Figure 4

Identification and functional characterization of TFs interacting with conserved cis -sequences. (A) Simplified similarity tree (based on STAMP analysis) displaying the main associations observed between the different groups of conserved cis-sequences and the different class of TFs identified in yeast one-hybrid (Y1H) experiments. (B-E) Transcriptional activity and interaction of selected TFs with sequences 3 (B), 7 (C), 5 (D) and 9 (E) in Y1H experiments (left panels). Six repeats of the tested sequence, or their mutated versions (bold characters indicate the mutated nucleotides), fused to the HIS3 auxotrophic gene were tested. Upper part, growth on control media deprived of W amino acid, allowing the selection of yeast that express the studied TFs (i.e. NAC, NAC42, PIF7, MYB61 and MYB31). Lower part, growth on selective media deprived of W and H amino acids. Physcomitrella patens protoplasts transient expression assays (right panels). Green fluorescent protein (GFP) intensity measured in P. patens protoplasts cotransfected with sequences 3, 7, 5 and 9 fused to the 35S cauliflower mosaic virus minimal promoter and the GFP reporter gene with either, the NAC (sequences 3 and 9) or the MYB31 (sequences 7 and 5) DNA binding domain fused to the VP16 activation domain. Error bars ± SE. t-test significance: *, P < 0.05 and ***, P < 0.001.