Figure 5

GT-4 interacts with TEM2 in vivo and in vitro. (a) Dimerization of Arabidopsis GT-4 in yeast two-hybrid assay. The yeast cells harboring the plasmid combinations were grown on DDO plates (SD medium without Leu and Trp) and QDO/X/A plates (SD/–Ade/–His/–Leu/–Trp medium containing 40 μg mL-1X-α-Gal and 125 ng mL-1 Aureobasidin A) for 3 d. The cells generating blue color indicate positive interactions between the two proteins. (b) GT-4 and C-terminal of GT-4 interact with TEM2 in yeast two hybrid assay. (c) GT-4 and TEM2 co-localized in the nucleus. Tobacco leaves were co-infiltrated with 35S:GFP-GT-4 and 35S:RFP-TEM2. Signals were observed directly under a confocal microscope after 3 days. (d) Bimolecular fluorescence complementation (BiFC) assay. The fusion constructs were co-transformed into Arabidopsis protoplasts and the cells were observed 16 h later under a confocal microscope. YFP fluorescence was excited at a wavelength of 488 nm. (e) Co-IP assay. Nuclei were isolated from tobacco leaves expressing 35S:GT-4-FLAG and 35S:TEM2-MYC. Then proteins were extracted and incubated with anti-c-Myc agarose beads. The proteins were then eluted and followed by western blotting analysis with anti-flag or anti-Myc antibodies.