elongator is required for root stem cell maintenance by ... · vious observations of the elp2...

Elongator Is Required for Root Stem Cell Maintenance byRegulating SHORTROOT Transcription1[OPEN]

Linlin Qi,a,b,2,3 Xiaoyue Zhang,b,c,2 Huawei Zhai,b,c,2 Jian Liu,a,2 Fangming Wu,b Chuanyou Li,b,4 andQian Chena,4,5

aState Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Taian,Shandong 271018, ChinabUniversity of the Chinese Academy of Sciences, Beijing 100049, ChinacState Key Laboratory of Plant Genomics, National Centre for Plant Gene Research, Institute of Genetics andDevelopmental Biology, Chinese Academy of Sciences, Beijing 100101, China

ORCID IDs: 0000-0001-5187-8401 (L.Q.); 0000-0003-4358-5881 (X.Z.); 0000-0003-0202-3890 (C.L.); 0000-0002-0300-3931 (Q.C.).

SHORTROOT (SHR) is essential for stem cell maintenance and radial patterning in Arabidopsis (Arabidopsis thaliana) roots, buthow its expression is regulated is unknown. Here, we report that the Elongator complex, which consists of six subunits (ELP1 toELP6), regulates the transcription of SHR. Depletion of Elongator drastically reduced SHR expression and led to defective rootstem cell maintenance and radial patterning. The importance of the nuclear localization of Elongator for its functioning, togetherwith the insensitivity of the elp1 mutant to the transcription elongation inhibitor 6-azauracil, and the direct interaction of theELP4 subunit with the carboxyl-terminal domain of RNA polymerase II, support the notion that Elongator plays important rolesin transcription elongation. Indeed, we found that ELP3 associates with the premessenger RNA of SHR and that mutation ofElongator reduces the enrichment of RNA polymerase II on the SHR gene body. Moreover, Elongator interacted in vivo withSUPPRESSOR OF Ty4, a well-established transcription elongation factor that is recruited to the SHR locus. Together, theseresults demonstrate that Elongator acts in concert with SUPPRESSOR OF Ty4 to regulate the transcription of SHR.

Root growth in higher plants relies on a group ofpluripotent, mitotically active stem cells residing in theroot apical meristem. In the root apical meristem of themodel plant Arabidopsis (Arabidopsis thaliana), the mi-totically less active quiescent center (QC) cells, togetherwith their surrounding stem cells, constitute the root

stem cell niche (SCN), which continues to provide cellsfor all root tissues (van den Berg et al., 1995). Pioneeringstudies have identified several key regulators that helpdetermine the specification and functioning of the SCN.Among these, the gibberellic acid insensitive, repressorof gibberellic acid, and SCR (SCARECROW) familytranscription factors SHORTROOT (SHR) and SCRprovide positional information along the radial axis,whereas the plant hormone auxin, together with itsdownstream components, the PLETHORA (PLT) classof transcription factors, provide longitudinal informa-tion (Di Laurenzio et al., 1996; Helariutta et al., 2000;Aida et al., 2004; Aichinger et al., 2012).

In addition to regulating the positional specificationof the QC, SHR also controls the formative division ofthe cortex/endodermis initial (CEI) stem cell and itsimmediate daughter cell (CEID), which generates theseparate endodermis and cortex cell layers constitutingroot ground tissue (van den Berg et al., 1995). Interest-ingly, SHR is transcriptionally expressed in the stele,and its encoded protein moves into the outer adjacentcell layer, where its partner SCR sequesters SHR to thenucleus by forming the SHR-SCR complex (Nakajimaet al., 2001; Cui et al., 2007). Recent efforts have suc-cessfully identified important transcriptional targets ofthe SHR-SCR complex. Among these are a group of so-called BIRD family genes encoding zinc finger proteins(Levesque et al., 2006; Welch et al., 2007; Long et al.,2015) and the cell cycle gene CYCLIN D6;1 (CYCD6;1;Sozzani et al., 2010). The spatiotemporal activation of

1This work was supported by the National Basic Research Pro-gram of China (2015CB942900 and 2013CB967301), the Tai-ShanScholar Program from the Shandong Province, China, the State KeyLaboratory of Plant Genomics of China, the State Key Lab of CropBiology of China, and the National Natural Science Foundation ofChina (31320103910).

2These authors contribute equally to the article.3Current address: State Key Laboratory of Veterinary Etiological

Biology, Lanzhou Veterinary Research Institute, Chinese Academy ofAgricultural Sciences, Lanzhou 0931, China.

4Senior authors.5Author for contact: [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Qian Chen ([email protected]).

L.Q. and X.Z. carried out genetic assays and genotyped the mu-tants; X.Z. andH.Z. undertook the confocal microscopy; J.L. made theconstructs and prepared the transgenic plants; F.W. carried out thebiochemical assays; X.Z. and Q.C. designed the project and draftedthe article with contributions of all the authors; C.L. and Q.C. super-vised and complemented the writing.

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CYCD6;1 is controlled by a bistable switch involvingSHR, SCR, and the cell differentiation factor RETINOBLASTOMA-RELATED, which also is regulated by theformation of a dynamic MED31-SCR-SHR ternarycomplex (Cruz-Ramírez et al., 2012; Zhang et al., 2018).Despite these advances, how the master regulator geneSHR itself is regulated remains largely unknown.In eukaryotic cells, protein-coding genes are transcribed

by RNA polymerase II (RNAPII). The multifunctionalprotein complex, Elongator, was first identified as aninteractor of hyperphosphorylated (elongating) RNAPII inyeast and laterwas purified fromhuman andArabidopsiscells (Otero et al., 1999; Hawkes et al., 2002; Nelissen et al.,2010). Elongator consists of six subunits, designated ELP1to ELP6, with ELP1 and ELP2 functioning as scaffolds forcomplex assembly, ELP3 acting as the catalytic subunit,and ELP4 to ELP6 forming a subcomplex important forsubstrate recognition (Versées et al., 2010; Glatt et al., 2012;Woloszynska et al., 2016). In yeast, the loss of Elongatorsubunits leads to altered sensitivity to stresses includingsalt, caffeine, temperature, and DNA-damaging agents(Otero et al., 1999; Krogan and Greenblatt, 2001; Esberget al., 2006). Since Elongator was copurified with elon-gating RNAPII and the ELP3 subunit showed histoneacetylation activity, it was initially proposed that Elon-gator functions mainly as a transcription elongation fac-tor, a process that occurs in the nucleus (Otero et al., 1999;Wittschieben et al., 1999; Winkler et al., 2002). Shortlythereafter, this proposition was questioned, as severalstudies show that yeast Elongator has diverse functionsrelated to its tRNAmodification activity that take place inthe cytoplasm (Huang et al., 2005; Esberg et al., 2006; Liet al., 2009; Chen et al., 2011; Bauer et al., 2012; Fernández-Vázquez et al., 2013).The physiological functions of Elongator in mammals

are exemplified by the finding that impaired Elongatoractivity in humans is correlated with the neurologicaldisorder familial dysautonomia (Anderson et al., 2001)and that mutations in Elongator subunits are lethal inembryotic mice (Chen et al., 2009). Like its yeast coun-terpart, human Elongator also has Lys acetyltransferaseactivity. Among the major substrates for the Lys acetyl-transferase activity of human Elongator are Histone H3and a-tubulin, reflecting the distinct functions of Elon-gator in the nucleus and cytoplasm. While, in the nu-cleus, the acetylation of Histone H3 is linked to thefunction of Elongator in transcription (Svejstrup, 2007),the cytoplasmic acetylation of a-tubulin by Elongatorunderlies the migration and maturation of neurons(Creppe et al., 2009).Genetic studies have demonstrated that Elongator

plays an important role in regulating multiple aspectsof plant development and adaptive responses to bioticand abiotic stresses (Nelissen et al., 2005, 2010; Zhouet al., 2009; Wang et al., 2013; Jia et al., 2015). Recentstudies reveal the role of plant Elongator in regulatingmicroRNA biogenesis and tRNA modification (Fanget al., 2015; Leitner et al., 2015).Here, we report the action mechanism of plant

Elongator in regulating root SCN and radial patterning.

We show that the root developmental defects of Elon-gator mutants are largely related to drastically reducedSHR expression. We provide evidence that Elongatoracts as a transcription regulator of SHR.

RESULTS

Elongator Is Required for SCN Maintenance and GeneralRoot Growth

To systematically evaluate the role of Elongator inregulating root growth, we investigated mutants of allsix Elongator subunits (elp1 to elp6; see “Materials andMethods”) and several double mutants, including elp1elp2, elp1 elp4, elp1 elp6, elp2 elp4, elp2 elp6, and elp4 elp6.Each of the single mutants exhibited similar reductionsin root growth, and none of the investigated doublemutant lines showed additive effects (SupplementalFig. S1A), implying that each subunit is essential for thefunctioning of Elongator and that Elongator acts as anintegral complex that regulates root growth. Therefore,we used elp1 as a representative mutant for detailedphenotypic analyses.Cytological observations revealed that both cell di-

vision and cell elongation were reduced in elp1(Supplemental Fig. S1, B–H). In a Lugol’s iodine starchstaining assay of wild-type roots expressing the QC-specific marker QC25, one layer of columella stemcells (CSCs) without starch staining was visible be-tween the QC and the columella cell layers, hinting at awell-organized and functional SCN (Fig. 1A). By con-trast, in elp1 root tips, QC25 expression was weak in theQC, but its expression pattern expanded downwardand merged with that of starch staining, and the CSCscould not be discerned clearly (Fig. 1B), suggesting theloss of QC cell identity and CSC differentiation. Consis-tently, in RNA in situ hybridization assays of wild-typeroots,WUSCHEL-RELATEDHOMEOBOX5 (WOX5) wasexpressed specifically in theQC, but its expression patternwas diffuse and merged with neighboring cells in elp1roots (Fig. 1, C and D). These results, together with pre-vious observations of the elp2 mutant (Jia et al., 2015),indicate that Elongator is required for root SCN mainte-nance and general root growth.

Elongator Regulates Radial Patterning in Roots throughthe SHR Pathway

To investigate the genetic relationship betweenElongator and the SHR pathway, we generated anelp1 shr-2 double mutant line. At 5 d after germination,general root growth and the meristem cell number ofthe double mutant were similar to those of shr-2 (Fig. 1,E and F), indicating that elp1 and shr-2 do not haveadditive effects on root growth. These results supportthe notion that Elongator acts genetically in the SHRpathway to regulate root growth.In parallel experiments, the elp1 plt1-1 plt2-4 triple mu-

tant line appeared to show an additive effect comparedwith its parental lines, elp1 and plt1-1 plt2-4 (Supplemental

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Fig. S2, A–F). Consistently, the elp1 mutation had only aminor effect (if any) on PLT1 and PLT2 expression(Supplemental Fig. S2, G–J). These results support thenotion that Elongator acts genetically in parallel with thePLT pathway.

In addition to having a defective SCN, shr mutantsalso exhibit irregular radial patterning and reducedstele width (Levesque et al., 2006). Hence, we examinedwhether elp1 shows similar phenotypes. The anatomi-cal organization showed that the cortex and endoder-mal cellular patterns of elp1were disordered comparedwith the wild type (Supplemental Fig. S3, A and B):CEI/CEID did not divide correctly and formed a singlecell layer in shr-2, and elp1 shr-2 showed similar phe-notypes with shr-2 (Supplemental Fig. S3, C and D).

Using the cortex-specific marker pCO2:H2B-YFP andthe endodermis-specific marker pSCR:GFP-SCR, weclearly distinguished these well-organized cell layers inwild-type roots (Fig. 1, G and I), whereas irregularpatterning in certain regions of the cortex and/or en-dodermis cell layers was observed frequently in elp1roots (Fig. 1, H and J). Consistently, the expressionlevels of CO2 and SCR were lower in elp1 than in thewild type (Fig. 1, G–N). In elp1, the expression of thecortex and endodermal marker J0571 also was dis-turbed and almost undetectable in some ground tissuecells, which demonstrates that these cells in elp1 losttheir cell identity (Supplemental Fig. S3, E and F).Moreover, the stele width at the transition zone alsowas reduced significantly in elp1 compared with the

Figure 1. Elongator functions in root SCNmaintenance and radial patterning throughthe SHR pathway. A and B, Double stainingof the QC-specific marker QC25 (blue) andstarch granules (dark brown) in the wildtype (WT; A) and elp1 (B) at 5 d after ger-mination (DAG). C and D, WOX5 expres-sion in 5 DAG in the wild type (C) and elp1(D) revealed by whole-mount RNA in situhybridization with a WOX5 antisenseprobe. E, Photograph of 5-DAG wild-type,elp1, shr-2, and elp1 shr-2 seedlingsshowing the involvement of the geneticrelationship of Elongator and SHR in regu-lating root growth. F, Quantification ofmeristem cell number of the indicatedplants. Data shown are averages and SD

(n = 20). Samples with different letters aresignificantly different at P , 0.01 (Fisher’sLSD mean separation test). G to J, Expressionof the cortex-specific marker pCO2:H2B-YFP (G and H) and the endodermis-specificmarker pSCR:GFP-SCR (I and J) in wild-type (G and I) and elp1 (H and J) roots.White rectangles highlight the disorga-nized cell layers in the elp1 mutant, andhorizontal white bars indicate the stelewidth (including pericycle cells). K to N,Transverse confocal sections showing theexpression of the endodermis-specificmarker pSCR:GFP-SCR at the CEI/CEIDposition (K and M) and the transition zone(L andN) in thewild type (K and L) and elp1(M and N). Horizontal white bars indicatethe stele width (including pericycle cells).O, Quantification of stele width in the wildtype and elp1. The stele width (includingthe pericycle cells) at the transition zoneposition in the longitudinal confocal im-ages was measured with ImageJ software.Data shown are averages and SD (n = 20),and asterisks denote Student’s t test signif-icance compared with the wild type: **,P , 0.01. Bars = 50 mm (A, B, and G–N)and 20 mm (C and D).

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Elongator Regulates SHORTROOT Transcription










wild type (Fig. 1, G–O). Together, the phenotypic sim-ilarity between elp1 and shr strengthens the idea thatElongator acts in the SHR pathway to regulate radialpatterning in roots.

Depletion of Elongator Impairs the Expression of SHR andIts Target Genes

Using the SHR promoter fusion line pSHR:erGFP andthe SHR protein fusion line pSHR:SHR-GFP, we foundthat SHR expression levels were reduced drastically inelp1 compared with the wild type (Fig. 2, A–D). Thisobservation was confirmed by RNA in situ hybridiza-tion (Fig. 2, E and F) and reverse-transcription quanti-tative PCR (RT-qPCR) assays (Fig. 2G). Not surprisingly,as revealed by RT-qPCR, the expression levels of severalSHR transcriptional targets, including SCR, BR6ox2,CYCD6;1, MGP, NUTCRACKER (NUC), RECEPTOR-LIKE KINASE (RLK), and SNEEZY/SLEEPY2 (SNE), alsowere reduced substantially in elp1 compared with thewild type (Fig. 2G). We then investigated whether theelp1 mutation impairs the expression of the cell cyclegene CYCD6;1 in the CEI/CEID. Previous studies haveelegantly demonstrated that the spatiotemporal activa-tion of CYCD6;1 coincides with the formative division ofCEI/CEID and that this process is strictly controlled bySHR and the related transcription factor SCR (Sozzaniet al., 2010; Cruz-Ramírez et al., 2012). As expected, the

spatiotemporal expression of CYCD6;1 in the CEI/CEIDwas largely disrupted in the elp1 mutant (Fig. 2, H and I).Together, these results indicate that the depletion of Elon-gator impairs the expression of SHR and its target genes.To visualize the expression pattern of the Elongator

subunit ELP1, we fused the ELP1 promoter with theGUS reporter and generated pELP1:GUS transgenicplants. GUS staining revealed that, like SHR, ELP1washighly expressed in the stele of the root tip (Fig. 2J). Thisobservation strengthens the notion that Elongator reg-ulates root development through the SHR pathway.

Nuclear Localization of Elongator Is Important for ItsFunction in Regulating Root Development

To determine the subcellular localization of Elon-gator, we introduced the pELP3:ELP3-GFP fusionconstruct into elp3 plants. The functionality of the ELP3-GFP fusion protein was verified by its ability to rescuethe root growth defects of the elp3mutant (SupplementalFig. S4A). Confocal microscopy of elp3;pELP3:ELP3-GFPplants indicated that, in stele cells of themeristem region,ELP3-GFP was localized predominantly to the cyto-plasm and, to a lesser extent, the nucleus (Fig. 3, A andB). Interestingly, we observed more obvious nuclear lo-calization in columella cells and epidermis cells at theelongation zone in the previously reported line, p35S:GFP-ELP3 (Supplemental Fig. S4B).

Figure 2. Effect of Elongator depletion on the expression of SHR and its target genes. A to F, SHR expression is compared betweenthe elp1 mutant (B, D, and F) and the wild type (WT; A, C, and E), as revealed by the expression of the marker constructs pSHR:erGFP (A and B) and pSHR:SHR-GFP (C and D) and bywhole-mount RNA in situ hybridizationwith a SHR antisense probe (E andF). G, RT-qPCR analysis showing the relative expression levels of SHR and its target genes in thewild type and elp1. Total RNAwasextracted from 0.5-cm root tip sections of 5-DAG seedlings. Transcript levels were normalized to the reference gene PP2AA3.Error bars represent SD (Student’s t test, **, P, 0.01). The experiments were repeated three times, yielding similar results. H and I,Representative images showing the location-specific expression and the reduced expression of pCYCD6;1:GFP-GUS in CEI/CEIDcells of the wild type and the elp1mutant, respectively. J, GUS staining of pELP1:GUS showing the expression pattern of ELP1 inroot tips. Bars = 50 mm (A–F, H, and I) and 100 mm (J).


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We then employed a cell fractionation approach todetermine the subcellular localization of endogenousELP1 and ELP3. For these experiments, protein extractsof wild-type seedlings were fractionated and probedwith antibodies that specifically recognize endogenousELP1 or ELP3 protein (Supplemental Fig. S5, A and B).Histone H3 was detected exclusively in the nuclearcompartment, whereas phosphoenolpyruvate carbox-ylase (PEPC) was detected only in the cytoplasmiccompartment, validating our approach. Consistentwith the above cytological observations, endogenousELP1 and ELP3 were detected clearly in both the cyto-plasmic and nuclear fractions (Fig. 3C). We obtainedsimilar results from cell fractionation experiments usingelp3;p35S:ELP3-myc transgenic plants, in which the rootgrowth defects of the elp3 mutant had been rescued(Supplemental Fig. S5C). These results help confirm thefinding that Arabidopsis Elongator is located in boththe cytoplasm and the nucleus.

To determine if the nuclear localization of ELP3 iscritical for its function, we artificially confined ELP3 tothe nuclear compartment and investigated whether itwould still retain its function. For these experiments,ELP3-GFP was fused with the efficient SV40 nuclear

localization sequence (NLS; van der Krol and Chua,1991) to generate the p35S:NLS-ELP3-GFP construct.Analysis of the resulting transgenic plants indicatedthat the NLS-ELP3-GFP fusion protein was trans-located successfully into the nucleus and, more im-portantly, the nucleus-localized NLS-ELP3-GFP fusionprotein was functional, as it fully rescued the rootgrowth defects of elp3 (Fig. 3, D–F). The HistoneH3 Lys-14 acetylation level is reduced slightly in elp1and elp3, which is consistent with the nuclear localiza-tion and the reported histone modification function ofthe Elongator complex (Supplemental Fig. S5D). Theseresults support the notion that the nuclear localizationof Elongator is important for its function in regulatingroot development.

Elongator Functions as a Transcription Elongation Factor toRegulate SHR Transcription

Our finding that the nuclear localization of plantElongator is important for its biological function sug-gested that Elongator might act as a transcriptionelongation factor involved in RNAPII-dependent tran-scription. To investigate this notion, we first examined

Figure 3. Importance of the nuclear localization of Elongator for its function. A and B, Representative images showing thelocalization of ELP3-GFP in 5-DAG elp3;pELP3:ELP3-GFP transgenic plants. B shows a magnification of the image in the whiterectangle in A. C, Cell fractionation assay of Columbia-0 (Col-0) seedlings. Ten-day-old Col-0 seedlings were collected for cellfractionation. Proteins from different fractions were immunoblotted with antibodies against ELP1, ELP3, H3, and PEPC. H3 andPEPC were used as nucleus- and cytoplasm-specific marker proteins, respectively. Asterisks indicate the positions of the specificbands. T, Total extracts; C, cytoplasmic fraction; N, nuclear fraction. The experiments were repeated three times, yielding similarresults. D and E, Representative images showing the subcellular localization of ELP3-GFP (D) and NLS-ELP3-GFP (E), indicatingthe nuclear localization of NLS-ELP3-GFP. F, Photograph of 5-DAG seedlings showing that nucleus-localized ELP3 fully com-plemented the short-root phenotype of the elp3mutant. ELP3was fusedwith an efficient SV40NLS to artificially translocate it intothe nucleus. The resulting constructs were transformed into the elp3 background to determine phenotype complementation. WT,Wild type. Bars = 50 mm (A, D, and E) and 10 mm (B).








whether the Elongator subunits interact with the con-served C-terminal domain (CTD) of RNAPII, an inter-action platform between RNAPII and other proteinsinvolved in transcription.The RNAPII CTD interacted with ELP4 and ELP5 in

yeast two-hybrid (Y2H) assays (Fig. 4A). To determinewhether ELP4 interacts with CTD in planta, we con-ducted firefly luciferase (LUC) complementationimaging (LCI) assays inNicotiana benthamiana leaves. Inthese experiments, ELP4 was fused to the N-terminalhalf of LUC (nLUC) to produce ELP4-nLUC, whereasCTDwas fused to the C-terminal half of LUC (cLUC) toproduce cLUC-CTD. N. benthamiana cells coexpressingELP4-nLUC and cLUC-CTD displayed strong lumi-nescence signals, whereas those coexpressing nLUCand cLUC-CTD or ELP4-nLUC and cLUC displayed nosignal (Fig. 4, B and C), confirming that the ELP4-CTDinteraction occurs in vivo.We then examined the response of elp1 to 6-azauracil

(6-AU), an inhibitor of enzymes involved in purine andpyrimidine biosynthesis. In yeast, 6-AU is a widelyused inhibitor of transcription elongation, as it altersnucleotide pool levels in vivo (Exinger and Lacroute,1992). Strikingly, elp1 was more resistant to 6-AU-in-duced root growth inhibition than the wild type(Fig. 4D), providing another line of evidence thatElongator functions as a transcription elongation factorin Arabidopsis.Next, we investigated whether Elongator partici-

pates in RNAPII-dependent transcription elongationof SHR using chromatin immunoprecipitation(ChIP)-qPCR assays. In wild-type plants, CTD washighly enriched on both the transcription start siteand the gene body of SHR (Fig. 4E). In the elp1 mu-tant, however, CTD levels on the SHR locus werereduced significantly (Fig. 4E), revealing that Elon-gator is important for the recruitment of RNAPII tothe SHR locus during SHR transcription. This linealso demonstrates that Elongator is involved mainlyin transcription elongation rather than transcriptioninitiation.We then performedRNA immunoprecipitation assays

to investigatewhether Elongator associates with the pre-mRNA of SHR. Specifically, we used the GFP antibodyto immunoprecipitate ELP3-GFP from extracts of theelp3;p35S:NLS-ELP3-GFP transgenic line. The resultingELP3-GFP immunoprecipitates then were reversetranscribed into cDNAs and subjected to RT-PCRwith primers specific for SHR, SCR, PLT2, orACTIN7 (ACT7). Pre-mRNA of SHR was detected inimmunoprecipitates from the elp3;p35S:NLS-ELP3-GFP line but not from those of the wild type (Fig. 4F),confirming that ELP3 indeed associates with SHR pre-mRNA. As a control, SCR, PLT2, and ACT7 pre-RNAswere not detected in the same ELP3-GFP immuno-precipitates (Fig. 4F), suggesting that ELP3 associatesspecifically with the pre-mRNA of SHR. Together,these results led us to conclude that Elongator regu-lates the transcription of SHR through associatingwithits pre-mRNA.

ELP1 Associates with the Transcription Elongation FactorSUPPRESSOR OF Ty4, Which Is Recruited to theSHR Locus

Our results support the notion that Elongator regu-lates the transcription elongation of SHR through as-sociating with the pre-mRNA of SHR. Intriguingly,however, we failed to detect Elongator enrichment onthe SHR locus in the ChIP experiments. We speculatedthat Elongator might act in concert with other knowntranscription elongation factors to regulate the elonga-tion of SHR transcript. Indeed, ELP1 and ELP3 recentlywere affinity copurified with SUPPRESSOR OF Ty4(SPT4) and several other conserved transcription elon-gation factors in eukaryotic cells (Dürr et al., 2014;Antosz et al., 2017). In Arabidopsis, SPT4 is encoded bytwo redundant genes, designated SPT4-1 and SPT4-2(Dürr et al., 2014). In a coimmunoprecipitation (Co-IP)assay using p35S:SPT4-2-GFP plants and anti-ELP1antibodies, SPT4-2-GFP pulled down native ELP1, in-dicating that ELP1 associates with SPT4-2 in vivo(Fig. 5A). The in vivo association of ELP1 with SPT4-2was confirmed further in LCI assays: N. benthamianacells coexpressing ELP1-nLUC and cLUC-SPT4-2 dis-played strong luminescence signals (Fig. 5, B and C).To demonstrate that SPT4-2 plays a role in root de-

velopment, we generated SPT4-RNAi plants in whichthe expression of both SPT4-2 and SPT4-1was knockeddown by RNA interference (RNAi; Supplemental Fig.S6). Like elp1 and the other elp mutants (SupplementalFig. S1), the SPT4-RNAi plants also showed reducedroot growth (Fig. 5D) and irregular patterning in certainregions of the cortex and/or endodermis cell layers(Supplemental Fig. S6, C–E). These results are consis-tent with a previous observation, and they suggest thatthe interplay between Elongator and SPT4/SPT5 mighthelp regulate root development (Dürr et al., 2014; VanLijsebettens et al., 2014; Woloszynska et al., 2016;Antosz et al., 2017). Furthermore, like the elp1 mutant,SPT4-RNAi plants were less sensitive to 6-AU-inducedroot growth inhibition than wild-type plants (Fig. 5D),implying that the function of SPT4-2 in regulating rootgrowth is related to transcription elongation. Indeed, theChIP-qPCR assays revealed that SPT4-2 was enriched onthe SHR locus and, importantly, that the levels of SPT4-2on the SHR gene body regions were higher than those onthe SHRpromoter region (Fig. 5E), suggesting that SPT4 isinvolved mainly in SHR transcription elongation. Takentogether, our results support the notion that Elongatoracts in concert with SPT4/SPT5 to regulate the tran-scription of SHR, thereby regulating root development.

DISCUSSION

Elongator Is Required for Root SCN Maintenance andRadial Patterning through Regulating SHRGene Expression

In addition to the reduced primary root growth anddefective SCN reported previously (Jia et al., 2015), weobserved irregular radial patterning and reduced stele


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width in Elongator single and double mutants, indi-cating that Elongator functions as an integral complexin the regulation of root development. Several lines ofevidence indicate that the developmental defects in

roots of the Elongator mutants are largely due to animpaired SHR pathway. Indeed, the elp1 root pheno-types resemble those of shr (Fig. 1) and coincide withthe similar developmental gene expression patterns in

Figure 4. Elongator functions as a transcription elongation factor to regulate SHR expression. A, Interactions of different Elongatorsubunits with the RNAPII CTD in a Y2H assay. The yeast transformants were dropped onto synthetic dextrose (SD)/-Ade/-His/-Trp/-Leu (SD/-4) medium to assess protein-protein interactions. The experiments were repeated three times with similar results. B and C,Firefly LCI assay showing the interaction of the ELP4 subunit with the RNAPII CTD inN. benthamiana.N. benthamiana leaves wereinfiltratedwith Agrobacterium tumefaciens containing the indicated construct pairs (B). The image was obtained 3 d after infiltration(C). The colored bar indicates the relative signal intensity. The experiments were repeated three times, yielding similar results. D,Photographs of 5-DAG seedlings showing the insensitivity of the elp1mutant to 6-AU.Wild type (WT) and elp1 seeds were sown onone-half-strength Murashige and Skoog (1/2 MS) medium without or with 0.5 mg L21 6-AU, and the plates were photographed at 5DAG. E, RNAPII enrichment at various regions of the SHR locus by ChIP-qPCR. Chromatin was extracted from Col-0 and elp1seedlings at 5 DAG and precipitated with an anti-CTD antibody (Abcam). Precipitated DNA was amplified with primers corre-sponding to the different regions of SHR as shown. The no-antibody (No Ab) precipitates served as negative controls. The ChIP signalwas quantified as the percentage of total input DNA by qPCR and arbitrarily set to 1 in the no-antibody samples. TSS, Transcriptionstart site. The experiments were repeated three times, yielding similar results. Error bars represent SD. Asterisks indicate significantdifferences between Col-0 and the elp1mutant according to Student’s t test (**, P, 0.01). F, RNA immunoprecipitation PCR resultsshowing the association of Elongator with SHRmRNA but not with PLT2 or SCRmRNA. Protein-RNA complexeswere isolated fromCol-0 and elp3;p35S:NLS-ELP3-GFP seedlings at 5 DAG and precipitatedwith an anti-GFPantibody (Abcam). The precipitated RNAwas reverse transcribed and then amplified with primers targeting the respective coding sequence (CDS) regions. The RNA im-munoprecipitation signal was quantified as the percentage of total input RNA by qPCR. Samples before precipitation were taken asinput, and the no-antibody (NA) precipitates served as negative controls. The experiments were repeated three times with similarresults. Asterisks indicate significant differences according to Student’s t test (***, P , 0.01)..





Figure 5. Elongator functions in concert with SPT4/SPT5. A, Co-IP assay showing that Elongator associates with SPT4-2 in plantcells. Protein extracts from 10-d-old Col-0 and p35S:SPT4-2-GFP seedlings were immunoprecipitated with an anti-GFPantibody(Abcam). Samples before (Input) and after immunoprecipitation (IP) were blotted with anti-GFP and anti-ELP1 antibodies. Theexperiments were repeated three times with similar results. B and C, Firefly LCI assay showing the interaction of the ELP1 subunitwith SPT4-2 in N. benthamiana. N. benthamiana leaves were infiltrated with A. tumefaciens containing the indicated constructpairs (B). The image was obtained 3 d after infiltration (C). The colored bar indicates the relative signal intensity. The experimentswere repeated three times, yielding similar results. D, Photographs of 5-DAG seedlings showing the insensitivity of SPT4-RNAi to6-AU.Wild type (WT) and SPT4-RNAi seedswere sown on 1/2MSmediumwithout or with 0.5mg L21 6-AU, and the plateswerephotographed at 5 DAG. E, ChIP-qPCR results showing the enrichment of SPT4-2 on the SHR locus. Sonicated chromatin from 5-DAGCol-0 and p35S:SPT4-2-GFP seedlingswas precipitatedwith an anti-GFPantibody (Abcam). The precipitatedDNAwas usedas a template for qPCR analysis with primers targeting different regions of the SHR locus as shown. The promoter region of ACT7(ACT7-P) was used as a negative control. The ChIP signal was quantified as the percentage of the total input DNA and was ar-bitrarily set to 1 in Col-0. TSS, Transcription start site. The experiments were repeated three times, yielding similar results. Errorbars represent SD. Asterisks indicate significant differences according to Student’s t test (**, P, 0.01). F, Proposed mechanism inwhich Elongator acts in concert with SPT4/SPT5 to regulate the transcription elongation of SHR. During the transcription elon-gation process of SHR, Elongator interacts directly with the RNAPII CTD and the nascent SHRmRNA and associates with SPT4/SPT5.Meanwhile, SPT4/SPT5 is in close contact with the chromosomal region harboring the SHR locus, the RNAPII subunits, andElongator.


Qi et al.



the primary root, with the highest expression in thestele tissue of root tips. Elongator acts upstream of SHRand independently of the PLT pathway, as the expres-sion levels of SHR and its target genes SCR andCYCD6;1 were reduced drastically in elp1 (Fig. 2, A–I),whereas the expression of PLT1 and PLT2 was less af-fected in this mutant (Supplemental Fig. S2). Thus,Elongator regulates root development mainly throughits impact on the SHR pathway. In contrast to ourknowledge about SHR target genes and interactingproteins, little had been known about how SHR itself isregulated, except that SHR expression is not regulatedby PLT1, PTL2, SCR, or SHR itself (Helariutta et al.,2000; Aida et al., 2004). Our results indicate that Elon-gator is a regulator of SHR expression. The develop-mental and external stimuli that regulate the expressionof the genes encoding the six Elongator subunits, suchas hormones and temperature (Woloszynska et al.,2016), might affect the production of Elongator subu-nits, thus affecting the assembly and accumulation ofthis crucial complex and thereby controlling (to someextent) SHR gene expression. Elongator might serve asan interface between these stimuli and SHR, and theregulation of the SHR gene at the transcription elon-gation stage might render its expression more flexibleand responsive, which is important for the spatial andtemporal control of root development.

Elongator Regulates the Transcription of SHR in Concertwith SPT4/SPT5

The role of Elongator as a transcription elongationfactor is highly controversial in yeast and humans(Glatt et al., 2012) and was suggested recently in plants(Woloszynska et al., 2016). Here, using various experi-mental approaches, we provide direct evidence for thetranscription elongation activity of Elongator. Usingconfocal microscopy analysis of various transgeniclines combined with cell fractionation, we showed thatthe Elongator subunits are localized partially to thenucleus, although major proportions of these subunitsare localized to the cytoplasm (Fig. 3). Moreover, thenuclear localization of these subunits is important fortheir biological function, as artificially nucleus-localizedELP3 was still able to complement the elp3 mutantphenotype (Fig. 3, D–F); a similar experiment was per-formed in yeast, but with contrasting results (Rahl et al.,2005). Our protein purification assays revealed a directinteraction of the ELP4 subunit with RNAPII CTD(Fig. 4, A–C) and the association of ELP1 with the well-established transcription elongation factor SPT4/SPT5(Fig. 5, A–C), indicating that Elongator is involved intranscription elongation.However,we failed to detect aninteraction between ELP4 and RNAPII in a Co-IP assay.This result confirms a previous report showing interac-tions between the different subunits of Elongator but nointeractions between Elongator subunits and RNAPIIusing tandem affinity purification-mass spectroscopy(Nelissen et al., 2010) but disagrees with a more recentreport showing copurification of the two largest subunits

of RNAPII with the Elongator subunits ELP1 and ELP3using SPT4 as bait (Dürr et al., 2014). Hence, the associ-ation of Elongator with RNAPII in vivo might be tran-sient and dynamic (Van Lijsebettens et al., 2014).

Yeast mutants defective in transcription elongationexhibit an altered sensitivity to 6-AU (Nakanishi et al.,1995; Wu et al., 2003). We found that both elp1 andSPT4-RNAi plants were highly resistant to 6-AU treat-ment (Figs. 4D and 5D), suggesting that Elongator playsa similar role in transcription elongation to that ofSPT4/SPT5. Finally, we detected a significant reductionin the enrichment of RNAPII CTD on the SHR locus inelp1 compared with the wild type (Fig. 4E) as well as anassociation of ELP3 with SHR mRNA (Fig. 4F). More-over, its associated protein, SPT4, also was recruited toSHR chromatin (Fig. 5E). These findings indicate thatboth Elongator and SPT4/SPT5 are involved directly inthe transcription regulation of SHR. However, we failedto detect a significant enrichment of Elongator on theSHR region via ChIP, possibly due to a lack of DNA-binding activity and/or the dynamic properties of itsinteraction with other proteins. No ChIP data areavailable currently for the enrichment of Elongator ontranscriptional regulatory regions in Arabidopsis.Hence, we propose a model in which Elongator acts inconcert with SPT4/SPT5 to maintain the transcriptionof SHR. In this model, Elongator interacts directly withthe RNAPII CTD through the ELP4 subunit and asso-ciates with SHRmRNA,whereas SPT4/SPT5 associateswith RNAPII and Elongator as well as the chromo-somal region harboring SHR (Fig. 5F).

Transcription elongation is a tightly controlled, dy-namic process that can be divided into three distinctstages: promoter escape, promoter-proximal pausing,and productive elongation. Based on their activities,transcription elongation factors can be categorized aspositive or negative. In yeast, mutations of positivetranscription elongation factors often are associatedwith hypersensitivity to 6-AU, whereas the disruptionof negative transcription elongation factors renders thecells less sensitive to 6-AU (Wu et al., 2003). We dem-onstrated that Elongator associates with SPT4/SPT5(Fig. 5, A–C) and that both elp1 and SPT4-RNAi arehighly insensitive to 6-AU (Figs. 4D and 5D), suggest-ing that Elongator and SPT4/SPT5 are negative tran-scription elongation factors, as in yeast. Indeed, humanSPT4/SPT5, known as DSIF (for 5,6-dichloro-1-b-D-ri-bofuranosylbenzimidazole sensitivity-inducing factor),is a negative transcription elongation factor that con-tributes to promoter-proximal pausing (Yamaguchiet al., 2013). Transcriptional pausing also is thought toparticipate in mRNA synthesis, possibly through theformation of a preactivated state (Yamaguchi et al.,2013). Hence, Elongator and SPT4/SPT5 might playan (as yet) unknown role in promoter-proximal pausingin Arabidopsis. Moreover, our ChIP-qPCR results in-dicated that CTD enrichment on the SHR promoter(around the transcription start site region), which also isassociated with SPT4, was reduced in the elp1 mutant(Figs. 4E and 5E). These findings imply that the






ELP-SPT4-polymerase II complex not only regulatesSHR transcription elongation but also transcription in-itiation, which is consistent with the abolished expres-sion of pSHR:erGFP (Fig. 2, A and B) and also supportsthat Elongator and SPT4/SPT5 may function inpromoter-proximal pausing.

Functional Diversification of Elongator in Eukaryotes

In yeast, tRNA modification appears to be the direct,unequivocal biochemical function of Elongator, be-cause its cytoplasmic localization (Rahl et al., 2005)excludes the possibility of this complex being a tran-scription elongation factor. In addition, overexpressingtwo related tRNA species rescued almost all of thereported phenotypes of yeast Elongator mutants, in-cluding reduced H3K14Ac levels, suggesting that evenhistone acetylation might be an indirect effect of Elon-gator activity (Esberg et al., 2006). Various biologicalprocesses are modulated through Elongator tRNAmodifications, such as telomeric gene silencing (Chenet al., 2011), cell cycle control (Bauer et al., 2012), andoxidative stress responses (Fernández-Vázquez et al.,2013). However, in animal cells, Elongator is localizedpartially to the nucleus (Hawkes et al., 2002; Creppeet al., 2009), and its role in transcription elongation issupported by the enrichment of Elongator subunits oncertain genes related to cell migration. Here, we pro-vided direct evidence that Elongator acts as a tran-scription regulator in Arabidopsis. Since Elongator wasfirst copurified with elongating RNAPII from total cellextracts in yeast (Otero et al., 1999) and their interactionwas confirmed further in human cells (Hawkes et al.,2002) and here in Arabidopsis, it is reasonable to sus-pect that the association of Elongator with RNAPII inyeast is notmerely a coincidence. Thus, we propose thatboth tRNA modification and RNAPII association aretwo functions of Elongator. However, the cytoplasmiclocalization of this complex in yeast precludes it frombeing a transcriptional regulator; therefore, the tRNAmodification activity of Elongator contributes the mostto its function. By contrast, in human and plant cells, theacquisition of a partial nuclear localization for thiscomplex might have caused it to develop a capacity fortranscriptional regulation. In animals, the cytoplasm-localized Elongator evolved various other activities,such as acetylation of a-tubulin in the mouse cortex(Creppe et al., 2009). In plants, Elongator also playsroles in the cytoplasm; namely, its tRNA modificationactivity is conserved in Arabidopsis (Versées et al.,2010), but how this localization contributes to its bio-logical function is still uncertain, although auxin re-sponses depend on Elongator tRNA activity (Leitneret al., 2015).Transcriptional regulation is a major, delicate regu-

latory mechanism involving numerous proteins. Thekey players in postembryonic root development inplants, such as PLT1, PLT2, SHR, SCR, and WOX5, areall transcription factors (Aichinger et al., 2012). Severalother transcriptional regulators also are required for

this process, such as chromatin-remodeling factors(Aichinger et al., 2012), histone acetyltransferases, andsplicing factors. In plants, Elongator might play a role inthe cross talk between environmental and develop-mental stimuli to flexibly control SHR transcription,thereby modulating root growth and developmentthroughout the plants’ life cycle.

MATERIALS AND METHODS

Plant Material and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) Elongator mutants elp1 (abo1-2;SALK_004690), elp2, elp4 (SALK_079193), and elp6 and the double mutants elp1elp2, elp1 elp4, elp1 elp6, elp2 elp4, elp2 elp6, and elp4 elp6 (Zhou et al., 2009) wereobtained from Zhizhong Gong. The mutants elp3 (elo3-6; GABI_555H06;Nelissen et al., 2010) and elp5 (GABI_700A12) were ordered from the Arabi-dopsis Biological Resource Center. The plant materials used in this study weredescribed previously: plt1-4 plt2-2 (Aida et al., 2004), shr-2 (Helariutta et al.,2000), pCYCB1;1:GUS (Colón-Carmona et al., 1999), QC25 (Sabatini et al., 1999),pSHR:erGFP (Koizumi et al., 2012), pSHR:SHR-GFP (Nakajima et al., 2001),pSCR:GFP-SCR (Sabatini et al., 1999), pCO2:H2B-YFP (Heidstra et al., 2004), andpCYCD6;1:GFP-GUS (Sozzani et al., 2010). The triple mutant elp1 plt1-4 plt2-2,the double mutant elp1 shr-2, and different marker lines in the mutant back-ground were all obtained by genetic crossing.

Seeds ofArabidopsiswere surface sterilizedwith 10% (v/v) bleach for 10minand washed three times with sterile water. Sterilized seeds were suspended in0.1% (w/v) agarose and plated on 1/2 MS medium (PhytoTechnology Labo-ratories). After stratification for 2 d at 4°C, they were transferred to the growthchamber at 22°C with a 16-h-light/8-h-dark cycle.

Plasmid Construction and Plant Transformation

An approximately 2-kb fragment including the promoter region and thecoding sequences for theN-terminal 29 amino acids of ELP1was amplified fromgenomic DNA by PCR and cloned into the PacI/AscI sites of the binary vectorpMDC162, resulting in the pELP1:GUS construct, inwhich the coding sequenceswere fused in frame with GUS. For the pELP3:ELP3-GFP construction, the re-gion containing the GFP-coding sequence and the NOS-T fragment from thepGFP-2 vector as well as the coding sequence (CDS) of ELP3 and its promotersequences were sequentially cloned in frame into the binary vector pCAM-BIA1300 with the restriction enzyme sites XbaI/BamHI and SalI/XbaI, respec-tively. To generate the p35S:ELP3-myc or p35S:ELP3-GFP plasmid, the ELP3CDS was first cloned with the pENTR Directional TOPO Cloning Kit (Invi-trogen) and then recombined with the binary vector pGWB17 or pGWB5 withthe Gateway LR Clonase Enzyme Mix (Invitrogen). The p35S:NLS-ELP3-GFPconstruct was generated the same as p35S:ELP3-GFP except that the sequenceencoding the functional SV40 NLS (van der Krol and Chua, 1991) was attachedto the forward primer used to clone the ELP3 CDS. The constructionmethod forthe p35S:SPT4-2-GFP plasmid was the same as that for p35S:ELP3-GFP. For theSPT4-RNAi construct, the SPT4-2 CDS was cloned into pHellsgate 2 in bothforward and reverse directions with a one-step BP reaction. All the primersused for the molecular cloning are listed in Supplemental Table S1.

All the constructs were transformed into Agrobacterium tumefaciens strainGV3101 (pMP90), which was used for plant transformation with the vacuuminfiltration method.

Histology and Microscopy

Phenotypic analysis, Lugol staining, GUS staining, microscopic observation,and confocal microscopy all were done as described previously (Zhou et al.,2010). For marker expression control, at least 15 seedlings were used for eachsample, and representative images are shown. For quantitative measurements,20 seedlings of each sample were analyzed, and the statistical significance wasevaluated by Student’s t test. For multiple comparisons, an ANOVA was fol-lowed by Fisher’s LSD test (SPSS) on the data.

Whole-Mount RNA in Situ Hybridization

Whole-mount RNA was hybridized in situ according to the method de-scribed previously, and the probes forWOX5, PLT1, and PLT2 had already been


Qi et al.




synthesized (Zhou et al., 2010). The antisense and sense probes for SHR weresynthesized with digoxigenin-11-UTP (Roche Diagnostics) by T7 RNA poly-merase from an SHR-specific fragment with the T7 promoter sequence either atthe reverse primer or at the forward primer, respectively (Supplemental TableS1). To enhance the probe permeability for SHR detection, SHR probes werehydrolyzed to an approximately 100-bp size, the proteinase K concentrationwas increased to 80mgmL21, and the incubation timewas prolonged to 20min.

Gene Expression Analysis

For RT-qPCR analysis, approximately 0.5-cm root tips were harvested from5-d-old seedlings for RNA extraction with TRIzol reagent (Invitrogen). First-strand cDNA was synthesized from 2 mg of total RNA with the Moloney mu-rine leukemia virus reverse transcriptase (Promega) and oligo(dT) primer andwas quantified with the LightCycler 480 II apparatus (Roche) and the SYBRGreen Kit (Takara) according to each manufacturer’s instructions. The expres-sion levels of the target genes were normalized to the reference gene PP2AA3.The statistical significance was evaluated by Student’s t test. Primers used forRT-qPCR analysis are listed in Supplemental Table S1, some of which had beendescribed previously (Sozzani et al., 2010).

Cell Fractionation

Cell fractionation was performed with the Plant Nuclei Isolation/ExtractionKit (Sigma). Briefly, 4 g of 10-d-old wild-type seedlings was harvested and fullyground in liquid nitrogen. The powder was transferred to 8 mL of precooledNIBA buffer (Sigma) and filtered through a nylon membrane. Triton X-100 wasadded to a final concentration of 0.5% (v/v), and the sample was kept on ice for15 min, followed by centrifugation at 2,000g at 4°C for 10 min. The extractsbefore centrifugation were collected as total proteins, whereas the supernatantsafter centrifugation were collected as cytoplasmic fractions. The pellets wereresuspended in 1 mL of NIBA and applied on top of an 800-mL cushion of 1.5 M

Suc, followed by centrifugation at 12,000g at 4°C for 10 min. The pellets werewashed twice by resuspension in 1 mL of NIBA and centrifugation at 12,000gfor 5 min. Thereafter, the pellets were resuspended in 600 mL of NIBA as thenuclear fraction. For each fraction, samples of 20 mL of protein were used forimmunoblot analysis.

Antibody Preparation

The partial CDS encoding the 400 amino acids of the C terminus of ELP1(BamHI/XhoI) and the full-length CDS of ELP3 (BamHI/HindIII) were clonedinto the pET-28a vector to express the recombinant proteins in Escherichia colistrain BL21. The primers used for cloning are listed in Supplemental Table S1.The recombinant proteins were used to raise polyclonal antibodies in mice.

Immunoblot Analysis

Protein extraction and immunoblotting were done according to standardprotocols. Seedlings were ground into a fine powder in liquid nitrogen and thentransferred to extraction buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% [v/v]Nonidet P-40, 1mM phenylmethylsulfonyl fluoride, 10mMMG132, and proteaseinhibitor cocktail [Roche]). For immunoblot analysis, protein samples wereboiled for 5 min after mixing with SDS loading buffer, separated by SDS-PAGE,and transferred to polyvinylidene fluoride membranes. Immunoblots wereprobed with the following antibodies: a-CTD (Abcam; 1:2,000), a-H3 (Abcam;1:4,000), a-PEPC (Rockland; 1:2,000), a-myc (Abmart; 1:2,000), a-ELP1(1:2,000), and a-ELP3 (1:4,000). Ponceau S-stained membranes are shown asloading controls.

Y2H Assays

Y2H assays were based on the MATCHMAKER GAL4 Two-Hybrid System(Clontech). The full-length CDS of each of the six Elongator subunits (SmaI/SacI) was cloned into pGADT7, whereas the sequence encoding the RNAPIICTD (EcoRI/BamHI) was cloned into pGBKT7. The primers used for cloning arelisted in Supplemental Table S1. Constructs were cotransformed into the yeaststrain Saccharomyces cerevisiae AH109. The presence of the transgenes wasconfirmed by growth on SD/-Leu/-Trp plates. For protein interaction assess-ment, the transformed yeast was suspended in liquid SD/-Leu/-Trp mediumand cultured to anOD of 1. Five microliters of suspended yeast was dropped on

plates containing SD/-Ade/-His/-Leu/-Trp medium. Interactions were ob-served after 3 d of incubation at 30°C.

LCI Assays

LCI assays were done with Nicotiana benthamiana leaves as described pre-viously (Song et al., 2011). Briefly, the full-length CDS of the two proteins werecloned into pCAMBIA1300-NLUC (BamHI/SalI or SacI/SalI) andpCAMBIA1300-CLUC (KpnI/SalI). The primers used for vector construction areshown in Supplemental Table S1. The resulting constructs were introduced intoA. tumefaciens strain GV3101. The N. benthamiana leaves were coinfiltrated withcombinations of strains as described and incubated for 3 d before observationwith the NightOWL II LB 983 (Berthold) imaging system.

Co-IP Assays

The Co-IP assays were done according to the publishedmethod (Chen et al.,2012) with minor modifications. Briefly, total proteins were extracted from 10-d-old Col-0 and p35S:SPT4-2-GFP seedlings with the protein lysis buffer (50mM

Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% [v/v] Triton X-100, 0.2% [v/v] Nonidet P-40, 0.6mM phenylmethylsulfonyl fluoride, 20mMMG132, and protease inhibitorcocktail [Roche]). To preclear 2 mg of protein extracts, 20 mL of protein A/Gplus agarose (Santa Cruz) was used. Thereafter, the supernatants were incu-bated with 2 mL of GFP antibody (Abcam) overnight and further precipitatedwith another 20 mL of protein A/G plus agarose (Santa Cruz). The precipitatedsampleswere washed four timeswith the lysis buffer and then eluted by boilingfor 5 min with SDS loading buffer. Immunoblots were detected with a-ELP1(1:2,000) and a-GFP (Abmart; 1:2,000) antibodies.

ChIP-qPCR Assays

ChIP assays were done according to the published protocol (Chen et al.,2012). Briefly, 2 g of 5-d-old seedlings was cross-linked in 1% (v/v) formalde-hyde for chromatin isolation. For immunobinding, 2 mL of CTD antibody(Abcam) or GFP antibody (Abcam) was used. The protein-DNA complex wascaptured with 50 mL of protein A agarose/salmon sperm DNA (Millipore). Theeluted DNAwas purified with the QIAquick PCR purification kit (Qiagen) andused for qPCR analysis. Primers used for ChIP-qPCR are listed in SupplementalTable S1.

RNA Immunoprecipitation PCR Assays

The RNA immunoprecipitation assays described previously (Zheng et al.,2009) were modified slightly. Five-day-old seedlings of elp3;p35S:NLS-ELP3-GFPwere harvested for RIP assays. The seedlingswere cross-linked in 1% (v/v)formaldehyde. Subsequently, protein-RNA complexes were isolated andimmunoprecipitated according to published procedures. The associated RNAswere detected with semiquantitative reverse transcription PCRwith the primerpairs listed in Supplemental Table S1.

Accession Numbers

The sequence data can be found in the Arabidopsis Genome Initiative underthe following accession numbers: ELP1 (At5g13680), ELP2 (At1g49540), ELP3(At5g50320), ELP4 (At3g11220), ELP5 (At2g18410), ELP6 (At4g10090), SHR(At4g37650), SCR (At3g54220), BR6ox2 (At3g30180), CYCD6;1 (At4g03270),MGP (At1g03840), NUC (At5g44160), RLK (At5g67280), SNE (At5g48170),PP2AA3 (At1g13320), PLT1 (At3g20840), PLT2 (At1g51190), WOX5(At3g11260), ACT7 (At5g09810), SPT4-1 (At5g08565), and SPT4-2 (At5g63670).

Supplemental Data

The following supplemental materials are available

Supplemental Figure S1. Elongator acts as an integral complex to regulateroot growth.

Supplemental Figure S2. Elongator acts independently of the PLTpathway.
















Supplemental Figure S3. Elongator functions in root radial patterningthrough the SHR pathway.

Supplemental Figure S4. Subcellular localization of ELP3.

Supplemental Figure S5. Antibody characterization, cell fractionation as-say, and histone acetylation detection.

Supplemental Figure S6. Construction of SPT4-RNAi.

Supplemental Table S1. Primers used in this study.

ACKNOWLEDGMENTS

We thank Zhizhong Gong, Ben Scheres, and Klaus Palme for sharing theirresearch materials and Martine De Cock for helping us prepare the article.

Received May 3, 2018; accepted October 29, 2018; published November 6, 2018.

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