Skip to main content
. 2020 Aug 28;19(2):300–310. doi: 10.1111/pbi.13462

Figure 6.

Figure 6

OsRE1 functions cooperatively with OsRIP1 to negatively regulate Ehd1 expression. (a) EMSA showed that OsRIP1 cannot directly bind to but facilitate OsRE1 to bind to the A‐box motif of the Ehd1 promoter. The hot probe was a biotin‐labelled A‐box motif‐containing fragment in the Ehd1 promoter (sequence showed in Figure 1c). (b) ChIP‐qPCR assay showed in vivo binding of OsRIP1 to the Ehd1 promoter is dependent on OsRE1. Cross‐linked chromatin samples were extracted from Flag‐OsRIP1 and osre1/Flag‐OsRIP1 transgenic plants and then precipitated with Flag antibody. NoAb (No antibody) served as a negative control. Values are means ± SD; n = 3. (c) Schematic diagram of various constructs used in the transient transformation assay. 35S: REN‐ProEhd1: LUC was constructed as the reporter. 35S: OsRE1‐GFP and 35S:3 × Flag‐OsRIP1 were constructed as effectors. Free GFP and Flag were used as negative controls. (d) In vivo luciferase assay verified the inhibitory effect of OsRE1 and OsRIP1 on Ehd1 expression in N. benthamiana leaves. Expression level of Renilla (REN) was used as an internal control. The LUC/REN ratio represents the relative activity of the Ehd1 promoter. Values are means ± SD; n = 3. Asterisks indicate significantly different values (*, P < 0.05; **, P < 0.01).