Fig. 2.

WRKY21 binds to PHT1;1 promoter region in vivo and in vitro. (a, b) Diagram of the 1053‐bp promoter region F0 of PHT1;1 showing the relative positions of the W‐boxes. Three W‐boxes Wx, Wy, Wz (corresponding to the sequences in red (b)) are marked by black vertical bars and relative positions and sizes of two synthesised DNA probes F1, F2 (b) are indicated by red rectangles under the W‐box cis‐elements (a). (c, d) WRKY21 binds to the PHT1;1 promoter region in the Y1H assay. Yeast cells were transformed with a bait vector containing a promoter fragment F0, F1 or F2 fused to AUR1‐C reporter gene, and a prey vector, containing WRKY21 fused to a GAL4 activation domain. Yeast cells were grown in liquid medium to an OD₆₀₀ of 1.0 and diluted in a 10× dilution series (10⁻¹ to 10⁻³). From each dilution, 5 μl was spotted onto SD/−Leu medium to select for plasmids, and SD/−Leu supplemented with 100, 200 or 600 ng ml⁻¹ aureobasidin A (AbA) to select for interaction. (e) Electrophoretic mobility shift assay (EMSA) to test the binding of WRKY21 to PHT1;1 promoter fragments. Each biotin‐labelled probe was incubated with MBP‐WRKY21 protein. An excess amount of unlabelled probes (cold probe) only was added to compete with labelled F2 DNA probes. Biotin‐labelled probes alone or biotin‐labelled probes incubated with MBP protein served as negative controls. The WRKY21–DNA complex (bound probes) and free DNA probes are indicated by black arrows, respectively. (f) Chromatin immunoprecipitation (ChIP)‐qPCR analysis of WRKY21 binding to the PHT1;1 promoter region. Rice seeds of Pro35S:GFP and Pro35S:WRKY21‐GFP transgenic plants were germinated in deionised H₂O and supplied with sufficient Pi. The whole plants were harvested for ChIP analysis. Enriched DNA fragments (F1 and F2) in the PHT1;1 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. P‐values were determined using Student’s t‐test. Pro35S:WRKY21‐GFP vs Pro35S:GFP; **, P < 0.01.