Figure 5.

Interactions between Different Forms of ROP10 and the NF Receptor NFP of M. truncatula.

(A) GAL4-based Y2H assays. ROP10 and mutant forms (ROP10CA and ROP10DN) were fused with Gal4 DNA BD in pGBKT7 as baits, and NFP full length (FL), the intracellular kinase domain of NFP (PK), LYK3 PK, and LYK3 FL were fused with the Gal4 AD in pGADT7 as preys. Yeast cells cotransformed with bait and prey constructs were selected on SD medium lacking His, Leu, and Trp supplemented with X-α-Gal (SD/-3/X-α-Gal) and more stringent SD medium lacking His, Ade, Leu, and Trp supplemented with X-α-Gal (SD/-4/X-α-Gal) for 5 d. The results indicate an interaction between NFP PK and ROP10 or ROP10CA. The interaction between mammalian p53 and SV40 served as a positive control, whereas the coexpression of lamin (Lam) and SV40 served as a negative control.

(B) The strength of interaction was quantified by assaying β-galactosidase activities in yeast colonies with chlorophenol red-β-d-galactopyranoside as substrate. Error bars indicate se. Data presented are representative of at least three independent experiments.

(C) Colocalization of ROP10 and NFP at the PM in N. benthamiana leaf epidermal cells. Bars = 50 µm.

(D) BiFC analysis to test interactions between ROP10 or mutant forms and NFP PK in N. benthamiana leaf epidermal cells. Bars = 50 µm.