Premium
Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐ GFP association
Author(s) -
Liu TzuYin,
Chou WenChun,
Chen WeiYuan,
Chu ChingYi,
Dai ChenYi,
Wu PeiYu
Publication year - 2018
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13874
Subject(s) - nicotiana benthamiana , green fluorescent protein , intein , bimolecular fluorescence complementation , subcellular localization , arabidopsis , microbiology and biotechnology , biology , fusion protein , protein–protein interaction , computational biology , arabidopsis thaliana , protein subcellular localization prediction , agroinfiltration , chemistry , biochemistry , mutant , gene , cytoplasm , recombinant dna , rna , rna splicing
Summary Despite the great interest in identifying protein–protein interactions ( PPI s) in biological systems, only a few attempts have been made at large‐scale PPI screening in planta . Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPI s in vivo at subcellular resolution. However, when the non‐fluorescent fragments are highly expressed, spontaneous and irreversible self‐assembly of the split halves can easily generate false positives. The recently developed tripartite split‐ GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β‐estradiol‐inducible expression cassette, for the detection of membrane PPI s in planta . Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split‐ GFP association in plant cells and affirm that the tripartite split‐ GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPI s, including the membrane PPI s implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPI s in various cellular compartments in planta . Moreover, the technique combining the tripartite split‐ GFP association and dual‐intein‐mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof‐of‐concept implementation of the tripartite split‐ GFP system as a potential tool for membrane PPI screens in planta .