Invsc1 strain

The plasmids pYES2/NT-C, pY762-889GFP, pY762-1062GFP and pYES2-cycle3GFP were transformed individually to yeast INVSc1 strain (Invitrogen, Life Technologies, Carlsbad, CA, USA). Individual colonies were selected and cultivated in SC-U medium containing 2% dextrose at 30°C for 16 h. The overnight cultures were pelleted to remove the medium and resuspended in SC-U induction medium containing 2% galactose to an OD₆₀₀ of 0.5 and incubated for additional 4 h. The promoter activities were determined by measuring the GFP fluorescence and the OD₆₀₀ of individual constructs at 0 h and 4 h, with four replicates, as described above.
For western blot assays, individual colonies of each plasmid were selected and cultivated in 1 ml SC-U medium containing 2% dextrose at 30°C for 20 h. The overnight cultures were pelleted and resuspended in 100 µl 1× SDS loading dye and boiled at 100°C for 10 min, followed by SDS-PAGE and transferred to PVDF membrane. The GFP proteins in the samples were detected by using a GFP monoclonal antibody (SIGMA-Aldrich, St. Louis, MO, USA), followed by a rabbit anti-mouse IgG AP-conjugated antibody. Transiently expressed GFP in N. benthamiana at 3 dpi was used as a positive and protein size control, in which the GFP gene was driven by the CaMV 35S promoter.