Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐GFP association

Despite the great interest in identifying protein-protein interactions (PPIs) 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 PPIs 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 PPIs 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 PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs 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.