Fig. 8

ONAC066 bound to the JBSL cis-element in OsDREB2A promoter. a A diagram showing putative NAC core-binding sequences and JBSL element in the promoter of OsDREB2A. P1, P2 and P3 were the probes used in ChIP-PCR assays. b Schematic diagram of the different constructs used in ONAC066-pOsDREB2A or ONAC066-pOsDREB2A-JBSL binding assays. c ONAC066 bound to OsDREB2A promoter and JBSL cis-element in OsDREB2A promoter in vivo. Rec2-ONAC066 and Rec2 empty vectors were co-transformed with the reporter vectors pHis-pOsDREB2A or pHis-pOsDREB2A-JBSL into yeast and co-transformed yeasts were dropped by a series of 10-fold dilutions on medium of SD/−Trp-Leu, SD/−Trp-Leu-His/50 mM 3-AT and SD/−Trp-Leu-His/100 mM 3-AT. Binding activity was estimated according to the growth status of the co-transformed yeasts on different medium at 2 days after plating. d ONAC066 bound to a JBSL-containing sequence in OsDREB2A promoter. ChIP of ONAC066-GFP transgenic line using GFP antibody (α-GFP) or pre-immune (Pre) serum was performed and precipitated DNA fragments were subject to PCR analysis with OsDREB2A promoter primers. 10% of chromatin amount before IP was used as positive controls (input) and IP sample with pre-immune serum was used as a negative control. e Binding activity of ONAC066 to JBSL cis-element. Biotin-labeled wJBSL and mJBSL probes or biotin-labeled wJBSL probe in combination with unlabeled wJBSL or mJBSL probe were incubated with GST-fused ONAC066 protein or a purified GST preparation as a negative control. Specific protein-DNA complexes and free probes are indicated by the arrowheads on left. Experiments in (c-e) were repeated for three times with similar results