Fig. 1

Comparison and characterization of promiscuous protein biotinylation by different biotin ligases in plant. a Diagram of the expression cassettes used for the expression of three biotin ligases. Citrine was fused to the N-terminus while a MYC tag was added to the C-terminus of BioID, BioID2 and TurboID. Expression was under the control of Arabidopsis ubiquitin-10 promoter (pUBQ) and nopaline synthase terminator (NOSt). b The biotin ligases promiscuously biotinylate endogenous proteins in plant cells with varying efficiencies. N. benthamiana leaves were agroinfiltrated with the agrobacterium containing citrine-BioID-3xMYC (BioID), citrine-BioID2-3xMYC (BioID2) and citrine-TurboID-3xMYC (TurboID), or the empty vector control (vector), respectively. 36 h post-agroinfiltration (hpi), medium containing the buffer (−) or 200 µM biotin (+) were infiltrated into the previously agroinfiltrated leaves. Infiltrated N. benthamiana plants were directly incubated at room temperature (RT) or in a 37 °C chamber. Western blot analysis was performed on tissue collected 12 h after infiltration of biotin. Streptavidin-HRP and anti-MYC antibodies were used for detection of biotinylated proteins (top panel) and different biotin ligases (middle panel), respectively. PEPC served as loading control for equal protein loading (bottom panel). The molecular weight size markers in kDa are indicated at the left of each panel. An additional repeat of this experiment is shown in Supplementary Fig. 1b. c Determination of the optimal biotin concentrations required for TurboID-based proximity labeling in planta. Different concentrations of biotin, as indicated above the panels, were infiltrated into the N. benthamiana leaves expressing the citrine-TurboID. These plants were then incubated in the 37 °C chamber or at room temperature (RT) for 8 h followed by western blot analysis as described in (b). PEPC served as loading control for equal protein loading (bottom panel). The molecular weight size markers in kDa are indicated at the left of each panel. Additional repeat of this experiment is shown in Supplementary Fig. 1c. d Effect of incubation time on TurboID-based proximity labeling in plants. N. benthamiana leaves expressing citrine-TurboID were infiltrated with 200 µM biotin, plants were then incubated under 37 °C or RT followed by collection of leaves at different time points as indicated above the panels. Western blot analysis was then carried out as described in Fig. 1b. Due to the instability of rbcL protein levels at different time points after biotin treatment (Supplementary Fig. 2), the protein bands below that of the rbcL in the Coomassie Brilliant Blue (CBB)-stained gel is shown as a loading control (bottom panel). The molecular weight size markers in kDa are indicated at the left of each panel. Source data are provided as a Source Data file