Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Summary

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Abstract

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay¹ allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)² and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Protocol

I. Plasmid preparation

The plasmid DNA used for bombardment must be of high purity and at a concentration of 500ng/μl or greater. For BiFC involving two plasmid constructs, 25 μg of each plasmid is needed. In other words, 1:1 molar ratio of the two plasmids should be mixed to give rise to 50 μg total plasmid DNA for the preparation of cartridges. Be mindful that adding more DNA than 50 μg may cause agglomeration of the gold particles and should be avoided. Commercially available plasmid DNA extraction kits are recommended for isolating plasmid DNAs.

Plasmid vectors should be relatively small in size such as pUC19....

Discussion

1. For first time users, we recommend practicing the entire procedure, from cartridge preparation to observation, with a single plasmid constitutive reporter such as 35S::GFP or 35S::GUS. We used a 35S::GFP plasmid obtained from Drs. Steve Mount and Xiaoning Zhang⁶ (Figure 1A, D). The gene shot control cartridges sold at RioRad (Cat 165-2244) are not suitable for plants.
2. For BiFC, a second positive control is necessary, which should consist of two known interacting partners fused to the C-terminal and N-terminal f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Yellow onion	Any Supplier
Helios Gene Gun	Bio-Rad
Fluorescent microscope with appropriate filters for GFP or YFP	Zeiss, Nikon, Olympus