci in representative Proteobacteria, one per genus, and their location adjacent to T6SS genes and within genomes that encode T6SS. Hyde2 was found based on blast search against the nr db with Acav_4635 as the query.

Supplementary Table 24

Divergence of Jekyll gene operon. An analysis of the Jekyll gene cluster that is presented in Figure 6b. Control genes are shown in Figure S26c. The table summarizes a comparison between multiple sequence alignments of the Jekyll locus (Figure S24b) and the control genes (Figure S24c).

Supplementary Table 25

Toxicity of Hyde proteins and recovery of prey cells confronted with Hyde-encoding Acidovorax and different mutants. Includes primers used to make Acidovorax deletion strains, strains used as prey and their antibiotic resistance, raw results for cell toxicity and competition assays.

Supplementary Table 26

Significant orthogroups (orthofinder clusters) supported by three statistical approaches: either hypergbin, phyloglmbin, and Scoary, or hypergcn, phyloglmcn, and Scoary

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Levy, A., Salas Gonzalez, I., Mittelviefhaus, M. et al. Genomic features of bacterial adaptation to plants. Nat Genet 50, 138–150 (2018). https://doi.org/10.1038/s41588-017-0012-9

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