Figure 7.

GmWRKY46 binds to DEG6 promoter region in vivo and in vitro. (A) Diagram of the 2000-bp promoter region of DEG6 showing the relative positions of the W-boxes. Four W-boxes F1, F2, F3, and F4 were marked by yellow triangle. Numbers indicated the position of starting nucleotide of each W-box relative to translation start. (B) GmWRKY46 binds to the DEG6 promoter region in the Y1H assay. Yeast cells were transformed with a bait vector containing a promoter fragment F1, F2, F3, or F4 fused to pAbAi vector, and a prey vector containing GmWRKY46 fused to pGADT7 vector. Yeast cells were grown in liquid medium to an OD600 of 1.0 and diluted in a 10× dilution series (10⁻¹-10⁻³). From each dilution, 5 μl was spotted onto SD/-Ura/-Leu medium to select for plasmids, and SD/-Ura/-Leu supplemented with 300 ng/ml Aureobasidin A (AbA) to select for interaction. Empty pGADT7 was used as control. (C) ChIP-qPCR analysis of GmWRKY46 binding to the DEG6 promoter region. Arabidopsis seeds of WT and Pro35S:GmWRKY46-GFP transgenic plants were germinated in medium and supplied with sufficient Pi. The whole plants were harvested for ChIP analysis. Enriched DNA fragments (F1 to F4) in the DEG6 promoter were quantified using RT-qPCR. Enrichment was calculated as the ratio of immunoprecipitation to input. Values represent means ± SD (n = 3). Data significantly different from the control are indicated (Student's t-test, *P < 0.05; **P < 0.01).