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).