Fig 1: TIE1 interacts with BRC1 in vitro and in vivo.(A) Yeast two-hybrid assays of TIE1 with BRC1 and BRC2. The N-t of TIE1 (1 to 108 residues) was fused to GAL4–DBD to generate the bait protein. The transcription factors BRC1 and BRC2 were fused with the GAL4–AD to generate the prey protein. AD, activation domain; DBD, DNA binding domain. Co-transformed yeast cells were grown on medium lacking Leu and Trp (SD-Leu-Trp) and selected on medium lacking Leu, Trp and His (SD-Leu-Trp-His) with 5 mM 3-amino-1,2,4-triazole (3-AT). The empty vectors PDEST32 or PDEST22 were used as negative controls. The three spots are triplicates of the experiment. (B) Pull-down assays to test TIE1 interaction with BRC1 in vitro. The MBP-BRC1 was used to pull down TIE1-His with the MBP as a negative control. The mixtures MBP-BRC1/TIE1-His and MBP/TIE1-His were incubated with Amylose resin (NEB) and the beads were washed six times to avoid the false positive. The “elution 1” and “elution 6” mean the supernatant in the first wash and sixth wash of the MBP-BRC1/TIE1-His and MBP/TIE1-His beads, respectively. In the sixth wash, the TIE1-His was not detected in the supernatant, indicating the amylose resin was cleaned up and avoiding the false positive. After the six washes, the bound proteins in the beads (precipitate) were eluted with 2×SDS buffer and detected with anti-His6. The TIE1-His could be pulled down by MBP-BRC1 (the lane designated MBP-BRC1 output) but not by MBP control (the lane designated MBP output). (C) BiFC assay of the interaction between TIE1 and BRC1. GFP signal was observed in N. benthamiana leaves co-transformed with nYFP-TIE1 and cCFP-BRC1 vectors. cCFP-BRC1/empty nYFP and nYFP-TIE1/empty cCFP were used as negative controls. (D) Interaction between TIE1 and BRC1 in vivo tested by firefly LUC complementation imaging (LCI) assay. The signal of LUC activity was detected in the cLUC-BRC1/TIE1-nLUC combination. cLUC/TIE1-nLUC, cLUC-BRC1/nLUC and nLUC/cLUC were used as negative controls (E) In vivo confirmation of TIE1-BRC1 interaction by FRET-acceptor photobleaching assay. E-FRET% is calculated as relative increase in GFP fluorescence intensity after photobleaching of the mCherry acceptor. Intramolecular FRET (both fluorophores were in the same protein, TIE1-GFP-mCherry; BRC1-GFP-mCherry) and background signal measured using a non-interacting protein (epitope MYC-GFP) are included as positive and negative controls, respectively. Results show mean ± SEM (n = 10 cells from at least 3 different leaves); EFRET% values obtained for the BRC1-GFP/TIE1-mCherry and TIE1-GFP/BRC1-mCherry interaction are significantly different from values obtained for both negative controls (MYC-G/BRC1-C and MYC-G/TIE1-C). *** p-value<0.001 in a two-tailed Student’s t-test. (F) TIE1 interacts with the TCP domain in yeast two-hybrid assays. Left, schematic representation of the different BRC1 deletions and BRC1 mutant proteins (in the TCP domain: tcpWL, tcpDRV, tcpRDR or in the R domain: BRC1mR) that were fused to the GAL4 prey (AD). Yeast two-hybrid assays were carried out between these BRC1-AD proteins and an N-t of TIE1 (1 to 108 residues) fused to GAL4–DBD bait. The empty vectors pGADT7 or pGBKT7 were used as negative controls. The three spots are triplicates of the experiment.