Plasmid Isolation, Protoplast Isolation, and Transient Transfection

Plasmids of the reporter and effector constructs were extracted using a GoldHi EndoFree Plasmid Maxi Kit (CWBIO) according to the manufacturer’s instructions. Protoplasts were isolated and transfected by following a procedure previously described (Xiong et al., 2019). Briefly, protoplasts were isolated from trifoliate leaves of 2-week-old soil pot-grown Wm82 wild-type plants, plasmids were transfected or co-transfected into the protoplasts isolated, and transfected protoplasts were incubated under darkness at room temperature. For subcellular localization assays, the transfected protoplasts were incubated for 16–18 h, and then GFP fluorescence was examined under an Olympus BX61 fluorescence microscope. For transcription activity assays, the transfected protoplasts were incubated for 22–24 h, and then GUS activities were measured by using a Synergy^TM HT fluorescence microplate reader (BioTEK).