Fig. 3. SP8 and RSV P2 promote interaction of OsGID1 with SLR1.

Co-IP analyses of the interactions between viral proteins SP8 (a) and RSV P2 (b) with OsGID1 in N. benthamiana leaves. Each experiment was repeated three times with similar results. c, d. Pull-down assays for analysis of the interaction between OsGID1 and SP8 (c) or P2 (d). An equal amount of MBP-His-OsGID1 was incubated with immobilized GST and GST-SP8 (c) or GST-P2 (d) separately, and then the bound proteins were detected by Western blotting using anti-His and anti-GST antibodies. Each experiment was repeated three times with similar results. Co-IP assays showing that SP8 and RSV P2 promote interaction of OsGID1 with SLR1 in planta. OsGID1-flag and SLR1-GFP/SLR1-myc were transiently infiltrated using Agrobacterium together with/without increasing SP8-myc (e) or P2-GFP (f) in leaves of N. benthamiana, while leaves expressing HA-GFP or GFP-flag were used as negative controls. Cultures were pelleted to a final OD₆₀₀ of 0.5, increasing amounts of SP8 and P2 following agrobacterium infection with final OD₆₀₀ = 0.5 or OD₆₀₀ = 1.0, respectively. Proteins were immunoprecipitated with Flag- paramagnetic beads. The red asterisks point to the specific band. The co-infiltrated leaves were treated with MG132 (50 µM) or DMSO at 24 hpi and then harvested for coimmunoprecipitation 24 h later. Each experiment was repeated three times with similar results. In vitro pull-down assays showing the effects of SP8 (g) and RSV P2 (h) on the activation of the interaction between OsGID1 and SLR1. The indicated proteins were purified from E. coli and pulled down by GST beads. Immunoblots were performed using anti-GST and anti-His antibodies to detect the associated proteins. Each experiment was repeated three times with similar results. Source data including uncropped scans of gels (a–h) are provided in the Source data file.