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A plant genetic network for preventing dysbiosis in the phyllosphere

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Abstract

The aboveground parts of terrestrial plants, collectively called the phyllosphere, have a key role in the global balance of atmospheric carbon dioxide and oxygen. The phyllosphere represents one of the most abundant habitats for microbiota colonization. Whether and how plants control phyllosphere microbiota to ensure plant health is not well understood. Here we show that the Arabidopsis quadruple mutant (min7 fls2 efr cerk1; hereafter, mfec)1, simultaneously defective in pattern-triggered immunity and the MIN7 vesicle-trafficking pathway, or a constitutively activated cell death1 (cad1) mutant, carrying a S205F mutation in a membrane-attack-complex/perforin (MACPF)-domain protein, harbour altered endophytic phyllosphere microbiota and display leaf-tissue damage associated with dysbiosis. The Shannon diversity index and the relative abundance of Firmicutes were markedly reduced, whereas Proteobacteria were enriched in the mfec and cad1S205F mutants, bearing cross-kingdom resemblance to some aspects of the dysbiosis that occurs in human inflammatory bowel disease. Bacterial community transplantation experiments demonstrated a causal role of a properly assembled leaf bacterial community in phyllosphere health. Pattern-triggered immune signalling, MIN7 and CAD1 are found in major land plant lineages and are probably key components of a genetic network through which terrestrial plants control the level and nurture the diversity of endophytic phyllosphere microbiota for survival and health in a microorganism-rich environment.

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Fig. 1: Total and endophytic leaf microbiota in Col-0 and mfec plants.
Fig. 2: Endophytic leaf microbiota in Col-0, fec, min7 and mfec plants.
Fig. 3: Functional effect of SynComCol-0 and SynCommfec on plant health.
Fig. 4: Microbiota phenotypes in the ben3 mutant.

Data availability

Raw source 16S rRNA gene sequences from this project are available in the Sequence Read Archive database under BioProject PRJNA554246, accession numbers SAMN12259846 to SAMN12260169. Bacterial genome source data are available in the Sequence Read Archive database under the BioProject PRJNA555902. Source Data for Figs. 14 and Extended Data Figs. 3, 4, 68 are provided with the paper.

Code availability

Scripts used in the microbiota analyses are available at https://github.com/godlovexiaolin/A-genetic-network-for-host-control-of-phyllosphere-microbiota-for-plant-health. All other software used in this study are cited in the text.

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Acknowledgements

We thank members, including B. Hsueh, of the He laboratory for technical help and insightful discussions; T. Gong for help with testing leaf necrosis phenotypes of plants grown on MS agar plates; and H. Tanaka and J. Friml for ben3/cad1S205Fseeds. This project was supported by funding from National Institutes of Health (GM109928), the Department of Energy (the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science; DE–FG02–91ER20021 for ben3/cad1S205F mutant characterization) and Plant Resilience Institute at Michigan State University for support of optimization of the GnotoPot system (to S.Y.H.) and by funding from the CAS Center for Excellence in Molecular Plant Sciences and the Institute of Plant Physiology and Ecology, Chinese Academy of Sciences (to X.-F.X.).

Author information

Affiliations

Authors

Contributions

X.-F.X. and S.Y.H. conceptualized, designed the experiments and co-supervised the project. T.C. and K.N. performed most of experiments; X.-F.X. performed initial 16S sequencing set up and sample collection while at Michigan State University; R.S. performed GnotoPot experiments; X.W. performed 16S bioinformatics analysis; J.X. performed bacterial genome analysis; L.Y. performed the MS plate assay for Col-0 and the mfec mutant; B.C.P. performed 16S bioinformatics analysis. L.M. was involved in cad1-related experiments; J.K. was involved in initial 16S RNA gene sequencing design; Y.C. was involved in mapping the cad1 mutation; L.Z. performed phylogenetic analysis of CAD1 and MIN7 genes and advised on statistical analyses; N.W. and E.W. advised on bioinformatics and statistical analyses. T.C., X.-F.X. and S.Y.H. wrote the manuscript with input from all co-authors. X.W. and R.S. contributed equally as co-second authors.

Corresponding authors

Correspondence to Xiu-Fang Xin or Sheng Yang He.

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The authors declare no competing interests.

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Peer review information Nature thanks Steven Lindow and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Leaf and root appearance of soil-grown Col-0 and mfec plants.

a, Leaf appearance of Col-0 and mfec plants grown in Arabidopsis mix soil (soil A) and Michigan State University (MSU) community agricultural soil (soil B; equal parts MSU community soil, medium vermiculate and perlite) or organic seed starter premium potting mix (Espoma) (soil C) for 6.5 weeks. Images were taken 5 days (soil A) or 11 days (soil B and soil C) after plants were shifted to high humidity (~95%). Representative leaf images are shown. b, Root appearance of Col-0 and mfec plants grown in Arabidopsis mix soil for five weeks and shifted to high humidity (~95%) for 5 days. Representative root images are shown. Experiments in a, b, were repeated three times with similar results.

Extended Data Fig. 2 Observed OTUs of total and endophytic leaf bacteria in different plant genotypes and requirement of microbiota for appearance of dysbiosis symptoms in mfec leaves.

a, b, Observed OTUs of total (a) and endophytic leaf bacteria (b) in Col-0 and mfec plants, which were grown in Arabidopsis mix soil and shifted to high humidity for 5 days. c, Observed OTUs of endophytic leaf microbiota in Col-0, fec, min7 and mfec plants supplemented with SynComCol-0. In box plots, the centre line represents the median, box edges show the 75th and 25th percentiles, and whiskers extend to 1.5× the interquartile range. Two-tailed Mann–Whitney U-test. n = 15 (Col-0) and n = 15 (mfec) biological replicates passing quality control for analysis of total leaf bacterial microbiota across 3 independent experiments; n = 18 (Col-0) and n = 20 (mfec) biological replicates passing quality control for analysis of leaf endophytic bacterial microbiota across 4 independent experiments. n = 20 (Col-0), n = 19 (fec), n = 19 (min7) and n = 19 (mfec) biological replicates passing quality control for analysis of leaf endophytic bacterial microbiota with SynComCol-0 across 4 independent experiments. d, Leaf appearance of Col-0 and mfec plants grown in sterile MS agar plates. Pictures were taken 5 days after shifting plates to high humidity (~95%). e, Leaf appearance of Col-0 and mfec plants grown in GnotoPots in the absence (axenic) or presence of SynComCol-0 for 6.5 weeks. Plants were then shifted to high humidity (~95%) for 10 days, before images were taken. Rosette leaf images are representative of at least four replicated experiments.

Extended Data Fig. 3 A Maximum-likelihood phylogenetic tree for genome-sequenced bacterial isolates in SynComCol-0 and SynCommfec.

a, Tree was constructed on the basis of the full-length 16S rRNA gene using MEGA7. A total of 100 bootstrap replicates were made, and bootstrap values are indicated at the branch points. Colours represent bacterial isolates from different plant genotypes: mfec mutant (purple); Col-0 (green). In total, 48 strains were derived from healthy Col-0 endophytic leaves and 52 strains were derived from mfec endophytic leaves displaying dysbiosis symptoms. b, Col-0 leaves were syringe-infiltrated with SynComCol-0 and SynCommfec at 1 × 107 CFU ml−1; infiltrated plants were kept under ambient humidity for 1 h for water to evaporate. Bacterial populations were then determined after plant leaves returned to pre-infiltration appearance. Colony-forming units were normalized to tissue fresh weight (left) and leaf disk areas (right). Statistical significance was determined by two-tailed Mann–Whitney U-test. n = 6 biological replicates, data are mean ± s.e.m. Experiments were repeated three times with similar results. c, Col-0 plants were syringe-infiltrated with SynComCol-0, SynCommfec or SynComCol-0-38 (with 10 Firmicutes removed from SynComCol-0) at 1 × 107 CFU ml−1. Inoculated plants were kept under high humidity (~ 95%), and leaf images were taken 7 days after infiltration. Experiments were repeated three times with similar results. Images are representative of leaves from four plants. Source Data

Extended Data Fig. 4 Multiplication- and dysbiosis-symptom phenotypes of bacterial strains in Col-0 leaves.

a, Population sizes (log10CFU/cm2 leaf area) of bacterial strains in Col-0 leaves on day 0 (1 h after leaf infiltration) and day 5 after leaf infiltration with each strain at 1 × 106 CFU ml−1. The experiment was carried out at ~95% humidity. DC3000, Pst DC3000 (pathogenic on Col-0 plants); hrcC, a nonpathogenic mutant of DC3000 defective in type III secretion; Col-0-33, mfec-20 and mfec-1, control strains that do not induce dysbiosis symptoms (Supplementary Table 1); other mfec strains, induce dysbiosis symptoms (Supplementary Table 1). Statistical analysis was performed by two-way ANOVA with Tukey’s test. n = 3 biological replicates, data are mean ± s.e.m. Experiments were repeated twice with similar results. b, Leaf dysbiosis symptoms 7 days after infiltration of leaves of 4.5-week-old Col-0 plants with indicated mfec strains or SynCommix 5 at 1 × 107 CFU ml−1. The experiment was carried out at ~95% humidity. SynCommix 5 is a mix of mfec-10, mfec-23, mfec-41, mfec-48 or mfec-51 with equal OD600 values. Experiments were repeated three times with similar results. Source Data

Extended Data Fig. 5 Binary inter-bacterial inhibition.

a, Examples of inhibitory halos are labelled with strong, weak or no inhibition. b, Binary inhibition assays (2,116 combinations) on a R2A plate of 46 strains that represent all bacterial species identified in SynComCol-0 and SynCommfec. Target bacterial strains are presented along the horizontal axis, whereas attacker bacterial strains are listed vertically. A large or clear halo, indicative of strong binary inhibition, is represented by a red-filled cell; a small or less transparent halo, indicative of weaker binary inhibition, is represented by a pink-filled cell; the absence of halo is represented in white. Strains labelled with a star were used for the in planta binary inhibition assay in Extended Data Fig. 6. Experiments were repeated three times with similar results.

Extended Data Fig. 6 In planta binary inhibition.

a, In planta inhibition of Firmicutes by Proteobacteria strains that displayed a strong inhibitory effect in R2A agar plate assay. Leaves of Col-0 plants were syringe-infiltrated with Paenibacillus chondroitinus (C3; a Firmicutes) alone, Comamonas testosteroni (C13, a Proteobacterium) alone or C3 and C13 together at 1 × 104 CFU ml−1, corresponding to approximately 1 × 102 CFU cm−2 leaf area; or 1 × 106 CFU ml−1, corresponding to approximately 1 × 104 CFU cm−2 leaf area. After infiltration plants were maintained under high humidity (~95%) for 5 days before bacterial populations (log10CFU/cm2 leaf area) were determined. b, Similar to a, but with a non-inhibitory binary interaction between strains C3 and Variovorax sp. C52 (a Proteobacterium). c, Leaves of Col-0 plants were syringe-infiltrated with P. chondroitinus (C3; a Firmicutes) alone, Stenotrophomonas maltophilia (C45, a Proteobacterium) alone or C3 and C45 together at 1 × 104 CFU ml−1 or 1 × 106 CFU ml−1. d, Leaves of Col-0 plants were syringe-infiltrated with P. chondroitinus (C41; a Firmicutes) alone, C. testosteroni (C13, a Proteobacterium) alone or C41 and C13 together at 1 × 104 CFU ml−1 or 1 × 106 CFU ml−1. After infiltration, plants were maintained under high humidity (~95%) for 5 days before bacterial populations were determined. One-way ANOVA with Tukey’s test. n = 4 biological replicates, data are mean ± s.e.m. Experiments were repeated three times with similar results. Source Data

Extended Data Fig. 7 Appearance and bacterial populations in Col-0 and cad1S205F plants before and after shift to 95% humidity.

a, Leaf appearance of 5-week-old Col-0 and cad1S205F plants grown in the absence (axenic) or presence of SynComCol-0 in the FlowPot gnotobiotic system (see Methods). Images were taken before (day 0) and 5 days after plants were shifted to high humidity (~95%). b, Levels of endophytic bacterial community (log10CFU/cm2 leaf area) in the presence of SynComCol-0 in the FlowPot gnotobiotic system. One-way ANOVA with Tukey’s test. Data are mean ± s.e.m., n = 6 biological replicates. Experiments were repeated three times with similar results. c, Leaf appearance of Col-0 and cad1S205F plants grown in enclosed sterile LS agar plates for 4 weeks. d, Leaf appearance of 5-week-old Col-0, deps and cad1S205F plants grown in Arabidopsis mix 5 days after plants were shifted to ~95% relative humidity. e, Levels of endophytic leaf microbiota (log10CFU/cm2 leaf area) in 5-week-old Col-0, deps and cad1S205F plants 5 days after plants were exposed to high humidity (~95%). One-way ANOVA with Tukey’s test. Data are mean ± s.e.m., n = 4 biological replicates. Experiments were repeated three times with similar results. Source Data

Extended Data Fig. 8 Identification of a cad1 mutation responsible for dysbiosis in the cad1 mutant.

a, Leaf appearance of 4.5-week-old Col-0, cad1S205F and big2 plants grown in redi-earth potting soil. Images were taken at day 5 after plants were shifted to 95% humidity. b, Bacterial populations (log10CFU/cm2 leaf area) of the endophytic bacterial community. One-way ANOVA with Tukey’s test. Data are mean ± s.e.m., n = 6 biological replicates. Experiments were repeated three times with similar results. Two independent T-DNA insertion lines of BIG2 were analysed with similar results (big2-1, SALK_033446 and big2-2, SALK_016558). c–e, Appearance (c) and endophytic bacterial populations (d; log10CFU/cm2 leaf area) in Col-0, cad1S205F and cad1S205F/35S::CAD1 plants at day 5 after plants were shifted to high humidity. Plants were grown in redi-earth potting soil for 4.5 weeks before they were shifted to high humidity. One-way ANOVA with Tukey’s test. Data are mean ± s.e.m., n = 6 biological replicates. Experiments were repeated three times with similar results. e, Two independent different complementation lines (cad1S205F/35S::CAD1 line 1 and cad1S205F/35S::CAD1 line 2) were analysed with similar results and protein levels were confirmed by western blot with the CAD1 antibody. Uncropped gel image is shown in Supplementary Fig. 2. f, cad1S205F genomic mapping. Green and brown dots indicate wild type-like and cad1S205F-like allele frequencies, respectively (detailed information in Supplementary Table 6). g, Schematic of mutations in big2 and cad1S205F mutants. h, i, Quantitative PCR analyses of CAD1 transcript in Col-0 (h) and min7 (i) plants grown in Arabidopsis mix soil. Five-week-old Col-0 and min7 leaves were infiltrated with 1 μM flg22 and collected at the indicated time points. Transcript levels were normalized to that of the PP2AA3 gene. One-way ANOVA with Tukey’s test. Data are mean ± s.e.m., n = 3 biological replicates. Experiments were repeated three times with similar results. Source Data

Extended Data Fig. 9 A model for plant control of endophytic phyllosphere microbiota.

a, The 16S rRNA gene-sequence profiles of endophytic leaf bacteria in Col-0 and cad1S205F plants supplemented with SynComCol-0. Data presentation and statistical analysis as in Fig. 1d. n = 20 (Col-0) and n = 20 (cad1S205F) biological replicates. b, A simplified diagram depicting pattern-triggered immune signalling, MIN7 and CAD1 as three components of a putative genetic framework for controlling endophytic bacterial microbiota, which live outside a plant cell. MIN7 has previously been shown to be involved in regulating callose deposition51,52 and aqueous microenvironment in the leaf apoplast (that is, extracellular space)1. c, Large shifts in the level and composition of endophytic leaf microbiota in wild-type Col-0 versus mfec (or cad1S205F) leaves in part via competition between Proteobacteria and Firmicutes. Some components in b and c were drawn using tools in biorender.com.

Extended Data Fig. 10 Phylogenetic trees of protein sequences of MIN7 and CAD1 homologues from different plant species.

a, b, Protein sequences of A. thaliana AtMIN7 (also known as AtBIG5) (AT3G43300.1) (a) and AtCAD1 (also known as AtNSL2) (AT1G29690.1) (b) were used for comparisons by Blast search against the proteome of Arabidopsis and seven other plant species (https://phytozome.jgi.doe.gov/). Homologues with E values lower than E100 were selected to generate phylogenetic trees across taxa, and only homologues specific to the AtMIN7 or AtCAD1 clade were presented with selected proteins from Arabidopsis as outgroups. Bootstrap values were obtained from 1,000 replicates using the maximum-likelihood algorithm using MEGA7. The scale bar represents 0.2 substitutions per amino acid site. The genes are listed in Supplementary Table 7. AtMIN7 and AtCAD1 are highlighted with red stars. Abbreviations: BIG, BREFELDIN A-INHIBITED GUANINE NUCLEOTIDE-EXCHANGE PROTEIN; NSL, NECROTIC SPOTTED LESIONS; At, A. thaliana; Mp, Marchantia polymorpha; Os, Oryza sativa; Pp, Physcomitrella patens; Pt, Populus trichocarpa; Sm, Selaginella meollendorffii; Sl, Solanum lycopersicum; Zm, Zostera marina.

Supplementary information

Supplementary Information

Supplementary Discussion. A discussion on the cad1 mutant phenotypes and involvement of other plant processes such as plant defense hormones, in microbiota assembly and/or dysbiosis.

Reporting Summary

48-member SynCom

Supplementary Table 1Col-0 derived from healthy wild-type Col-0 leaves and 52-member SynCommfec derived from mfec leaves displaying dysbiosis symptoms. This table describes SynCom strains, their taxonomical names and their ability or inability to induce dysbiosis symptoms.

Differential ASVs of endophytic phyllosphere microbiota between Col-0 and

Supplementary Table 2 mfec mutant plants. This table describes relative abundance of amplicon sequence variants (ASVs) representing unique bacterial 16S rRNA gene sequences in Col-0 vs. mfec mutant plants.

Raw sequencing data and genome assembly for SynCom

Supplementary Table 3Col-0 and SynCommfec bacterial strains. This table describes a summary of raw sequence data and assembly information about the genomes of SynCom strains.

Taxonomic information for SynCom

Supplementary Table 4Col-0 and SynCommfec bacterial strains based on average nucleotide identity (ANI) with references, phylogenetic tree using 120 marker genes, and 16S rRNA gene. This table summarizes molecular analyses that lead to taxonomical assignment of SynComCol-0 and SynCommfec bacterial strains.

The ANI values between the strains within the same genus for the SynCom

Supplementary Table 5Col-0 and SynCommfec bacterial genomes. This table shows genome-wide average nucleotide identity (ANI) between SynComCol-0 and SynCommfec strains within the same genus.

Mutations in chromosome 1 surrounding the

Supplementary Table 6 CAD1 gene in the ben3(cad1S205F) mutant. This table shows single nucleotide polymorphisms between Col-0 and the cad1S205F mutant in chromosome 1.

Supplementary Table 7

Protein list for generating MIN7 and CAD1 phylogenetic trees. This table lists proteins used to generate MIN7 and CAD1 phylogenetic trees.

Supplementary Figure 1

This figure contains Source data for Fig. 4d and 4e.

Supplementary Figure 2

This figure contains Source data for Fig. 8e.

Source data

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Chen, T., Nomura, K., Wang, X. et al. A plant genetic network for preventing dysbiosis in the phyllosphere. Nature 580, 653–657 (2020). https://doi.org/10.1038/s41586-020-2185-0

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