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.