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Detection of protein–protein interactions in plants using bimolecular fluorescence complementation

Detection of protein–protein interactions in plants using bimolecular fluorescence complementation Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein–protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non‐overlapping N‐terminal (YN) and C‐terminal (YC) fragments. Each fragment was cloned in‐frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the α and β subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION‐INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co‐expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co‐expression of free non‐fused YN and YC or non‐interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT α and β subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non‐split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein–protein interactions in plants. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Plant Journal Wiley

Detection of protein–protein interactions in plants using bimolecular fluorescence complementation

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References (31)

Publisher
Wiley
Copyright
Copyright © 2004 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0960-7412
eISSN
1365-313X
DOI
10.1111/j.1365-313X.2004.02206.x
pmid
15469499
Publisher site
See Article on Publisher Site

Abstract

Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein–protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non‐overlapping N‐terminal (YN) and C‐terminal (YC) fragments. Each fragment was cloned in‐frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the α and β subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION‐INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co‐expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co‐expression of free non‐fused YN and YC or non‐interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT α and β subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non‐split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein–protein interactions in plants.

Journal

The Plant JournalWiley

Published: Nov 1, 2004

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