Figure 9.

The RING E3 Ligase HUB1 Interacts with the MED21 Subunit of Arabidopsis Mediator Complex.

(A) HUB1 interacts with MED21 in the yeast two-hybrid assay. Yeast strains containing the MED21 in the prey vector (pAD-MED21) and HUB1 in the bait prey (pBD-HUB1) were assayed for growth on selective medium (-Leu, -Trp, and -His) (left) and β-galactosidase activity (right) showing interaction between MED21 and HUB1 in yeast. The β-galactosidase activity was assayed from yeast cells grown on synthetic complete medium. The positive (+) and negative (−) controls from the Stratagene kit were also assayed in parallel.

(B) HUB1 interacts with MED21 in vivo. Bimolecular fluorescence complementation assay showing in vivo interaction between HUB1 and MED21. pHUB1-cYFP and pMED21-nYFP were transiently coexpressed or were coexpressed with the vector alone in N. benthamiana leaf cells. YFP fluorescence was detected when pHUB1-cYFP was coexpressed with pMED21-nYFP. Images were examined under the bright field (left column), fluorescence (YFP), and as a merged image (bottom) showing either no interaction or interaction in the nucleus. DAPI, 4',6-diamidino-2-phenylindole.

(C) Siliques from wild-type, MED21/med21, and med21/med21;35S:MED1 lines. Arrows indicate aborted embryos.