Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Summary

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Abstract

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Introduction

Studying the formation of protein complexes and their localization in plant cells in vivo is essential to investigate cellular networks, signaling and metabolic processes. BiFC allows visualization of protein-protein interactions in their natural environment directly within the living plant cell^1-5.

In the BiFC approach the complementation of two nonfluorescent N- and C-terminal fragments of a fluorescent protein lead to a reconstituted fluorescent protein. Fragments of many different fluorescent proteins have been used to detect protein interactions, e.g. the green fluorescent protein (GFP) the chromophore of w....

Protocol

1. Transformation of BiFC Constructs in Agrobacteria

1. Cloning of BiFC constructs
   1. Amplify the gene of interest from an appropriate template using oligonucleotides containing flanking attB-sites. Perform a PCR using a proofreading polymerase. Adapt the length of the annealing step to the designed primer combination and the length of the elongation step according to the fragment size. Check the PCR product by agarose gel electrophoresis and purify it by using a PCR Clean-up Kit.
   2. Perform the BP reaction with the obtained fragment and the entry vector using a BP recombinase. Mix 15-150 ng of the attB-PCR product with 150 ng of the entry ve....

Results

In this example we used the BiFC method to monitor the interaction of the cytosolic molecular chaperone HSP90 with the membrane docking proteins AtTPR7 and Toc64. AtTPR7 is part of the Sec translocon and interacts with cytosolic chaperones, which possibly deliver secretory preproteins for post-translational translocation to the ER membrane. Likewise, Toc64 at the chloroplast outer envelope acts in post-translational import by receiving HSP90 associated chloroplast preproteins. Both proteins comprise a cytosolic exposed T.......

Discussion

Upon planning a BiFC experiment several points should be considered. Although no structural information about the proteins of interest is required, the topology has to be known when working with membrane spanning proteins. The fluorescent proteins have to reside in the same subcellular compartment or face the same side of a membrane to allow interaction. Naturally, when analyzing proteins which require an N-terminal targeting sequence, only a C-terminal tag can be considered. Since it is possible that the tag interferes .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3',5'-Dimethoxy-4'-hydroxyacetophenone	Sigma-Aldrich	D134406	Acetosyringone
Cellulase, Onozuka-R10	Serva	16419	from Trichoderma viridae
Macerozyme R-10	Serva	28302	from Rhizopus sp.
GATEWAY, BP Clonase II, Enzyme Kit	Invitrogen	11789-(020)
GATEWAY, LR Clonase II, Enzyme Kit	Invitrogen	11791-(020)
QIAprep Spin Miniprep Kit	Qiagen	27106
NucleoSpin Gel and PCR Clean-up Kit	Macherey-Nagel	740609-250
pDEST-GWVYNE	Invitrogen		Gateway-cloning
pDEST-VYNE(R)GW	Invitrogen		Gateway-cloning
pDEST-SCYCE(R)GW	Invitrogen		Gateway-cloning