C2 er confocal microscope

SiMYBS3 was amplified by PCR using primers SiMYBS3-F2 and SiMYBS3-R2 and subsequently subcloned into the pBWA(V)HS-GLosgfp expression vector. The recombinant plasmid was transferred into GV3101 and then transformed into tobacco leaves for subcellular localization using a Nikon C2-ER confocal microscope (Nikon, Tokyo, Japan).
The coding regions were cloned into the pGBKT7 vector for yeast autoactivation assays using primers SiMYBS3-F3 and SiMYBS3-R3. To assess the reliability of the autoactivation, different concentrations of the surviving clones grown on a SD/-Trp medium were dotted on SD/-Trp/-His medium. The resulting yeast growth was photographed after incubation at 30 °C for 3 d. Primer information is listed in the Supplementary Materials, Table S1.

Complete coding sequence of BnaA01.GSK3 was amplified from the B. napus cv. Zhongshuang11 (ZS11). The purified DNA fragment was fused with green fluorescent protein (GFP) in the backbone vector pBWA(V)HS-gfp, resulting in the plasmid 35S:BnaA01.BIN2-GFP via the ClonExpressMultiS One Step Cloning Kit C113-01 (Vazyme). The 35S:GFP plasmid was used as the mock control. These plasmids were transiently transformed into Arabidopsis protoplast cells using the Agrobacterium-mediated method. The subcellular localization of BnaA01.BIN2 was determined by observing GFP using a Nikon C2-ER confocal microscope (Nikon, Japan) (Zhao et al., 2021 (link)). The primers used for amplification of BnaA01.BIN2 are listed in Supplementary Table 4.