Dataset Information

Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

ABSTRACT: Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C-terminal fragments of enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), and monomeric red fluorescent protein (mRFP1). Using a combination of N- and C-terminal fragments from ECFP, EGFP and EYFP, we observed a shift in the emission wavelength, enabling the simultaneous detection of multiple protein-protein interactions. Moreover, we developed these vectors as binary vectors for use in Agrobacterium infiltration and for the generate transgenic plants. We verified that the binary vectors functioned well in tobacco cells. The results demonstrate that the BiFC vectors facilitate the design of various constructions and are convenient for the detection of multiple protein-protein interactions simultaneously in plant cells.

SUBMITTER: Kamigaki A

PROVIDER: S-EPMC4973907 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

Kamigaki Akane A Nito Kazumasa K Hikino Kazumi K Goto-Yamada Shino S Nishimura Mikio M Nakagawa Tsuyoshi T Mano Shoji S

PloS one 20160804 8

Bimolecular fluorescence complementation (BiFC) is widely used to detect protein-protein interactions, because it is technically simple, convenient, and can be adapted for use with conventional fluorescence microscopy. We previously constructed enhanced yellow fluorescent protein (EYFP)-based Gateway cloning technology-compatible vectors. In the current study, we generated new Gateway cloning technology-compatible vectors to detect BiFC-based multiple protein-protein interactions using N- and C- ...[more]

PMID: 27490375

Similar Datasets

Project description:BackgroundThe bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein-protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial. Furthermore, the challenges of performing BiFC in restricted cellular compartments has not been specifically addressed.ResultsHere we describe the development of a flexible modular cloning-based toolkit for BiFC (MoBiFC) and proximity labelling in the chloroplast and other cellular compartments using synthetic biology principles. We used pairs of chloroplast proteins previously shown to interact (HSP21/HSP21 and HSP21/PTAC5) and a negative control (HSP21/ΔPTAC5) to develop standardised Goldengate-compatible modules for the assembly of protein fusions with fluorescent protein (FP) fragments for BiFC expressed from a single multigenic T-DNA. Using synthetic biology principles and transient expression in Nicotiana benthamiana, we iteratively improved the approach by testing different FP fragments, promoters, reference FPs for ratiometric quantification, and cell types. A generic negative control (mCHERRY) was also tested, and modules for the identification of proximal proteins by Turbo-ID labelling were developed and validated.ConclusionsMoBiFC facilitates the cloning process for organelle-targeted proteins, allows robust ratiometric quantification, and makes available model positive and negative controls. Development of MoBiFC underlines how Goldengate cloning approaches accelerate the development and enrichment of new toolsets, and highlights several potential pitfalls in designing BiFC experiments including the choice of FP split, negative controls, cell type, and reference FP. We discuss how MoBiFC could be further improved and extended to other compartments of the plant cell and to high throughput cloning approaches.

Dataset Information

Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

Publications

Gateway Vectors for Simultaneous Detection of Multiple Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets