ERF12 Binds Directly to the DRE/CRT Element in the DOG1 Promoter. ERF12 Binds Directly to the DRE/CRT Element in the DOG1 Promoter. (A) EMSA of the oligonucleotide DRE/CRT with GST-tagged ERF12 protein. The 22-bp wild-type DRE/CRT fragment labeled with biotin probe was shifted by the GST-ERF12 protein, but this shift disappeared when unlabeled probe was added. The arrow shows the shifted band; free probe is distributed at the bottom. Biotin-labeled probe was used at a final concentration of 20 nM. (B) Schematic diagram of the DOG1 promoter structure. The dotted line indicates the ∼2000-bp promoter sequence, and the black line between the vertical dashes represents the first 500 bp of the open reading frame. The relative positions of the PCR-amplified fragments (P1 to P5) for each tested region are depicted below the gene structure. P6 in a 211-bp fragment located in the untranslated region of AT5G45820, which is located 4397 bp away from ATG of DOG1 (AT5G45830); this fragment was used as the negative control. Bar represents 500 bp. (C) ChIP-qPCR analysis of YFP-ERF12 levels on DOG1. Immunoprecipitates were obtained from freshly harvested seeds of Col-0 and Pro12S:YFP-ERF12 using YFP antibody. ACTIN8 was used as a negative control. The experiment was repeated three times with independent samples. The numbers on the x axis represent the PCR-amplified sites described in (B). **P < 0.01, based on Student’s t test. (D) Relative activity of the ProDOG1:LUC reporter in Arabidopsis protoplasts cotransformed with the indicated effector constructs. M represents a mutated DRE/CRT element (GGC to TTC), which was introduced into ProDOG1:LUC. The activity of the LUC reporter driven by the DOG1 promoter was measured by assaying LUC/REN ratios × 1000. Data are shown as means ± sd, different letters denote significant differences (n = 3, one-way analysis of variance test, P < 0.05). Xiaoying Li et al. Plant Cell 2019;31:832-847 ©2019 by American Society of Plant Biologists