Fig 1. DRN competitively interacts with WUS.
(A) Y2H assays exhibiting the interaction of WUS-DRN. BD and AD empty vectors were used as negative controls. Yeast cells were grown on the selective medium (SD/−Leu/−Trp/−His/−Ade) in a series of dilutions of 10−1, 10−2, and 10−3. Two independent experiments were performed with similar results. (B) BiFC exhibiting that the interaction of WUS-DRN occurs in tobacco leaves. BF, bright field. Scale bars, 100 μm. Five leaves were analyzed for each group. Two independent experiments were performed with similar results. (C) Co-IP showing the interaction of WUS and DRN in Arabidopsis. 35S::DRN-Flag and 35S::WUS-3×HA were transformed into Arabidopsis protoplasts. The anti-Flag antibody was used for IP. Instead of the anti-Flag antibody, IgG was used for IP as the negative control. Two independent experiments were performed with similar results. (D) Pull-down assays showing the interaction of WUS and DRN directly. Recombinant proteins were expressed in E. coli. Anti-His and anti-GST antibodies were used for immunoblot analysis. Two independent experiments were performed with similar results. (E) WUS–WUS interaction is disrupted by DRN in Y3H. Selective media (SD/−Leu/−Trp/−His) and (SD/−Leu/−Trp/−His/−Ade) were used to test the interaction of WUS-WUS. Selective media (SD/−Leu/−Trp/−His/−Met) and (SD/−Leu/−Trp/−His/−Ade/−Met) were used to test the competitive binding of WUS-DRN. Two independent experiments were performed with similar results. (F) Pull-down assays were used to determine the interruption of WUS homodimer by DRN. The binding group demonstrates the interaction of WUS-WUS, which is compromised by introducing 8His-MBP-DRN. Two independent experiments were performed with similar results. (G) Split-luciferase complementation assays in tobacco leaves were conducted to test that DRN competitively interacts with WUS. Five leaves were analyzed in each independent replicate. Two independent experiments were performed with similar results. BiFC, bimolecular fluorescence complementation; Y2H, yeast two-hybrid; Y3H, yeast three-hybrid.