Skip to main content
. 2022 Aug 16;34(11):4366–4387. doi: 10.1093/plcell/koac250

Figure 4.

Figure 4

OsIAA1/9 and OsIAA21/31 regulate OsEIL1-activated OsTAR2 promoter activity through interacting with OsEIL1. A, Regulatory activity of OsIAA1/9/21/31 on OsEIL1-activated OsTAR2pro:LUC activity in rice protoplasts. Data are means ± sd (n = 3). Immunoblot assay shows the levels of the proteins encoded by the co-transfected constructs, and Coomassie Brilliant Blue Staining served as a loading control. **P < 0.01, as determined by a two-tailed Student’s t test compared to OsEIL1. B, Co-IP assay showing the interaction between OsIAA1/9/21/31 and OsEIL1. Nucleus-located protein OsMYB60 (Liu et al. 2018) served as a negative control. C, BiFC assay showing the interaction between OsIAA1/9/21/31 and OsEIL1 in rice protoplasts. More than five cells were observed in different microscope fields, and representative cells are shown. Scale bars, 10 μm. D, Yeast two-hybrid assay for the interaction between OsIAA1/9/21/31 and OsEIL1. AD-OsEIL1N and AD-OsEIL1C contain aa 1–350 and aa 251–641 of OsEIL1, respectively. E and F, Co-IP assay for the interaction between the C-terminus of OsEIL1 and the PB1 domain of OsIAA1/9/21/31. EIL1N-nYFP-Flag and EIL1C-nYFP-Flag contain aa 1–350 and aa 351–641 of OsEIL1, respectively. PB1 domains are aa 93–199, aa 91–182, aa 126–264, and aa 71–197 regions of OsIAA1, OsIAA9, OsIAA21, and OsIAA31, respectively. “*” indicates IAA21PB1-cYFP-Myc or EIL1C-nYFP-Flag band according to the predicted protein size. G, Enhanced effect of OsIAA1/9/21/31 on OsEIL1 binding to the EBS1 and EBS2 sites in the OsTAR2 promoter by EMSA-supershift assay. H, Activity of PB1 domain-deleted OsIAA1/9/21/31 in regulating OsEIL1-activated OsTAR2 promoter activity. Domains I and II are aa 1–92, aa 1–90, aa 1–125, and aa 1–70 regions of OsIAA1, OsIAA9, OsIAA21, and OsIAA31, respectively. Data are means ± sd (n = 3). **P < 0.01; ***P < 0.001, as determined by a two-tailed Student’s t test compared to the corresponding control.