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SEUSS integrates transcriptional and epigenetic control of root stem cell organizer specification

2020; Springer Nature; Volume: 39; Issue: 20 Linguagem: Inglês

10.15252/embj.2020105047

1460-2075

Huawei Zhai, Xiaoyue Zhang, Yanrong You, Lihao Lin, Wenkun Zhou, Chuanyou Li,

Plant tissue culture and regeneration

Article14 September 2020Open Access Source DataTransparent process SEUSS integrates transcriptional and epigenetic control of root stem cell organizer specification Huawei Zhai Huawei Zhai State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiaoyue Zhang Xiaoyue Zhang orcid.org/0000-0003-4358-5881 State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yanrong You Yanrong You State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Lihao Lin Lihao Lin State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai'an, Shandong Province, China Search for more papers by this author Wenkun Zhou Corresponding Author Wenkun Zhou [email protected] orcid.org/0000-0002-2480-2644 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Frontier Science Center for Molecular Design and Breeding, China Agricultural University, Beijing, China Search for more papers by this author Chuanyou Li Corresponding Author Chuanyou Li [email protected] orcid.org/0000-0003-0202-3890 State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Huawei Zhai Huawei Zhai State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiaoyue Zhang Xiaoyue Zhang orcid.org/0000-0003-4358-5881 State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yanrong You Yanrong You State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China Search for more papers by this author Lihao Lin Lihao Lin State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai'an, Shandong Province, China Search for more papers by this author Wenkun Zhou Corresponding Author Wenkun Zhou [email protected] orcid.org/0000-0002-2480-2644 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Frontier Science Center for Molecular Design and Breeding, China Agricultural University, Beijing, China Search for more papers by this author Chuanyou Li Corresponding Author Chuanyou Li [email protected] orcid.org/0000-0003-0202-3890 State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Author Information Huawei Zhai1,2,‡, Xiaoyue Zhang1,‡, Yanrong You1, Lihao Lin3, Wenkun Zhou *,4,5 and Chuanyou Li *,1,2 1State Key Laboratory of Plant Genomics, National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Beijing, China 2CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China 3State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai'an, Shandong Province, China 4State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China 5Frontier Science Center for Molecular Design and Breeding, China Agricultural University, Beijing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 13426014658; E-mail: [email protected] *Corresponding author (lead contact). Tel: +86 10 64806612; E-mail: [email protected] The EMBO Journal (2020)39:e105047https://doi.org/10.15252/embj.2020105047 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Proper regulation of homeotic gene expression is critical for stem cell fate in both plants and animals. In Arabidopsis thaliana, the WUSCHEL (WUS)-RELATED HOMEOBOX 5 (WOX5) gene is specifically expressed in a group of root stem cell organizer cells called the quiescent center (QC) and plays a central role in QC specification. Here, we report that the SEUSS (SEU) protein, homologous to the animal LIM-domain binding (LDB) proteins, assembles a functional transcriptional complex that regulates WOX5 expression and QC specification. SEU is physically recruited to the WOX5 promoter by the master transcription factor SCARECROW. Subsequently, SEU physically recruits the SET domain methyltransferase SDG4 to the WOX5 promoter, thus activating WOX5 expression. Thus, analogous to its animal counterparts, SEU acts as a multi-adaptor protein that integrates the actions of genetic and epigenetic regulators into a concerted transcriptional program to control root stem cell organizer specification. Synopsis The evolutionarily conserved multi-adaptor protein SEUSS (SEU) promotes root stem cell organizer specification by integrating genetic and epigenetic regulators to drive expression of the quiescent center (QC) determinant WOX5 in Arabidopsis thaliana. SEU promotes QC specification in the root. Transcription factor SCARECROW (SCR) recruits SEU to the WOX5 promoter to promote its expression. SEU directly recruits the histone methyltransferase SDG4 to induce methylation and activation of the WOX5 promoter. SEU acts as an interface to coordinate the formation of a SCR-SEU-SDG4 transcriptional complex. Introduction Despite the huge evolutionary distance between plant and animal kingdoms, stem cell niches in both living forms contain organizer cells that maintain the adjacent stem cells (Dolan et al, 1993; Scheres, 2007; Dinneny & Benfey, 2008). In the model plant Arabidopsis thaliana, root stem cells are maintained by a small group of slowly dividing organizer cells called the quiescent center (QC) (Aichinger et al, 2012; Petricka et al, 2012). The QC generates signals that prevent differentiation of abutting stem cells, and it also acts as a reservoir to replace injured stem cells (van den Berg et al, 1995; Xu et al, 2006; Cruz-Ramirez et al, 2013). In turn, the pluripotent stem cells undergo formative asymmetric division to generate specific tissue layers of the whole root system (Aichinger et al, 2012; Petricka et al, 2012). QC is first initiated in the embryo by asymmetric division of the hypophyseal cell during the early-to-mid-globular embryo stage; the upper lens-shaped daughter cell acquires QC identity, whereas the lower daughter cell becomes columella stem cells (CSCs) (Jürgens et al, 1994; Scheres & Benfey, 1999; Jürgens, 2001; Weigel & Jürgens, 2002). Remarkably, QC and the entire root stem cell niche can be readily re-established in response to internal cues and external stresses during the lifelong post-embryonic growth, which, as in the case of some trees, can extend beyond several thousand years (Chen et al, 2011; Marhava et al, 2019; Zhou et al, 2019). Decades of molecular genetic studies have identified key transcription factors that regulate the acquisition of QC identity. Among these, the homeodomain transcription factor WUSCHEL (WUS)-RELATED HOMEOBOX 5 (WOX5) is the best-studied molecular marker of QC identity (Sarkar et al, 2007). WOX5 expression coincides with the embryonic formation of QC progenitors and persists specifically in the QC during post-embryonic root growth (Haecker et al, 2004; Sarkar et al, 2007). WOX5 suppresses CYCLIN D activity to establish the quiescence of the QC and coordinates with several hormonal signals and transcriptional regulators to maintain the identity of CSCs (Stahl et al, 2009; Ding & Friml, 2010; Chen et al, 2011; Stahl et al, 2013; Forzani et al, 2014; Pi et al, 2015). Given that WOX5 is exclusively expressed in the QC, extensive research has been conducted to identify factors that confine WOX5 expression to such a narrow domain (Zhang et al, 2015; Long et al, 2017). However, the molecular mechanism underlying WOX5 expression activation remains largely unknown. In contrast to WOX5, which is expressed exclusively in the QC, genes encoding the SCARECROW (SCR)/SHORT ROOT (SHR) transcription factors (belonging to the GRAS family) and PLETHORA (PLT) transcription factors [belonging to the APETALA2 (AP2) family] are expressed in larger domains including the QC (Di Laurenzio et al, 1996; Helariutta et al, 2000; Wysocka-Diller et al, 2000; Sabatini et al, 2003; Aida et al, 2004; Heidstra et al, 2004; Galinha et al, 2007). The scr single mutant and plt1 plt2 double mutant displayed similar root stem cell defects (Sabatini et al, 2003; Aida et al, 2004), leading to the hypothesis that the SCR/SHR and PLT pathways converge to specify the QC identity as follows: The SCR/SHR pathway provides positional information along the radial axis, while the PLT pathway provides apical–basal information (Scheres, 2007; Dinneny & Benfey, 2008). Consistent with this hypothesis, a recent study proposed that PLT and SCR form a protein complex through their interaction with the teosinte-branched cycloidea PCNA (TCP) transcription factor, and the PLT–TCP–SCR complex is essential for WOX5 expression and QC specification (Shimotohno et al, 2018). However, a previous study showed that the expression of WOX5 was reduced or undetectable in shr and scr mutants, but expanded to regions abutting the QC in the plt1 plt2 double mutant (Sarkar et al, 2007), suggesting that the SCR/SHR and PLT1/2 transcription factors might regulate WOX5 expression via distinct modes of action. The glutamine (Q)-rich SEUSS (SEU) protein contains a conserved domain, which shares high sequence similarity with the dimerization domain of the LIM-domain-binding (LDB) transcriptional co-regulator proteins in animals (Franks et al, 2002). The animal LDB proteins, such as LDB1 in mouse and Chip in Drosophila, play fundamental roles in the transcriptional regulation of cell-fate determination in versatile developmental processes (Agulnick et al, 1996; Morcillo et al, 1997; Matthews & Visvader, 2003; van Meyel et al, 2003; Bronstein et al, 2010; Bronstein & Segal, 2011; Love et al, 2014; Liu & Dean, 2019). Similar to its animal counterparts, SEU associates with cis-regulatory elements through its interaction with specific transcription factors to regulate gene expression during multiple developmental processes (Franks et al, 2002; Pfluger & Zambryski, 2004; Sridhar et al, 2004, 2006; Grigorova et al, 2011; Gong et al, 2016; Huai et al, 2018). Here, we report that SEU assembles a transcriptional complex to regulate root stem cell-fate determination. SEU functions in the SCR signaling pathway to promote WOX5 expression for QC specification. SCR physically interacts with and recruits SEU to the WOX5 promoter. Then, SEU recruits the ASH1-RELATED 3 (ASHR3) methyltransferase SET DOMAIN GROUP 4 (SDG4) (Cartagena et al, 2008; Kumpf et al, 2014) to the WOX5 promoter, which induces trimethylation of histone H3 lysine (K) 4 (H3K4me3), leading to WOX5 expression activation. Thus, SEU plays a fundamental role in the cell-fate determination of root stem cell organizers by coordinating the formation of a functional SCR–SEU–SDG4 transcriptional complex. Results SEU positively regulates WOX5 expression and QC specification To investigate the role of SEU in root stem cell determination, we generated transgenic plants expressing SEU fused to the green fluorescence protein (GFP) gene under the control of the SEU promoter (pSEU::SEU-GFP). The SEU-GFP fusion was localized to the nucleus and broadly expressed in the root meristem of post-embryonic seedlings (Fig EV1A). During embryogenesis, the expression of SEU-GFP was initiated early at the dermatogen stage and enriched broadly in different cells of the developing embryo (Fig 1A). Click here to expand this figure. Figure EV1. SEU regulates QC specification A. Expression pattern of pSEU::SEU-GFP in WT at 5 DAG. B–D. Root apical meristem phenotypes of the indicated genotypes. E, F. Expression pattern of pWOX5::WOX5-GFP in the indicated genotypes at 5 DAG. Insets: GFP channel (Insets scale bars: 10 μm). G. Quantification of the QC number in the indicated genotypes at 5 DAG. Data represent mean ± SD of three independent replicates. Different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01). Data information: In (A–F), scale bars: 20 μm. Download figure Download PowerPoint Figure 1. Ablation of SEU reduces WOX5 expression and impairs quiescent center (QC) specification A. Expression patterns of pSEU::SEU-GFP and pWOX5::GFP in the embryos of the indicated genotypes at dermatogen, early globular, and heart stages. White arrows indicate the QC precursor cell, and white dashed lines indicate embryos. Scale bars: 10 μm. B. Quantification of pWOX5::GFP GFP fluorescence in wild-type (WT) and seu-3 mutant embryos. Fluorescence intensity at the early globular stage of WT embryos was set to 1. C, D. Expression pattern of pWOX5::GFP in WT (C) and seu-3 (D) embryos at 5 days after germination (DAG). Scale bars: 20 μm. E. Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic plants, as shown in (C) and (D). Fluorescence intensity was normalized to the WT. F. RT–qPCR analysis of the relative expression levels of WOX5 in WT and seu-3 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. G–J. Modified pseudo-Schiff propidium iodide (mPS-PI) staining of stem cell niche areas in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indicate the columella stem cells (CSCs). The numbers denote total number of scored samples, with similar phenotypes showing in (G–J). Scale bars: 20 μm. K. Quantification of the CSC layer in the indicated genotypes at 5 DAG. L, M. Double staining of the QC184 β-glucuronidase (GUS) marker (light blue) and starch granules (dark brown) in WT (L) and seu-3 (M) seedlings at 5 DAG. N, O. Expression pattern of J2341 in WT (N) and seu-3 (O) seedlings at 5 DAG. P, Q. Expression pattern of CS9227 in WT (P) and seu-3 (Q) seedlings at 5 DAG. Data Information: In (B), (E), (F), and (K), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test). In (L–Q), white arrows indicate the CSCs. Scale bars: 20 μm. Download figure Download PowerPoint We then investigated the effect of SEU on the expression of WOX5, which is specifically expressed in and required for QC specification and function (Sarkar et al, 2007), by expressing the pWOX5::GFP construct (Blilou et al, 2005) in wild-type (WT) and seu-3 mutant (Pfluger & Zambryski, 2004) plants. In WT embryos, pWOX5::GFP expression was initiated in the QC progenitors at the early globular stage (Fig 1A). However, in seu-3 mutant embryos, pWOX5::GFP expression initiation was delayed to the heart stage, and the level of pWOX5::GFP expression was significantly reduced compared with the WT (Fig 1A and B). At 5 days after germination (DAG), seu-3 seedlings displayed markedly reduced pWOX5::GFP expression compared with the WT (Fig 1C–E). Consistently, reverse transcription-quantitative PCR (RT–qPCR) assays showed that the WOX5 transcript levels were significantly reduced in seu-3 seedlings compared with WT seedlings (Fig 1F). Together, these results indicate that SEU plays an essential role in promoting WOX5 expression during both embryogenesis and post-embryonic development. Next, we investigated whether the observed delay and reduction in WOX5 expression in seu-3 were accompanied by defects in QC specification and function. Similar to the wox5-1 mutant (Sarkar et al, 2007), seu-3 seedlings showed supernumerary cells with nonstereotyped shapes in the QC position (Fig 1C, D and G–I). Consistently, expression of the QC-specific marker QC184 was markedly reduced in seu-3 seedlings compared with the WT (Fig 1L and M), suggesting a loss of QC identity. The CSCs adjacent to the QC in seu-3 seedlings were also abnormal in shape and size and showed ectopic accumulation of starch granules, indicating that they had undergone differentiation (Fig 1L and M). Consistently, expression of the CSC-specific marker J2341 was largely abolished (Fig 1N and O), while that of the columella marker CS9227 expanded to the CSCs (Fig 1P and Q). Complementation of the seu-3 mutant by the introduction of the pSEU::SEU-GFP construct confirmed that the seu-3 mutation caused the observed phenotype (Fig EV1A–D). Together, these observations revealed that SEU positively regulates WOX5 expression and QC specification. To determine the genetic relationship between SEU and WOX5, we generated a seu-3 wox5-1 double mutant line. The seu-3 wox5-1 double mutant exhibited similar QC defects and CSC differentiation phenotypes as the wox5-1 mutant (Fig 1G–K). In addition, expression of pWOX5::WOX5-GFP (Pi et al, 2015) in seu-3 partially rescued the QC defects of the mutant (Fig EV1E–G). These results collectively support that SEU acts in the same pathway with WOX5 to regulate QC specification. SEU functions in the SHR/SCR pathway to promote WOX5 expression and QC specification Next, we asked whether and how SEU interacts with the master transcription factors SHR/SCR and PLTs, which converge in the root stem cell niche and play essential roles in QC specification (Sabatini et al, 2003; Aida et al, 2004; Scheres, 2007; Dinneny & Benfey, 2008). In the scr-3 mutant embryo, the expression of the pWOX5::GFP construct did not initiate until the heart stage, and the level of pWOX5::GFP expression was significantly lower than that in the WT embryo (Fig EV2A and B). By contrast, the plt1-4 plt2-2 double mutant showed higher pWOX5::GFP expression compared with the WT (Fig EV2A and B). Similarly, at 5 DAG, pWOX5::GFP expression was strongly reduced in scr-3 seedlings compared with the WT (Fig 2A, B and D) but expanded to the CSCs surrounding the QC in plt1-4 plt2-2 double mutant seedlings (Fig 2A, C and D). Consistently, RT–qPCR assays showed that the WOX5 transcript levels were significantly reduced in the scr-3 mutant but increased in the plt1-4 plt2-2 double mutant compared with the WT (Fig 2E). These observations uncovered that, similar to SEU, SCR positively regulates WOX5 expression, whereas PLT1/2 negatively regulate WOX5 expression. Click here to expand this figure. Figure EV2. SCR and PLT1/2 differentially regulate WOX5 expression during embryogenesis, and the SHR transcript levels were reduced in the seu-3 mutant A. Expression pattern of pWOX5::GFP in WT, scr-3, and plt1-4 plt2-2 embryos at the globular and heart stages. The white dashed lines indicate embryos. Scale bars: 10 μm. B. Quantification of GFP fluorescence in pWOX5::GFP transgenic and WT embryos shown in (A). Fluorescence intensity in the WT early globular stage embryos was set to 1. Data represent mean ± standard deviation (SD) of three independent replicates. C. RT–qPCR analysis of the relative expression levels of SHR in WT and seu-3 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test). D–G. Representative confocal images of the indicated genotypes at 5 DAG. Scale bars: 20 μm. H. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein–protein interactions. Download figure Download PowerPoint Figure 2. SEU acts in the SHR/SCR pathway to promote WOX5 expression and QC specification A–C. Expression pattern of pWOX5::GFP in WT (A), scr-3 (B), and plt1-4 plt2-2 (C) at 5 DAG. D. Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic seedlings, as shown in (A–C). GFP signal intensity in each genotype was normalized relative to that in the WT. E. RT–qPCR analysis of the relative expression levels of WOX5 in WT, scr-3, and plt1-4 plt2-2 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. F. Primary root length of the indicated genotypes at 5 DAG. G–J. Representative confocal images of the indicated genotypes at 5 DAG. White arrows indicate the QC. K. Quantification of GFP fluorescence in the QC of pSCR::GFP and pSCR::GFP-SCR transgenic seedlings, as shown in (G–J). GFP signal intensity of each genotype was normalized relative to that of the WT. L–Q. Root stem cell niche phenotypes of the indicated genotypes at 5 DAG. White dashed lines indicate the QC region. The numbers denote total number of scored samples, with similar phenotypes showing in (L–Q). Data information: In (D), (F), and (K), n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (D) and (F), different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01). In (E) and (K), **P < 0.01 (Student's t-test). Scale bars: 20 μm. Download figure Download PowerPoint The finding that both SEU and SCR promote WOX5 expression suggests that these proteins function in the same pathway. To test this possibility, we investigated the genetic interaction between SEU and SCR by comparing the root growth defects of the seu-3 scr-3 double mutant with its parental lines. While the seu-3 scr-3 double mutant displayed similar root growth defects as the scr-3 single mutant, seu-3 significantly enhanced the root growth defects of the plt1-4 plt2-2 (Fig 2F), indicating that SEU and SCR act genetically in the same pathway, which is independent of the PLT pathway. Consistent with these results, the expression of pSCR::GFP-SCR and pSCR::GFP was dramatically reduced in the QC region of seu-3 roots (Fig 2G–K), revealing that mutation of the SEU gene leads to a significant reduction in SCR expression in the QC, which affects QC identity. We also observed reduced expression of pSHR::GFP and pSHR::SHR-GFP in seu-3 roots compared with WT (Fig EV2D–G). Consistently, RT–qPCR assays showed that the SHR transcript levels were reduced in the seu-3 mutant (Fig EV2C). These results are in line with recent observations that SEU acts as an upstream transcriptional regulator of SHR (Gong et al, 2016; Clark et al, 2020). The seu-3 shr-2 double mutant displayed comparable root growth defects as shr-2 (Fig 2F). Additionally, the QC defects observed in the seu-3 scr-3 and the seu-3 shr-2 double mutants were similar to those observed in their parental lines, scr-3 and shr-2, respectively (Fig 2L–Q). Together, our results support that SEU acts in the SHR/SCR pathway to regulate QC specification. SCR physically recruits SEU to promote WOX5 expression and QC specification The finding that SEU and SHR/SCR functionally and genetically interact to regulate WOX5 expression and QC specification prompted us to test their possible physical interaction. Yeast two-hybrid (Y2H) assays showed that SEU interacts with SCR (Fig 3A) but not SHR (Fig EV2H). To confirm this observation, we performed in vitro pull-down experiments using purified maltose-binding protein (MBP)-tagged SEU (SEU-MBP) and FLAG epitope-tagged SCR (SCR-FLAG) or SHR (SHR-FLAG). SEU-MBP pulled down SCR-FLAG but not SHR-FLAG (Fig 3B), indicating that SEU interacts specifically with SCR in vitro. Furthermore, in co-immunoprecipitation (Co-IP) experiments performed in Nicotiana benthamiana leaves, GFP-tagged SCR (SCR-GFP) was immunoprecipitated by SEU-myc (Fig 3C). In Co-IP assays performed in transgenic Arabidopsis plants expressing SCR-GFP using anti-SEU antibody, SCR-GFP pulled down endogenous SEU (Fig 3D), confirming that SEU interacts with SCR in planta. Figure 3. SCR recruits SEU to the WOX5 promoter to promote its expression A. Yeast two-hybrid (Y2H) assays showing the interaction between SEU and SCR. The yeast transformants were plated on synthetic defined (SD) media lacking Leu and Trp (SD/-2) or lacking Ade, His, Leu, and Trp (SD/-4) to assess protein–protein interactions. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. B. In vitro pull-down assays showing that SEU directly interacts with SCR but not with SHR. SCR-FLAG was pulled down by SEU-MBP immobilized on amylose resin. Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti-FLAG antibody. The asterisk indicates the position of SEU-MBP. C. Verification of in vivo interactions between SCR and SEU in Nicotiana benthamiana leaves via Co-IP assays. SCR-GFP and SEU-myc were transiently coexpressed in N. benthamiana leaves. Protein samples were immunoprecipitated using anti-myc antibody. D. Co-IP assays of SEU with SCR in Arabidopsis. Protein extracts from WT and SCR-GFP roots were isolated at 5 DAG and immunoprecipitated with anti-GFP antibody. E. Schematic diagram of the WOX5 and PCR amplicons (indicated as letters A, B, and I) used for ChIP-qPCR. TSS, transcription start site. F, G. SCR and SEU physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP antibody. The precipitated DNA was used as a template for qPCR analysis. A, B, and I indicated the PCR amplicons as shown in (E). ACT7, control. H. Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was extracted from SEU-GFP and SEU-GFP scr-3 seedlings at 5 DAG and precipitated with anti-GFP antibody. ChIP signals were quantified by qPCR as a percentage of total input DNA. I. SEU stimulated SCR-mediated WOX5 promoter activation in transient expression assays in Nicotiana benthamiana leaves. The pWOX5::LUC reporter was cotransformed with the indicated effector constructs. The pWOX5::LUC activity was normalized relative to the internal control [LUC/Renilla luciferase (REN)]. The schematic diagram shows the construct used in the transient expression assays. Arrows indicate promoter regions, and boxes indicate coding sequences. Different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01). J–M. Representative confocal images of the indicated genotypes at 5 DAG. Scale bars: 20 μm. Insets show the QC region in which the solid white lines indicate the QC of WT, and the dashed white lines indicate the QC of seu-3. Insets scale bars: 5 μm. N. Quantification of GFP signal intensity in the QC of WT and seu-3 seedlings expressing pWOX5::SEU-GFP and pWOX5::SCR-GFP. GFP signal intensity in seu-3 seedlings was normalized relative to the WT. Individual values (black dots) are shown. n denotes the total number of scored samples. Data information: In (F), (G), (H), (I), and (N), data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test). Source data are available online for this figure. Source Data for Figure 3 [embj2020105047-sup-0003-SDataFig3.pdf] Download figure Download PowerPoint The above results suggest that SEU is physically recruited by SCR to the WOX5 promoter to promote its expression. Consistent with this presumption, chromatin immunoprecipitation (ChIP)-qPCR assays using pSCR::GFP-SCR transgenic Arabidopsis plants and anti-GFP antibody showed the enrichment of WOX5 promoter at approximately −1,100 bp (Fig 3E, fragment B and 3F). Parallel ChIP-qPCR assays using pSEU::SEU-GFP transgenic Arabidopsis roots and anti-GFP antibody revealed a similar enrichment pattern of the WOX5 promoter as that obtained using pSCR::GFP-SCR plants (Fig 3E, fragment B and 3G). Together, these results indicate that SEU and SCR are recruited to the same region of the WOX5 promoter. However, in scr-3 mutant plants expressing the pSEU::SEU-GFP construct, the enrichment of the WOX5 promoter was markedly reduced (Fig 3E, fragment B and 3H), indicating that th