*4.7. Subcellular Localization in Tobacco*

The pCAMBIA1300-c-GFP vector was used for the subcellular localization of the *RbSWEET* genes in *Nicotiana benthamiana.* The CDS sequences of three *RbSWEET* genes were amplified using a forward primer containing a HindIII restriction site and a reverse primer containing a KpnI restriction site. The amplification products were recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme Bio Co., Nanjing, Jiangsu Province, China) and then recombined with the digested plasmid by using the ClonExpress® Ultra One Step Cloning Kit (Vazyme). The recombined plasmids were later transformed into *Agrobacterium tumefaciens* strain GV3101. The infection solution was prepared with sterile water containing 10 mM MgCl2, 10 mM 2-Morpholinoethanesulfonic Acid (MES), and 0.1 M Acetosyringone (AS), and then the *A. tumefaciens* was resuspended to an optical density (OD600) of 1.0. After standing in the dark for 3 h, the infection solution was injected into the leaf of *N. benthamiana* with a syringe. Two days after infiltration, the transient expression position of the *RbSWEET* proteins was observed by a Leica TCS SP8 fluorescence microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA) using filter blocks to select for spectral emission at 488 nm, and the empty vector was used as a control.
