Subcellular localization of GFP fusion proteins

The open reading frame of GeSUT4 without the stop codon was amplified from the pDRf1-GW-GeSUT4 plasmid with specific primers (GeSUT4-5-BP and GeSUT4-3- BP-fusion ). The purified cDNA was cloned into pDONR221-f1 and transferred into p2GWF7 by using Gateway technology (Karimi et al. 2007) thereby encoding a GsSUT4-GFP fusion protein. Arabidopsis mesophyll protoplasts were transformed with the p2GWF7-GeSUT4 plasmid as described previously (Ho et al.2019). To localize the plasma membrane and tonoplast, protoplasts were co-transformed with either the membrane marker, At PIP2A-RFP (Nelson, Cai & Nebenführ 2007) or the tonoplast markerAt γTIP-RFP (Jauh, Phillips & Rogers 1999), always in 1:1 ratio.