Fig. 4: Identification of the genome-wide direct targets of OsMYB8.

a Nuclear localization of OsMYB8-GFP in rice protoplasts. D53-mCherry was used as a nuclear marker. Scale bars, 20 μm. b Yeast two-hybrid assay showing the transcriptional activity of OsMYB8. Left is the diagrams of full-length and truncated OsMYB8 proteins, which were fused with the DNA-binding domain (BD). c The core binding motif of OsMYB8 protein identified using MEME-ChIP. d Venn diagrams showing a comparison of DAP-seq binding genes with the identified DEGs in Osmyb8 mutants by RNA-seq. e GO enrichment analysis of 345 overlap genes in (d). MF indicates molecular function, BP indicates biological process. f ChIP-qPCR analysis showing that OsMYB8 binds to the OsJAR1 promoter in vivo. The upper panel is a diagram of OsJAR1 promoter with the indicated regions used for detection by ChIP-qPCR. The red lines indicate the position of “TTHGGY” motifs in the OsJAR1 promoter regions. Immunoprecipitation was performed with anti-GFP antibody, ZH11 was used as a negative control. (n = 3 technical replicates). g EMSA assay showing that GST-OsMYB8 recombinant protein directly binds to the “TTHGGY” motif-containing regions of the OsJAR1 promoter. Unlabeled probes were used as competitors. GST was used as a negative control. h Transient dual-LUC assay showing that OsMYB8 induces the transcriptions of OsJAR1 promoter in rice protoplasts. The upper panel is diagrams of various constructs used in the transient expression assay. The expression level of REN was used as an internal control. The LUC/REN ratio represents the relative activity of the OsJAR1 promoter. (n = 3 technical replicates). The Values in (f) and (h) are means ± SEM. Significance is evaluated by the two-sided Student’s t-test, and P values are indicated. The subcellular localization experiments in (a) and the EMSA experiments in (g) were independently repeated three times with similar results. Source data are provided as a Source Data file.