Tripartite Fluorescence Complementation Assay

We modified BiFC vectors pUC19-YN and pUC19-YC to generate TriFC assays vectors with a MS2 system^{32 (link),56 (link)}. Briefly, MSCP was fused to pUC19-YN, HeFP was fused to pUC19-YC, 12×MS2-MISSEN-S or 12×MS2-MISSEN-AS was fused to pUC19-YN without the sequence of nYFP. PEG-mediated transfections were performed as previously described^{57 (link)}. Briefly, Aliquots of protoplasts (200 μL) were transferred into a 2-mL round-bottom microcentrifuge tube and mixed gently with 20 μg plasmid DNA. Transfected protoplasts were collected by centrifugation for 5 min at 100×g, resuspended and then incubated at 28 °C in the dark for 20 h. Protoplasts were observed using a Zeiss7 DUO NLO LSM880 confocal laser microscope (Carl Zeiss, Germany) with excitation/emission wavelengths of 514/519–563 nm.