Figure 4.

In Vitro Binding of TGA2 to the TGACG Motif in the Promoter of the Arabidopsis PDF1.2 Gene.

(A) Oligonucleotides used in the gel mobility shift assay. The as-1 probe contains two inverted TGACG motifs and represents the SA-responsive as-1 element in the promoter of the Arabidopsis PR-1 gene. The pdf probe resembles the wild-type sequence surrounding the TGACG motif in the JA-responsive PDF1.2 gene. The mpdf probe is similar to the pdf probe but contains three point mutations in the TGACG motif.

(B) Gel mobility shift assay to test the binding of TGA2 to the TGACG motif in the PDF1.2 promoter. Binding reactions contained 7 × 10⁴ cpm of ³²P-labeled probe incubated with 1 μg of either a control protein preparation (lanes 1, 3, and 14) or partly purified TGA2 (lanes 2, 4 to 13, and 15). The specificity of the binding of TGA2 to the TGACG motif in the PDF1.2 promoter was tested by the addition of 100×, 50×, 10×, 1×, or 0.5× molar excess amounts of unlabeled pdf (lanes 5 to 8) or mpdf (lanes 9 to 13) competitor probes. The double asterisks indicate specific binding of TGA2 to the oligonucleotide probe, and the single asterisk indicates specific TGA2 binding and dimerization as a result of the presence of two TGACG motifs in the oligonucleotide probe. FP, free probe.