Yeast two hybrid and 1-by-1 assays

To identify host proteins interacting with CTV-p23, the coding sequence of this protein from isolate T36 was cloned in frame with the LexA DNA binding domain (DBD) into plasmid pB27, derived from the original pBTM116 (Vojtek and Hollenberg 1995). The DBD construct was checked by sequencing the entire insert. Clones of the N. benthamiana library (Hybrygenics) were arranged in frame with the Gal4 activation domain (AD) into plasmids pP6 or pP7 that derive from the original pGAD GH (Bartel et al. 1993).

Identification of N. benthamiana proteins interacting with p23 as bait was based on the reporter gene HIS3 (growth assay without histidine). The interaction pairs were tested using a mating protocol with L40ΔGal4 (MATa) and Y187 (MATα) yeast strains, and in duplicate since two independent clones from each co-transformation were picked for the growth assay. For each interaction, several dilutions (10⁻¹, 10⁻², 10⁻³ and 10⁻⁴) of the diploid yeast cells culture, normalized at 5 × 10⁴ cells and expressing both bait and prey constructs, were spotted on several selective media. The DO-2 selective medium lacking tryptophan and leucine was used as a growth control and to verify the co-transformation of the bait and prey plasmids. The different dilutions were also spotted on a selective medium without tryptophan, leucine and histidine (DO-3). Four different concentrations (1, 5, 10 and 100 mM) of 3-aminotriazol (AT), an inhibitor of gene HIS3 product, were added to the DO-3 plates to increase stringency and reduce possible autoactivation by p23. The following interaction pairs, empty pB27-empty pP7, empty pB27-pP7-GAPDH, empty pB27-pP7-transducin/WD40 without stop codon and empty pB27-pP7-transducin/WD40, were included as negative controls and tested using a mating protocol with L40ΔGal4 (MATa) and Y187 (MATα) yeast strains (Fromont-Racine et al. 1997).