Gal4 ad

The N. benthamiana cDNA library was generated with an activation domain (AD) having GAL4-AD (Clontech Laboratories Inc., Mountain View, CA, USA). It was generated at Plant Virology Lab at The University of Arizona, USA. Concentrated bait culture of pGBKT7-βC1 working as binding domains (BD) was prepared and combined with library vial AD, after mating the culture was spread on an array of selective agar plates. Initially on 150 mm double dropout SD/–Leu/–Trp (DDO), quarter dropout SD/-Ade/His/Leu/-Trp QDO/X-α-Gal agar plates following incubation at 30 °C for 3–5 days.
Mating efficiency was determined by the following formula: ${\displaystyle \text{Mating efficiency}\left(\text{Diploids \%}\right)=\frac{\text{Number of cfu}/\text{ml of diploids}}{\text{Number of cfu}/\text{ml of limiting partner}}\times 100}$ whereas, the number of colony forming units per mL (cfu/mL) of diploid indicated the diploid viability that was equal to number of cfu/mL on SD/–Leu/–Trp, number of cfu/mL of limiting partner was estimated as viability of the prey library that was equal to the number of cfu/mL on SD/–Leu, while the viability of bait was equal to the number of cfu/mL on SD/–Trp.

Proteins from agroinfiltrated N. benthamiana leaves and co-transformed yeast cells were extracted as described [25 (link)]. Proteins in whole-cell lysates were subjected to sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (12% or 15% polyacrylamide gels) and transferred to a Hybond-P nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting. Polyclonal anti-HCpro (courtesy of F. Rabenstein, Julius Kühn-Institut, Quedlinburg, Germany) or polyclonal anti-eIF4E (courtesy of K. Browning, University of Texas, Austin, TX, USA) was used for plant samples, and a monoclonal antibody against the GAL4 AD or GAL4 DNA BD (1:50,000; Clontech, Mountain View, CA, USA) was used for yeast samples. Bound polyclonal antibodies were detected with horseradish peroxidase–conjugated anti-rabbit serum (1:250,000; GE Healthcare, Buckinghamshire, UK), and monoclonal antibodies were detected with horseradish peroxidase-conjugated anti-mouse serum (1:200,000; GE Healthcare). Detection was carried out with the Super Signal West Femto Chemiluminescent Substrate for detection of horseradish peroxidase (Thermo Scientific, Rockford, IL, USA) and visualized by exposure to X-ray film. Equal loading was controlled by Coomassie staining.