The TOPLESS Interactome: A Framework for · PDF fileThe TOPLESS Interactome: A Framework for...

The TOPLESS Interactome: A Framework for GeneRepression in Arabidopsis1[W][OA]

Barry Causier, Mary Ashworth, Wenjia Guo2, and Brendan Davies*

Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom

Transcription factors activate or repress target gene expression or switch between activation and repression. In animals andyeast, Groucho/Tup1 corepressor proteins are recruited by diverse transcription factors to induce context-specific transcrip-tional repression. Two groups of Groucho/Tup1-like corepressors have been described in plants. LEUNIG and LEUNIG_HOMOLOG constitute one group and TOPLESS (TPL) and the four TPL-related (TPR) corepressors form the other. To discoverthe processes in which TPL and the TPR corepressors operate, high-throughput yeast two-hybrid approaches were used toidentify interacting proteins. We found that TPL/TPR corepressors predominantly interact directly with specific transcriptionfactors, many of which were previously implicated in transcriptional repression. The interacting transcription factors revealthat the TPL/TPR family has been coopted multiple times to modulate gene expression in diverse processes, includinghormone signaling, stress responses, and the control of flowering time, for which we also show biological validation. Theinteraction data suggest novel mechanisms for the involvement of TPL/TPR corepressors in auxin and jasmonic acid signaling.A number of short repression domain (RD) sequences have previously been identified in Arabidopsis (Arabidopsis thaliana)transcription factors. All known RD sequences were enriched among the TPL/TPR interactors, and novel TPL-RD interactionswere identified. We show that the presence of RD sequences is essential for TPL/TPR recruitment. These data provide aframework for TPL/TPR-dependent transcriptional repression. They allow for predictions about new repressive transcriptionfactors, corepressor interactions, and repression mechanisms and identify a wide range of plant processes that utilize TPL/TPR-mediated gene repression.

Differential gene expression is fundamental to anorganism’s ability to respond to its environment andunderpins the processes of development and differ-entiation. The majority of genes that have been asso-ciated with the regulation of plant developmentencode transcription factors that control the expres-sion of downstream target genes (Kaufmann et al.,2010). These factors often act as positive regulators ofgene expression by activating the expression of down-stream target genes. However, it is increasingly beingrecognized that repression or context-dependentswitching between activation and repression plays avital role in gene regulatory networks (Krogan andLong, 2009; Kagale and Rozwadowski, 2011). Core-pressors are transcriptional regulators that are incapa-ble of independent DNA binding, being recruiteddirectly or indirectly by DNA-binding transcriptionfactors to repress target gene expression. The Groucho

(Gro)/Tup1 family, first identified in Drosophila andSaccharomyces, respectively, represents an archetypalclass of corepressors that are recruited by a range ofDNA-binding transcription factors to elicit a repressedchromosomal state, thereby shutting off gene expres-sion (Liu and Karmarkar, 2008).

The first Gro/Tup1 family member to be isolated inplants was LEUNIG (LUG; Conner and Liu, 2000).LUG was identified due to its repressive activity,restricting the expression domain of the floral home-otic gene AGAMOUS (Conner and Liu, 2000). LUGand its close homolog LEUNIG_HOMOLOG (LUH)share the characteristic C-terminalWD-40 andN-terminalGln-rich motifs of the Gro/Tup1 family but havean additional N-terminal domain, known as LUFS,that includes a previously defined LisH (for Lis1-homologous) motif. LisH domains have been shown topromote protein-protein interaction (Cerna andWilson,2005). The LUFS domain is required for interactionwith the SEUSS protein that serves as an adaptor tolink LUG and LUH to a range of transcription factors(Pfluger and Zambryski 2004; Sridhar et al., 2004, 2006;Gregis et al., 2006; Stahle et al., 2009). As well asdirectly interacting with YABBY transcription factors(Stahle et al., 2009), LUG/LUH corepressors indirectlyinteract with transcription factors via the SEUSS adap-tor protein or related SEUSS-LIKE proteins (Frankset al., 2002; Sridhar et al., 2004). Another Gro/Tup1corepressor family, TOPLESS (TPL; including TPLand TPL-related [TPR]), has recently been described inplants that interacts both directly and indirectly with

1 This work was supported by the Biotechnology and BiologicalSciences Research Council.

2 Present address: Sir Alexander Fleming Building, South Ken-sington Campus, Imperial College London, London SW7 2AZ, UK.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Barry Causier ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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transcription factors (Kieffer et al., 2006, Long et al.,2006). TPL/TPR corepressors have been shown tointeract with transcription complexes involved inauxin and jasmonate signal transduction, meristemmaintenance, and defense responses (Kieffer et al.,2006; Szemenyei et al., 2008; Gallavotti et al., 2010;Pauwels et al., 2010; Zhu et al., 2010; ArabidopsisInteractome Mapping Consortium, 2011).

TPL/TPR corepressors were first described as directinteractors of the Arabidopsis (Arabidopsis thaliana)homeodomain transcription factor WUSCHEL (WUS;Laux et al., 1996; Kieffer et al., 2006). WUS is expressedin the organizing center at the base of the apicalmeristem, where it signals to the overlying stem cellsto maintain their meristematic fate as part of a well-studied feedback loop that controls meristem homeo-stasis (Brand et al., 2000; Schoof et al., 2000; Sablowski,2007). Within this network, WUS represses the expres-sion of type A Arabidopsis response regulator genes(Leibfried et al., 2005). Two members of the TPL/TPRfamily of corepressors were identified as interactors ofWUS (TPL and TPR4, previously known asWSIP1 andWSIP2), and the interaction was shown to require theC-terminal domain of WUS, which contains threeshort conserved protein sequences, and the N-terminalregion of TPL/TPR, which contains a LisH domain aswell as a “C-terminal to LiSH” (CTLH) domain (Kiefferet al., 2006). By analogy to Gro/Tup1, TPL/TPR-mediated repression by WUS was hypothesized toact via histone deacetylation, a conclusion supportedby the WUS-like meristem defects observed in plantsgrown on histone deacetylase inhibitor (Kieffer et al.,2006). More recently, it has been shown that in maize(Zea mays), the zinc (Zn)-finger transcription factorRAMOSA1 interacts with the TPL/TPR factor REL2 torepress indeterminate meristem fate (Gallavotti et al.,2010), thus demonstrating another involvement ofTPL/TPR corepressors in meristem maintenance, al-though acting with a different transcription factor andwith an opposite developmental outcome.

TPL was identified as the gene affected in thetemperature-sensitive, semidominant tpl-1 embryo de-velopment mutant of Arabidopsis. tpl-1 plants showsevere polarity defects, ranging from fused cotyledonsto the complete replacement of the shoot with a secondroot (Long et al., 2002, 2006). TPL defines a family offive genes in Arabidopsis (TPL and TPR1 to -4; Longet al., 2006). The dominant negative nature of the tpl-1mutation was demonstrated by the fact that a quintu-ple loss of function, in which all five TPL/TPR geneswere inactivated by mutation or RNA interference, isrequired to phenocopy the original tpl-1 phenotype,suggesting that the five members of the TPL/TPRfamily act redundantly (Long et al., 2006). Consistentwith a histone deacetylation repression mechanism,tpl-1 is enhanced by a histone deacetylase mutant andsuppressed by a histone acetyl transferase mutant(Long et al., 2006).

The auxin/indole-3-acetic acid (AUX/IAA) proteinBODENLOS (BDL) functions in root development and

was recently shown to interact with TPL. AUX/IAAproteins, including BDL, interact with activatingAUXIN RESPONSE FACTOR (ARF) transcription fac-tors, thereby converting them to transcriptional re-pressors (Szemenyei et al., 2008). In the presence ofauxin, the AUX/IAA proteins are ubiquitinated anddegraded, freeing the ARFs to activate the expressionof their auxin-induced target genes. A similar mech-anism has also been demonstrated for jasmonic acid(JA) signaling, where the activating MYC2 transcrip-tion factors recruit JAZ proteins, which, in turn, recruitTPL/TPR corepressors via an accessory protein knownas NINJA (Pauwels et al., 2010). In this system,JA promotes the ubiquitination and degradation ofthe JAZ proteins, preventing the recruitment of TPL/TPR and activating JA-responsive gene expression(Pauwels et al., 2010).

The roles of TPL/TPR corepressors are not confinedto development. Recently, a genetic suppressor of aconstitutively active disease resistance gene SUPPRES-SOR OF NPR1-1, CONSTITUTIVE1 (SNC1) was iden-tified as TPR1, and the two proteins were shown tointeract to repress downstream target gene expressionduring the response to pathogen infection (Zhu et al.,2010). Taken together, these findings suggest thatTPL/TPR corepressors are involved in a wide rangeof processes and that they interact directly or indi-rectly with DNA-binding transcription factors to re-press the expression of downstream target genes.

Interaction between the TPL/TPR corepressors andWUS, AUX/IAA, NINJA, and RAMOSA1 is mediatedby a small conserved protein motif known as theethylene response factor (ERF)-associated amphiphilicrepression (EAR) domain (Ohta et al., 2001), with theconsensus sequence (L/F)DLN(L/F)xP, which has alsobeen identified in several other transcription factorswith repressive activity (Ohta et al., 2001; Hiratsuet al., 2004; Hill et al., 2008). EAR repression domains(RDs) have been shown to convert activating tran-scription factors into dominant repressors even whenas few as six amino acids (DLELRL) are added and canovercome strong transactivating motifs such as VP16from Herpes simplex virus (Ohta et al., 2001; Hiratsuet al., 2002). A recent bioinformatic study attemptedto predict the complete repertoire of the EAR repress-ome in Arabidopsis, identifying 219 candidate pro-teins belonging to 21 transcription regulator families(Kagale et al., 2010).

To discover the extent of the processes in which theTPL/TPR family of corepressors operates in plants, weused high-throughput yeast two-hybrid approaches toestablish the framework of TPL/TPR protein-proteininteractions, with the aim of providing a resource thatwill inform future studies. TPL/TPR proteins interactoverwhelmingly with specific transcription factorfamilies, many of which have previously been impli-cated in transcriptional repression. The framework ofTPL/TPR interactions reveals the widespread use ofTPL/TPR corepressors in diverse processes, includinghormone signaling, stress responses, and the control of

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flowering time, and makes new predictions about themechanism of repression in both auxin and JA signaltransduction. We show that all known plant RD se-quences are enriched among the TPL/TPR interac-tome and that these domains are necessary for TPL/TPR recruitment. These data constitute an experimen-tally derived framework for the repressive control ofgene expression in Arabidopsis and facilitate theidentification of further regulatory factors that represstranscription in plants. Finally, to demonstrate thebiological relevance of the TPL/TPR interactions, weshow that TPL contributes to WUS function and isrequired for the repression of FLOWERING LOCUS T(FT) expression by TARGET OF EAT1 (TOE1).

RESULTS

TPL/TPR Corepressors Interact with SpecificTranscription Factor Families

Each of the five TPL/TPR proteins was individuallytested for protein interaction partners in two-hybridlibrary screens, testing a total of 43 million clones. Listsof interactors are presented in Figure 1 and Supple-mental Table S1. Most of the detected interactingproteins were transcription factors (Supplemental Ta-ble S2). The Arabidopsis Information Resource 9 lists27,379 proteins encoded by the Arabidopsis genome,of which approximately 1,922 (approximately 7%) aretranscription factors or regulators of transcription(based on information from the Database of Arabi-dopsis Transcription Factors [DATF]; Guo et al., 2005).Transcription factors were significantly enriched in allscreens, representing between 88% and 51% of theinteractors (Supplemental Table S2).Since the majority of all TPL/TPR interactions were

with transcription factors, we attempted to determinethe range of transcription factor interactions by screen-ing an arrayed yeast two-hybrid library of 1,296Arabidopsis transcription factors (Paz-Ares and TheREGIA Consortium, 2002; Castrillo et al., 2011) witheach TPL/TPR protein, making a total of 6,480 indi-vidual interaction tests. Screens were conducted intriplicate, with only interactions seen in at least tworeplicates being considered bona fide. The combina-tion of two screening approaches, using different yeastand vector systems, provides a robust interactionframework. The screens show that TPL/TPR proteinsinteract with transcriptional regulators, representingat least 17 distinct families (Fig. 1; Table I). AP2/ERF,Zn finger, AUX/IAA, and MYB factors were found tointeract with multiple TPL/TPR proteins and werehighly represented in the yeast two-hybrid screens(Fig. 1; Supplemental Fig. S1). Proteins from the ARF,JAZ, class II LBD, MADS box, TCP, NAC domain,WOX, and other families were also found to interactwith the TPL/TPR proteins (Fig. 1; Table I), suggestingthat TPL/TPRs are recruited as corepressors by di-verse transcription factors.

Current evidence suggests that the TPL/TPR familymediates transcriptional repression by acting withhistone deacetylases to induce a repressive chromatinstate at the target locus (Kieffer et al., 2006; Long et al.,2006; Zhu et al., 2010). In addition, Gro/Tup1 proteinsfrom animals and yeast, and LUG from Arabidopsis,also interact with components of the mediator com-plex, disrupting its function (Courey and Jia, 2001;Malave and Dent, 2006; Gonzalez et al., 2007). We didnot detect direct interactions between TPL and thehistone deacetylase HDA19 or the mediator complexcomponent HEN3 (Supplemental Fig. S2), suggestingthat additional factors might mediate such associations.An alternative mechanism for TPL-mediated gene si-lencingwas suggested by the interaction between TPL/TPRs and the SET domain protein SDG19 (Fig. 1;Supplemental Table S3), which is a histone methyl-transferase that induces a repressive chromatin state.

Gro/TLE proteins form homotetramers or heterote-tramers (for review, see Chen and Courey, 2000).We see evidence for the formation of oligomers be-tween the TPL/TPR proteins (Fig. 1). Furthermore,TPL has previously been reported to interact withitself (Szemenyei et al., 2008; Arabidopsis InteractomeMapping Consortium, 2011). Together, the interactiondata indicate that the formation of TPL/TPR com-plexes is also important for gene silencingmediated byplant Gro/TLE corepressors.

TPL/TPR Interacting Transcription Factors Are Enrichedfor RDs

Of the 1,922 Arabidopsis transcription factors listedin DATF, only 2.2% are associated with the GeneOntology (GO) term “transcription repressor activity”(GO:0016564 and children; Gene Ontology Consor-tium [www.geneontology.org]), whereas 9% of thetranscriptional regulators that interact with TPL/TPRare associated with this GO term. When DATF GOdata are combined with knowledge of repressive tran-scription factors gathered from the literature, func-tional repressors are further enriched among the TPL/TPR interactors (41% of interactors annotated as re-pressors; Fig. 1). Known repressive transcription fac-tors, therefore, are highly represented among the TPL/TPR interactors. As more functional information be-comes available for the remaining interactors, it seemslikely that further enrichment will be seen, suggestingthat the TPL/TPR family has the potential to be in-volved inmuch of the transcription repression in plants.

Several RDs have been identified in Arabidopsistranscription factors, including the EAR domain [de-fined as (L/F)DLN(L/F)xP, including LxLxL, DLNxP,and DLNxxP, which also includes FDLNI] and the(R/K)LFGV and TLxLF sequences (Ohta et al., 2001;Matsui et al., 2008; Ikeda and Ohme-Takagi, 2009;Ikeda et al., 2009; Kagale et al., 2010). All of these RDsare present among the TPL/TPR interactors (Fig. 1),and they are enriched among the “gene regulator”class of interactors (Supplemental Table S4). Approx-

Transcriptional Repression and the TOPLESS Interactome

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Figure 1. A summary of the TPL/TPR protein-protein interactions. A, Interactions detected in the whole plant yeast two-hybridexperiments. B, Interactions identified from the arrayed transcription factor library. In each case, the first column provides theArabidopsis Genome Initiative (AGI) number for each factor. The second column indicates the identity of the factor, if known.Factors highlighted in gray in this column represent those that are known to act as transcriptional repressors. The third columnindicates the transcription factor family to which each factor belongs, where dark green represents the AP2/ERF family, purple theMYB family, red the AUX/IAAs, pink the ARFs, light blue the C2H2 Zn fingers, dark blue the homeodomain family, brown theNAC proteins, orange the MADS box family, and yellow the TCP factors. Interactions between these factors and the TPL/TPRproteins are represented by dark gray boxes in the appropriate columns. For the whole plant yeast two-hybrid experiment,numbers represent the frequency (%) at which each factor was isolated. The RD column shows the sequence of any knownfunctional RD found in the TPL/TPR-interacting factors. Black boxes to the right side of each table indicate those factors isolatedin both the whole plant library and arrayed transcription factor screens. Factors and RDs highlighted in pale green in the arrayedtranscription factor library table represent those proteins in which mutagenesis of the RD was performed to evaluate itsrequirement for interaction with the TPL protein.

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Table I. Comparison of the major transcription factor families isolated in the yeast two-hybrid experimentsTranscription factor families are as defined in the DATF. Seventeen families in total were identified in the

large-scale yeast two-hybrid experiments. Five additional proteins (AT2G31720, AT3G63180, AT4G03250,AT5G55040, and AT5G63470) were also isolated that did not belong to any of the DATF families. Factors inparentheses represent putative weak interactions.

Transcription Factor Family Whole Plant Library Screen Arrayed Transcription Factor Screen

Class II LBDa LBD37LBD41

AP2/ERFAP2 family TOE1 TOE1

TOE2 TOE2AP2

ANT family BBM1ERF family ERF3 ERF3

ERF4 ERF4ERF7 ERF7ERF8 ERF8ERF11 ERF10

ERF11ERF12(ERF6)(ERF13)

RAV family TEM1 (TEM1)(TEM2)RAV1NGA2AT1G13260AT1G50680AT1G51120AT5G06250

DREB family CBF4(RAP2.1)(TINY)

Zn fingerC2H2 ZAT5

ZAT6 ZFP4STZ ZFP8ZFP11 ZFP10WIP3 AT4G35610

GATA GATA9GATA18 GATA16

OthersFRS3 AT2G44410AT1G30810 AT3G21890

AUX/IAA IAA1IAA2 IAA2IAA7 IAA3/SHY2IAA8 IAA4IAA16 IAA7IAA17/AXR3 IAA10IAA18 IAA11IAA27 IAA13

(IAA18)IAA19IAA26IAA28(IAA34)

ARF ARF2ARF9 ARF17ARF18 (ARF1)

(ARF3)(ARF4)(ARF19)

(Table continues on following page.)





Table I. (Continued from previous page.)


JAZa JAZ5JAZ6

TCP AT1G58100 AT1G58100TCP2TCP3TCP4TCP14TCP16AT1G35560

HSF HSF4HSFB2AHSFB2B

bZIP bZIP9bZIP45bZIP67bZIP-AbZIP-BOBF5

NAC NAC1/52NAC3NAC50NAC052NAC96

MYB AS1EPR1 EPR1-LIKEEPR1-LIKE MYB15MYB7 MYB20MYB32 MYB73MYB44 MYB97MYB77 MYB98AT3G52250 KAN

MYBL2MYB3R2GT-2AT1G25550AT1G68670AT2G38300AT3G12730AT3G16350

HD WUS/WOX WOX4WOX2(WOX3)(WOX4)(WOX6)(WOX7)

MADS SOC1AGL15(AGL18)(AGL21)(AGL24)(AGL43)(AGL78)(MAF5)(SEP1)(SEP4)(SHP2)

WRKY WRKY6WRKY30WRKY32WRKY61

BES1 BEH4

(Table continues on following page.)

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imately 65% of interactors belonging to the generegulator class have a complete, known RD and afurther 29% have a partial RD (such as LxL and FxLxF,which is related to the FDLNI EAR motif). Only 5% ofthe gene regulator class of interactors appears tocontain no known RD sequence (Supplemental TableS4). Characterized RDs, therefore, are highly overrep-resented among the TPL/TPR interactors. The LxLxLmotif is found in 22% of transcription factors in theDATF database but is present in 48% of the 130interactors (Supplemental Table S5), confirming sig-nificant enrichment for this domain among TPL/TPRinteracting factors. Enrichment for many of the otherRD motifs was also observed among the TPL/TPRinteractors (Supplemental Table S5).Previous data have demonstrated that the LxLxL

motif of BDL is necessary for interaction with TPL(Szemenyei et al., 2008). To test whether the DLNxxPand RLFGV RD sequences are also necessary for TPLinteraction, we mutated these motifs in ERF3 andRAV1, respectively, and tested interaction with TPL.Conversion of the DLNFPP motif to AHNFPP in ERF3(and ERF7) abolished the interaction (Fig. 2). Similarly,mutation of the RLFGV motif to RSSGV in RAV1prevented interaction with TPL (Fig. 2). The TPL/TPRlibrary screens also identified interacting proteins withvariations of the R/KLFGV RD sequence, includingMLFGV, RLFGI, and RLFGF (Fig. 1). To determinewhether such variants are necessary for TPL interac-tion, we mutated the MLFGV sequence to MQCGV inthe AP2/B3 factor AT1G51120 and found that themutated protein was unable to interact with TPL (Fig.2). These findings indicate that all the RD motifsidentified in the TPL/TPR screens are necessary forthe observed interactions. This confirms that TPL isable to interact via diverse peptide sequences, allow-ing for the prediction of novel RDs and thus newfactors that can function as transcriptional repressors.While the direct interactions between transcription

factors and the TPL/TPR proteins are mediated by thepresence of a conserved RD in the transcription factor,interactions between TPL/TPR factors and other factorsinvolved in the repression mechanism might involvevery different motifs. Consistent with this, presumedchromatin-remodeling factors isolated in the screens,such as SDG19, EMF1, VRN5, FVE, and the TPRs them-selves, lack any known repression motifs (Fig. 1).

Validation of TPL Interactions

False positives and false negatives can be reportedin yeast two-hybrid screens. Although we used a nor-

malized whole plant library to increase the sensitivityof the assay and to minimize the presence of falsepositives, heat shock factor (HSF) and ribosomal pro-teins, both of which have previously been reported asfalse positives in yeast two-hybrid screens (for review,see Causier and Davies, 2002), were isolated in eachscreen. However, the overwhelming predominance oftranscriptional regulators among the positives was anunusual and striking feature of all TPL/TPR screens(Supplemental Table S2).

To test further the specificity of the two-hybridscreens, we took a protein closely related to the TPL/TPR family and tested it for interactions against thearrayed transcription factor library. The protein, hereindesignated TPR-like (AT2G25420), is shorter thanother family members; it has fewer WD-40 domainsat its C terminus and a repeat of the LisH/CTLHdomains at its N terminus. Phylogenetic and syntenystudies reveal that TPR-like is the closest protein to

Figure 2. Short RD sequences are necessary for interaction with TPL.The predicted RDs of ERF3 (FDLNFPP to FAHNFPP), RAV1 (RLFGV toRSSGV), and AP2-L (MLFGV to MQCGV) were mutagenized in thecontext of the full-length prey proteins and tested for interaction withthe TPL bait in a yeast two-hybrid assay. Transformed yeast were spottedonto medium that selects for protein-protein interactions (2His) orcontrol mediumwithout selection (+His). Wild-type prey proteins (WT)were compared with mutant prey proteins (M1 and M2) for interactionwith TPL.

Table I. (Continued from previous page.)


ABI3-VP1 AT2G36080SBP SPL1 SPL1

aClass II LBD and JAZ proteins are not present in the arrayed transcription factor library.





the TPL/TPR family of corepressors in the Arabidop-sis genome. No interactions were detected betweenthe TPR-like bait and the 1,296 transcription factors.This further suggests that our yeast two-hybrid ap-proaches are specific and that this relative of theTPL/TPR corepressors does not act as a transcrip-tional corepressor by interacting directly with tran-scription factors. We cannot rule out an indirect linkageto transcription via an as yet unidentified adaptorprotein.

To provide further validation for the detected TPL/TPR interactions with specific transcription factor fam-ilies, two example interactions were chosen to deter-mine whether they are of biological significance: theinteraction between TPL and the AP2 protein TOE1and the interaction between TPL and the WOX proteinWUS.

TPL/TPR-AP2 Factor Interactions in Floral Induction

AP2/ERF transcription factors fall into two generalclasses, and proteins from each of these groups inter-act with members of the TPL/TPR family (Fig. 1).Interesting among these were several known repres-sors of the flowering-time gene FT, including TOE1,TOE2, and TEM1 (Jung et al., 2007; Castillejo andPelaz, 2008). While TOE1 and TOE2 were recentlyshown to interact with TPL and TPR3 (ArabidopsisInteractome Mapping Consortium, 2011), our dataextend the interactions seen between the TOE proteinsand the TPL family, as both TOE1 and TOE2 interactwith all five TPL/TPR proteins.

To determine the biological significance of the TPL/TPR-TOE1 interaction, we examined the dependenceof TOE1 on TPL activity. Consistent with a previousreport (Jung et al., 2007), constitutive expression of theTOE1 protein (35S::TOE1; n = 15 independent lines)delays flowering by approximately 6 d compared withwild-type Landsberg erecta (Ler) control plants (n = 14;Fig. 3A). However, this delay in flowering is abolishedin a tpl-1 mutant background, where 35S::TOE1 plants(n = 22 independent lines) flower at the same time asnontransgenic tpl-1 plants (n = 16; Fig. 3A). In wild-type plants, TOE1 represses the transcription of FT toprevent precocious flowering (Jung et al., 2007). To testwhether this repression requires TPL, we compared FTexpression in the leaves of Ler, Ler 35S::TOE1, tpl-1,and tpl-1 35S::TOE1 lines. As expected, high levels ofTOE1 in the leaves of Ler 35S::TOE1 plants (Fig. 3B)resulted in a 16.4-fold reduction in the abundance ofthe FT transcript compared with wild-type Ler controlplants (Fig. 3C), correlating with the late floweringphenotype. In contrast, despite high levels of TOE1expression in the leaves of tpl-1 35S::TOE1 lines (Fig.3B), relative FT expression levels were approximately64% of those found in nontransgenic tpl-1 plants (Fig.3C), indicating that at least part of the repression of FTby TOE1 requires TPL. The slight but statisticallysignificant (P # 0.05; Welch’s t test) differences in FTexpression between tpl-1 and tpl-1 35S::TOE1 probably

reflects residual TPL/TPR activity in tpl-1, but it is alsopossible that TOE1 can repress FT through a TPL/TPR-independent mechanism. Interestingly, FT levelswere found to be consistently higher in transgenic andnontransgenic tpl-1 lines compared with Ler controls(Fig. 3C). The TPL/TPR interactome data identified anumber of other interactors involved in FT repression,including TEM1, AP2, and AGL15 (Fig. 1), suggestingthat loss of TPL disrupts the function of several

Figure 3. Comparison of flowering-time phenotypes and FT expressionin wild-type and tpl-1 plants constitutively expressing TOE1. A, Left toright: wild-type (Ler) plants, Ler plants overexpressing TOE1 (TOE1oe),tpl-1mutant, and tpl-1 overexpressing TOE1. For Ler lines, photographswere taken 30 d after germination; for tpl-1 lines, photographs weretaken 27 d after germination. B, qRT-PCR analysis of TOE1 expressionin Ler, Ler TOE1oe, tpl-1, and tpl-1 TOE1oe leaves relative to the EIF4Acontrol. C, qRT-PCR analysis of FT expression in Ler, Ler TOE1oe, tpl-1,and tpl-1 TOE1oe leaves relative to the EIF4A control. Values representmeans of three independent biological replicates.

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pathways leading to FT repression. Comparison offlowering time between Ler and tpl-1 lines was notpossible due to the developmental defects of tpl-1plants. Together, these data suggest that TOE1 recruitsTPL to repress FT in the leaves of wild-type plants andprovide a role for TPL/TPR corepressors in the floraltransition.

TPL/TPR-WOX Interactions

We originally isolated TPL using theWUS protein asbait in a yeast two-hybrid library screen (Kieffer et al.,2006; Supplemental Fig. S2). In the screens employedin this study, we found further interactions betweenthese two families. Using TPL as bait, we recoveredWOX4 and WOX2 interactions as well as potentialinteractions between a number of other WOX proteinsand the TPL/TPRs (Fig. 1; Supplemental Table S3). Toanalyze the biological significance of TPL/TPR-WOXinteractions, we carried out complementation assaysin a wus-1 mutant background using full-length WUSand variants thereof under the control of the WUSpromoter (Baurle and Laux, 2005; Fig. 4A), whichgives an appropriate expression pattern in GUS assays(Fig. 4B). Full-length WUS (pWUS::WUS) rescued es-sentially all aspects of the wus-1 mutant phenotype(Fig. 4C). wus-1 mutants flower rarely and produceflowers with reduced reproductive organ number,often terminating after the development of a singlestamen and never producing carpels (Laux et al., 1996;Table II). pWUS::WUS wus-1 inflorescences producednumerous flowers, similar towild-type plants. Approxi-mately 50% of the lines analyzed produced flowerswith a full complement of organs (strong rescue),including fully fertile carpels that later set viable seed.In the remaining lines, flowers terminated after thedevelopment of several stamens and did not producecarpels (weak rescue; Fig. 4C; Table II). The pWUS::WUSD construct, in which the conserved TPL/TPRinteraction domain of WUS has been deleted (Kiefferet al., 2006), was unable to rescue any aspect of themutant phenotype (Fig. 4C), indirectly suggesting thatthe loss of the WUS-TPL interaction impairs WUSfunction.To test this, we generated a third WUS construct

expressing a WUSD-TPL hybrid protein (pWUS::WUSD-TPL), recapitulating the WUS-TPL interaction.As with the pWUS::WUSD construct, pWUS::WUSD-TPL was unable to rescue any aspect of the vegetativephenotype of wus-1 (Fig. 4C), perhaps indicating thatTPL does not play a role in WUS function duringvegetative stages of development. However, duringthe reproductive phase, partial rescue of wus-1 by thehybrid construct was observed. Inflorescences of wus-1bear very few flowers before the inflorescencemeristemterminates, while in pWUS::WUSD-TPL wus-1 plants,inflorescences are indeterminate, producing manyflowers (Fig. 4C). In contrast to wus-1 flowers, pWUS::WUSD-TPL wus-1 flowers produce between three andfive stamens (Fig. 4C; Table II) but never produce

carpels. Thus, the phenotype of pWUS::WUSD-TPLwus-1 flowers closely resembles that of wus-1 flowersexpressing full-length WUS, in which weak rescue wasobserved (Fig. 4C; Table II). The pWUS::WUSD-TPLwus-1 phenotype is also similar to that of the weakwus-3 mutant allele, the inflorescences of which pro-duce many flowers that terminate after the productionof approximately three stamens (Mayer et al., 1998; Fig.4D). Thus, wus-3 reproductive meristems are main-tained longer than those ofwus-1mutants.We predictedthat complementation of wus-3 with the pWUS::WUSD-TPL construct wouldmaintain floral meristemslong enough for carpels to develop. Transgenic wus-3plants expressing WUSD from the native WUS pro-moter were not rescued and resembled the wus-3mutant (Fig. 4D). In contrast, pWUS::WUSD-TPL wus-3 flowers showed a range of phenotypes. In a smallproportion of lines (less than 10% of primary trans-formants), no obvious rescue was observed. In themajority of lines (approximately 80%), stamen numberincreased to an average of four per flower (Table II). Inthe remaining lines (approximately 10%), not only wasstamen number increased, but approximately 30% offlowers also developed carpels in the fourth whorl (Fig.4D; Table II).

In summary, the C-terminal domain of the WUSprotein that is required for TPL interaction is essen-tial for WUS function. By fusing TPL to an inactive,truncated WUS protein, both inflorescence and floralmeristems are maintained for longer than in wusmutants, suggesting that TPL is required for WUSfunction during the reproductive stages of develop-ment.

DISCUSSION

The Specificity and Sensitivity of the YeastTwo-Hybrid Approaches

Several factors indicate the robustness of the inter-action framework. First, we found an abundance ofrepressive transcription factors among the identifiedTPL/TPR interactors. Our screens support most of thepreviously reported interactions by identifying TPL/TPR interactions with WOX, AUX/IAA, Zn-finger,AP2/ERF, JAZ, AFP, and TIR-NB-LRR proteins(Kieffer et al., 2006; Szemenyei et al., 2008; Gallavottiet al., 2010; Pauwels et al., 2010; Zhu et al., 2010;Arabidopsis Interactome Mapping Consortium, 2011).The recent Arabidopsis interactome lists 20 non-AUX/IAA interactors for TPL and two for TPR3 (Arabidop-sis Interactome Mapping Consortium, 2011). We con-firm both of the TPR3 interactors and seven of the TPLinteractors, with other family members being identi-fied for a further four interactors. Second, there was anoverrepresentation of functional RDs among the iso-lated transcription factors. Finally, we chose two ex-amples of TPL-transcription factor interactions toshow the importance of TPL in the biological function





of those transcription factors. Although the resultssuggest that false positives are likely to be limited inthese screens, there is evidence that false negatives aremore common. The relatively small degree of overlapbetween the library screens and the transcription fac-tor array screens (35%overlap; Fig. 1), which is commonwhen comparing experimentally distinct screens (Vidaland Legrain, 1999; Rajagopala et al., 2009), highlightsthe fact that no single screen can detect the full rangeof interactions. However, the combination of yeasttwo-hybrid approaches used in this study also en-hances the sensitivity of the screens by reporting moreinteractions than are obtained in a single screen. Al-though the overlap between TPL/TPRs and individ-ual transcription factors in the different screens was

only 35%, at the family level, the overlap was muchgreater (60%; Table I).

TPL/TPR Act as Corepressors in ManyDevelopmental Programs

The TPL proteins appear to have been cooptedmultiple times in evolution to cause transcriptionalrepression. Database analyses indicate that the expres-sion of TPL/TPR genes is largely constitutive; there-fore, our data suggest that this family of proteins act asan “always-on” hub through which repression can beutilized in diverse pathways and in response to envi-ronmental cues (Fig. 5). The framework of TPL/TPRinteraction allows us to propose a number of addi-

Figure 4. Complementation of wusmutants. A, WUS constructs used inthe complementation experiments. B,GUS staining patterns for plants ex-pressing GUS from the WUS promoter(3,351 bp) used to prepare the con-structs for the complementation exper-iment. The left panel shows staining inseedlings, and the right panel showsstaining in an early flower and theanthers of a later flower. C, Comple-mentation of the strong wus-1 allele.The first column shows mature wild-type plants, the second column showswus-1 plants with the pWUS::WUSconstruct, the third column shows wus-1plants with the pWUS::WUSDTPL con-struct, the fourth column shows wus-1plants with the pWUS::WUSD construct,and the fifth column showswus-1mutantplants. In all cases, the top panel showsmature plants, the middle panel showstypical inflorescences, and the bottompanel shows typical flowers. For wus-1pWUS::WUS, the bottom panel shows atypical weak rescue flower on the leftwith a strong rescue flower on the right.D, Complementation of the weak wus-3allele. The left panel shows a typicalwus-3mutant flower, the center panel shows awus-3 flower with the pWUS::WUSDconstruct, and the right panel shows awus-3 flower with the pWUS::WUSDTPLconstruct.

Causier et al.




tional developmental mechanisms and programs inwhich these corepressors function and to predict novelaspects of the auxin and JA signaling pathways.

Hormone and Stress Responses

TPL is recruited by AUX/IAA proteins to suppressthe expression of auxin-responsive genes in the ab-sence of auxin (Szemenyei et al., 2008). Our data,together with those of two earlier studies (Szemenyeiet al., 2008; Arabidopsis Interactome Mapping Con-sortium, 2011), identify 20 of the 29 Arabidopsis AUX/IAA proteins as interaction partners of the TPL/TPRs(AUX/IAA1, -2, -3, -4, -6, -7, -8, -9, -10, -11, -12, -13, -14,-16, -17, -18, -19, -26, -27, and -28). The AUX/IAA-TPLcomplex is tethered to the promoters of auxin-responsivegenes through interaction between AUX/IAA proteinsand activating ARF transcription factors. However, adistinct class of ARFs represses transcription withoutinteracting with AUX/IAA proteins. The mechanismof repression by this class has not been determined.Here, we show that repressive ARF proteins, such asARF2 and ARF9, interact directly with TPL/TPR pro-teins (Fig. 1), suggesting a mechanism for repressionand implicating TPL/TPR corepressors in both formsof ARF-mediated repression (Fig. 5).Our data, together with other recent findings, also

provide compelling evidence for the involvement ofTPL in JA signaling (Pauwels et al., 2010; ArabidopsisInteractome Mapping Consortium, 2011; Fig. 5). It hasbeen reported that the adaptor protein NINJA linksTPL to JAZ proteins recruited to the promoters ofJA-responsive genes (Pauwels et al., 2010). Althoughwe did not identify NINJA as an interactor in ourscreens, like Pauwels et al. (2010) we identified AFPproteins interacting with the TPL/TPRs, which sug-gests that many combinations of interactions betweenthe TPL and AFP families may be possible. Consistentwith recent data (Arabidopsis Interactome Map-

ping Consortium, 2011), we also found direct interac-tions between TPL/TPRs and JAZ proteins (Fig. 1),which may reveal a novel way to achieve target generepression in JA signaling.

JA is important for plant responses to woundingand pathogen attack. The hormone ethylene is alsoinvolved in biotic and abiotic stress responses, inwhich ERFs play a role. For example, ArabidopsisERF3 and ERF4, both of which interact with TPL/TPRcorepressors, are induced by ethylene, high-salt con-ditions, drought stress, and pathogen attack. The TPL/TPR interaction framework also indicates corepressorinteraction with several other stress response tran-scription factors. The Zn-finger protein STZ (ZAT10)plays a role in salt tolerance and other stress responses,while ZAT6 is involved in nutrient stress responses(Sakamoto et al., 2004; Mittler et al., 2006; Devaiahet al., 2007). Many MYB factors, including MYB32 andMYB44, are induced in response to wounding orabscisic acid (ABA) treatment (Preston et al., 2004;Jung et al., 2008), while the MYB protein AS1 has beenshown to be involved in phytopathogen response(Nurmberg et al., 2007). From our data, we predictthat aspects of the function of these and other repres-sive MYB proteins will require TPL/TPR. ABA alsoplays a key role in stress responses, seed germination,and development (for review, see Hubbard et al.,2010). ABI5-related bZIP transcription factor proteinsregulate ABA signaling and are found to interact withAFP proteins to mediate the ABA response (Lopez-Molina et al., 2003; Garcia et al., 2008). Since interac-tions between the TPL family and the ArabidopsisAFP proteins have been identified in independentstudies (Pauwels et al., 2010; this study), it is temptingto speculate that AFPs may link TPL to the ABAsignaling pathway (Fig. 5). TPL/TPRs were recentlyshown to be involved in the SNC1-mediated immuneresponse (Zhu et al., 2010), and we also show interac-tion between TPR3 and a TIR-NB-LRR protein related

Table Il. Analysis of floral organ number in complemented wus-1 and wus-3 flowers

Plant naOrgan No. 6 SD

Sepals Petals Stamens Carpels

Wild type 15 4.0 6 0.0 4.0 6 0.0 6.0 6 0.0 2.0 6 0.0wus-1 pWUS::WUS FL (strong) 42 4.0 6 0.0 4.0 6 0.0 5.6 6 0.71 1.7 6 0.2wus-1 pWUS::WUS FL (weak) 54 4.0 6 0.0 4.0 6 0.0 4.5 6 0.54 0.75 6 0.3wus-1 pWUS::WUSD-TPL 96 4.0 6 0.0 4.0 6 0.0 3.9 6 0.56 0.0 6 0.0wus-1 14 3.7 6 0.62 3.4 6 0.71 1.4 6 0.36 0.0 6 0.0wus-3 16 n.d.b n.d. 2.8 6 0.45 0.0 6 0.0wus-3 pWUSD 18 n.d. n.d. 2.8 6 0.38 0.0 6 0.0wus-3 pWUSD-TPL (weak) 30 n.d. n.d. 4.0 6 0.56 0.0 6 0.0wus-3 pWUSD-TPL (strong) 37 n.d. n.d. 4.3 6 0.79 0.6 6 0.93

an = total number of flowers analyzed from the following numbers of plants (independent lines):pWUS::WUS FL (strong), seven lines; WUS FL (weak), nine lines; WUSD-TPL, 12 lines; wus-1, sevenplants; wus-3, four plants; wus-3 pWUS::WUSD , three lines; wus-3 pWUS::WUSD-TPL (weak), five lines;wus-3 pWUS::WUSD-TPL (strong), two lines. Note that, like wus-1 mutants, wus-1 pWUS::WUSD plantsrarely flowered. Any flowers that were produced were essentially identical to those of the wus-1mutant. bn.d., Not determined.





to SNC1 (Fig. 1). Thus, the interaction frameworkpoints to a widespread involvement of the TPL/TPRfamily in biotic and abiotic stress responses (Fig. 5).

Transition to Flowering

The TPL/TPR genes are expressed in apical tissues,particularly during the transition to flowering, and infloral tissues (data from the Arabidopsis eFP Browserat bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi;Winter et al., 2007), suggesting that these proteins

may have roles to play during flowering. TPL/TPRcorepressors interact with AP2 family transcriptionfactors, including AP2, TEM1, TOE1, and TOE2, all ofwhich have been implicated in flowering-time controlthrough the repression of FT. For example, Arabidop-sis plants overexpressing TOE1 show significantlydelayed flowering (Jung et al., 2007; Fig. 3). We showthat this late-flowering phenotype requires TPL/TPRactivity (Fig. 3), providing evidence to support the rolein floral induction that was predicted by the interac-tion framework (Fig. 5). Our data also reveal interac-

Figure 5. The TPL family acts as general corepressors in diverse biological pathways. The interactome data place the TPL/TPRfamily of corepressors at the center of many biological processes. TPL is implicated in hormone responses, with the interactiondata suggesting that the TPL/TPR family is involved in novel mechanisms of auxin and JA signaling. TPL/TPRs appear to beinvolved in a broad range of stress and plant immune responses in addition to numerous developmental pathways, such as floraltransition and leaf and flower development. Colored arcs represent the different processes in which the TPL family may act andshow how aspects of these pathways overlap. TF, Transcription factor.

Causier et al.




tions between TPL/TPRs and proteins such as SOC1,EMF1, VRN5, and FVE (Fig. 1), suggesting that TPL/TPRs could be involved in the regulation of floweringat multiple control points.

TPL/TPR Interact via Short Protein Motifs

Short, specific peptide motifs of certain transcriptionfactors are required for the recruitment of Gro/TLEcorepressors (Courey and Jia, 2001; Swingler et al.,2004). Similarly, the EAR RD conserved in the AUX/IAA, NINJA, and WUS proteins is required for inter-action with plant TPL/TPR corepressors (Kieffer et al.,2006; Szemenyei et al., 2008; Pauwels et al., 2010). EARmotifs were significantly enriched among the tran-scriptional regulators found to interact with the TPL/TPR proteins (Supplemental Table S5). In addition tothe EAR motif, the factors found in the TPL/TPRinteraction framework contain a number of differentRDswith quite diverse sequences (Supplemental TableS5), which we demonstrate are also required to recruitTPL (Fig. 2). Together, these data demonstrate that allknown functional RDs described in Arabidopsis areable to recruit the TPL/TPR proteins and that allsuch transcriptional repression might be mediated,at least in part, by this family of corepressors. Asignificant number of proteins isolated in the proteininteraction screens contained only a partial, or noknown, RD, suggesting that other RD sequences re-main to be discovered. For example, we identifiedthe MLFGV motif in an AP2/B3 factor (AT1G51120)and showed that it is necessary for TPL interaction(Fig. 2). Our data already indicate that TPL/TPR-mediated transcriptional repression is a widespreadmechanism, but the discovery of new RD sequencesfound in the interaction partners of the TPL/TPRcorepressors allows for predictions about new repressivepathways.Recently, Kagale et al. (2010) defined the Arabidop-

sis EAR repressome using a bioinformatic approachto identify transcription regulators containing EAR(LxLxL and/or DLNxxP) domains. They identified219 proteins belonging to 21 families of regulatoryfactors. By analyzing our yeast two-hybrid data of in-teracting proteins containing LxLxL and/or DLNxxP(73 in total), we find 48 in common with the data set ofKagale et al. (2010), representing approximately 22%of the 219 factors. While the overlap was modest forindividuals, at the family level, we found factorsbelonging to 17 of the 21 families identified by Kagaleet al. (2010). This indicates that these two very differentapproaches provide similar broad frameworks forEAR-mediated transcriptional repression in Arabi-dopsis and suggests that family-level data can beused to infer possible interactions.TPL/TPR proteins are recruited by LxLxL-containing

proteins, but there is at least one other unrelatedArabidopsis corepressor that also utilizes this motif.AtSAP18 is the ortholog of human SAP18, whichinteracts with LxLxL-containing proteins, including

ERFs and MADS box factors, involved in flowering(Song and Galbraith, 2006; Hill et al., 2008; Liu et al.,2009), suggesting that AtSAP18 repression may over-lap with repression mediated by TPL/TPR. SAP18is conserved in higher eukaryotes and suppressestranscription through interaction with histone deace-tylases. Thus, SAP18 may be part of a conservedrepression mechanism, while TPL/TPR, and the re-lated factors LUG and LUH (Conner and Liu, 2000;Sitaraman et al., 2008), may represent gene repressionsystems that evolved specifically in plants. Our dataextend the repressome beyond EAR to incorporateproteins with other RDs such as (R/K)LFGV andTLxLF, providing a means of identifying whetheruncharacterized factors may act as transcriptionalrepressors.

Mechanisms of Repression

The Gro/Tup1 group of corepressors induces tran-scriptional repression using several mechanisms. Inparticular, they induce local chromatin compaction attarget sites through an association with chromatinremodelers such as histone deacetylases. ArabidopsisGro/Tup1 proteins belonging to the LUG and TPLfamilies have also been shown to function togetherwith histone deacetylases (Sridhar et al., 2004; Longet al., 2006; Gonzalez et al., 2007), indicating that therecruitment of these corepressors to target genes re-sults in histone deacetylation, chromatin condensa-tion, and gene silencing. In the case of LUG, repressionis mediated by direct association between the LUGand HDA19 proteins (Gonzalez et al., 2007). How-ever, we and others have failed to detect an associa-tion between TPL and HDA19 (Supplemental Fig. S2;Kagale and Rozwadowski, 2011). Genetic evidencesuggests that TPL acts through HDA19 (Long et al.,2006), and interactions between TPR1 and HDA19were observed in pull-down experiments from plantextracts (Zhu et al., 2010), which might suggest thatroutes to histone modification require additional fac-tors to bridge between TPL/TPR proteins and histonedeacetylases.

Chromatin modifications such as methylation of his-tone H3 Lys residue 9, which is catalyzed by SUV39Hhistone methyltransferases, also result in nucleosomecompaction and gene silencing (for review, see Zhaoand Shen, 2004; Pontvianne et al., 2010). Interestingly,we identified a SUV39H-like protein, SUVH3, as aTPL/TPR interactor (Fig. 1; Supplemental Table S3).Furthermore, we also identified a protein related to thePICKLE (PKL) CHD3/Mi-2-like chromatin remodeler(Ogas et al., 1999), PKR1, as an interaction partner ofTPR2 (Fig. 1). PKL acts to repress the expression ofseed-associated genes during germination by pro-moting the methylation of histone H3 Lys residue 27(Zhang et al., 2008). The protein interaction data,therefore, imply that the TPL/TPR proteins can usemultiple chromatin-remodeling mechanisms to inducetranscriptional repression, and it will be interesting to





discover whether different mechanisms are utilizeddepending on the developmental context.

MATERIALS AND METHODS

Yeast Two-Hybrid Whole Plant Library Screens

A normalized, random-primed whole plant Arabidopsis (Arabidopsis

thaliana) yeast two-hybrid library, prepared in the pGADT7-Rec plasmid

and transformed into yeast strain AH109 (Clontech), was used for each screen.

TPL/TPR coding sequences were cloned into bait vectors pGBKT7 or pGBT9,

which were transformed to yeast strain Y187 (Clontech), using standard

protocols (Gietz and Woods, 2002). Library screens were performed by yeast

mating between the bait and library strains according to the manufacturer’s

instructions (Clontech Matchmaker3 system). In each case, over 1 million

diploids were screened for interactions (Supplemental Table S2), and putative

positives were isolated on minimal medium plates lacking His but containing

2.5 mM 3-amino-1,2,4-triazole (3-AT). Interactions were validated by the use of

the ADE2 and/or MEL1 reporters.

Yeast Two-Hybrid Transcription Factor Library Screens

TPL, TPR2, TPR3, and TPR4 coding sequences were cloned into the bait

vector pDEST32 (Invitrogen). The TPR1 bait construct was the same as that

used in the whole plant library screen. Baits were transformed to yeast strain

PJ69-4a. Each bait was mated against the arrayed yeast two-hybrid library (in

vector pDEST22 and yeast strain PJ69-4a) of Arabidopsis transcription factors

(Paz-Ares and The REGIA Consortium, 2002; Castrillo et al., 2011) in triplicate

on yeast peptone dextrose plates. Diploids were selected on minimal medium

lacking Trp and Leu, and interactions were detected by patching the diploids

onto minimal medium lacking His but containing 2.5 mM 3-AT.

Yeast Two-Hybrid Analysis of Putative RDs

RD sequences were mutated using the Phusion Site-Directed Mutagenesis

Kit (Finnzymes) according to the manufacturer’s instructions. Point mutations

were introduced by PCR using primers E3mF (5#-GTTTCAATTCGC-

TCATAATTTTCCACCGTTGG-3#) and E3R (5#-GGCGGATTCCGTCGC-

CGTGAAGACGATGCGATATC-3#) for the ERF3 gene (AT1G50640);

RAVmF (5#-GGTTTTGAGATCGTCCGGAGTTAACATTTCACC-3#) and RAVR

(5#-CGACCCGCATCTAAATCTGACCCGGATCTCGAC-3#) for the RAV1 gene

(AT1G13260); and A2mF (5#-GAGGGTTTATGCAGTGTGGTGTTAGGATC-

CAATAG-3#) and A2R (5#-CTTTCTTCTCCTCTGATTTGGTTTCTTCTTC-

TACC-3#) for the AP2-like gene (AT1G51120). Ligated PCR products were

propagated in Escherichia coli, and plasmid DNA was isolated from at least

five independent colonies. Of these, at least four verified constructs for each

prey were transformed into the pDEST32-TPL bait yeast strain. As a control,

the TPL bait strain was also transformed with the appropriate wild-type

prey construct. Interactions were assessed by growth on minimal medium

lacking His but containing 2.5 mM 3-AT.

Bioinformatics

Putative RD sequences (LxLxL, DLNxxP/FDLNI, K/RLFGV, and TLxLF)

were identified manually among the sequences of TPL/TPR interactors. To

identify all Arabidopsis proteins (The Arabidopsis Information Resource 9)

containing these RDs, we used the Patmatch algorithm at www.arabidopsis.

org (default settings). To identify the complement of Arabidopsis transcription

factors containing the RDs, we compared the DATF (datf.cbi.pku.edu.cn;

Guo et al., 2005) with the Patmatch list of Arabidopsis proteins containing the

same motifs using the COUNTIF function in Microsoft Excel. Arabidopsis

factors previously reported to act as transcriptional repressors were identified

using the Gene Ontology Consortium’s annotation and ontology toolkit

AmiGO (amigo.geneontology.org; Carbon et al., 2009) and through literature

surveys.

In Planta Analyses

All constructs were transformed into Agrobacterium tumefaciens strain

GV3101, and Arabidopsis plants were transformed using the floral dip

method (Clough and Bent, 1998). All plants were grown in glasshouses at a

constant temperature of 21�C with a 16/8-h photoperiod.

35S::TOE1 Transgenic Lines

The TOE1 coding sequence was amplified from Arabidopsis cDNA using

primers TOE1-F (5#-ggggacaagtttgtacaaaaaagcaggctTCGCTAGATTTGTAAT-

TTTCAGAG-3#; lowercase bases represent the Gateway sequence) and TOE1-R

(5#-ggggaccactttgtacaagaaagctgggtTTAAGGGTGTGGATAAAAGTAACC-3#)and cloned into pALLIGATOR III plasmid (Gateway-modified pFP101;

Bensmihen et al., 2004) to generate the 35S::TOE1 construct. 35S::TOE1 was

transformed to wild-type (Ler) and tpl-1 plant lines, and flowering time was

monitored under long-day conditions.

Expression levels of FT and TOE1 in leaves of transgenic and nontrans-

genic plants were measured by quantitative reverse transcription (qRT)-PCR.

cDNA was synthesized from 2 mg of total RNA extracted from leaves using

SuperScript II Reverse Transcriptase (Invitrogen). qPCRs were run in triplicate

on the Bio-Rad CFX96 Real-Time PCR System, using SsoFast EvaGreen

supermix (Bio-Rad), as follows: 30 s at 95�C, 39 cycles of 3 s at 95�C, 5 s at

60�C, and plate read, followed by a melt curve of 65�C to 95�C, with a plate

read at every 0.2�C increase. FT amplifications were performed using primers

described by Jang et al. (2009). TOE1 amplifications were performed using

primers qTOE-F (5#-GCTGAAGGGATGATGAGTAACTGG-3#) and qTOE-R

(5#-ACTGAGAACAATGGTGGTGGTTG-3#). For each genotype, expression

levels of TOE and FT were calculated relative to the EIF4A control and am-

plified using primers SR_elf4A_i_F (5#-GGTCATGCGTGCCCTTGGTGA-3#)and SR_elf4A_i_R (5#-ACCAGCCTGGAGAATGCGCTG-3#).

Complementation of wus Mutants

Plants heterozygous for the wus-1 or wus-3 mutation were transformed

with a number ofWUS constructs, each under the control of 3.3 kb of theWUS

promoter, prepared in the pALLIGATOR V vector (Gateway-converted

pFP100; Bensmihen et al., 2004). Full-length WUS was amplified from ge-

nomic DNA using Gateway-compatible primers WUS-F (5#-ggggacaagtttgta-caaaaaagcaggctATGGCTTTTTGGCAAGACGGATC-3#; lowercase bases

represent the Gateway sequence) and WUS-R (5#-ggggaccactttgtacaagaaa-gctgggtCTAGTTCAGACGTAGCTCAAGAG-3#). C-terminally truncated

WUS (WUSD) was amplified from genomic DNA using primers WUS-F

and WUSD*-R (5#-ggggaccactttgtacaagaaagctgggtCTAATGACCTTCTA-

GACCAAACAGAGG-3#). The WUSD-TPL construct was generated using

MultiSite Gateway, to generate a fusion between WUSD and the TPL coding

sequence. TheWUSD sequencewas amplified fromgenomic DNAusingWUS-F

and WUSD-R (5#-ggggacaacttttgtatacaaagttgtATGACCTTCTAGACCAAACA-

GAGG-3#). The TPL sequence was amplified from inflorescence first-strand

cDNA using primers TPL-F (5#-ggggacaactttgtatacaaaagttgtgTCTTCTCTTAG-

TAGAGAGCTCG-3#) and TPL-R (5#-ggggaccactttgtacaagaaagctgggtTCATCTC-TGAGGCTGATCAGATG-3#). The genotype of thewus-1 allele in all transgenics

was confirmed by PCR amplification of the endogenousWUS gene using primers

OL95 (5#-GATCTTGATTGGGGCAAACC-3#) and WUS-UTR (5#-CTAGCGAAG-

CATAGTTGTGAACATACG-3#) andDNA sequencing. The genotype of thewus-3

allele was confirmed by PCR using primers wus3F (5#-ATGGAGCCGCCACAG-

CATCAGCATC-3#) and WUS-UTR and DNA sequencing.

To ensure that the 3.3 kb ofWUS promoter gave the appropriate expression

pattern, it was cloned upstream of the GUS reporter gene in the pJawohl11-

GW-GUS vector using Gateway (Ulker et al., 2007).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Breakdown of transcription factors isolated in

library screens.

Supplemental Figure S2. Additional TPL interactions.

Supplemental Table S1. Non-TF TPL/TPR interactors.

Supplemental Table S2. Summary of TPL/TPR Y2H screens.

Supplemental Table S3. Putative weak TPL/TPR interactors.

Supplemental Table S4. Analysis of RDs among the TPL/TPR interactors.

Supplemental Table S5. Comparison of Arabidopsis RDs from various

sources.

Causier et al.




ACKNOWLEDGMENTS

We thank George Coupland and Franziska Turck at the Max Planck Institute

(Cologne, Germany) and Gerco Angenent and Richard Immink at Plant

Research International (Wageningen, The Netherlands) for the arrayed yeast

two-hybrid library of Arabidopsis transcription factors. The normalized Arabi-

dopsis whole plant yeast two-hybrid library was the generous gift of Hans

Sommer and Simona Masiero (Max Planck Institue, Cologne, Germany). Seed

for the tpl-1 allele was kindly provided by Kathy Barton (Department of Plant

Biology, Carnegie Institution, Stanford, CA), and wus-3 seed was the generous

gift of Thomas Laux (University of Freiburg, Freiburg, Germany). We are also

grateful to Stefan Kepinski and James Lloyd (Centre for Plant Sciences, Univer-

sity of Leeds, UK) for comments on the manuscript, Sam Rayson for her

assistance with qRT-PCR, and Katie Curnock, Amie Blinkhorn, and Lauren Hill

(University of Leeds, UK) for help with the yeast two-hybrid library screens.

Received September 12, 2011; accepted November 4, 2011; published

November 7, 2011.

LITERATURE CITED

Arabidopsis Interactome Mapping Consortium (2011) Evidence for net-

work evolution in an Arabidopsis interactome map. Science 333:

601–607

Baurle I, Laux T (2005) Regulation of WUSCHEL transcription in the stem

cell niche of the Arabidopsis shoot meristem. Plant Cell 17: 2271–2280

Bensmihen S, To A, Lambert G, Kroj T, Giraudat J, Parcy F (2004) Analysis

of an activated ABI5 allele using a new selection method for transgenic

Arabidopsis seeds. FEBS Lett 561: 127–131

Brand U, Fletcher JC, Hobe M, Meyerowitz EM, Simon R (2000) Depen-

dence of stem cell fate in Arabidopsis on a feedback loop regulated by

CLV3 activity. Science 289: 617–619

Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, Lewis S, AmiGO

Hub, Web Presence Working Group (2009) AmiGO: online access to

ontology and annotation data. Bioinformatics 25: 288–289

Castillejo C, Pelaz S (2008) The balance between CONSTANS and

TEMPRANILLO activities determines FT expression to trigger flower-

ing. Curr Biol 18: 1338–1343

Castrillo G, Turck F, Leveugle M, Lecharny A, Carbonero P, Coupland G,

Paz-Ares J, Onate-Sanchez L (2011) Speeding cis-trans regulation

discovery by phylogenomic analyses coupled with screenings of an

arrayed library of Arabidopsis transcription factors. PLoS ONE 6:

e21524

Causier B, Davies B (2002) Analysing protein-protein interactions with the

yeast two-hybrid system. Plant Mol Biol 50: 855–870

Cerna D, Wilson DK (2005) The structure of Sif2p, a WD repeat protein

functioning in the SET3 corepressor complex. J Mol Biol 351: 923–935

Chen G, Courey AJ (2000) Groucho/TLE family proteins and transcrip-

tional repression. Gene 249: 1–16

Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-

mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743

Conner J, Liu Z (2000) LEUNIG, a putative transcriptional corepressor that

regulates AGAMOUS expression during flower development. Proc Natl

Acad Sci USA 97: 12902–12907

Courey AJ, Jia S (2001) Transcriptional repression: the long and the short of

it. Genes Dev 15: 2786–2796

Devaiah BN, Nagarajan VK, Raghothama KG (2007) Phosphate homeo-

stasis and root development in Arabidopsis are synchronized by the

zinc finger transcription factor ZAT6. Plant Physiol 145: 147–159

Franks RG, Wang C, Levin JZ, Liu Z (2002) SEUSS, a member of a novel

family of plant regulatory proteins, represses floral homeotic gene

expression with LEUNIG. Development 129: 253–263

Gallavotti A, Long JA, Stanfield S, Yang X, Jackson D, Vollbrecht E,

Schmidt RJ (2010) The control of axillary meristem fate in the maize

ramosa pathway. Development 137: 2849–2856

Garcia ME, Lynch T, Peeters J, Snowden C, Finkelstein R (2008) A small

plant-specific protein family of ABI five binding proteins (AFPs) regu-

lates stress response in germinating Arabidopsis seeds and seedlings.

Plant Mol Biol 67: 643–658

Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/

single-stranded carrier DNA/polyethylene glycol method. Methods

Enzymol 350: 87–96

Gonzalez D, Bowen AJ, Carroll TS, Conlan RS (2007) The transcription

corepressor LEUNIG interacts with the histone deacetylase HDA19 and

mediator components MED14 (SWP) and CDK8 (HEN3) to repress

transcription. Mol Cell Biol 27: 5306–5315

Gregis V, Sessa A, Colombo L, Kater MM (2006) AGL24, SHORT VEGE-

TATIVE PHASE, and APETALA1 redundantly control AGAMOUS

during early stages of flower development in Arabidopsis. Plant Cell

18: 1373–1382

Guo A, He K, Liu D, Bai S, Gu X, Wei L, Luo J (2005) DATF: a database of

Arabidopsis transcription factors. Bioinformatics 21: 2568–2569

Hill K, Wang H, Perry SE (2008) A transcriptional repression motif in the

MADS factor AGL15 is involved in recruitment of histone deacetylase

complex components. Plant J 53: 172–185

Hiratsu K, Ohta M, Matsui K, Ohme-Takagi M (2002) The SUPERMAN

protein is an active repressor whose carboxy-terminal repression do-

main is required for the development of normal flowers. FEBS Lett 514:

351–354

Hiratsu K, Mitsuda N, Matsui K, Ohme-Takagi M (2004) Identification of

the minimal repression domain of SUPERMAN shows that the DLELRL

hexapeptide is both necessary and sufficient for repression of transcrip-

tion in Arabidopsis. Biochem Biophys Res Commun 321: 172–178

Hubbard KE, Nishimura N, Hitomi K, Getzoff ED, Schroeder JI (2010)

Early abscisic acid signal transduction mechanisms: newly discovered

components and newly emerging questions. Genes Dev 24: 1695–1708

Ikeda M, Mitsuda N, Ohme-Takagi M (2009) Arabidopsis WUSCHEL is

a bifunctional transcription factor that acts as a repressor in stem

cell regulation and as an activator in floral patterning. Plant Cell 21:

3493–3505

Ikeda M, Ohme-Takagi M (2009) A novel group of transcriptional repres-

sors in Arabidopsis. Plant Cell Physiol 50: 970–975

Jang S, Torti S, Coupland G (2009) Genetic and spatial interactions

between FT, TSF and SVP during the early stages of floral induction in

Arabidopsis. Plant J 60: 614–625

Jung C, Seo JS, Han SW, Koo YJ, Kim CH, Song SI, Nahm BH, Choi YD,

Cheong JJ (2008) Overexpression of AtMYB44 enhances stomatal clo-

sure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant

Physiol 146: 623–635

Jung JH, Seo YH, Seo PJ, Reyes JL, Yun J, Chua NH, Park CM (2007) The

GIGANTEA-regulated microRNA172 mediates photoperiodic flower-

ing independent of CONSTANS in Arabidopsis. Plant Cell 19: 2736–2748

Kagale S, Links MG, Rozwadowski K (2010) Genome-wide analysis of

ethylene-responsive element binding factor-associated amphiphilic re-

pression motif-containing transcriptional regulators in Arabidopsis.

Plant Physiol 152: 1109–1134

Kagale S, Rozwadowski K (2011) EAR motif-mediated transcriptional

repression in plants: an underlying mechanism for epigenetic regulation

of gene expression. Epigenetics 6: 141–146

Kaufmann K, Pajoro A, Angenent GC (2010) Regulation of transcription in

plants: mechanisms controlling developmental switches. Nat Rev Genet

11: 830–842

Kieffer M, Stern Y, Cook H, Clerici E, Maulbetsch C, Laux T, Davies B

(2006) Analysis of the transcription factor WUSCHEL and its functional

homologue in Antirrhinum reveals a potential mechanism for their roles

in meristem maintenance. Plant Cell 18: 560–573

Krogan NT, Long JA (2009) Why so repressed? Turning off transcription

during plant growth and development. Curr Opin Plant Biol 12: 628–636

Laux T, Mayer KF, Berger J, Jurgens G (1996) The WUSCHEL gene is

required for shoot and floral meristem integrity in Arabidopsis. Devel-

opment 122: 87–96

Leibfried A, To JP, Busch W, Stehling S, Kehle A, Demar M, Kieber JJ,

Lohmann JU (2005) WUSCHEL controls meristem function by direct

regulation of cytokinin-inducible response regulators. Nature 438:

1172–1175

Liu C, Xi W, Shen L, Tan C, Yu H (2009) Regulation of floral patterning by

flowering time genes. Dev Cell 16: 711–722

Liu Z, Karmarkar V (2008) Groucho/Tup1 family co-repressors in plant

development. Trends Plant Sci 13: 137–144

Long JA, Ohno C, Smith ZR, Meyerowitz EM (2006) TOPLESS regulates

apical embryonic fate in Arabidopsis. Science 312: 1520–1523

Long JA, Woody S, Poethig S, Meyerowitz EM, Barton MK (2002)

Transformation of shoots into roots in Arabidopsis embryos mutant at

the TOPLESS locus. Development 129: 2797–2806

Lopez-Molina L, Mongrand S, Kinoshita N, Chua NH (2003) AFP is a





novel negative regulator of ABA signaling that promotes ABI5 protein

degradation. Genes Dev 17: 410–418

Malave TM, Dent SY (2006) Transcriptional repression by Tup1-Ssn6.

Biochem Cell Biol 84: 437–443

Matsui K, Umemura Y, Ohme-Takagi M (2008) AtMYBL2, a protein with a

single MYB domain, acts as a negative regulator of anthocyanin bio-

synthesis in Arabidopsis. Plant J 55: 954–967

Mayer KF, Schoof H, Haecker A, Lenhard M, Jurgens G, Laux T (1998)

Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot

meristem. Cell 95: 805–815

Mittler R, Kim Y, Song L, Coutu J, Coutu A, Ciftci-Yilmaz S, Lee H,

Stevenson B, Zhu JK (2006) Gain- and loss-of-function mutations in

Zat10 enhance the tolerance of plants to abiotic stress. FEBS Lett 580:

6537–6542

Nurmberg PL, Knox KA, Yun BW, Morris PC, Shafiei R, Hudson A, Loake

GJ (2007) The developmental selector AS1 is an evolutionarily con-

served regulator of the plant immune response. Proc Natl Acad Sci USA

104: 18795–18800

Ogas J, Kaufmann S, Henderson J, Somerville C (1999) PICKLE is a CHD3

chromatin-remodeling factor that regulates the transition from embry-

onic to vegetative development in Arabidopsis. Proc Natl Acad Sci USA

96: 13839–13844

Ohta M, Matsui K, Hiratsu K, Shinshi H, Ohme-Takagi M (2001) Repres-

sion domains of class II ERF transcriptional repressors share an essential

motif for active repression. Plant Cell 13: 1959–1968

Pauwels L, Barbero GF, Geerinck J, Tilleman S, Grunewald W, Perez AC,

Chico JM, Bossche RV, Sewell J, Gil E, et al (2010) NINJA connects the

co-repressor TOPLESS to jasmonate signalling. Nature 464: 788–791

Paz-Ares J, The Regia Consortium (2002) REGIA, an EU project on

functional genomics of transcription factors from Arabidopsis thaliana.

Comp Funct Genomics 3: 102–108

Pfluger J, Zambryski P (2004) The role of SEUSS in auxin response and

floral organ patterning. Development 131: 4697–4707

Pontvianne F, Blevins T, Pikaard CS (2010) Arabidopsis histone lysine

methyltransferases. Adv Bot Res 53: 1–22

Preston J, Wheeler J, Heazlewood J, Li SF, Parish RW (2004) AtMYB32 is

required for normal pollen development in Arabidopsis thaliana. Plant

J 40: 979–995

Rajagopala SV, Hughes KT, Uetz P (2009) Benchmarking yeast two-hybrid

systems using the interactions of bacterial motility proteins. Proteomics

9: 5296–5302

Sablowski R (2007) The dynamic plant stem cell niches. Curr Opin Plant

Biol 10: 639–644

Sakamoto H, Maruyama K, Sakuma Y, Meshi T, Iwabuchi M, Shinozaki

K, Yamaguchi-Shinozaki K (2004) Arabidopsis Cys2/His2-type zinc-

finger proteins function as transcription repressors under drought, cold,

and high-salinity stress conditions. Plant Physiol 136: 2734–2746

Schoof H, Lenhard M, Haecker A, Mayer KF, Jurgens G, Laux T (2000) The

stem cell population of Arabidopsis shoot meristems is maintained by a

regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100:

635–644

Sitaraman J, Bui M, Liu Z (2008) LEUNIG_HOMOLOG and LEUNIG

perform partially redundant functions during Arabidopsis embryo and

floral development. Plant Physiol 147: 672–681

Song CP, Galbraith DW (2006) AtSAP18, an orthologue of human SAP18, is

involved in the regulation of salt stress and mediates transcriptional

repression in Arabidopsis. Plant Mol Biol 60: 241–257

Sridhar VV, Surendrarao A, Gonzalez D, Conlan RS, Liu Z (2004)

Transcriptional repression of target genes by LEUNIG and SEUSS, two

interacting regulatory proteins for Arabidopsis flower development.

Proc Natl Acad Sci USA 101: 11494–11499

Sridhar VV, Surendrarao A, Liu Z (2006) APETALA1 and SEPALLATA3

interact with SEUSS to mediate transcription repression during flower

development. Development 133: 3159–3166

Stahle MI, Kuehlich J, Staron L, von Arnim AG, Golz JF (2009) YABBYs

and the transcriptional corepressors LEUNIG and LEUNIG_HOMO-

LOG maintain leaf polarity and meristem activity in Arabidopsis. Plant

Cell 21: 3105–3118

Swingler TE, Bess KL, Yao J, Stifani S, Jayaraman PS (2004) The proline-

rich homeodomainprotein recruitsmembers of theGroucho/Transducin-like

enhancer of split protein family to co-repress transcription in hematopoietic

cells. J Biol Chem 279: 34938–34947

Szemenyei H, Hannon M, Long JA (2008) TOPLESS mediates auxin-

dependent transcriptional repression during Arabidopsis embryogene-

sis. Science 319: 1384–1386

Ulker B, Shahid Mukhtar M, Somssich IE (2007) The WRKY70 transcrip-

tion factor of Arabidopsis influences both the plant senescence and

defense signaling pathways. Planta 226: 125–137

Vidal M, Legrain P (1999) Yeast forward and reverse ‘n’-hybrid systems.

Nucleic Acids Res 27: 919–929

Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007)

An “Electronic Fluorescent Pictograph” browser for exploring and

analyzing large-scale biological data sets. PLoS ONE 2: e718

Zhang H, Rider SD Jr, Henderson JT, Fountain M, Chuang K, Kandachar

V, Simons A, Edenberg HJ, Romero-Severson J, Muir WM, et al (2008)

The CHD3 remodeler PICKLE promotes trimethylation of histone H3

lysine 27. J Biol Chem 283: 22637–22648

Zhao Z, Shen WH (2004) Plants contain a high number of proteins showing

sequence similarity to the animal SUV39H family of histone methyl-

transferases. Ann N Y Acad Sci 1030: 661–669

Zhu Z, Xu F, Zhang Y, Cheng YT, Wiermer M, Li X, Zhang Y (2010)

Arabidopsis resistance protein SNC1 activates immune responses

through association with a transcriptional corepressor. Proc Natl Acad

Sci USA 107: 13960–13965

Causier et al.




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