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

Hann Ling Wong, Akira Akamatsu, Qiong Wang, Masayuki Higuchi, Tomonori Matsuda, Jun Okuda, Ken-ichi Kosami, Noriko Inada, Tsutomu Kawasaki, Takako Kaneko-Kawano, Shingo Nagawa, Li Tan, Yoji Kawano, Ko Shimamoto

Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles. We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images. Our protocol should be adaptable for monitoring activation for other plant small GTPases and protein-protein interactions for other FRET sensors in various plant cells.