*4.6. Subcellular Localization*

To determine its subcellular localization, the coding sequence of *YL* lacking its stop codon was amplified by PCR in a 50 μL volume that contained 200 ng cDNA from wild type, 1 × PCR buffer, 0.4 mM dNTPs, 1 U KOD FX Neo (Toyobo, Osaka, Japan) and 0.3 μM of both forward and reverse primers of *YL-GFP* listed in Table S3. The sample was heated to 94 ◦C for 2 min, followed by 45 cycles of denaturation at 94 ◦C for 15 s, annealing at 61 ◦C for 30 s, elongation at 68 ◦C for 1 min. The amplified product was cloned into the pCAMBIA 1302 vector between the cauliflower mosaic virus 35S promoter and the GFP-coding sequence. The construct of YL-GFP vector was sequencing confirmed by Sangon Biotech (Shanghai, China).

For transformation of *Agrobacterium*, 1 μg purified plasmid DNA was added to competent cells of thawing *Agrobacterium tumefaciens* strain *GV3101* on ice. After ice bath for 30 min, the cell/DNA mix was immersed in liquid nitrogen for 1 min and subsequently incubated at 37 ◦C for 5 min, then ice bath for 2 min. By adding 900 μL liquid growth medium (no antibiotics), the cell/DNA mix was shocked for at least 120 min at 28 ◦C. After centrifugation, the cells were resuspended and plated on an agar plate containing kanamycin (50 mg/L) for selection of transformants.

For infiltration of *Nicotiana benthamiana*, the *Agrobacterium tumefaciens* strains carrying the YL-GFP, pCAMBIA 1302 and p19 of tomato bushy stunt virus plasmids were grown at 28 ◦C in liquid growth medium with kanamycin (50 mg/L), rifampicin (50 mg/L) and gentamicin (25 mg/L) until OD600 reached 1.0. The *Agrobacterium tumefaciens* strains containing the YL-GFP and p19 or pCAMBIA 1302 and p19 plasmids were mixed. After centrifugation of the strain mixtures, the harvested cells were resuspended in 10 mM MES buffer containing 10 mM MgCl2 and 100 mM acetosyringone to a final OD600 of 1.0, followed by incubation at room temperature for 120 min. Strain mixtures were infiltrated into the abaxial surface of leaves of four-week-old *Nicotiana benthamiana* plants using a 1 mL syringe. The transformed epidermal cells were detected using a Zeiss LSM710 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).
