Subcellular localization
The full-length open reading frame (ORF) of SmGRAS5 was fused
with GFP in the pA7-GFP vector. Gene gun (Bio-Rad, USA) was used
to transform the pA7 -SmGRAS5-GFP and control plasmids
transiently into onion epidermis. The onion epidermis was stained with
4,6-diamidino-2-phenylindole dihydrochloride (Solarbio, China) for 20
min after 1 day of incubation. And then washed the epidermis twice with
PBS buffer (pH 7.2) and observed under a confocal laser scanning
microscope (Nikon A1R, Japan).
The pA7 0390-SmGRAS5-GFP and control plasmids were
transformed into Agrobacterium GV3101 . 4-week-old N.
benthamiana leaves were infiltrated with the GV3101 suspension
cultures, following the previously described method (Bai et al. ,
2018). The protoplasts were prepared after 2 days of co-culture, as
previously described (Li, 2011). The subcellular localization analysis
primers are listed in Supplemental Table S2.