Skip to main content
. 2022 Feb 16;34(5):1912–1932. doi: 10.1093/plcell/koac057

Figure 5.

Figure 5

WCR1 is regulated by OsDOF17. A, Y1H assay. DOFBS represents a 20-bp probe containing the AAAAG motif from BL130. Yeast strains were cultured on SD/-Trp-Ura + X-Gal selection medium. B, EMSA of OsDOF17 protein with the AAAAG motif in the WCR1 promoter. Probe A, a labeled 55-bp sequence containing a AAAAG motif from the WCR1 promoter (−1,737 to −1,683); Probe G, a labeled 55-bp sequence with a single nucleotide mutation (A–G; shown in red). The AAAAG motif is underlined. The OsDOF17-GST fusion protein was used for EMSA with the probes. The unlabeled competitive probe was added in 1-, 10-, and 100-fold molar excess. Lane 1, no protein; lanes 2–6, fusion protein with Probe A, competitive probe, and Probe G, as indicated. C and D, Transient expression assays of the transcriptional activity of OsDOF17 on the A/G site of WCR1 promoter in vivo. In (C), Left, the reporter vector contained the GAL4 binding cis-elements (UAS), the 35S promoter and the firefly LUC gene. The effectors consisted of the negative vector containing a GAL4 DNA-binding domain (GAL4DBD) and the positive vector GDOF17 containing a GAL4DBD and full-length OsDOF17. The internal control was the rLUC gene. Right, relative LUC activity (LUC/rLUC). P-value was determined by a two-tailed t test, **P ≤ 0.01. In (D), Left, the reporter vector containing the mini35S promoter and the LUC gene was inserted in the 20-bp sequence containing A (LUCA) or G (LUCG). The effectors consisted of the negative vector None and positive vector NDOF17 containing the full-length OsDOF17. Right, relative LUC activity. Significant differences were determined by Duncan’s test. All data in (C and D) were based on three replicate experiments. Error bars, sem. E, WCR1-YFP and OsDOF17-CFP co-localize in the nucleus. Scale bar, 20 μm. F, Spatiotemporal expression patterns of OsDOF17. FL: flag leaves at the heading stage; 5YP, 10YP and 15YP: young panicles 5, 10, and 15 cm long; 5E, 7E, 10E, and 14E: grain endosperm at 5, 7, 10, and 14 DAF. Samples were obtained from different plants. Data are means (n = 4) with error bars, sem.