TOO MUCH LOVE, a Novel Kelch Repeat-Containing F-box Protein, Functions in the Long-Distance...

TOO MUCH LOVE, a Novel Kelch Repeat-Containing F-boxProtein, Functions in the Long-Distance Regulation of theLegume–Rhizobium SymbiosisMasahiro Takahara1,2, Shimpei Magori3,6, Takashi Soyano2,6, Satoru Okamoto2, Chie Yoshida4,Koji Yano2, Shusei Sato5, Satoshi Tabata5, Katsushi Yamaguchi2, Shuji Shigenobu1,2, Naoya Takeda1,2,Takuya Suzaki1,2 and Masayoshi Kawaguchi1,2,*1Department of Basic Biology in the School of Life Science of the Graduate University for Advanced Studies, Aichi, Japan2National Institute for Basic Biology, Aichi, Japan3Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, NY, USA4Department of Biological Science, Graduate School of Science, The University of Tokyo, Tokyo, Japan5Kazusa DNA Research Institute, Chiba, Japan6These authors contributed equally to this work.*Corresponding author: E-mail, [email protected]; Fax, +81-564-55-7564.(Received January 12, 2013; Accepted January 29, 2013)

The interaction of legumes with N2-fixing bacteria collectivelycalled rhizobia results in root nodule development. Thenumber of nodules formed is tightly restricted through thesystemic negative feedback control by the host called auto-regulation of nodulation (AON). Here, we report the charac-terization and gene identification of TOO MUCH LOVE (TML),a root factor that acts during AON in a model legume Lotusjaponicus. In our genetic analyses using another root-regulated hypernodulation mutant, plenty, the tml-1 plentydouble mutant showed additive effects on the nodulenumber, whereas the tml-1 har1-7 double mutant did not,suggesting that TML and PLENTY act in different genetic path-ways and that TML and HAR1 act in the same genetic path-way. The systemic suppression of nodule formation byCLE-RS1/RS2 overexpression was not observed in the tmlmutant background, indicating that TML acts downstreamof CLE-RS1/RS2. The tml-1 Snf2 double mutant developedan excessive number of spontaneous nodules, indicatingthat TML inhibits nodule organogenesis. Together with thedetermination of the deleted regions in tml-1/-2/-3, the finemapping of tml-4 and the next-generation sequencinganalysis, we identified a nonsense mutation in the Kelchrepeat-containing F-box protein. As the gene knockdown ofthe candidate drastically increased the number of nodules, weconcluded that it should be the causative gene. An expressionanalysis revealed that TML is a root-specific gene. In addition,the activity of ProTML-GUS was constitutively detected in theroot tip and in the nodules/nodule primordia upon rhizobialinfection. In conclusion, TML is a root factor acting at the finalstage of AON.

Keywords: Autoregulation of nodulation � CLE � HAR1 �

Lotus japonicus � Systemic regulation � TOO MUCH LOVE.

Abbreviations: ANOVA, analysis of variance; AON, autore-gulation of nodulation; CaMV, Cauliflower mosaic virus;dCAPS, derived cleaved amplified polymorphic sequence;dpi, days post-inoculation; EMS, ethyl methanesulfonate;EST, expressed sequence tag; GFP, green fluorescent protein;Gm, Glycine max; GUS, b-glucuronidase; iPCR, inverse PCR;Lj, Lotus japonicus; LRR-RLK, leucine-rich repeat receptor-likekinase; Mt, Medicago truncatula; NLS, nuclear localizationsignal; ORF, open reading frame; Ps, Pisum sativum; qPCR,quantitative real-time reverse transcription–PCR; RNAi,RNA interference; SDI, shoot-derived inhibitor; SSR, sim-ple sequence repeat; TML, TOO MUCH LOVE; UBQ,ubiquitin.

Introduction

The interaction of legumes with N2-fixing bacteria collectivelycalled rhizobia results in root nodule development. Rhizobiasupply the host legumes with ammonia fixed from the air,and host plants provide the rhizobia with photosynthates inreturn. Because the formation of nodules is energetically expen-sive, the number of nodules and the nodulation zone are tightlyrestricted by the negative feedback regulation of host legumes,which is termed the autoregulation of nodulation (AON)(Bhuvaneswari et al. 1980, Pierce et al. 1983, Caetano-Anolleset al. 1991, Ferguson et al. 2010)

The surgical excision of nodules in Trifolium pratense led toan increase in the number of subsequently developed nodules,indicating that AON is activated by preceding nodulationevents and negatively regulates the subsequent nodule forma-tion (Nutman 1952). The pre-inoculated half of a split-root

Plant Cell Physiol. 54(4): 433–447 (2013) doi:10.1093/pcp/pct022, available FREE online at www.pcp.oxfordjournals.org! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

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system inhibited the subsequent nodule formation on theother half in Glycine max (L.) Merr., T. subterraneum,Medicago sativa, Phaseolus vulgaris and Lotus japonicus. Thesefindings suggest that AON is a systemic regulation and iscommon in a variety of legumes (Singleton 1983, Kosslaket al. 1984, Sargent et al. 1987, George 1992, Catford et al.2003, Suzuki et al. 2008).

In addition, recent studies have suggested that AON isactivated via a long-distance communication between theroot and shoot. Several mutants (i.e. Ljhar1/Mtsunn/Pssym29/Gmnark, Pssym28 and Ljklv) were identified as hypernodulatingmutants in which the number of nodules is increased (Saganet al. 1996, Szczyglowski et al. 1998, Krusell et al. 2002,Nishimura et al. 2002, Penmetsa et al. 2003, Searle et al. 2003,Oka-Kira et al. 2005, Schnabel et al. 2005, Suzaki et al. 2008,Miyazawa et al. 2010, Schnabel et al. 2010, Krusell et al. 2011).Reciprocal grafting experiments between these hypernodulat-ing mutants and wild-type plants revealed that the shoot geno-type rather than the root genotype determines thehypernodulation phenotype (Delves et al. 1986, Olsson et al.1989, Sheng et al. 1997, Wopereis et al. 2000, Krusell et al. 2002,Nishimura et al. 2002, Penmetsa et al. 2003, Oka-Kira et al.2005). Additionally, the genes responsible for the hypernodula-tion encode leucine-rich repeat receptor-like kinases(LRR-RLKs) (Krusell et al. 2002, Nishimura et al. 2002, Searleet al. 2003, Oka-Kira et al. 2005, Schnabel et al. 2005,Miyazawa et al. 2010). These findings indicate that the activa-tion of AON requires the LRR-RLKs acting in the shoot. Theorthologs of these LRR-RLKs in Arabidopsis thaliana (CLV1,CLV2 and RPK2) and Oryza sativa (FON1) have roles in main-taining the shoot apical meristem and are activated by receivingthe short-distance signaling molecule, i.e. the CLE family ofpeptides CLV3 (Ogawa et al. 2008) and FON2/FON4 (Chuet al. 2006, Suzaki et al. 2008, Suzaki et al. 2009), respectively.In legumes, the CLE family of peptides are strong candidates forthe root-derived signal molecules because the expression of CLEgenes such as LjCLE-RS1/RS2, MtCLE12/13 and GmRIC1/2 isinduced by rhizobial infection (Okamoto et al. 2009, Mortieret al. 2010, Mortier et al. 2011, Reid et al. 2011) and because theoverexpression of these CLE genes drastically reduces orabolishes nodulation in a HAR1/SUNN/NARK- or KLV-depend-ent manner (Okamoto et al. 2009, Miyazawa et al. 2010, Mortieret al. 2010, Lim et al. 2011, Reid et al. 2011). Together with therelationship between the LRR-RLKs and the CLE peptides in A.thaliana and O. sativa, AON-related CLEs might be root-derivedligands for the AON-related LRR-RLKs in the shoot.

In addition, there have been several reports on the relation-ship between cytokinins and AON. The gain-of-function muta-tion of the cytokinin receptor LjLHK1 in the har1 or klv mutantbackground develops an excessive number of spontaneousnodules without rhizobial infection, indicating that HAR1 andKLV inhibit the nodule formation induced by the cytokininsignaling. Moreover, a recent report has shown that a cytokininregulates MtCLE13 expression (Mortier et al. 2010). Thereforethe CLE-, HAR1/SUNN/NARK- and KLV-dependent suppression

of nodule formation may be induced by the cytokinin-inducednodule organogenesis itself, even without rhizobial infection.

To establish the systemic regulation of nodulation vialong-distance communication, the AON needs both theroot-to-shoot signal and a shoot-to-root signal (which is alsocalled a shoot-derived inhibitor; SDI) (Lin et al. 2010). Withrespect to the shoot-to-root signal, some research attemptingto isolate shoot-derived signals is in progress (Akashi et al. 2010,Lin et al. 2010, Yamaya and Arima 2010a, Yamaya and Arima2010b, Lin et al. 2011). Recent studies suggest that the SDI is anamphiphilic and low molecular weight compound. However, itremains unsolved as to how nodulation is inhibited down-stream of SDI in the final stage of AON in the root.

In a previous study, we have shown that too much love (tml),a hypernodulating mutant of L. japonicus, has a defect in nega-tive feedback regulation and that TML is a root factor actingdownstream of HAR1 (Magori et al. 2009). Therefore, TML mustbe a key factor in understanding what happens during the finalstage of AON to maintain proper nodulation. Hence, it is crucialto investigate the function of TML and identify the TML gene. Inthis paper, we clarify the point at which TML acts during AONthrough the characterization of the tml mutant. Genetic andmolecular analyses indicate that TML and PLENTY (anothermutant of the root factor that regulates the number of nodules)act in different genetic pathways; TML acts downstream ofLjCLE-RS1/2 and might suppress the nodulation signalingdownstream of the cytokinin receptor LHK1/CRE1. We alsoreveal that the TML gene encodes a Kelch repeat-containingF-box protein with two nuclear localization signals (NLSs) andpotentially functions in proteasome-mediated degradation ofits target protein. In conclusion, we identify the F-box proteinTML as a key factor in maintaining proper nodulation duringthe final stage of AON.

Results

TML and PLENTY function in different geneticpathways, whereas TML and HAR1 function inthe same genetic pathway

A previous study from our group showed that the har1 geno-type did not enhance the tml hypernodulation phenotype inthe grafting experiment or in the double mutant analysis, sug-gesting that TML functions downstream of HAR1. However, theresults in the previous report did not exclude the possibilitythat the observations reflect that the root of the tml-1 mutantdoes not have the developmental potential to produce anymore nodules. To clarify which explanation is reasonable, weexamined the genetic interaction between TML and PLENTY,another root factor that regulates the number of nodules. First,we observed the nodulation phenotype of the tml-1 plentydouble mutant. The tml-1 plenty double mutant showed anincreased number of nodules compared with the respectivesingle mutants, indicating that TML and PLENTY function in

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different genetic pathways (Fig. 1A–E). This finding alsoindicates that if TML and the other gene that is also involvedin the regulation of the nodule number act in different geneticpathways, the double mutant indeed illustrates the additionaleffect on the nodule number. We analyzed the tml-1 har1-7double mutants as well. In contrast, the tml-1 har1-7 doublemutant did not show an additive effect on nodulation(Fig. 1A–C, F, G), which is consistent with previous results.Taken together, we conclude that TML and HAR1 function inthe same genetic pathway.

TML is required for the suppression of nodulationcaused by CLE-RS1/RS2

The previous study demonstrated that either CLE-RS1 orCLE-RS2 systemically suppresses nodule formation in a HAR1-dependent manner. Therefore, to investigate the genetic inter-action between TML and CLE-RS1/RS2, we introduced the ex-pression construct of CLE-RS1/RS2 driven by the Cauliflowermosaic virus (CaMV) 35S promoter using the hairy root trans-formation method and examined whether the tml mutationaffects the suppression by CLE-RS1/RS2. As has been reported,the roots overexpressing either CLE-RS1 or CLE-RS2 in thewild-type plants developed a drastically decreased number ofnodules compared with the control roots overexpressing the

b-glucuronidase (GUS) gene. In contrast, in the tml mutantbackground, the roots overexpressing either CLE-RS1 orCLE-RS2 developed an excessive number of nodules comparedwith that in the wild-type background (Fig. 2A). In addition,there is no statistically significant difference between thenumber of nodules in the CLE-overexpressing roots and thecontrol GUS-overexpressing roots in the tml mutant back-ground (Fig. 2B). This result indicates that CLE-RS1/RS2 sup-presses nodulation in a TML-dependent manner. Therefore, weconclude that TML functions in nodule development down-stream of CLE-RS1/RS2.

TML inhibits the nodule organogenesis induced bythe LHK1-mediated cytokinin signaling

In L. japonicus, the gain-of-function mutant of the cytokininreceptor LHK1 (Snf2) develops nodule-like structures called‘spontaneous nodules’ in the absence of rhizobia (Tirichineet al. 2007). In previous reports, an excessive number of spon-taneous nodules was observed in the har1 Snf2 and klv Snf2double mutants, suggesting that AON functions downstreamof the cytokinin signaling-induced activation of root nodulefounder cells (Tirichine et al. 2007, Miyazawa et al. 2010). Toassess the role of TML in AON, we crossed the tml mutant withthe Snf2 mutant to generate the tml-1 Snf2 double mutant.

Fig. 1 The nodulation phenotypes in the tml-1 har1-7 and tml-1 plenty double mutants, each single mutant and the wild type. (A) The numberof nodules and nodule primordia counted at 21 dpi. The results are represented as the means ± SD. Columns with the same lower case charactersare not significantly different [Tukey’s test after an analysis of variance (ANOVA) (P< 0.001)]. (B–G) The nodulation phenotype of each mutantobserved at 21 dpi.


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In the F2 population, the number of spontaneous nodules in theSnf2 mutants that were homozygous at the TML locus (tml/tmlor TML/TML) was counted 5 weeks post-germination. The tml-1Snf2 double mutant spontaneously developed many small nod-ules similar to those generally observed in the tml mutant uponrhizobial infection, whereas the Snf2 single mutant developedspontaneous nodules similar to those produced in the wild-typeupon infection. The tml-1 Snf2 double mutant produced anaverage of 12.1 ± 6.6 spontaneous nodules, while the Snf2single mutant produced an average of 3.6 ± 1.7 (Fig. 3D), indi-cating that TML inhibits the nodule organogenesis induced bythe LHK1-mediated cytokinin signaling. In addition, this resultclearly indicates that the nodule formation itself, even withoutrhizobial infection, is enough to activate TML.

Additionally, the early symbiotic responses were analyzed inthe tml-1 mutant. Calcium (Ca2+) spiking is defined as periodiccalcium ion changes observed around the nucleus of the hostroot in response to the symbiotic signal Nod factor. The hyper-nodulating mutant sickle, as well as some mutants in the earlysymbiotic signaling pathway, showed a defect in or disturbanceof the oscillation of Ca2+ (Wais et al. 2000, Walker et al. 2000,Oldroyd et al. 2001, Miwa et al. 2006, Sun et al. 2006). However,in the tml-1 mutant, Ca2+ spiking was observed after treatmentwith Nod factor, and the frequency and shape of spikes werenormal compared with those in the wild-type plant(Supplementary Fig. S1). This result suggests that TML doesnot affect the early signaling events in nodule symbiosis.

TML encodes a Kelch repeat-containing F-boxprotein with two NLSs

A previous study demonstrated that the gene responsible forthe hypernodulation phenotype in the tml mutant is a rootregulator involved in the HAR1-mediated long-distance controlof nodule numbers (Magori et al. 2009). To unveil its precisefunction, it is crucial to isolate the gene. In an attempt to lo-calize the gene, the TML locus was mapped to the genomicregion between the simple sequence repeat (SSR) markersTM0805 and TM0356 on chromosome 1. To narrow downthe candidate region, inverse PCR (iPCR) was performed toidentify the deleted regions in the large deletion alleles tml-1[formerly tml (Magori et al. 2009)], tml-2 and tml-3 (Fig. 4A). Inall three alleles, the breakpoint was attached to repetitive re-gions. As a result, the deletions began at –2,857 bp (tml-1), –10,382 bp (tml-2) and –1 bp (tml-3) from the initial codon ofthe first open reading frame (ORF) of the deleted region intml-1. In addition, fine mapping was performed using theethyl methanesulfonate (EMS) allele tml-4 [formerly rdh1(Yokota et al. 2009)]. SSR and derived cleaved amplified poly-morphic sequence (dCAPS) markers were assessed forco-segregation in 1,958 F2 population. Consequently, the TMLlocus was confined to the region between the SSR markersTM0805 and TM2344. Together with the results of the finemapping and iPCR, the TML gene locus was delimited to aregion of approximately 117 kb (Fig. 4A). Because the region

Fig. 2 Nodulation phenotypes of CLE-RS1/RS2-overexpressing roots. (A) Nodulation phenotypes of GUS-, CLE-RS1- and CLE-RS2-overexpressingroots in the wild type, tml-1 and tml-4. Bars indicate 2 mm. (B) Number of nodules on CLE-RS1-, CLE-RS2- and GUS-overexpressing roots. Theresults are represented as the means ± SD. Asterisks indicate the statistically significant difference at P< 0.05 (*) using Student’s t-test.


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was newly assembled in the contig CM0064, we analyzed thesequence by GENSCAN. Of the 21 genes predicted in the region,the whole-genome resequencing of tml-4 using anext-generation sequencer found only one non-synonymoussingle nucleotide alteration in the gene corresponding to theexpressed sequence tag (EST) sequence (GenBank accessionNo. AK339024), which results in a premature stop codon(Fig. 4B). Therefore, to determine whether the loss of the can-didate gene is responsible for the tml hypernodulation pheno-type, we knocked down the expression of the candidate gene inroots by introducing the 340 bp target sequence driven by theLjUBQ promoter. We checked the decreased expression of thecandidate gene by quantitative real-time reverse transcription–PCR (qPCR) analysis. As expected, the number of nodules de-veloped on the candidate gene-silenced roots increased ap-proximately 7.5-fold compared with those on the controlroots (Fig. 4C–G). In addition, to verify that this result wasnot due to an off-target effect, we performed gene silencingtriggered by two other sequences of the candidate gene andobtained the same results (Supplementary Fig. S2). These re-sults clearly indicate that the gene corresponding to AK339024is indeed responsible for the tml hypernodulating phenotype.

The sequence analysis revealed that TML encodes a Kelchrepeat-containing F-box protein with three types of conserveddomains: the F-box domain, the Kelch-repeat domain and two

NLSs. An F-box domain is a motif that binds to the Skp1 familyof proteins (e.g. ASK1 and ASK2 in A. thaliana), resulting in theformation of the Skp1 Cullin F-box (SCF) E3 ubiquitin ligasecomplex. A Kelch-repeat domain is a motif involved in protein–protein interactions. In the well-studied Kelch repeat-containing F-box proteins (i.e. FKF1 and LKP2 in A. thalianaand JFK in Homo sapiens), the Kelch-repeat domains arerequired for the physical interaction with their target proteins(Imaizumi et al. 2005, Sun et al. 2009). Therefore, in the tml-4mutant, the transcript is predicted to encode a non-functionalprotein lacking the Kelch-repeat domain (Fig. 4B).

There are two NLSs predicted upstream of and within theF-box domain (Fig. 4B). To analyze the subcellular localizationof the TML protein, a TML–synthetic green protein (sGFP)fusion protein was expressed in the roots of wild-type plantsand the fluorescence of TML–sGFP was observed in the nucleus(Fig. 4H, I). Therefore, we conclude that TML is a gene encodinga Kelch repeat-containing F-box protein that acts in the nucleusto regulate the nodule number.

To obtain the supplemental information from the orthologs,we conducted a phylogenetic analysis. A BLAST search usingthe full-length peptide sequence of L. japonicus TML (LjTML) asa query against the G. max proteome database in thephytozome database (http://www.phytozome.net/) revealedtwo closely related sequences, i.e. Glyma16g06160.1 and

Fig. 3 Analyses of the genetic interactions between TML and LHK1. (A) Appearance of the spontaneous nodules on Snf2 single mutants. (B)Appearance of spontaneous nodules on tml-1 Snf2 double mutants. (C) A magnified image of (B). (D) The number of spontaneous nodules ontml-1 Snf2 double mutants and Snf2 single mutants counted 5 weeks post-germination. The 96 plants that were genotyped from the F2 progenyof F1 plants of tml-1 crossed with Snf2. tml-1 Snf2 double mutants and Snf2 single mutants were selected to assess the phenotype. Bars in (A–C)indicate 5 mm. Error bars in (D) represent SD. Asterisks indicate the statistically significant difference at P< 0.001 (***) using Student’s t-test.




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Glyma19g25770.1, with amino acid identities of 81% and 80% toLjTML, respectively. In addition, related sequences, includingAT3G27150 and AT5G40680, were collected from the com-parative gene analysis resource, GreenPhyl (http://greenphyl.cirad.fr/v2/cgi-bin/index.cgi). No orthologs of LjTML werefound in Cyanidioschyzon merolae, Ostreococcus tauri orChlamydomonas reinhardtii. A phylogenetic analysis using the

F-box domains of the collected sequences revealed that theTML-related Kelch repeat-containing F-box proteins werewidely conserved in embryophytes and were classified intothree groups (i.e. the TML clade, the TML-like clade and thebasal clade) (Supplementary Fig. S3). LjTML, G. max TMLorthologs (Glyma16g06160.1 and Glyma19g25770.1) andA. thaliana TML orthologs (AT3G27150 and AT5G40680)

Fig. 4 Identification of the TML gene. (A) Map-based cloning of the TML gene. (B) Annotated domains of the TML gene product. (C) Schematicillustrations of target sequences used in the subsequent gene knockdown approach. (D, E) Nodulation phenotypes of GUS- and TML-silencedroots. Trigger sequences were driven under the ubiquitin promoter. (F) Number of nodules on TML-silenced roots. (G) Relative expression levelof TML in the TML-silenced roots. (H, I) Subcellular localization of the TML protein. Arrowheads indicate TML localization in the nucleus.


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were included in the TML clade (Fig. 5). The phylogenetic treeshowed that at least one close ortholog of TML in each legumeplant exists in the TML clade, suggesting the conserved func-tion of TML among legume species.

TML expression is regulated in both aMesorhizobium loti infection-dependentand -independent manner

To characterize the TML gene, we analyzed the spatial andtemporal expression patterns of TML using samples from sev-eral tissues of wild-type plants 14 days post-inoculation (dpi)with rhizobium. The total RNA was extracted from whole rootswith nodules, nodules only, roots after the removal of noduleswith a razor, and shoots. The qPCR analysis showed that theexpression was not detected in shoots from nodulated plantsnor shoots from plants without rhizobial inoculation (Fig. 6A),demonstrating that TML is constitutively expressed in the rootsand nodules (Fig. 6B). Furthermore, we investigated the de-tailed spatial expression patterns of TML using a ProTML-GUSreporter construct in which a 6 kb DNA fragment from theputative translation initiation codon of TML was inserted up-stream of the GUS reporter. We introduced this construct intoL. japonicus roots by the Agrobacterium-mediated method andperformed a histochemical GUS staining analysis. The GUSsignal of ProTML-GUS was detected in the root apex transitionzone (Fig. 7A–C). The intensities of the GUS staining gradually

reduced toward the basal parts of the root (Fig. 7D). This ex-pression pattern at the root tip was not significantly changed byinoculation with M. loti or by nitrogen-rich conditions(Fig. 7A–C). In addition, we found GUS staining in developingroot nodules (Fig. 7E) and in nodule primordia before theemergence from roots (Fig. 7F). At the earlier stages ofnodule organogenesis, GUS expression was not detected inthe dividing cortical cells even beneath the epidermal cellwith the infection thread (Fig. 7G). These results indicatethat TML expression is regulated in both an M. lotiinfection-dependent and infection-independent manner.

Discussion

TML, HAR1 and CLE-RS1/RS2 negativelyregulate nodule organogenesis in thesame genetic pathway

In this study, we examined the detailed role of TML in AONusing tml mutants. First, we demonstrated that the tml mutantstill possesses the potential to develop more nodules whoseformation is inhibited at least by another mechanism involvingPLENTY. This result further supports our proposal that TML andHAR1 act in the same genetic pathway. Because our resultssuggested that TML acts downstream of HAR1, we assumedthat TML is the undiscovered root factor of AON that acts

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Fig. 5 The phylogenetic tree of the TML clade. Deduced amino acid sequences of the F-box domains in TML-related proteins were aligned andthe phylogenetic tree was constructed using the Neighbor–Joining method. A node was supported in 1,000 bootstrap pseudoreplications. Thetree is shown with bootstrap confidence values expressed as a percentage.




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downstream of CLE-RS1/RS2. The overexpression of CLE-RS1/RS2 in the roots of the tml mutant did not affect the number ofnodules, indicating that the suppression of nodulation byCLE-RS1/RS2 requires a functional TML.

Suzaki et al. (2012) reported that the overexpression ofCLE-RS1/RS2 affects auxin accumulation at root cortical cells,

resulting in the abortion of cell division, suggesting the relation-ship among TML, auxin accumulation and cortical cell divisionas the factors downstream of CLE-RS1/RS2.

Additionally, our results demonstrated that TML inhibits thenodule organogenesis induced by the LHK1-mediated cytokininsignaling. Moreover, no abnormalities in the Ca2+ spiking were

Fig. 7 GUS expression in roots transformed with ProTML-GUS. (A–C) GUS staining in root tips. Roots were cultured in 1/2 B&D medium with(B) or without (A) M. loti for 7 d. The medium was supplemented with 10 mM NH4NO3 (C). (D) A magnified image of the boxed region in(B). White lines represent sizes of cortical cells. (E–G) GUS expression in a developing root nodule (E) and in root nodule primordia (F, G). (F) and(G) show merged images of GUS staining with fluorescence from DsRed that is constitutively expressed in M. loti. Arrowheads in (G) indicatedivision planes of cortical cells. Bars indicate 0.1 mm in (A–C), 0.5 mm in (E) and 50 mm in (D, F, G).

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Fig. 6 Expression analyses of TML transcripts. (A) The relative expression levels of TML in various organs of L. japonicus. (B) The relativeexpression levels of TML in the whole root at 0–7 dpi. The results are represented as the means ± SD.


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observed in the tml mutant. As the Nod factor-induced earlysymbiotic signaling involving the Ca2+ spiking activates theLHK1-mediated cytokinin signaling (reviewed in Madsen et al.2010, Oldroyd et al. 2011), these results suggest that the targetof AON is not early symbiotic signaling for the bacterialinfection but rather the nodule organ formation.

Taken together, we conclude that TML acts at the final stageof AON downstream of CLE-RS1/RS2 and HAR1 to regulate thenodule number negatively by inhibiting the organogenesisinduced by the cytokinin signaling (Fig. 7A).

TML encodes a Kelch repeat-containingF-box protein

We narrowed down the candidate region responsible for thetml phenotype through fine mapping and identified a candi-date gene that has the nonsense mutation in the tml-4 mutant.

We then confirmed that the knockdown of the candidate geneinduced the same phenotype as the tml mutants. Hence, wefinally conclude that the Kelch repeat-containing F-box proteinwith two NLSs is the TML gene.

In A. thaliana, among >100 Kelch repeat-containing F-boxproteins, only five members (i.e. ZTL, FKF1, LKP2, AFR andSON1) are well characterized. ZTL, FKF1 and LKP2 act as bluelight receptors in the light-regulated growth and development(Ito et al. 2012), AFR is involved in the phytochromeA-mediated light signaling (Harmon et al. 2003) and SON1acts in the defense response (Kim et al. 2002). Therefore, ourfindings that TML acts in the long-distance control of organo-genesis provide novel insight into the function of the Kelchrepeat-containing F-box proteins. In this report, we foundthat the TML protein localizes to the nucleus. A previousstudy has reported that FKF1 and LKP2 localize to the nucleus

HAR1

A

CLE-RS1/RS2 TML

Nod factor perceptiond l

Nodule organogenesis

Nodulation

Preceding nodulation events

Nodule organogenesis

Subsequent nodulation

Cytokinin

and early responses(e.g. Ca2+ spiking)

Responses for bacterial infection

Nodulation

SCFTMLUb

Ubiquitination of the target Nucleus in a root cortical cell

B

Transcription factor?

UbUb

Target degradation

Inhibition of the subsequent nodulation

Ub

Fig. 8 Schematic illustration of the proposed models. (A) A model for the TML-mediated long-distance control of nodulation. (B) A proposed modelfor TML-mediated degradation of its downstream target indispensable for nodule formation.




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(Takase et al. 2011). In addition, ZTL, FKF1 and LKP2 bind totranscription factors (i.e. TOC1 or CDF1) and promote theirdegradation (Ito et al. 2012). Hence, it is inferred that TML actsin regulating the degradation of the transcription-associatedfactor involved in nodule formation. Furthermore, the transla-tional products of the closest orthologs of TML in A. thaliana(AT3G27150 and AT5G40680) are localized to the nucleus andthey are reported to bind to the Skp1 family of proteins, asubunit of the ubiquitin E3 ligase complex (Schumann et al.2011). These data support our hypothesis that TML and itsorthologs promote the degradation of transcription-associatedfactors.

An E3 ligase is involved in the degradation of a specific targetprotein by polyubiquitination. Therefore, TML might regulatethe stability of a positive and/or negative regulator of noduleorganogenesis by mediating polyubiquitination and 26Sproteasome-dependent degradation (Fig. 7B). Because the26S proteasome degrades the ubiquitinated proteins even ifthe process consumes ATP as an energy source, the ubiquitinproteasome-mediated degradation is often a biologically mean-ingful process. Indeed, in most of the known phytohormones(e.g. auxin, jasmonate, gibberellin, strigolactone and salicylicacid), the signalings are mediated by the components of E3ligase–substrate complexes (Gomi et al. 2004, Dharmasiriet al. 2005, Kepinski et al. 2005, Umehara et al. 2008, Yanet al. 2009, Fu et al. 2012, Yoshida et al. 2012). Intriguingly,the strigolactone signaling that is mediated by the LRR-typeF-box protein has recently been reported to regulate thenumber of nodules positively, in contrast to TML (Foo et al.2012). As it has been suggested that SDI is an amphiphilic andlow molecular weight compound, extrapolating from the rela-tionship between phytohormones and F-box proteins, it is pos-sible that SDI reception triggers the activation of the TMLprotein. As TML is an F-box protein that functions during thefinal stage of AON, identifying its target will unveil a detailedmechanism for establishing the well-suited symbiotic relation-ship. In the future, the identification of the proteins physicallyinteracting with TML will clarify the function of TML at themolecular level.

TML is bifunctional in nodulation events

The quantitative estimation of the TML expression leveldemonstrated that TML is a root-specific gene. Although theTML expression level was not altered upon rhizobial infectionwhen detected using qPCR, the detailed investigation of thepromoter GUS assay revealed that TML is constitutively ex-pressed not only in the root tip but also in the nodules andnodule primordia upon rhizobial infection. The expression pat-tern of ProTML-GUS in root nodule primordia suggests thatTML inhibits the nodule development after the initiation ofcortical cell division. This hypothesis is consistent with the ap-pearance of arrested root nodule primordia in roots overexpres-sing either CLE-RS1 or CLE-RS2 genes (Suzaki et al. 2012).However, the expression in root nodule primordia is not

sufficient for explaining why the tml mutant develops an ex-cessive number of infection threads (Magori et al. 2009). Wedemonstrated that TML is constitutively expressed in the rootapex transition zone, which is at an earlier developmental stagethan the susceptible region for M. loti infection. One hypothesisis that TML expressed in the transition zone indirectly inhibitsformation of infection threads prior to rhizobial infection.Therefore, TML may act bifunctionally, regulating infectionthread development and root nodule formation.

The function of TML in nodule numberregulation might be co-opted fromanother systemic regulation

TML-related F-box genes are conserved among land plants andare classified into three groups: the basal clade, the TML cladeand the TML-like clade. In L. japonicus, TML acts to coordinatethe legume–rhizobium symbiosis. A phylogenetic analysis re-vealed that other legumes have at least one TML ortholog thatbelongs to the TML clade, indicating that these orthologs play arole in nodule number regulation. However, angiosperms otherthan legumes have TML orthologs that belong to the TMLclade, suggesting that TML orthologs have a general function.

A recent study reported that the TML ortholog in A. thali-ana, AT3G27150, is regulated by the microRNA miR2111 inresponse to the nutrient status. Interestingly, althoughAT3G27150 is expressed specifically in the roots, a large quan-tity of miR2111 was detected in the phloem sap during Pi limi-tation, suggesting that AT3G27150 acts in the systemicregulation responsive to environmental stresses (Pant et al.2009). In addition, a recent study has shown that rootdevelopment is systemically regulated in response to theNO3

� status in the root (Ruffel et al. 2011). Moreover, severalreports have demonstrated that AON is activated in responseto nitrogen as an environmental cue (Okamoto et al. 2009,Jeudy et al. 2010, Reid et al. 2011). Furthermore, LjTML is con-stitutively expressed in the root tips. Taken together, TMLorthologs in angiosperms might act in the root in the systemicenvironmental response.

Interestingly, no TML orthologs belonging to the TML cladewere found in monocots (Supplementary Fig. S3). The rootsystem architecture differs between monocots and eudicots,especially with regard to the existence of lateral roots. Severalreports on the relationship between lateral root formation andnodule organogenesis (Mathesius et al. 2000, Gonzalez-Rizzoet al. 2006, Bishopp et al. 2009, Kuppusamy et al. 2009,Yendrek et al. 2010) suggest that the TML clade genes mightregulate the root system architecture that is required ineudicots.

Considering these instances, it has been suggested that thesystemic and long-distance regulation of root development viaTML orthologs has been co-opted to root nodule organogenesisin legume plants. Thus, we propose a hypothesis in which AONregulates nodule organogenesis through the 26S proteasomepathway and that this mechanism has evolved by co-opting


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the systemic regulation of root development responsive toenvironmental stress.

Materials and Methods

Plant materials and growth conditions

Lotus japonicus ecotype Miyakojima MG-20 was used as the wildtype. After overnight water absorption, plants were grown withor without M. loti MAFF 30-3099 in autoclaved vermiculite sup-plemented with Broughton and Dilworth (B&D) solution(Broughton et al. 1971) containing 0.5 mM KNO3 under 16 hlight/8 h dark cycles at a light intensity of 150 mE s–1 m–3 at22�C in a Biotron LH-300 growth cabinet (Nihon-ika).

Double mutant analysis

For the double mutant analysis, tml-1 and plenty mutants werecrossed. From the F2 population, the homozygous double mutantswere selected using PCR, which amplified the deleted region inplenty and tml-1. The number of nodules was counted at 21 dpi.To generate the tml-1 Snf2 double mutant, we crossed the tml-1mutant with the Snf2 mutant. For tml-1 Snf2 double mutantanalysis, the Snf2 mutants were selected from the F2 populationbased on spontaneous nodule formation and could have beeneither heterozygous or homozygous for the snf2 mutation, as themutant phenotype is dominant. The tm-1l genotypes were thenchecked using PCR, which amplified the region containing thejunction of the deleted region in tml-1. The number of spon-taneous nodules was counted 5 weeks post-germination. Allexperiments were performed in biological duplicates.

Hairy root transformation

Transgenic hairy roots were induced by Agrobacterium accord-ing to the method of Okamoto (2009). The CLE-RS1, CLE-RS2 orGUS overexpression construct, the TML RNA interference(RNAi) construct or the ProTML-GUS reporter construct wasintroduced to generate transgenic hairy roots. GFP driven bythe constitutive CaMV 35S promoter in the T-DNA region ofthe binary vector was used as the reporter for transformation.Plants with transformed roots were placed on vermiculite withB&D medium containing 1 mM KNO3 in the growth cabinet at24�C with a photoperiod of 16 h light and 8 h dark for 6 d beforethe plants were inoculated with M. loti. The total number ofnodules on the GFP-positive roots was counted at 21 dpi.

Constructs for RNA silencing

The 340 bp fragment of the TML gene and the 325 bp fragmentof the GUS gene were amplified using PCR, cloned into pENTRd-TOPO (Invitrogen) and subcloned into pUB-GWS-GFP(Maekawa et al. 2008). The hairy root transformation was per-formed as described above.

The primers used here were TML RNAi_F, 50-CACCATGGCCAATAAAAAAGCATT-30 and TML RNAi_R, 50-ACATGAACACTGAGGGCTCTTT-30, which amplified nucleotides 1–340 of the

TML gene; and GUS RNAi_F, 50-CACCTGAACCGTTATTACGGAT-30 and GUS RNAi_R, 50-CGAGTGAAGATCCCTTTCTTG-30, which amplified nucleotides 1,392–1,718 of the GUSgene.

Constructs for the ProTML-GUS reporter assay

The 6 kb DNA fragment from the putative translation initiationcodon of TML was inserted upstream of the GUS reporter inpGWB3 (Nakagawa et al. 2007).

The primers used here were ProTML_F, 50-GAAACACAAACCTCGACAACCACCA-30 and ProTML_R, 50-CATAATAAGTCAGGTACAGGCAAATGCTTC-30.

Measurement of Ca2+ spiking

Ca2+ spiking was measured using a transgenic plant containingthe calcium indicator protein yellow cameleon 2.1 (YC2.1). Thetml-1 mutant was crossed with the transgenic plant, and a tml-1mutant containing YC2.1 (F3) was obtained for calciumimaging. Calcium imaging was performed on the Nikon in-verted microscope ECLIPSE Ti equipped with a �40 dryobjective (numerical aperture 0.6) as described previously(Ehrhardt et al. 1996). The measurements were analyzedusing NIS elements (Nikon) and Microsoft Excel.

Map-based cloning of the TML gene

The tml-4 mutant was crossed with Gifu B-129, and F1 hybridswere selfed to obtain F2 seeds. A total of 1,958 F2 plants (614mutant phenotype (nod++) plants and 1,344 wild-type pheno-type (nod+) plants) were scored for the SSR markers TM0001or TM0805 and BM1852. Among the 614 mutant (nod++)population, the recombinants were further scored for SSR ordCAPS markers C28_1, TM2344, SNP5 and TM2548.

The primers for the dCAPS markers used here are listedbelow.

C28_1 [a 225 bp fragment (MG-20) or 17 + 208 bp fragment(Gifu B129) were obtained by MluI digestion]: C28_1_F, 50-CACCTTAGGAATCAAAGAGCCCTC-30and C28_1_R. 50-GTTTGTTTCCGCGTCAACGCG-30.

SNP5 [a 199 bp fragment (MG-20) or 27 + 172 bp fragments(Gifu B129) were obtained by HinfI digestion]; SNP5_F, 50-CCTTCACTGTCAACCCCCTC-30and SNP5_R; 50-GGACTATGTCTCTTGAAGAGTGTGATT-30.

iPCR-based deletion detection in tmldeletion alleles

The genomic DNA was isolated from 100 mg of fresh youngleaves of each tml mutant plant using the DNeasy Plant Minikit (Qiagen) after grinding using a mortar and pestle. A total of1mg of genomic DNA was digested with restriction enzymes(EcoRI or MseI). For the self-ligation reaction, 0.5mg of digestedDNA was diluted to 500ml with T4 ligase buffer (50 mM Tris–HCl, 10 mM MgCl2, 1 mM ATP, 10 mM dithiothreitol, pH 7.5 at25�C) containing 5ml of ligase (final 4 U ml–1). The reaction mix-ture was then incubated overnight at 16�C. A nested PCR was




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performed using the ligated DNA as a template. The obtainedfragment was sequenced to identify the junction sequence.

Expression analysis

The total RNA was isolated using the RNeasy Plant Mini Kit(Qiagen). First-strand cDNA was prepared using a QuantiTectReverse Transcription Kit (Qiagen). The real-time RT–PCR wasperformed using an ABI Prism 7000 (Applied Biosystems) with aQuantiTect SYBR Green RT-PCR Kit (Qiagen). Transcript amountsin different samples were normalized to those of ubiquitin (UBQ).The results are provided as the means ± SD of results for inde-pendent biological duplicates with technical triplicates.

The primers used here were: TML_qPCR_F, 50-ACAAACAGCTGGAGCCTAATTC-30; TML_qPCR_R, 50-AGAAGCATCAAGCGAGTAAAGC-30; UBQ_qPCR_F, 50-TTCACCTTGTGCTCCGTCTTC-30; and UBQ_qPCR_R. 50-AACAACAGCACACACAGACAATCC-30.

Phylogenetic analysis

To examine the phylogenetic relationships, the amino acid se-quences of Kelch repeat-containing F-box proteins were down-loaded from http://greenphyl.cirad.fr/v2/cgi-bin/index.cgi(family 36739) and http://www.phytozome.net/ (two EST se-quences from Medicago truncatula, one genomic sequencefrom Phaseolus vulgaris and two genomic sequences fromPhyscomitrella patens). All nucleotide data were translatedinto peptide sequences. The pseudogenes and fragmentedORFs were removed, resulting in seven genes from A. thaliana,three from Brachypodium distachyon, three from Caricapapaya, 17 from G. max, four from M. truncatula, four fromO. sativa, five from Populus trichocarpa, five from Ricinus com-munis, five from Sorghum bicolor, seven from Selaginella moel-lendorffii, six from Vitis vinifera and seven from Zea mays. Thepeptide sequences were analyzed using ClustalW multiple se-quence alignment programs. The phylogenic tree was calcu-lated using the Neighbor–Joining method after the alignmentof the sequences. A bootstrap analysis was performed on 1,000random samples taken from the multiple alignment. The treeis shown with bootstrap confidence values expressed as apercentage.

Whole-genome resequencing and thedata analyses

Genomic DNA of the tml-4 homozygous mutant was isolatedusing a DNeasy Plant Mini Kit (Qiagen). The genomic DNA wassheared into 100–150 bp fragments using the Covaris S2 system(Covaris, Inc.) in a 120 ml reaction containing 10 mM TE bufferin a Covaris microTube and the following program: 20% dutycycle, 5 intensity and 200 cycles per burst for 60 s at 5�C.Fragments were arranged into a SOLiD barcorded fragmentlibrary using the SOLiD Fragment Library Construction Kit(Life Technologies). The fragment libraries were amplified byemulsion PCR (ePCR) at a library concentration of 0.5 pM. AfterePCR, the beads were modified at the 30 terminus and

deposited onto a SOLiD sequencing slide, according to themanufacturer’s instructions. The libraries were sequenced to75 bp using the Applied Biosystems 5500xl SOLiD Systemwith an Exact Call Chemistry (ECC) module.

The obtained color space reads were mapped to theL. japonicus genome reference using LifeScope 2.1 software(Life Technologies) with default parameters. For the mapping,we built a custom reference sequence from the current re-lease of the L. japonicus draft genome assembly (release 2.5;http://www.kazusa.or.jp/lotus/summary2.5.html) and newlyassembled fragments (pre-release data by Kazusa DNAResearch Institute). In fact, causal mutation of tml-4 wasfound in the assembly gap of the release 2.5 data. Singlenucleotide polymorphisms (SNPs) were called using adiBayes SNP caller, a component of LifeScope. diBayes wasexecuted with a parameter setting defined as ‘medium callstringency’.

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by the Ministry of Education, Culture,Sports, Science and Technology of Japan [Grants-in-Aid forScientific Research (22128006 and 21027011 to M.K.)].

Acknowledgments

We thank Hiroshi Oyaizu (University of Tokyo) for providingthe rdh1 mutant plant with seeds, Tsuyoshi Nakagawa(Shimane University) for providing the pGWB3 plasmid, theNational BioResource Project Legume Base (MiyazakiUniversity) and Makoto Hayashi (National Institute ofAgrobiological Sciences) for providing the pUB-GWS plasmid,and Sachiko Funayama-Noguchi (University of Tokyo) for tech-nical advice on the statistical analysis. We also thank HisayoAsao [National Institute for Basic Biology (NIBB)] fornext-generation sequencing, and the members of theKawaguchi laboratory for helpful discussions. This study wascarried out under the NIBB Cooperative Research Program(#11-713) and the Model Plant Research Facility. Confocalimages were acquired at Spectrography and BioimagingFacility, NIBB Core Research Facilities.

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TOO MUCH LOVE, a Novel Kelch Repeat-Containing F-box Protein, Functions in the Long-Distance...

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