Fig. 3
Interaction between OsIAA29 and OsARF17. A Yeast two-hybrid assay. The full-length OsIAA29 cDNA was cloned into a vector bearing the DNA binding domain (BD), and the full-length cDNA of OsARF17 was cloned into a vector bearing an activation domain (AD). The transformants were grown on DDO (SD/-Leu/-Trp), QDO (SD/-Leu/-Trp/-His/-Ade), and QDO with X-α-Gal plates. B Pull-down assays showing that there was a direct interaction between GST-OsIAA29 and His-OsARF17 in vitro. The recombinant proteins were expressed in the Escherichia coli BL21 strain (DE3) and Glutathione beads were used for pull-down. GST-fused free protein was used as the control. Western blotting was performed using anti-GST or anti-His antibody. (Sigma-Aldrich). C BiFC assays of OsIAA29 and OsARF17. OsIAA29-cYFP and OsARF17-nYFP interacted to form a functional CFP in rice protoplast cells. Scale bars are 10 μm. D Subcellular localization of OsIAA29 and OsARF17. OsGhd7, the nuclear marker. Bar = 5 μm. E–f Transient expression analysis of 35S-5xGAL4-TATA-LUC activity in rice protoplasts. E The vector of luciferase combination. F Dual luciferase analysis of the activation activity of OsARF17. OsARF17 or OsARF17/OsIAA29 was co-transfected with 35S-5xGAL4-TATA-LUC. 35S-5xGAL4-TATA-LUC used as control. The LUC/REN ratio was shown to indicate the expression level of the 35S-5xGAL4-TATA-LUC. The values in each column are the mean (± SD) of three replicates. Significant differences were determined using Student’s t-test (* P < 0.05; ** P < 0.01)