Fig. 1

The SIAH domain of RIP1 interacts with RMD. a Schematic diagrams of the full-length RMD protein, indicating PTEN-C2, FH1, and FH2 domains; the truncated RMD P1, P2, and P3 constructs; the full-length RIP1 protein, indicating RING and SIAH domains; and the truncated SIAH1 construct. b Yeast 2 hybrid (Y2H) experiment revealing that the P3 section of RMD is essential for interaction with the RIP1 SIAH1 domain. Full-length and truncated RMD proteins were expressed from the BK bait vector (pGBKT7), and RIP1/SIAH1 proteins were expressed from the AD prey vector (pGADT7). Yeast cells co-transformed with each bait-prey pair were grown on selective media (-Leu-Trp). c Bimolecular fluorescence complementation (BiFC) in tobacco leaves reveals that RMD (fused to cYFP) interacts with the SIAH1 domain of RIP1 (fused to nYFP, middle panel) and full-length RIP1 (fused to nYFP, bottom panel). Bar = 50 µm. d Western blot showing that MBP-labelled RIP1 co-immunoprecipitates with eGFP-labelled RMD. RIP1 was purified from E. coli, and RMD was extracted from tobacco leaves. RMD was pelleted using anti-GFP immunomagnetic beads, and the presence of RIP1 in the supernatant and pellet, in the presence or absence of RMD, tested using anti-MBP antibody. Lane 1 is a positive control for MBP detection. e Phylogenetic analysis of 6 rice RIP and 6 Arabidopsis SIAH proteins. Bootstrap support values are indicated on nodes (%)