In vivo monitoring of plant small GTPase activation using a Förster resonance energy transfer biosensor

Abstract

[...]monitoring the activation state of a Rac/ROP GTPase within living cells is critical to further understanding its functions.[...]the 1990s, activation states of small GTPases were measured in vivo by labelling cells with inorganic [32P] phosphate, followed by immunoprecipitation of small GTPases and thin-layer chromatography to obtain quantitative data for their associated GDP and GTP levels.[...]this intramolecular FRET biosensor eliminates the problem of variability in the expression levels of donor and acceptor fluorescent proteins and is an ideal sensor for monitoring the activation states of small GTPases.Transfection of protoplasts with Raichu-OsRac1 plasmids In general, we are able to observe a 30–40% transfection efficiency with control green fluorescent protein (GFP), and it is very important to keep this efficiency high to obtain reproducible and reliable results.Since it is easy to generate CA and DN mutants of small GTPases, we strongly recommend adding Raichu-OsRac1 CA and DN vectors as positive and negative controls to ensure a wide dynamic range of the Venus/CFP ratio in each experiment. * All steps are carried out at 25 °C (room temperature). * Prepare plasmid DNA for transfection and fresh PEG solution.Recently, Xie et al. employed the modified version Raichu-OsRac1 and bimolecular fluorescence complementation (BiFC)-based FRET to demonstrate the ternary repressor complexes that modulate histone deacetylation and/or methylation [30].Since the principle of the intramolecular FRET biosensor appears to have universal applications for all of the small GTPases, we believe this Raichu system will become used in diverse research areas for understanding the spatiotemporal regulation of plant small GTPases and protein–protein interactions and this protocol can be adapted for for other FRET in various plants.