Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

被引：0

作者：

Kenneth Wayne Berendzen

Maik Böhmer

Niklas Wallmeroth

Sébastien Peter

Marko Vesić

Ying Zhou

Franziska KatharinaElisabeth Tiesler

Frank Schleifenbaum

Klaus Harter

机构：

[1] Universität Tübingen,

[2] ZMBP,undefined

[3] Plant Physiology,undefined

[4] University of California,undefined

[5] San Diego,undefined

[6] Division of Biological Sciences,undefined

[7] Cell and Developmental Biology Section,undefined

[8] Universität Tübingen,undefined

[9] ZMBP,undefined

[10] Biophysical Chemistry,undefined

来源：

Plant Methods | / 8卷

关键词：

FACS; BiFC; Protein-protein interaction screen; CPK3;

D O I：

暂无

中图分类号：

学科分类号：

摘要：

Understanding protein and gene function requires identifying interaction partners using biochemical, molecular or genetic tools. In plants, searching for novel protein-protein interactions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel protein partners in living plant cells. We demonstrate that it is possible to screen for novel protein-protein interactions from a random library in protoplasted Arabidopsis plant cells and recover some of the interacting partners. Our screen is based on capturing the bi-molecular complementation of mYFP between an YN-bait fusion partner and a completely random prey YC-cDNA library with FACS. The candidate interactions were confirmed using in planta BiFC assays and in planta FRET-FLIM assays. From this work, we show that the well characterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3, HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This is one of the first randomin planta screens to be successfully employed.

引用

共 287 条

[1] Braun P(2009)An experimentally derived confidence score for binary protein-protein interactions Nat Methods 6 91-97
[2] Tasan M(2009)The determination of protein-protein interactions by the mating-based split-ubiquitin system (mbSUS) Methods Mol Biol 479 217-233
[3] Dreze M(1998)Yeast two-hybrid: so many interactions, (in) so little time Biol Reprod 58 302-311
[4] Barrios-Rodiles M(2007)Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts Plant J 52 185-195
[5] Lemmens I(2009)Protein complementation assays: Approaches for the in vivo analysis of protein interactions FEBS Lett 583 1684-1691
[6] Yu H(2004)Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation Plant J 40 428-438
[7] Sahalie JM(2008)Bimolecular Fluorescence Complementation (BiFC) Analysis as a Probe of Protein Interactions in Living Cells Annu Rev Biophys 37 465-487
[8] Murray RR(2010)A high-throughput bimolecular fluorescence complementation protein-protein interaction screen identifies functional Arabidopsis CDKA/B-CYCD4/5 complexes Plant Signal Behav 5 1276-1281
[9] Roncari L(2010)Functional modules in the Arabidopsis core cell cycle binary protein-protein interaction network Plant Cell 22 1264-1280
[10] de Smet AS(2007)Detection of transient protein-protein interactions by bimolecular fluorescence complementation: the Abl-SH3 case Proteomics 7 1023-1036

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