N. benthamiana Protoplast Isolation and Transfection

Nicotiana benthamiana protoplasts were isolated from 21-day-old seedlings. In short, the young leaves were chopped up and immersed in enzyme solution (0.5 M mannitol, 1.5% cellulose RS (Yakult Honsha, Tokyo, Japan), 0.75% macerozyme R10 (Yakult Honsha), 1 mM CaCl₂, and 0.1% BSA). The mixture was incubated on a shaking incubator (60 rpm) in the dark at room temperature for 4–5 h and then filtered through Miracloth. The protoplasts were pelleted by centrifugation at 200 g for 5 min and then resuspended in an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl and 1.5 mM MES, adjusted to pH 5.7), followed by centrifugation and resuspension in MMG solution (0.4 M mannitol, 15 mM MgCl₂ and 4.7 mM MES, adjusted to pH 5.7). Plasmid DNA (10 or 20 μg) was added to the protoplast solution and transfected with 40% polyethylene glycol (PEG) solution [40% PEG4000 (Sigma-Aldrich, St. Louis, MO, United States, PCode Number, 102078930), 0.4 M mannitol, and 100 mM Ca(NO₃)₂] at room temperature for 20 min. W5 solution was gradually added to dilute the PEG solution and then discarded. The transfected protoplasts were incubated overnight at room temperature and then observed under a confocal microscope.