Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

Summary

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Abstract

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact ^1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible^8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays¹⁰. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Protocol

1. Preparation of Agrobacterium culture

1. Agrobacterium strain GV3101 previously transformed with Gateway compatible BiFC vector pEarleyGate201-YC and pEarleyGate202-YN, each containing a fusion construct of GOI.
2. Inoculate a 5-ml YEB (5g L^-1 Beef extract, 1g L^-1 Yeast extract, 5g L^-1 Peptone,5g L^-1 Sucrose, 2mM MgSO₄, pH 7.2) culture of BiFC fusion protein constructs transformed agrobacterium with the appropriate antibiotic selection. Grow culture overnight at 28°C shaking at 200 rpm.
3. Remove 1 ml of culture into a sterile 1.5 ml centrifuge tube.
4. Pe....

Discussion

BiFC assay is a powerful tool for studying protein interactions. Unlike the traditional Y2H assay, BiFC not only allows the visualization of protein-protein interactions, but also provides more information of the protein complex such as sub-cellular localization. Also, it is possible to detect indirect interactions as long as the two candidate proteins can be brought close enough by a third partner. Like any technology, BiFC has its limitations. Due to the requirement of molecular oxygen for fluorophore formation, BiFC .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetosyringone	Sigma-Aldrich	D134406
MES hydrate	Sigma-Aldrich	M2933
1 mL slip-tip tuberculin syringe	BD Biosciences	309602