Fig. 1. Phenotypic characteristics of plants, and OsNAC022 regulation of tiller number by interacting with CCA1 promoter. A, CRISPR/Cas9-mediated target mutagenesis of OsNAC022 (CR-osnac022-1 and CR-osnac022-2) in Nipponbare (NIP) background. PAM, Protospacer adjacent motif; sgRNA, Single guide RNA. B and C, Comparison of tiller numbers in NIP, CRISPR/Cas9-mediated (CR-osnac022), and overexpression (OE-OsNAC022) lines at the late tillering stage. Scale bars, 20 cm. Different lowercase letters above columns in C indicate significant differences at P < 0.05 by Tukey’s test. D, Heatmap of differentially regulated tiller number-related genes in NIP and CR-osnac022 lines from RNA-seq. Gene expression was indicated by log2(fold change of expression ratios relative to NIP) (FPKM), with red indicating high expression and blue indicating low expression. E, DAP-seq of OsNAC022 binding elements in the promoter of CCA1. Positions of the interactive elements were marked by different color triangles, as per the color legend given at the bottom. F, Yeast one-hybrid (Y1H) assay showing the binding of OsNAC022 to the promoter of CCA1. Two promoter fragments (-2000 bp and -1184 to -1041 bp) of CCA1 were fused to the pLacZi vector for the OsNAC022 binding test. The empty vector pB42AD was used as a negative control. CK+ refers to positive control. G, Electrophoretic mobility shift assay (EMSA) of OsNAC022 binding to the CCA1 promoter. H and I, Luciferase (LUC) transient transactivation assay in rice protoplasts. Constructs used in the transient expression assays are shown in H. Transcriptional activation assay of different fragments on the activity of CCA1pro::LUC are shown in I. REN, Renilla luciferase. J, Expression levels of CCA1 in the tiller base of NIP and CR-osnac022 lines. The rice Ubiquitin gene was used as an internal control. K, Schematic illustration of the target site in the OsNAC022 gene by CRISPR/Cas9. L, Tillering dynamic performance of Xiushui 11 (XS11) and osnac22. Data are presented as mean ± SD (n = 10). Different lowercase letters above bars indicate significant differences between wild type XS11 and osnac22 mutants at P < 0.05 using Tukey’s test. M‒O, Comparison of tiller number (M), effective panicle number (N), and grain yield per plant (O) between wild type XS11 and osnac022 mutant plants. P, Expression of tiller number-related genes in the tiller base of NIP and CR-osnac022 lines. The rice Ubiquitin gene was used as an internal control. Data are presented as mean ± SD (n = 3 in I, J, P and n = 10 in N, O). ** indicates P < 0.01, by Student’s t-test.