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Obligate biotroph downy mildew consistently induces near-identical protective microbiomes in Arabidopsis thaliana

Nature Microbiology volume 8, pages 2349–2364 (2023)Cite this article

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Abstract

Hyaloperonospora arabidopsidis (Hpa) is an obligately biotrophic downy mildew that is routinely cultured on Arabidopsis thaliana hosts that harbour complex microbiomes. We hypothesized that the culturing procedure proliferates Hpa-associated microbiota (HAM) in addition to the pathogen and exploited this model system to investigate which microorganisms consistently associate with Hpa. Using amplicon sequencing, we found nine bacterial sequence variants that are shared between at least three out of four Hpa cultures in the Netherlands and Germany and comprise 34% of the phyllosphere community of the infected plants. Whole-genome sequencing showed that representative HAM bacterial isolates from these distinct Hpa cultures are isogenic and that an additional seven published Hpa metagenomes contain numerous sequences of the HAM. Although we showed that HAM benefit from Hpa infection, HAM negatively affect Hpa spore formation. Moreover, we show that pathogen-infected plants can selectively recruit HAM to both their roots and shoots and form a soil-borne infection-associated microbiome that helps resist the pathogen. Understanding the mechanisms by which infection-associated microbiomes are formed might enable breeding of crop varieties that select for protective microbiomes.

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Fig. 1: Distinct Hpa cultures are enriched for identical ASVs that dominate the phyllosphere bacterial communities.
Fig. 2: Hpa cultures from Utrecht and Cologne are enriched for identical ASVs.
Fig. 3: Isogenic HAM bacterial genomes are present in metagenomes of geographically separated Hpa cultures.
Fig. 4: HAM ASV abundances diminish in the absence of Hpa.
Fig. 5: HAM bacteria benefit from the presence of Hpa in a gnotobiotic system and can reduce disease.
Fig. 6: HAM ASVs are selectively promoted in response to Hpa infection and associated with soil-borne legacy.

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Data availability

The data that support the findings of this study and isolates are available from the corresponding author upon reasonable request. Moreover, the raw amplicon sequence data generated by this study are available at https://www.ncbi.nlm.nih.gov/sra/PRJNA944652; raw WGS data are available at http://www.ncbi.nlm.nih.gov/bioproject/1011197; genome assemblies generated in this study are available at http://www.ncbi.nlm.nih.gov/bioproject/1011284. Whenever possible, post-processing amplicon sequencing (ASV) count tables are also included together with the processing code at the Zenodo-archived GitHub page (https://doi.org/10.5281/zenodo.8307753). Source data are provided with this paper.

Code availability

The code used to analyse data and generate figures can be found at https://doi.org/10.5281/zenodo.8307753. No unpublished algorithms or methods were used.

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Acknowledgements

This study was sponsored by the TopSector Horticulture and Starting Materials (TKI grant number 1605-106), the Dutch Research Council (NWO) through the Gravitation programme MiCRop (grant number 024.004.014) and the XL programme ‘Unwiring beneficial functions and regulatory networks in the plant endosphere’ (grant number OCENW.GROOT.2019.063). The TKI project was carried out in collaboration with four industrial partners; DSM, Enza Zaden, Pop Vriend Seeds and RijkZwaan Breeding B.V. We thank J. Parker for providing Col-0 RPP5 and Ler rpp5 Arabidopsis seeds, P. Bakker and R. de Jonge for valuable input on experimental designs and bioinformatic approaches, and J. Elberse, D. Duijker, L. Pronk, C. Molina Ruiz, M. Alderkamp, X. Pan, L. Wagenaar and T. Tarrant for excellent technical assistance.

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Author notes

  1. These authors contributed equally: Pim Goossens, Jelle Spooren.

Authors and Affiliations

  1. Plant–Microbe Interactions, Institute of Environmental Biology, Department of Biology, Science4Life, Utrecht University, Utrecht, the Netherlands

    Pim Goossens, Jelle Spooren, Kim C. M. Baremans, Nakisa Echobardo, Corné M. J. Pieterse & Roeland L. Berendsen

  2. Translational Plant Biology, Institute of Environmental Biology, Department of Biology, Science4Life, Utrecht University, Utrecht, the Netherlands

    Annemiek Andel, Dmitry Lapin & Guido Van den Ackerveken

  3. Plant–Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany

    Dmitry Lapin

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Contributions

P.G., J.S., G.A., C.M.J.P. and R.L.B. designed the experiments and wrote the paper. R.L.B., G.A. and C.M.J.P. supervised the project and edited the paper. P.G. performed the experiments and data analysis shown in Figs. 1–4. D.L. performed the experiments in Cologne, Germany (Fig. 2). P.G. and N.E. performed the experiments shown in Fig. 5a,b. J.S. performed the experiments shown in Fig. 5c–e. P.G. and J.S. performed the experiments and data analysis shown in Fig. 6. K.C.M.B. and A.A. provided technical support during the conduct of the experiments and in the maintenance of Hpa and gnoHpa cultures.

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Correspondence to Roeland L. Berendsen.

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Extended data

Extended Data Fig. 1 Effect of water- and wind-transmitted Hpa infections on bacterial phyllosphere community structure.

(a) Quantification of Hpa DNA relative to Arabidopsis DNA by qPCR for samples taken 7 days post-inoculation. Bars represent average Hpa abundance. Error bars show standard error. N = 7 biologically independent samples. PCoA ordination plot based on Bray-Curtis dissimilarities of the (b) bacterial phyllosphere communities and (c) fungal phyllosphere communities of Arabidopsis thaliana Col-0 untreated control plants (blue symbols), or plants inoculated with Hpa spores via wind (orange stroke) or water (orange fill).

Source data

Extended Data Fig. 2 Compatibility of Noco2 and Cala2 with susceptible and resistant Arabidopsis accessions.

qPCR quantification of Hpa abundance in the susceptible and resistant Hpa-Arabidopsis interactions indicated, confirming that C24 is resistant to both Noco2 and Cala2, that Col-0 is susceptible to Noco2, that Ler is susceptible to Cala2, and that Pro-0 is susceptible to both Noco2 and Cala2. qPCR quantification was performed on total genomic DNA from inoculated leaves that were also used for 16 S rDNA amplicon sequencing (Fig. 1). Hpa abundance was calculated as a ratio of the levels of ACTIN in Hpa and Arabidopsis. Bars represent average ratios, error bars represent standard error. N = 4 biologically independent samples, except for C24 mock-, Noco2-, and Cala2- treated (N = 3 biologically independent samples).

Source data

Extended Data Fig. 3 Schematic overview of the ‘9-passages experiment’, which tests the effect of the removal of Hpa on its associated microbiome.

A uniform HAM (uHAM) containing a mix of of Noco2 and Cala2 spore suspensions was spray-inoculated on different Arabidopsis genotypes to selectively remove Noco2 and/or Cala2 from the microbiome (HAM) that travels together with these Hpa isolates upon passaging to new host plants. One week post-inoculation, Col-0 (Lineage 1) and Ler plants (Lineage 2) sporulated with Noco2 and Cala2, respectively, and Col-0/RPP5 transgenic plants (Lineage 3) did not sporulate. From each Arabidopsis genotype, a leaf wash-off was obtained, containing Noco2, Cala2, or no Hpa, and sprayed on eight pots containing small fields of Ler/rpp5 mutant plants, which are susceptible to both Noco2 and Cala2. All pots were then placed in individual plastic containers, to prevent cross-contamination between pots. One week post-inoculation, the Ler/rpp5 plants that were inoculated with Noco2 (Lineage 1) or Cala2 (Lineage 2), sporulated, while the Ler/rpp5 plants that were inoculated with the leaf wash-off without Hpa spores (Lineage 3) did not display disease symptoms. From each individual pot, the leaf wash-off was sprayed on a new pot containing Ler/rpp5 plants, thereby propagating eight separate phyllosphere microbiomes or Hpa cultures per lineage. This process was maintained for nine consecutive weeks, allowing eight separate lineages of Noco2, Cala2, or the uHAM without Hpa to develop independently. Eight untreated control pots with Ler/rpp5 plants were included for all planting cycles.

Extended Data Fig. 4 The Hpa-culture bacterial community is largely unaffected by removal of Hpa, but nonetheless there are community shifts in the absence of Hpa.

PcoA plots based on Bray-Curtis dissimilarities of (a) all samples from Lineages 1–3 and untreated plants of passages 1 (circles), passage 5 (triangles) and passage 9 (diamonds); and of all inoculated samples of Lineage 1–3 from (b) passage 1, (c) passage 5, and (d) passage 9. Plants were left untreated (black symbols) or were inoculated with leaf wash-offs from Lineage 1 containing Noco2 (orange symbols), from Lineage 2 containing Cala2 (green symbols), or from Lineage 3 which remained Hpa free (blue symbols).

Extended Data Fig. 5 Setup of soil-borne legacy experiments.

A conditioning population of two-week-old Arabidopsis thaliana Col- seedlings was inoculated with mock, Hpa Noco2 or gnoHpa Noco2. After one week of infection, shoots were cut-off and a response population of plants was directly sown on the conditioned soil and again mock- Hpa- or gnoHpa-inoculated. Figure created with BioRender.com.

Supplementary information

Supplementary Information

Supplementary Figs. 1–18 and Tables 1–11.

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Source data

Source Data Fig. 1

(Grouped) ASV abundance data.

Source Data Fig. 2

(Grouped) ASV abundance data and ASV-of-interest abundance data.

Source Data Fig. 4

ASV abundance data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Source data.

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Goossens, P., Spooren, J., Baremans, K.C.M. et al. Obligate biotroph downy mildew consistently induces near-identical protective microbiomes in Arabidopsis thaliana. Nat Microbiol 8, 2349–2364 (2023). https://doi.org/10.1038/s41564-023-01502-y

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