Transient Expression of Proteins in N. benthamiana

The Agrobacterium strain GV3101 carrying the individual construct in the binary vector pCAMBIA1300 was incubated in LB media containing specific antibiotics (rifampin, 50 μg/mL; kanamycin, 50 μg/mL) at 28°C incubation. The bacteria were collected at 5,000 rpm for 5 min and resuspended in MMA buffer (10 mM MES, 10 mM MgCl₂, and 100 μM AS). The Agrobacteria containing the expression constructs were infiltrated into leaves of N. benthamiana for transient expression assay. Leaves were examined for image acquisition by using the Zeiss fluorescence microscope (Zeiss imager A2) between 36 and 72 hpi. Western blot analyses were performed following a previous protocol (Li et al., 2017 (link)). Total proteins were extracted from equal amounts of leaves with the protein extraction buffer with 1 × loading buffer (0.05 mg/mL Bromophneil blue; 0.065 M Tris-HCl, pH 6.8; 0.02 g/mL SDS; 0.05 mL/mL 2-mercaptoethanol; 0.1 mL/mL glycerol; 1 × protease inhibitor cocktail EDTA-free, Bimake, b14002). Total proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore) by using Trans-Blot Turbo (BIO-RAD). Protein blot was hybridized with the rabbit anti-GFP to determine YFP accumulation, and membranes were stained with Ponceau S as the loading control.