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Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues

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Abstract

Proteins are the cell’s functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For Oryza sativa, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from Arabidopsis thaliana toward Oryza sativa, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on re-occurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUBUNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-of-the-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ.

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Abbreviations

AP–MS:

Affinity purification coupled to mass spectrometry

TAP:

Tandem affinity purification

APC10:

ANAPHASE PROMOTING COMPLEX SUBUNIT 10

CDKD:

CYCLIN-DEPENDENT KINASE D

PPI:

Protein–protein interaction

Y2H:

Yeast two-hybrid

ORF:

Open reading frame

PCA:

Protein complementation analysis

RING:

REALLY INTERESTING NEW GENE

CCS52A:

CELL CYCLE SWITCH PROTEIN 52 A

CAK:

CDK-activating kinase

CYCH:

CYCLIN H

MAT1:

MENAGE A TROIS

TFIIH:

General transcription factor II H

XPB:

XERODERMA PIGMENTOSUM B

XPD:

XERODERMA PIGMENTOSUM D

SBP:

Streptavidin-binding peptide

MS:

Mass spectrometry

TEV:

Tobacco etch virus

IgG:

Immunoglobulin G

RAP:

Rice annotation project

MSU:

Michigan State University

CBP:

Calmodulin-binding peptide

EGTA:

Ethylene glycol tetra-acetic acid

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Acknowledgments

Maarten Dedecker is supported by a predoctoral fellowship from the Agency for Innovation by Science and Technology and CropDesign N.V. (‘Baekeland’ funding, IWT 100222). The authors thank Annick Bleys for help in preparing the manuscript.

Author contributions

M.D., S.V. and G.D.J. designed the research. N.D.W., G.P., E.V.D.S, B.C., L.V., L.D. and N.W. performed experiments. M.D., D.E. and G.D.J. analyzed the data. K.G. provided protocols for LC–MS/MS analysis. J.V.L. and K.G. commented on the manuscript. M.D. and G.D.J. wrote the manuscript.

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Dedecker, M., Van Leene, J., De Winne, N. et al. Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues. Plant Mol Biol 91, 341–354 (2016). https://doi.org/10.1007/s11103-016-0471-x

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