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Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research

Abstract

The complex structure and function of a plant microbiome are driven by many variables, including the environment, microbe–microbe interactions and host factors. Likewise, resident microbiota can influence many host phenotypes. Gnotobiotic growth systems and controlled environments empower researchers to isolate these variables, and standardized methods equip a global research community to harmonize protocols, replicate experiments and collaborate broadly. We developed two easily constructed peat-based gnotobiotic growth platforms: the FlowPot system and the GnotoPot system. Sterile peat is amenable to colonization by microbiota and supports growth of the model plant Arabidopsis thaliana in the presence or absence of microorganisms. The FlowPot system uniquely allows one to flush the substrate with water, nutrients and/or suspensions of microbiota via an irrigation port, and a mesh retainer allows for the inversion of plants for dip or vacuum infiltration protocols. The irrigation port also facilitates passive drainage, preventing root anoxia. In contrast, the GnotoPot system utilizes a compressed peat pellet, widely used in the horticultural industry. GnotoPot construction has fewer steps and requires less user handling, thereby reducing the risk of contamination. Both protocols take up to 4 d to complete with 4–5 h of hands-on time, including substrate and seed sterilization. In this protocol, we provide detailed assembly and inoculation procedures for the two systems. Both systems are modular, do not require a sterile growth chamber, and cost less than US$2 per vessel.

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Fig. 1: Schematic illustration of the FlowPot system.
Fig. 2: Schematic illustration of the GnotoPot system.
Fig. 3: A. thaliana grown in FlowPots with axenic or holoxenic substrate.
Fig. 4: A. thaliana grown in GnotoPots with axenic or holoxenic substrate.
Fig. 5: Application for microbiota function studies in planta.
Fig. 6: Application for microbial community studies.

Data availability

All data are presented in this paper are available from the authors without restrictions. Information about the input soil microbial communities is available in Supplementary Table 1. Raw source 16S rRNA gene sequences from this project (for Fig. 6 and Supplementary Table 2) are available in the Sequence Read Archive database under BioProject PRJNA689857, accession numbers SAMN17220890 to SAMN17220933.

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Acknowledgements

We thank C. Griffin, A. Bonifer, A. Mundakkal, F. Dion, T. Ulrich, J. Martz and T. Johnson for assistance with FlowPot assembly and workflow optimization, M. A. Hassani and S. Hacquard for critical reading and helpful comments on the manuscript, and B. Kvitko and J. P. Jerome for their contributions to FlowPot growth system development. Figure 2 was partially created with Biorender.com. This project was supported by funding from the Gordon and Betty Moore Foundation (GBMF3037), the National Institutes of Health (GM109928) and the Plant Resilience Institute, Michigan State University. P.S.-L. was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) in the Priority Programme SPP 2125 DECRyPT.

Author information

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Authors

Contributions

J.M.K., J.T. and S.Y.H. designed the FlowPot experiments. R.S. and S.Y.H. designed the GnotoPot experiments. J.M.K., R.S., B.P., D.R., C.T. and J.F. performed and/or analyzed the experiments. P.S.-L. supervised J.M.K. during FlowPot protocol optimization at Max Planck Institute for Plant Breeding Research, Cologne. J.M.K., R.S., B.P. and S.Y.H. wrote and finalized the manuscript with input from all coauthors.

Corresponding authors

Correspondence to James M. Tiedje or Sheng Yang He.

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Competing interests

The FlowPot system has the following patent: Methods and apparatus for gnotobiotic plant growth, J. Kremer, J. M. Tiedje, S. Y. He, US Patent 10,645,886 (2020). However, this patent did not influence the writing of this paper.

Additional information

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

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Related links

Key references using this protocol

Durán, P. et al. Cell 175, 973–983 (2018): https://doi.org/10.1016/j.cell.2018.10.020

Chen, T. et al. Nature 580, 653–657 (2020): https://doi.org/10.1038/s41586-020-2185-0

Supplementary information

Supplementary Information

Supplementary Methods.

Reporting Summary

Supplementary Table 1

Geographic locations of soils used in this study

Supplementary Table 2

OTU information for the example community profiling workflow used to generate the heatmap in Fig. 6

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Kremer, J.M., Sohrabi, R., Paasch, B.C. et al. Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research. Nat Protoc 16, 2450–2470 (2021). https://doi.org/10.1038/s41596-021-00504-6

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