Figure 1.

(A) Principles of the binary transactivation system. A reporter gene linked to a novel promoter is silent when first introduced into reporter plants. Transgene expression is induced by crossing to activator lines that express a heterologous transcription factor that specifically recognizes the transgene promoter. The pattern of reporter gene expression will reflect the pattern of activator expression, allowing a gene of interest to be expressed under a variety of regimes simply by crossing to an appropriate activator line. (B) Schematic representation of activator and reporter construct. In the reporter construct the reporter or gene-of-interest is linked to a minimal promoter (TATA) that lacks intrinsic transcriptional activity. Upstream of this promoter are binding sites for a transcription factor with a DNA-binding specificity that is not found in plants. The activator construct expresses a transcription factor that possesses this novel DNA-binding specificity and also has the ability to activate transcription in plants. This transcription factor is expressed from a plant promoter that will give the desired characteristics of reporter expression. (C) Schematic illustration of the plasmids used to test a lac-repressor-based transactivation system. pTA-GUS contains a GUS reporter under control of a minimal CaMV 35S-promoter (−50 to +8). pOpGUS contains two optimized lac operator sequences inserted upstream of the minimal promoter but is otherwise identical to pTA-GUS. The 35S-GUS plasmid expresses the GUS reporter gene from the CaMV 35S-promoter. The T-DNAs of all these reporter and control plasmids contain a selectable hygromycin resistance marker. Binary vector pLh-0 contains the coding sequence of the lacI^His mutant under control of the CaMV 35S-promoter. pLh-Gal4 is identical but contains transcription activation domain-II of Gal4 (residues 768–881) inserted in-frame in the lacI^His C-terminal coding region. pL-VP16 contains a wild-type lac-repressor coding region with the herpes simplex virus VP16 transcription activation domain fused in-frame in the C-terminal-coding region and the nuclear targeting signal of simian virus 40 large T antigen inserted at the N terminus. The T-DNAs of all these activator and control plasmids also contain a CaMV-35S-dihydrofolate reductase cassette encoding methotrexate resistance in transgenic plants.