Volume 11, Issue 5, Pages 720-735 (May 2018) The Intronic cis Element SE1 Recruits trans-Acting Repressor Complexes to Repress the Expression of ELONGATED UPPERMOST INTERNODE1 in Rice  Yongyao Xie, Yaling Zhang, Jingluan Han, Jikai Luo, Gousi Li, Jianle Huang, Haibin Wu, Qingwei Tian, Qinlong Zhu, Yuanling Chen, Yoji Kawano, Yao-Guang Liu, Letian Chen  Molecular Plant  Volume 11, Issue 5, Pages 720-735 (May 2018) DOI: 10.1016/j.molp.2018.03.001 Copyright © 2018 The Authors Terms and Conditions

Figure 1 Characterization of the dEui1 Mutant. (A) The dwarf phenotype of the dEui1 mutant. Scale bar, 20 cm. (B) The severe panicle enclosure phenotype of dEui1. The arrowheads indicate the position of neck-panicle node. Scale bar, 10 cm. (C) The upper internodes (III and IV) of dEui1 failed to elongate properly. Scale bar, 10 cm. (D) The dEui1 mutation was caused by a T-DNA insertion in the Eui1 intron. P1–P3 indicate primers used for genotyping and expression analysis; P5 and P6 indicate primers used for qRT–PCR; R1–R5 indicate chromatin regions included in the histone acetylation and H3K27me3 analysis shown in Figure 6. (E) Co-segregation of the T-DNA insertion with the dominant dEui1 phenotype in T1 progeny. Primers HPTF/R amplify HPT, a selectable marker gene in the transgenic plants; WT, wild-type for both genotype and phenotype; T-, hemizygous for the T-DNA insertion; TT, homozygous for the T-DNA insertion; M, the mutant dwarf phenotype. (F) Expression of Eui1 in WT and dEui1 plants. L, leaf; S, stem; R, root; P, panicle; IV, uppermost internode; III, third internode. Data are shown as means ± SD (n = 3).*p < 0.05 and **p < 0.01, significant differences determined by t-tests between the WT and dEui1. (G) The length of the leaf sheath of dEui1 seedlings was rescued by treatment with exogenous bioactive GA3. Data are shown as means ± SD (n = 12). Molecular Plant 2018 11, 720-735DOI: (10.1016/j.molp.2018.03.001) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Identification of the Genomic Silencing Element SE1 by Analysis of the dEui1 Mutant. (A) An intronic fragment (365–499 bp) was replaced by the inserted T-DNA in dEui1. S1–S4 indicate four small sequences deleted in the binary constructs containing the genomic sequence of Eui1 including its native promoter (ΔS1–ΔS4) that were used for complementation tests. The RY element (underlined in S2) is the conserved cis element bound by B3 domain proteins (see Figure 3) leading to silencing of Eui1. (B) Transformation of WT rice with a construct where Eui1 cDNA expression is driven by the Eui1 native promoter (Eui1c) caused a dwarf phenotype. The arrowheads indicate the position of neck-panicle node. Scale bar, 20 cm. (C) The ΔS1–ΔS4 constructs were transferred into the eui1-3 line, which has a loss-of-function mutation of Eui1. Of the transgenic plants, only those carrying ΔS2 showed a dwarf phenotype similar to that of the dEui1 mutant; therefore, the S2 region functions as a genomic silencing element, which we termed SE1. Arrowheads indicate the neck-panicle node. Scale bar, 20 cm. (D) Height of the transgenic (T1) and control plants. Data are shown as means ± SD (n = 10). (E) The transcript levels of Eui1 in leaves of different genetic materials. The transcript levels were normalized to the level in WT (set as 1). Data are shown as means ± SD (n = 3). (F) Levels of endogenous GA1 in seedlings of different genetic materials. UD, undetectable; FW, fresh weight. Data are shown as means ± SD (n = 3). *p < 0.01, significant difference determined by t-tests between the WT and individual genotypes. Molecular Plant 2018 11, 720-735DOI: (10.1016/j.molp.2018.03.001) Copyright © 2018 The Authors Terms and Conditions

Figure 3 The Silencing Element SE1 Recruits the Repressor OsVAL2 via the B3 Domain. (A) Diagram of the conserved domains in the OsVAL2 and OsGD1 proteins and the core sequence of different RY mutants (se1 and M1–M3). PBZE stands for the initials of the four conserved domains in full-length proteins. The base pairs written in lowercase indicate point mutations in the RY element. (B) Yeast one-hybrid (Y1H) assays testing the interaction of the B3 domain-containing proteins OsVAL2 and OsGD1 with DNA fragments carrying the WT RY element (SE1) or RY elements with different point mutations (se1 and M1–M3). (C) Y1H assays showing that OsVAL2 interacts with SE1 via the B3 domain. The truncated fragments are indicated by the initials of the domains they contain. (D) In vitro interaction between SE1 and the recombinant B3 domain proteins rOsVAL2-B3 and rOsGD1-B3 was confirmed by the CAPS-based binding assay. M1–M3 indicate the three mutant forms of SE1 with the point mutations in the RY motif as described in (A). The asterisks indicate minimum amount of protein that inhibits restricion digestion. Molecular Plant 2018 11, 720-735DOI: (10.1016/j.molp.2018.03.001) Copyright © 2018 The Authors Terms and Conditions

Figure 4 OsVAL2, OsGD1, OsSAP18, and OsHDA710 Form Core Repressor Complexes. (A) OsGD1 interacts with OsVAL2 and the co-repressor OsSAP18 in yeast two-hybrid assays. Yeast cells were grown on synthetic dropout medium lacking Trp and Leu (-WL) as a control, and on medium lacking Trp, Leu, His, and adenine (-WLHA) to stringently select for positive interactions. (B) BiFC assay demonstrating that the co-repressor OsSAP18 interacts with OsHDA710 in vivo. The merged image includes combined images from the YFP and differential interference contrast channels. Scale bar, 10 μm. (C) Quantification of the FRET efficiency. The interactions of OsGD1 with OsVAL2 or OsSAP18 were individually confirmed by FRET assays. Interactions between OsGD1, OsSAP18, and OsHDA710 were demonstrated by the BiFC-based FRET assays. GEAR, EAR of OsGD1; GPHD, PHD of OsGD1; VPHD, PHD of OsVAL2; VB3, B3 of OsVAL2; S, OsSAP18; H, OsHDA710. NC, Raichu-DN-OsRac1 (negative control); PC, Raichu-CA-OsRac1 (positive control). FRET efficiency stands for the change in the fluorescence of the donor fluorophore (CFP) after photobleaching of the acceptor fluorophore (Venus). The background FRET efficiency indicates the stability of CFP fluorescence before photobleaching. Data are shown as means ± SE (18 ≤ n ≤ 24). *p < 0.01, significance determined by t-tests between the negative control and tested combinations. (D–F) In vivo CoIP assays indicate that GFP-tOsVAL2 interacts with tOsGD1-FLAG, GFP-OsSAP18 interacts with tOsGD1-FLAG, and GFP-OsSAP18 interacts with OsHDA710-FLAG. The recombinant proteins were expressed in rice protoplasts and the protein complex was precipitated using an anti-FLAG antibody, then GFP-tOsVAL2 (105 kDa) and GFP-OsSAP18 (40 kDa) were detected using an anti-GFP antibody. Ten percent of input protein serves as a control. (G) SE1-biotin DNA pull-down assays showed that OsHDA710-FLAG was present in the SE1-associated complex in WT cells, and the abundance of OsHDA710-FLAG decreased in osval2 mutants, thus suggesting the presence of the repressor complex in vivo. Ten percent of input protein serves as a control. Molecular Plant 2018 11, 720-735DOI: (10.1016/j.molp.2018.03.001) Copyright © 2018 The Authors Terms and Conditions

Figure 5 The Function of OsVAL2 and OsHDA710 in the Regulation of Eui1 Expression. (A) Schematic of OsVAL2 gene structures with the two different sites targeted by CRISPR/Cas9, T1 and T2, (see Supplemental Figure 8) indicated by asterisks. The coding sequence of OsVAL2 encodes a protein containing PHD, B3, Zf-CW, and EAR domains. osval2-T1-1 (shown in B) encodes a truncated protein, due to a nucleobase (A in red) insertion in the coding sequence of OsVAL2 resulting in a premature stop codon (TGA in boldface red). (B) Genotyping the osval2 plants. The sequences of the CRISPR/Cas9-targeted sites were determined by sequencing. The underlined DNA sequences indicate protospacer adjacent motifs for single guide RNAs. The positions highlighted in red show the targeted mutations. (C) The phenotype of WT, osval2-T1-1, ΔS2, and osval2-T1-1/ΔS2 plants. The osval2-T1-1/ΔS2 and ΔS2 plants exhibited similar phenotypes, indicating that SE1 and OsVAL2 may function in the same pathway to regulate Eui1 expression and plant height. Scale bar, 20 cm. (D) The height of WT, osval2-T1-1, ΔS2, and osval2-T1-1/ΔS2 plants. Data are shown as means ± SD (n = 10). (E) Level of Eui1 expression in osval2-T1-1, ΔS2, and osval2-T1-1/ΔS2 plants. Data are shown as means ± SD (n = 3). (F) Phenotype of WT plants with or without (mock) TSA treatment. Scale bar, 2 cm. (G) Height of WT rice seedlings treated with different concentrations of TSA for 6 days. Data are shown as means ± SD (n = 17). (H) Eui1 expression levels after TSA treatment for 6 days. Data are shown as means ± SD (n = 3). *p < 0.05, and **p < 0.01, significant differences determined by t-tests between the mutants (treatments) and WT (mock). Molecular Plant 2018 11, 720-735DOI: (10.1016/j.molp.2018.03.001) Copyright © 2018 The Authors Terms and Conditions

Figure 6 Histone Acetylation and Methylation States at Five Chromatin Regions of Eui1. (A) The histone H4 acetylation (H4ac) level of the Eui1 chromatin region R3 (see Figure 1D) is greatly enhanced in dEui1. “No data”: data for R2 were not available for testing in dEui1 due to its replacement by the T-DNA insertion. (B) The H4ac levels in the R1–R5 regions in the ΔS1–ΔS4 transgenic plants. (C) The H4ac levels at R2 and R3 are significantly increased in the osval2-knockout plants. (D) The H4ac levels at R2 and R3 are significantly increased in TSA-treated WT plants in a dose-dependent manner. (E) The H3K27me3 level of ΔS2 transgenic plants is lower than that of ΔS1 transgenic plants in the R1–R3 regions of Eui1. (F) The H3K27me3 levels at R1–R3 are decreased in the dEui1 and osval2 knockout plants. “No data”: data for R2 were not available for testing in dEui1 due to its replacement by the T-DNA insertion. *p < 0.05 and **p < 0.01, significant differences determined by t-tests between the mutants (treatments) and WT (mock) for each region. (G) Sequencing of genomic DNA containing SE1 after bisulfite treatment. The original Cs in SE1 and converted Ts in SE1 (SE1-CT) after bisulfite treatment are highlighted in red. The underlined sequence shows the RY element. PCR-amplified fragments were cloned into the pEasy-T1 vector and sequenced. The methylation rate was calculated by Web-based Kismeth software. Data in (A) to (F) are shown as means ± SD (n = 3). Molecular Plant 2018 11, 720-735DOI: (10.1016/j.molp.2018.03.001) Copyright © 2018 The Authors Terms and Conditions

Figure 7 Working Model for the Repressor Complex Recruited by the Silencing Element SE1 in Eui1. The intronic SE1 participates in negative regulation of Eui1 expression via core repressor complexes, which at least consist of repressors (OsVAL2 and OsGD1), a co-repressor (OsSAP18), and a histone deacetylase (OsHDA710). The repressor complexes may function in histone deacetylation and/or H3K27me3 methylation of Eui1 chromatin, which achieves a close chromatin state leading to low levels of Eui1 expression and GA homeostasis and thus normal plant growth. The biological function of OsVAL2 (solid-line oval) has been confirmed by genetic and molecular evidence, while other components (dashed-line ovals) of the repressor complex are only supported by molecular interaction evidence. Molecular Plant 2018 11, 720-735DOI: (10.1016/j.molp.2018.03.001) Copyright © 2018 The Authors Terms and Conditions