Bimolecular-fluorescence complementation

BiFC constructs were expressed in mesophyll protoplasts, which were generated from the leaves of Arabidopsis Col-0 by the Tape-Arabidopsis Sandwich method⁶². Briefly, the lower epidermal surface of a leaf was removed by peeling with a strip of tape fixed to it. The mesophyll cells remaining on the tape were incubated in 20 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.7) containing 1% cellulose (Yakult, Tokyo, Japan), 0.25% macerozyme (Yakult), 10 mM CaCl₂, 20 mM KCl, 0.1% bovine serum albumin (BSA), and 0.4 M mannitol with gentle agitation for 20–60 min until the protoplasts were released into the solution. The protoplasts were washed twice with W5 solution (2 mM MES, pH 5.7, 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, and 5 mM glucose), incubated on ice for 30 min, centrifuged, and resuspended into MMg solution (4 mM MES, pH 5.7, 15 mM MgCl₂, and 0.4 M mannitol). Protoplasts were transfected with plasmids in a U-bottom 96-well plate by incubating for 5 min at room temperature under the presence of 20% (w/v) PEG4000. Following two washings with W5 solution, the protoplasts were incubated in the dark at 25 °C overnight. The protoplasts were incubated with MitoTracker orange CMTMRos (Thermo Fisher Scientific) for mitochondrial staining at 37 °C for 10 min, followed by 26 °C for 20 min.

The constructs were also transformed into Agrobacteria and infiltrated into Arabidopsis leaves for BiFC analysis²⁵. Briefly, the Agrobacterium was streaked onto YEB agar plates containing 0.1 mM acetosyringone and antibiotics. After 2 days, the agrobacteria were put into wash solution (10 mM MgCl₂ and 0.1 mM acetosyringone) for checking OD, and then diluted into an infiltration buffer containing ¼ strength MS with 1% sucrose, silwet L-77, and 0.1 mM acetosyringone at OD 0.5. The transformed agrobacteria were infiltrated into 3-week-old Arabidopsis leaves, then kept in the dark for 24 h prior to being left in the greenhouse to recover for 2 days²⁵. Confocal images were taken using a DM6000B/SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). BiFC fluorescence was imaged with a 488-nm laser excitation and emission fluorescence was captured by 500–520-nm band-pass emission filters, respectively.