Characterization and localization of TRS120:GFP phosphovariants. (A) Western blot depicting protein expression of the phosphovariants TRS120-SαβγA and TRS120-SαβγD in trs120-4/trs120-4. Presence of the GFP-fused phosphovariants was detected with an anti-GFP antibody (upper panel). Shown are the expression levels in different primary transformants for each variant. Loaded protein amounts are shown by the corresponding CBB staining (Coomassie stain, lower panel). Protein sizes are given in kDa on the left. (B) Complementation analysis. The TRS120-SαβγA and TRS120-SαβγD phosphovariants in trs120-4/trs120-4 could rescue the null trs120-4 seedling lethal phenotype (third seedling from the left; white arrow) and did not differ from the wild-type Col-0 control in the T1 and T2 generations. Kanamycin selection was used to select for the presence of the phosphovariant construct. Images taken 13 days after stratification. Scale bar is 1 cm. (C and D) Silencing of the phosphovariant constructs upon propagation beyond the T2 generation. 2-week-old seedlings were imaged using a camera-equipped binocular microscope. (C) TRS120-SαβγA and (D) TRS120-SαβγD seedlings grown on kanamycin had some white sectors due to loss of chlorophyll in the shoot apical meristem and first true leaves; this was presumably due to gene silencing. Scale bars represent 200 µm. (E) Localization patterns of TRS120:GFP phosphovariants in root tip cells of light-grown seedlings. Confocal Scanning Laser Microscopy. P_TRS120::TRS120:GFP (Rybak et al., 2014) and phosphovariants thereof. Scale bars represent 5 µm. Wild-type TRS120 resides in the cytosol and at the TGN, it labels endomembrane compartments (white arrowheads) and the cell plate (yellow arrowhead); the phosphovariants have a similar appearance. Sample numbers n = interphase or cytokinetic cells imaged. 44 TRS120-WT, 13 -SαβγA, and 10 -SαβγD root tips were imaged. Source data are available for this figure: SourceData FS11.