OsSHI1 Binds Directly to the Promoter Regions of OsTB1 and OsDEP1. OsSHI1 Binds Directly to the Promoter Regions of OsTB1 and OsDEP1. (A) and (B) RT-qPCR analysis of OsTB1(A) and OsDEP1(B) expression levels in wild-type and shi1 axillary buds or young panicle tissues, respectively. Values are presented as mean ± sd, and the statistically significant differences were determined by Student’s t test (n = 4, *P < 0.05, **P < 0.01). WT, wild type. (C) and (D) Y1H assays to dissect the binding regions of OsSHI1 in the promoter regions of OsTB1(C) and OsDEP1(D). Series of promoter fragments of OsTB1 and OsDEP1 were fused to the upstream region of the LacZ reporter gene and tested for OsSHI1 binding.The empty pB42AD was used as the negative control. (E) to (G) EMSAs to test OsSHI1 binding to the two TCTCTAC (E) and (F) and one GCTCTAC (G) motifs in the OsTB1 and OsDEP1 promoters, respectively. The T/GCTCTAC motif was mutated into T/GAAAAAC to test for sequence specificity. The triangles indicate increased amounts of competing probes. GST or MBP proteins were used as the negative controls. The symbols “–” and “+” indicate the absence and presence of the corresponding proteins or probes. (H) and (I) ChIP-qPCR analyses of the P3 promoter regions of OsTB1(H) and OsDEP1(I) in ChIP samples precipitated by anti-OsSHI1–specific polyclonal antibodies. The fold enrichment was calculated as IP/Input. Values are presented as mean ± sd, and the statistically significant differences were determined by Student’s t test (n = 4, **P < 0.01). (J) ChIP-reChIP analysis of IPA1 and OsSHI1 co-occupy common target promoters. The chromatin of wild-type plants was first immunoprecipitated by anti-OsSHI1–specific polyclonal antibodies and then by anti-IPA1–specific polyclonal antibodies, and the precipitated DNA was quantified by qPCR analysis. The fold enrichment was calculated as IP/Input and normalized to that of the UBIQUITIN promoter region as an internal control. Values are presented as mean ± sd, and the statistically significant differences were determined by Student’s t test (n = 4, **P < 0.01). Erchao Duan et al. Plant Cell 2019;31:1026-1042 ©2019 by American Society of Plant Biologists