Figure 7.

OsDIS1 interaction with OsNek6 in vitro and in vivo. A, Yeast two-hybrid assay. OsDIS1 and OsNek6 were cloned into pGBKT7 and pGADT7 vectors, respectively. The derived constructs were transformed into the yeast strain HF7c. The transformed yeast cells were plated onto the SD/−His/−Trp/−Leu medium including 3 mm 3-amino-1,2,4,-triazole (3-AT). AD, pGADT7 vector that contains an activation domain; BD, pGBKT7 vector that contains a binding domain. B, In vitro GST pull-down assay for OsDIS1 and OsNek6. The [³⁵S]OsNek6 protein was in vitro transcribed and translated using the TNT Coupled Wheat Germ Extract Systems. GST-OsDIS1 protein was expressed in E. coli BL21 and purified. [³⁵S]OsNek6 protein (3 μL) was incubated with 1 μg of GST-OsDIS1 protein at room temperature for 1 h. The GST protein was used as a negative control, and 1 μL of [³⁵S]Met-labeled OsNek6 protein was used as input. C, In vivo coimmunoprecipitation assay for OsDIS1 and OsNek6. The protein of the N. benthamiana tissues coinfiltrated with plasmids of the DsRed/DsRed-OsDIS1/DsRed-OsDIS1(H71Y) and GFP-OsNek6 combinations was extracted and immunoprecipitated with anti-GFP antibody. Immunoblot analysis was performed using anti-DsRed antibody and anti-GFP antibody. The numbers at left denote the molecular masses of marker proteins in kilodaltons.