Abstract
Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.
Highlights
The function of a protein in living plant cells is typically carried out and tightly modulated through interactions with other proteins including cognite substrates, scaffolding proteins and activity or stability regulators
The split firefly luciferase complementation (SFLC) assay is ideal for in vivo protein-protein interactions (PPIs) studies in plant cells In comparison with existing PPI assays, the SFLC assay provides several advantages: (i) This assay provides a noninvasive analysis of in vivo PPIs in plant cells since the substrate D-luciferin is cell permeable and other co-factors in the reaction such as ATP and O2 are supplied from endogenous pools
This merit is important in that a PPI identified under undisturbed native conditions is more likely to be biologically relevant. (ii) This assay can quantitatively examine a large number of PPIs within 7– 8 hr from protoplast transfection to data collection
Summary
The function of a protein in living plant cells is typically carried out and tightly modulated through interactions with other proteins including cognite substrates, scaffolding proteins and activity or stability regulators. A large number of proteins need to dimerize or form higher-order oligomers for proper function [1]. It is the integrative network of all protein-protein interactions (PPIs) in the cell that virtually determines its developmental fate as well as its responses to the ever-changing extracellular environment. Only a few of them, including yeast two-hybrid (Y2H), affinity purification combined with mass spectrometry (AP-MS), bimolecular fluorescence complementation (BiFC) and protein microarray, have been successfully used for large-scale PPI studies [4,5,6,7,8]. A novel approach that could study in vivo PPIs in plant cells with the promise of high-throughput application would be highly valuable to the plant research community
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
