Co-Immunoprecipitation of PP2C15, BAK1, and P19 in N. benthamiana

Firefly LCA was performed as described (Zhou et al., 2018 (link)). For Co‐IP experiments, PP2C15‐HAHA, BAK1‐GFP, GFP, or the silencing suppressor P19 (Voinnet et al., 2003 (link)) were transformed into Agrobacterium tumefaciens GV3101 that was cultured in YEB liquid medium containing rifampicin and kanamycin for 18 h at 28°C. The strains were resuspended in 10 mM MgCl₂ and adjusted to OD₆₀₀ = 1. Combinations of PP2C15‐HAHA + P19, PP2C15‐HAHA + PP2C15‐GFP + P19, and PP2C15‐HAHA + BAK1‐GFP + P19 were mixed in equal proportions and added to 150 mM acetosyringone. Five‐week‐old leaves of N. benthamiana were injected with the above solutions using a needleless 1 mL syringe and cultured for 3 days. Thereafter, the leaves were ground to a powder with liquid nitrogen in the presence of a protein extraction buffer (Couto et al., 2016 (link)). anti‐GFP magnetic beads (YaMei Biology) were added to the supernatant, and Co‐IP experiments were performed according to the manufacturer's instructions. Finally, the Co‐IP results were analysed by western blotting using anti‐GFP (GenScript) and anti‐HA (GenScript) antibodies.