Fig. 1.

TARGET-tU identifies actively transcribed TF targets. Schematic of the TARGET-tU system. a Protoplasts (plant cells dissociated from whole roots) transfected with a 35S::GR::TF construct are sequentially treated with: i) the nitrogen (N) signal transduced by the TF, ii) cycloheximide (CHX) to block translation, allowing RNA synthesis of only primary TF targets, iii) dexamethasone (DEX) to release the GR-TF fusion from the cytoplasmic heat shock complex (HSP), inducing nuclear import. Five hours after DEX-induction of TF nuclear localization, cells were exposed to iv) 4-thiouracil (4tU) so that thio-labeled UTP nucleotides are incorporated into newly synthesized RNA (see also c and Additional file 2: Figure S1). b Thiol-specific biotinylation and pull-down with streptavidin-coated magnetic beads enable selection of newly synthesized transcripts apart from pre-existing transcripts. c Timeline of the sequential treatments described in this study. Cell protoplasts were exposed to 4tU nucleobase 5 h after bZIP1 nuclear activation, to show the continued transcription of “hit-and run” targets