2.11 Subcellular localization analysis using GFP and BiFC assay
The cDNA sequences containing the full length coding region ofCRCT (amino acid 1-308) and the CCT domain (amino acid 235-308) were amplified by RT-PCR using gene specific primer pairs (Table S1). The amplified cDNAs were each cloned into a GFP expression vector pHTII-CaMV35S-sGFP(S65T)-nos (Chiu et al., 1996). The GFP expression vectors were introduced into the epidermis of onion (Allium cepa ) by particle bombardment using 1-µm gold particles and a gene delivery system (PDS-1000, Bio-Rad, https://www.bio-rad.com). After a 24 h incubation in the dark at room temperature, the epidermis was peeled from the bombarded segment, and the fluorescence from GFP and DAPI were observed with a fluorescence microscope (ECLIPSE 80i, Nikon, https://www.nikon.co.jp) through FITC and V-2A cubes, respectively.
A BiFC experiment was carried out as described in Kodama, & Wada, (2009), except superfolder GFP (sfGFP) was used in this experiment (Fujii and Kodama, 2015). The full length coding regions of CRCTand GF14A were amplified by RT-PCR using gene-specific primers (Table S1). The PCR fragments were inserted into the pBsfGN155-MXMT vector containing the N-terminal region of sfGFP (sfGN) with 7-methylxanthine methyltransferase from Coffea arabica (MXMT) and the pBsfGC155-MXMT vector with the C-terminal region of sfGFP with MXMT by replacing MXMT (pBsfGN155-MXMT and pBsfGC155-MXMT were generous gifts from Dr. Kodama and used as a arbitrary control for BiFC in this study). Appropriate combinations of BiFC expression vectors were introduced into the epidermis of onion (Allium cepa ) as described above. As a control for transformation, a vector containing CaMV35S::DsRed (pHTII-CaMV35S-DsRed) was introduced together with the BiFC expression vectors. The fluorescence from sfGFP, DsRed and DAPI were observed with a fluorescence microscope (ECLIPSE 80i, Nikon) through FITC, G-2A and V-2A cubes, respectively.