Localization and bimolecular fluorescence complementation (BiFC)
assays
The full-length cDNA fragments of AtMTM1 , AtMTM2 , andAtMSD1 were PCR amplified and cloned into the pCR8/GW/TOPO vector
(Invitrogen). Constructs were recombined into the pEarleyGate101 vector
for subcellular localization, or into the pEarleyGate201-YN and
pEarleyGate202-YC for BiFC analysis (Lu et al., 2010). Four-week-old
plants were used for protoplast preparation and transfection, as
described in the earlier report (Yoo, Cho, & Sheen, 2007). 2 ×
104 protoplasts and 15 μg DNA were used for
transfection, and YFP signals were observed by using a TCS SP5 confocal
microscope (Leica).