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Fig. 1. ARF protein interactions. (A) ARF-ARF and ARF-Aux/IAA protein interaction in yeast two hybrid assays. Relative reporter gene expression levels (ß-galactosidase units, right) in yeast cells co-expressing C-terminal regions of the indicated ARF gene-coding sequences (left) fused to DNA binding (BD) and activation (AD) domains. Line one shows ß-galactosidase expression levels in yeast cells harboring both empty vectors (pAS2-1, bait; pACT2, prey). Line two shows ß-galactosidase expression in response to the CLONTECH^TM positive control. Bars represent the mean±s.e. (B) Auxin-regulated interference of Aux/IAA expression with ARF transcriptional activation in carrot suspension cell protoplasts. Quantification of GUS reporter gene activity after transfection of the reporter gene and indicated effector genes in the absence (gray) and presence (black) of 10 µM 1-NAA. Columns represent the mean±s.d. Both ARF effector genes (MP/ARF5 and NPH4/ARF7) comprise the DNA-binding domain (DBD) of yeast GAL4 (GAL4DBD) and the middle region (MR), and C-terminal domain (CTD) of the respective ARF. IAA effector genes encode full-length wild-type IAA4, IAA9, IAA19 or BDL/IAA12 proteins. Expression of effector genes was driven by the CaMV 35S promoter (Tiwari et al., 2003). The GUS reporter gene is under control of the GAL4 response element and contains a -46 minimal CaMV 35S promoter with four GAL4-binding sites fused just upstream (see Ulmasov et al., 1995; Tiwari et al., 2003).

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