Abstract
BackgroundThe rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis.ResultsA split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1.ConclusionsA method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome.
Highlights
The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways
We previously showed with the split luciferase complementation (SLC) assay that protein-protein interactions in Arabidopsis protoplasts are sensitive to environmental conditions (Kato and Bai 2010), indicating that the SLC assay would be well-suited for analyzing regulated protein-protein interactions in plant cells
We established a method in which rice protoplasts are transformed with polyethylene glycol (PEG) in a 96-well plate, based on the method previously developed for Arabidopsis leaf protoplasts (Kato and Jones 2010)
Summary
The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Interactome analysis collects data of protein-protein interactions occurring in cells. A useful application of the analysis is to identify functional modules of signal transduction pathways in which a network of protein-protein interactions mediate the signals (Barabasi et al 2011; Pawson and Nash 2000). Constitutive protein interactions occur all the time in cells. On the other hand, regulated protein interactions occur when cells are exposed to selected environments such as stress
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