Clade 2 ERFs Differentially Bind and Activate a PMT2 Promoter

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Clade 2 ERFs Differentially Bind and Activate a PMT2 Promoter Clade 2 ERFs Differentially Bind and Activate a PMT2 Promoter.(A) The purity of recombinant ERF189, ERF115, ERF179, ERF163, ERF91, and ERF32 proteins was analyzed by separation on a 12% SDS-PAGE gel and subsequent staining with Coomassie Brilliant Blue (CBB). Clade 2 ERFs Differentially Bind and Activate a PMT2 Promoter.(A) The purity of recombinant ERF189, ERF115, ERF179, ERF163, ERF91, and ERF32 proteins was analyzed by separation on a 12% SDS-PAGE gel and subsequent staining with Coomassie Brilliant Blue (CBB). The molecular mass of marker proteins is shown on the left in kilodaltons. Clade numbers are indicated in parentheses.(B) EMSA of recombinant ERF proteins with the probe P3 of the PMT2 promoter (see Figure 8A). Recombinant proteins were used at either 2.4 or 0.6 μg. The arrow on the right indicates a missing ERF32-P3 complex.(C) Transactivation of the PMT2pro236-GUS reporter with various ERF effectors. Cultured tobacco cells were bombarded with a combination of the reporter plasmid, a luciferase-expressing control plasmid, and either an ERF-expressing effector plasmid or an empty plasmid (EV). GUS activities in the cell extracts were shown relative to the luciferase activities. Error bars indicate the sd for three biological replicates. Significant differences among the effectors were determined at P < 0.05 by one-way ANOVA, followed by the Tukey-Kramer test and are indicated by different letters. Tsubasa Shoji et al. Plant Cell 2010;22:3390-3409 ©2010 by American Society of Plant Biologists