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Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase

Abstract

The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.

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Fig. 1: Identification of TOR-dependent phosphorylation sites by quantitative phosphoproteomics.
Fig. 2: Phosphopeptide intensity plots uncovering diverse classes of TOR-dependent phosphorylation patterns.
Fig. 3: Detection of known and novel TOR-regulated phosphoproteins in plants.
Fig. 4: TOR protein complex analysis by pull-down.
Fig. 5: Integration of phosphoproteomics and interactomics pinpoints candidate substrates of the TOR and S6K1 kinases.
Fig. 6: Selection of the TOR signalling network, revealing upstream and downstream TOR signalling events.

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Code availability

The custom code used for the phosphosite conservation analysis is available on request from the corresponding author.

Data availability

The data that support the findings of this study are available from the corresponding author on reasonable request. The protein interactions from this publication have also been deposited to the IMEx consortium (http://www.imexconsortium.org) through IntAct and assigned the identifier IM-26172. The MS phosphoproteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD008355. The Cytoscape file covering the integrated TOR kinase signalling network as well as all discussed subnetworks is available as Supplementary Dataset.

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Acknowledgements

We thank A. Bleys for help in preparing the manuscript, K.-i. Hayashi (Okayama University of Science, Japan) for providing PEO-IAA, M. Vuylsteke for help with statistical analyses and D. Inzé, S. Vanneste, N. Besbrugge and M. Bontinck for the fruitful discussions. C.H. was funded by a Belspo Grant.

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Authors and Affiliations

Authors

Contributions

J.V.L. wrote the manuscript. J.V.L., C.H., A.G., D.E., I.D.S. and G.D.J. conceived the research. J.V.L., C.H., A.G., B.C., C.M., N.D.W., G.P., E.V.D.S. and B.V.d.C. performed the experiments. D.E., F.I. and K.G. performed the MS data analysis. V.S. performed the statistical analysis of the phosphoproteome. M.V.B. and K.V. analysed the phosphosite conservation. E.S. helped with the phosphoproteomics protocol.

Corresponding author

Correspondence to Geert De Jaeger.

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The authors declare no competing interests.

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Journal peer review information: Nature Plants thanks Leslie Hicks and Christian Meyer and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary information

Supplementary Figures 1–8, Supplementary Table Legends, Supplementary Dataset Legends, Supplementary Notes, Supplementary Methods and Supplementary References.

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Supplementary Table 1

Supplementary Tables 1a–e.

Supplementary Table 2

Supplementary Tables 2a–e.

Supplementary Table 3

Supplementary Tables 3a and b.

Supplementary Table 4

Supplementary Tables 4a–d.

Supplementary Table 5

Supplementary Tables 5a and b.

Supplementary Table 6

List of oligos used for cloning.

Supplementary Dataset 1

Cytoscape file corresponding to the integrated TOR kinase signalling network.

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Van Leene, J., Han, C., Gadeyne, A. et al. Capturing the phosphorylation and protein interaction landscape of the plant TOR kinase. Nat. Plants 5, 316–327 (2019). https://doi.org/10.1038/s41477-019-0378-z

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