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Plant Methods. 2018 Jul 7;14:56. doi: 10.1186/s13007-018-0325-4. eCollection 2018.

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

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1Laboratory of Plant Molecular Genetics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, 630-0192 Japan.
2Present Address: Department of Biological Science, University Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900 Kampar, Perak Malaysia.
3Present Address: Department of Bioscience, Kwansei Gakuin University, 2-1 Gakuen, Sanda, 669-1337 Japan.
4Present Address: Signal Transduction and Immunity Group, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences, No. 3888 Chenhua Road, Shanghai, 201602 China.
5University of Chinese Academy of Sciences, Beijing, 100049 China.
6College of Life, Environment, and Advanced, Osaka Prefecture University Sciences, Sakai, Osaka 599-8531 Japan.
7Present Address: Department of Advanced Bioscience, Kindai University, 3327-204 Nakamachi, Nara, 631-8505 Japan.
8College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Japan.
Core Facility of Cell Biology, Shanghai Center for Plant Stress Biology, No. 3888 Chenhua Road, Shanghai, 201602 China.
10Present Address: FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, Fujian China.
11Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka, Totsuka, Yokohama, Kanagawa 244-0813 Japan.
Contributed equally



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.


Bioimaging; FRET sensor; Plant immunity; Small GTPase

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