In Vitro Dynamic and Quantitative Monitoring of Strigolactone-signaling Complex Formation by Time-resolved FRET

Strigolactones (SLs) are a class of plant hormones that play a critical role in the suppression of shoot branching. Furthermore, they are exuded from roots and act as signaling molecules for inter-organism communication in the rhizosphere. Strigolactones trigger those responses by inducing protein–protein interactions (PPIs) of signaling components and subsequent proteolysis of transcriptional repressors. The sequential event involves SL hydrolysis mediated by SL receptors belonging to an α/μ-hydrolase family, although the physiological role of SL hydrolysis is a subject of debate. To date, SL-induced PPIs have been analyzed by methods such as yeast-two hybrid, pull-down, and AlphaScreen assays. However, the kinetic aspect of PPI profiles has not been well studied. Here, we developed an in vitro method to monitor the formation of the SL signaling complex based on Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology. Our TR-FRET-based assay system allows us to analyze the mode of action of SL analogs from kinetic and quantitative perspectives. Notably, our method revealed differences in the intensity and time-dependency of PPI signals among different SL analogs with a range of hydrolyzabilities. In addition, we found that tolfenamic acid, an antagonist of the SL receptor, inhibited the SL-induced PPI but could not disrupt the already-formed signaling complex. The TR-FRET system was also used to rapidly and specifically detect naturally occurring SLs from root exudates containing many impurities. This work provides insights into the molecular mechanism of SL perception as well as a powerful tool for activity-based screening of SL signaling modulators.