A Laser Dissection-RNAseq Analysis Highlights the ... · SCRIPTION FACTOR Y (MtNF-YA1) and MtNF-YA2 (Laloum et al., 2014). It was shown earlier that two other transcriptional regulators,

A Laser Dissection-RNAseq Analysis Highlights theActivation of Cytokinin Pathways by Nod Factors in theMedicago truncatula Root Epidermis1[OPEN]

Marie-Françoise Jardinaud 2, Stéphane Boivin2, Nathalie Rodde3, Olivier Catrice, Anna Kisiala,Agnes Lepage, Sandra Moreau, Brice Roux, Ludovic Cottret, Erika Sallet, Mathias Brault,R.J. Neil Emery, Jérôme Gouzy, Florian Frugier, and Pascal Gamas*

LIPM, Université de Toulouse, Institut National de la Recherche Agronomique, Centre National de laRecherche Scientifique, 31326 Castanet-Tolosan, France (M.-F.J., N.R., O.C., A.L., S.M., B.R., L.C., E.S., J.G.,P.G.); INPT-Université de Toulouse, ENSAT, 31326 Castanet-Tolosan, France (M.-F.J.); Institute of PlantSciences-Paris Saclay University, Centre National de la Recherche Scientifique/Institut National de laRecherche Agronomique/Universités Paris-Sud/Paris-Diderot/d’Evry, 91190 Gif-sur-Yvette, France (S.B., M.B.,F.F.); Biology Department, Trent University, Peterborough, Ontario, Canada K9J 7B8 (A.K., R.J.N.E.); andDepartment of Plant Genetics, Physiology, and Biotechnology, University of Technology and Life Sciences,85–789 Bydgoszcz, Poland (A.K.)

ORCID IDs: 0000-0002-4084-2312 (S.B.); 0000-0003-3361-4730 (N.R.); 0000-0002-5820-1660 (O.C.); 0000-0002-4918-7836 (A.K.);0000-0001-6923-0902 (B.R.); 0000-0001-6062-5043 (E.S.); 0000-0003-1304-6430 (R.J.N.E.); 0000-0002-6253-4249 (P.G.).

Nod factors (NFs) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4 and24 h of treatment) in the root epidermis of the model legume Medicago truncatula. Tissue-specific transcriptome analysis wasachieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detectedin the epidermis, among which 1,070 were found to be regulated by NF addition, including previously characterizedNF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, includingperception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, withnumerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways wasindicated, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promotenodulation. These transcriptional activations were independently validated using promoter:b-glucuronidase fusions with theMtCRE1 CK receptor gene and a CK response reporter (TWO COMPONENT SIGNALING SENSOR NEW). A CK pretreatmentreduced the NF induction of the EARLY NODULIN11 (ENOD11) symbiotic marker, while a CK-degrading enzyme(CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased NF induction ofENOD11 and nodulation. Therefore, CK may play both positive and negative roles in M. truncatula nodulation.

The first step of nitrogen-fixing symbiosis consists ofthe mutual recognition of plants and bacteria by anexchange of diffusible signals during the so-calledpreinfection stage. This step enables later bacterial in-fection of root tissues, which, for legumes, generallytakes place via plant tubular structures that originate inroot hairs (RHs), the infection threads. Concomitantly,cell divisions are activated in the root to initiate theformation of specific organs, the nodules, in whichbacteria released from infection threads differentiate inbacteroids and fix atmospheric nitrogen for the plant’sbenefit (for review, see Desbrosses and Stougaard,2011; Oldroyd et al., 2011; Popp and Ott, 2011).

In most documented rhizobium-legume symbioses,key signals for triggering the infection and nodulationprocesses in specific host plants are bacterial lip-ochitooligosaccharides (LCOs) known as Nod factors(NFs), structurally related to signals involved in the

more ancient plant-arbuscular mycorrhizal fungi sym-biosis and transduced via a common set of genestermed the common symbiotic signaling pathway(Oldroyd, 2013). Members of the LYSM receptor-likekinase (RLK) family, namely NOD FACTOR RECEP-TOR1 (NFR1) and NFR5 in Lotus japonicus and NODFACTOR PERCEPTION (NFP) and LYSM RECEPTORKINASE3 (LYK3) in Medicago truncatula, play essentialroles in the perception of NFs (Limpens et al., 2003;Madsen et al., 2003; Radutoiu et al., 2003; Arrighi et al.,2006). Rhizobium spp. infection is strictly dependent onLYK3 andNFP, in contrast to theNF-induced responses(Mitra et al., 2004; Smit et al., 2007), for which only NFPis necessary. These LYSM receptors likely act in com-plexes with the symbiosis Leu-rich repeat receptor ki-nase (LRR RK) SYMRK in L. japonicus (Antolín-Lloveraet al., 2014), orthologous to DOES NOT MAKE IN-FECTIONS2 (DMI2) inM. truncatula, as well as proteins

2256 Plant Physiology�, July 2016, Vol. 171, pp. 2256–2276, www.plantphysiol.org � 2016 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon August 2, 2020 - Published by Downloaded from

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http://orcid.org/0000-0002-4084-2312

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http://orcid.org/0000-0002-4918-7836

http://orcid.org/0000-0001-6923-0902

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associated with particular membrane microdomains,the symbiotic remorins (Lefebvre et al., 2010) andFLOTILLIN-LIKE2 (FLOT2) and FLOT4 (Haney et al.,2011). SYMRK and DMI2 interact with proteins shownto be important for nodulation, which are, respectively,a mitogen-activated protein kinase kinase (MAPKK)called SYMRK-INTERACTING2 (Chen et al., 2012) anda 3-HYDROXY-3-METHYLGLUTARYL COENZYMEA REDUCTASE1 (MtHMGR1; Kevei et al., 2007).MtHMGR1 is thought to generate a secondary signalinvolved in triggering calcium oscillations within andaround the nucleus, a key step of LCO signaling(Venkateshwaran et al., 2015), which involves cationchannels (Ané et al., 2004; Charpentier et al., 2008;Venkateshwaran et al., 2012), a calcium pump (Capoenet al., 2011), and nucleoporins (Kanamori et al., 2006;Saito et al., 2007; Groth et al., 2010). Calcium oscillationsare decoded by a calcium- and calmodulin-dependentSer/Thr protein kinase (CCamK, also known as DMI3in M. truncatula), which interacts with and phosphory-lates CYCLOPS (called INTERACTING PROTEIN OFDMI3 in M. truncatula), a DNA-binding transcriptionalactivator (Singh et al., 2014).CYCLOPS transactivates the expression of the

NODULE INCEPTION (NIN) gene, encoding a tran-scriptional regulator that plays distinct roles in differentMedicago spp. root tissues (Vernié et al., 2015). NIN isrequired to initiate nodule formation in the cortex(Schauser et al., 1999) and to transcriptionally activateNF-YA1 (Soyano et al., 2013; Laloum et al., 2014), atranscription factor (TF) involved in various nodulationsteps (Combier et al., 2006; Soyano et al., 2013; Laporteet al., 2014; Xiao et al., 2014). In the epidermis, NIN is

necessary for the onset of rhizobium infection (Xie et al.,2012; Fournier et al., 2015) but restricts the expression ofEARLY NODULIN11 (ENOD11), a marker of the pre-infection and infection steps (Marsh et al., 2007; Verniéet al., 2015). ENOD11 is up-regulated in response to NFby ETHYLENE RESPONSE FACTOR REQUIRED FORNODULATION1 (ERN1; Andriankaja et al., 2007;Middleton et al., 2007) as well as NUCLEAR TRAN-SCRIPTION FACTOR Y (MtNF-YA1) and MtNF-YA2(Laloum et al., 2014). It was shown earlier that twoother transcriptional regulators, NODULATION SIG-NALING PATHWAY1 (NSP1) and NSP2, are essentialfor nodulation (Kaló et al., 2005; Smit et al., 2005) andform a complex binding the promoter of NIN, ERN1,and MtENOD11 (Hirsch et al., 2009). Thus, there is arequirement of apparently overlapping transcriptionalregulators during NF signaling (preinfection stage),rhizobium infection, and nodule initiation that can bemobilized using different protein complexes or pro-moter regions (Cerri et al., 2012).

The NF signaling and nodulation pathways arestrongly interconnected with hormonal cues and alsotrigger rapid and dynamic modifications in reactiveoxygen species (ROS; Cárdenas et al., 2008), probablyimpacting the regulation of symbiotic genes (Ramuet al., 2002; Andrio et al., 2013). Early responses to NF,notably calcium oscillations, and consequently nodu-lation, are negatively regulated by ethylene, jasmonicacid, and abscisic acid (Penmetsa and Cook, 1997;Oldroyd et al., 2001; Suzuki et al., 2004; Sun et al., 2006;Ding et al., 2008). By contrast, auxins and cytokinins(CKs) play positive roles in nodule initiation and de-velopment. Rhizobium spp. inoculation or LCO appli-cation modulates auxin fluxes in the root, with localauxin accumulation at the site of cortical cell divisionsleading to nodule initiation (Mathesius et al., 1998; Nget al., 2015). Auxin also is involved in controlling theprogression of rhizobium infections (Breakspear et al.,2014; Laplaze et al., 2015). The positive role of CK innodule initiation was demonstrated by the phenotypeof mutants affecting the CK receptors MtCHK1/CRE1and LjLHK1. Loss-of-function alleles are defective innodule formation (Gonzalez-Rizzo et al., 2006; Murrayet al., 2007; Plet et al., 2011), while the spontaneous noduleformation2 (snf2) mutant carrying a gain-of-functionmutation in LHK1 produces nodules in the absence ofrhizobia (Tirichine et al., 2007). An autoactive form ofCCamK/DMI3 (snf1 mutant) requires LHK1 to pro-duce spontaneous nodules, while snf2 does not requireCCamK/DMI3 to induce spontaneous nodules, indi-cating that CK/LHK1 acts downstream from CCamK(Tirichine et al., 2007). The CK biosynthesis genesISOPENTENYL TRANSFERASE3 (IPT3) in L. japonicusand LONELY GUY1 (LOG1) and LOG2 in M. truncatulaare up-regulated during nodulation via CRE1/LHK1,while decreasing MtLOG1 or LjIPT3 expression byRNA interference leads to decreased nodulation (Chenet al., 2014; Mortier et al., 2014). The CK-CRE1/LHK1pathway controls polar auxin transport (Plet et al., 2011;Suzaki et al., 2012) and the Rhizobium spp. induction of

1 This work was supported by the Centre National de la RechercheScientifique, the ANR Labex Saclay Plant Science, and the Lidex PlantPhenotyping Pipeline; by the French Région Ile de France ASTREAprogram (Ph.D. fellowship to S.B.); by the ANR (grant no. ANR–08–GENO–106 to P.G.) and the French Laboratory of Excellence projectTULIP (grant nos. ANR–10–LABX–41 and ANR–11–IDEX–0002–02);and by the Natural Sciences and Engineering Research Council ofCanada (Discovery Grant to R.J.N.E.).

2 These authors contributed equally to the article.3 Present address: Institut National de la Recherche Agronomique-

CNRGV, 24 Chemin de Borde Rouge-Auzeville CS 52627,31326 Castanet-Tolosan cedex, France.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

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

M.-F.J., F.F., and P.G. conceived the research plans; S.B., B.R., S.M.,N.R., O.C., M.-F.J., A.K., and M.B. performed most of the experi-ments; A.L. provided technical assistance to M.-F.J.; E.S. and L.C.performed bioinformatic analyses; J.G. supervised bioinformaticanalyses; M.-F.J. did the statistical tests; R.J.N.E., M.-F.J., F.F., andP.G. supervised the experiments and analyzed the data; P.G. con-ceived the transcriptomic project and wrote the article with F.F.and contributions of A.K., M.-F.J., M.B., S.B., L.C., and N.E.

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Plant Physiol. Vol. 171, 2016 2257

Root Epidermal Cytokinins and Nod Factor Signaling

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specific flavonoids that act as polar auxin transportinhibitors, able to rescue nodulation defects of the cre1mutant (Ng et al., 2015). Exogenous CK treatments in-duce NIN and NF-YA1 in L. japonicus (Heckmann et al.,2011) and NIN, NSP2, and ERN1 in M. truncatula (Pletet al., 2011), while many M. truncatula genes requireMtCRE1 for a full induction by NF (van Zeijl et al.,2015), consistent with a role of CRE1 and CK in NFsignaling.

Even though the different CK receptors were allshown to have positive roles in nodule initiation (Heldet al., 2014; Boivin et al., 2016), CK also may play neg-ative roles at later nodulation stages. Indeed, CKs havebeen shown in L. japonicus to mimic the activity of theshoot-derived signal that inhibits nodule formationduring systemic autoregulation of nodulation (Sasakiet al., 2014), while CKs induce, via CRE1/LHK1 in M.truncatula and L. japonicus, the expression ofCLAVATA3-LIKE (CLE) peptides promoting systemicautoregulation of nodulation (Mortier et al., 2012a;Soyano et al., 2014). In addition, the observations that,first, the expression of a CK-responsive reporter (TWOCOMPONENT SIGNALING SENSOR [TCS]; Müllerand Sheen, 2008),increaseswith time in the epidermis ofrhizobium-infected L. japonicus roots and, second, thatthe lhk1-1mutant is hyperinfected led to the suggestionthat CK might be involved in locally restricting thenumber of infections in L. japonicus (Held et al., 2014).

Molecular mechanisms linking NF signaling in theroot epidermis and the activation of cell divisions inthe root cortex are still unclear. A central role for NIN inthe coordination of epidermal and cortical responseswasproposed recently, with NIN promoting MtCRE1 ex-pression in a positive feedback loop (Vernié et al., 2015).The pMtCRE1:GUS construct is up-regulated a fewhours after Sinorhizobium meliloti inoculation in corticalcells (Lohar et al., 2006), while expression of the primaryCK-response gene MtRR4 (encoding a type-A responseregulator or RRA; Heyl et al., 2013) is detected in peri-cycle and cortical cells (Plet et al., 2011; Vernié et al.,2015). This led to the conclusion that the primary sites ofCK action are inner root tissues, and notably the cortex,consistent with the snf phenotypes induced by a gain-of-function LHK1mutation. Thiswas supported recently bythe use of the TCS:GUS reporter, which was detected atearly time points only in inner root tissues following NFaddition or Rhizobium spp. inoculation and not in theepidermis (Held et al., 2014; van Zeijl et al., 2015).

Transcriptome analysis is a powerful way to inves-tigate genes associated with signaling pathways. NF-induced gene expression reprogrammingwas analyzedrecently in M. truncatula using whole roots or rootsegments (Czaja et al., 2012; Rose et al., 2012; van Zeijlet al., 2015) as well as isolated RHs (Breakspear et al.,2014). Two of these studies used Affymetrix micro-arrays representing approximately 70% of the genome(Czaja et al., 2012; Breakspear et al., 2014), while the twoothers used RNA sequencing (RNAseq), with se-quencing readsmapped on the partial (Mt3.5) or the full(Mt4.0) M. truncatula genome sequence (Rose et al.,

2012; van Zeijl et al., 2015). In addition, genes whosetranscriptional activation by Rhizobium is NF depen-dent were identified by root RNAseq analysis(Larrainzar et al., 2015). Laser-capture microdissection(LCM) coupled to RNAseq enables a sensitive andgenome-wide analysis of gene expression in specifictissues or organ regions, as shown for M. truncatulanodule zones (Roux et al., 2014). In this study, we usedLCM-RNAseq to analyze early NF signaling in the rootepidermis. Hundreds of genes were found to beup-regulated within a few hours and involved in avariety of cellular processes. Strikingly, an early epi-dermal activation of the CK pathway was evidencedbased on the induction of CK metabolic and signalinggenes and independently validated using both the re-cently improved CK-responsive reporter TWO COM-PONENT SIGNALING SENSOR NEW (TCSn):GUS(Zürcher et al., 2013) and a pCRE1:GUS fusion. Thefunctional relevance of this epidermal activation of CKsignaling pathways was explored by testing the impactof CK on the NF induction of ENOD11 and the nodu-lation efficiency.

RESULTS

LCM-RNAseq Analysis of the Root Epidermal Response toNF Treatment

To identify genes associated with early NF signalingin the epidermis,M. truncatula roots treated with 1028

M

NF (4 and 24 h) were compared with mock-treatedroots. The root region responsive to NF was definedusing the stable pENOD11:GUS M. truncatula L416transgenic line (Journet et al., 2001), grown in parallel.Root segments (approximately 1 cm long) were col-lected for laser dissection of epidermal cells(Supplemental Fig. S1), and RNAseq analysis was car-ried out following a procedure described previously(Roux et al., 2014). About 247 million read pairs wereunambiguouslymapped on the fullM. truncatula genomesequence Mt20120830 (accessible at the SYMbiMICSWeb site https://iant.toulouse.inra.fr/symbimics/; down-load section), with an average of 61.9 million read pairsper condition (Supplemental Table S1). Using athreshold statistically defined following Rau et al.(2013), these data enabled the detection of 17,191 genes,which included MtEXP7, a gene specifically expressedin the epidermis (Murray et al., 2011). Complete results,including correspondence with Mt4.0 gene models andannotation data, are provided in Supplemental Table S2or at the SYMbiMICS Web site, where epidermis andprevious nodule RNAseq data can be easily queried usingvarious requests (M. truncatula gene or Affymetrix oligo-nucleotide identifiers, keywords, and BLAST search).

A set of 1,070 differentially expressed genes wasidentified (adjusted P, 0.05 and fold change. 2), with722 up-regulated and 348 down-regulated genes(670 and 310 corresponding genes in Mt4.0, respec-tively). To validate the experimental setup, we com-pared expression data obtained in this study versus

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recent transcriptome analyses of NF responses for a setof 19 NF-induced marker genes (Table I). Differentconditions were used in these studies, with differentNF concentrations, durations of NF treatment, andeither all root tissues or isolated RH (see Fig. 1 legend).Data also were compared with those obtained with S.meliloti-inoculated RH in the hyperinfected sickle (skl)mutant (affected in the ETHYLENE INSENSITIVE2gene; Penmetsa et al., 2008), used to maximize theplant symbiotic response (Breakspear et al., 2014). Pair-wise comparisons are shown in Supplemental Figure S2,which also includes data from Sinorhizobium medicae-inoculated roots (expression groups G1, G2, and G3,predicted to be NF induced; Larrainzar et al., 2015).Table I shows that the LCM-RNAseq approach used inour study enabled the detection of strong differentialexpression for all 19 NF-induced marker genes, vali-dating both the samples and the technical approach.Sets of 413 and 499 genes (386 and 457 corresponding

genes identified in Mt4.0, respectively) were scored asup-regulated in response to 4 or 24 h of NF treatment,respectively (for complete data, including comparisonwith other recently published transcriptome analyses,see Supplemental Table S3). Strikingly, a large num-ber of them exhibited a high level of up-regulation

(greater than 10-fold for 314 genes and greater than50-fold for 143 genes). About half of the NF-inducedgenes at 4 or 24 h had not been identified in the above-mentioned transcriptomics studies (Fig. 1; 33.4% at 4 hwhen also taking into account S. meliloti-inoculated sklRH). Only 45% of the genes induced at 4 h were stillup-regulated at 24 h (Supplemental Fig. S2), showing arelatively large proportion of transiently inducedgenes. We identified a set of genes found in all or moststudies of the NF-regulated transcriptome, with in total76 and 42 genes found in five and six conditions, re-spectively, and thus representing highly robust NF-in-duced marker genes (Supplemental Table S3).

The situation was somewhat different for NF down-regulated genes, with both a lower number of genes(247 and 139 at 4 and 24 h of NF treatment, respectively,with 225 and 98 corresponding genes in Mt4.0,respectively; Supplemental Table S4) and a higherproportion of genes not found in other studies(Supplemental Fig. S3). Only 14.7% (33 genes) of the NFdown-regulated genes at 4 h were still scored down-regulated at 24 h, indicating a very high proportion oftransient regulation, while only seven genes werefound in common among the LCM, RH, and all roottissues 24-h down-regulated genes (Supplemental

Table I. Detection of known NF-induced genes in this study and in recent transcriptomic analyses

Values correspond to the regulation levels (fold change) following NF treatment for the indicated times or S. meliloti inoculation at 5 d postinoculation (RH-5dpi; skl mutant background), determined in this study versus previous studies: a, Rose et al. (2012); b, van Zeijl et al. (2015); c,Czaja et al. (2012); and d, Breakspear et al. (2014). For entries in parentheses, differences were not statistically significant (adjusted P . 0.05). ART,All root tissues from whole root systems (a) or 1- to 2-cm-long root segments (b and c); NA, not applicable (no corresponding gene in the M.truncatula genome sequence used); NF, RNAseq reads were detected only in the NF-treated samples.

Mt20120830 MtV4.0 Annotation LCM-4h LCM-24hART-1h

(a)

ART-3h

(b)

ART-6h

(c)

ART-24h

(c)

RH-24h

(d)

RH-5dpi

(d)Gene Reference

Mt0004_00313 Medtr1g056530 MtNF-YA1 3513.9 NF 294.7 1.9 4.5 53.7 557.7 Laloum et al. (2014)Mt0017_10456 Medtr3g415670 MtENOD11 2560.1 NF NA 1,915.0 8.1 29.6 166.0 759.6 Journet et al. (2001)Mt0039_00030 Medtr1g090807 MtRPG 526.0 NF NA 372.3 20.1 23.1 78.7 121.8 Arrighi et al. (2008)Mt0006_10188 Medtr3g086320 MtNPL NF NF 1,456.7 2.6 3.2 36.5 217.2 Xie et al. (2012)Mt0017_10454 Medtr3g415650 MtENOD12 NF (NF) NA 96.9 68.4 469.2 Journet et al. (1994)Mt0012_10641 none MtENOD40-1 339.2 10.2 NA NA 21.8 127.2 Fang and Hirsch

(1998)Mt0037_10123 Medtr8g038210 MtAnn1 319.3 31.6 97.5 306.3 de Carvalho et al.

(1998)Mt0010_00289 Medtr5g083030 MtPUB1 106.9 10.7 9.6 14.3 14.3 6.0 3.6 4.0 Mbengue et al.

(2010)Mt0005_10038 Medtr5g005290 Mt NMN1 104.8 NF 1,090.5 18.7 12.0 36.8 40.3 Libault et al. (2011)Mt0001_00292 Medtr4g129010 MtSPK1 52.3 13.2 228.2 10.5 4.8 64.9 68.5 Andrio et al. (2013)Mt0001_00813 Medtr4g116990 MtNFH1 34.9 NF NA 40.0 Tian et al. (2013)Mt0010_01109 Medtr5g099060 MtNIN 23.8 16.5 2.5 13.8 10.3 16.4 34.0 116.8 Marsh et al. (2007)Mt0033_10061 Medtr6g027840 MtVAPYRIN 10.4 4.5 NA 81.4 8.0 8.1 7.4 9.6 Murray et al. (2011)Mt0011_00459 Medtr7g085810 MtERN1 (0.97) 21.1 2.3 4.0 4.4 3.8 2.5 9.2 Middleton et al.

(2007)Mt0033_10028 Medtr6g029180 MtERN2 (1.22) 5.8 2.2 1.5 2.5 Andriankaja et al.

(2007)Mt0028_00160 Medtr8g020840 MtNSP1 7.1 7.4 5.4 6.1 13.6 Smit et al. (2005)Mt0005_10559 Medtr5g016320 MtGH3 6.4 (0.2) 4.0 21.9 34.1 Mathesius et al.

(1998)Mt0027_10212 Medtr1g094960 MtARF16a 5.2 13.5 2.1 2.9 2.4 14.4 Breakspear et al.

(2014)Mt0008_10953 Medtr5g074860 MtRIP1 3.2 2.9 NA 1.5 2.1 Cook et al. (1995)












Table S4). Repressing gene expression, therefore, seemsto be a less critical component for the preinfection thangene induction.

A Variety of Functional Classes Is Observed among NFUp-Regulated Genes

An enrichment analysis of Gene Ontology functionalcategories among the NF-regulated genes was performed

(Supplemental Figs. S4 and S5), revealing a predominanceof extracellular, cell wall, and cytoskeleton components,as well as protein kinases, membrane transport, and sig-naling components. A functional classification was inde-pendently performed, based upon the best BLAST hits inSWISSPROT and The Arabidopsis Information Resourcedatabases (Fig. 2). Selected relevant genes in relation to thebiology of rhizobial infections in RH and to the activationof signaling pathways are detailed below.

Genes Encoding Proteins Involved in Cell Dynamics

NF perception leads to morphological modificationsof RH in the nodulation-competent zone of the root,with swelling followed by branching associated with areorientation of RH growth (for review, see Gage, 2004).RH growth is governed by multiple factors: vesicle andorganelle movement using myosin-based motors trav-eling along actin filaments, microtubules that are no-tably involved in nuclear positioning, and the buildupof a tip-focused calcium gradient, modulated by ROSand small GTPase activity (Gage, 2004). This studyrevealed 11 genes encoding proteins related to the actinand microtubule cytoskeleton that are strongly NF in-duced (Table II; Supplemental Table S3), such as thekinesin-4 microtubule-binding motor proteins, themicrotubule-associated proteins MAP65 and MAP70,the TPX2 microtubule-targeting protein, and the tubu-lin- or actin-binding formin-like proteins. Four of theseproteins have been reported previously to be phos-phorylated 1 h after NF addition (Rose et al., 2012),indicating a combination of transcriptional and post-transcriptional regulation.

RH swelling and branching require cell wall relaxa-tion followed by a redirection of cell wall material se-cretion. Acidification of the apoplast caused byP-ATPases triggers hydration and cell wall loosening, aprocess facilitated by expansins and different enzymes(Sablowski and Carnier Dornelas, 2014). The genesencoding the H+-ATPase MtHA1 and the NODULIN26aquaporin are rapidly induced by NF and thus couldplay a role in RH swelling, along with genes encod-ing expansins and enzymes involved in cell wall re-modeling (pectin methyl esterases, cellulase, xyloglucantransglycosylases, pectin lyases, polygalacturonases,and endoglucanases), as well as specific Pro-rich pro-teins (ENOD11 and ENOD12 and the Pro-rich proteinMtPRP4-like; Table II; Supplemental Table S3). Inter-estingly, a gene likely encoding a plasmodesmata-located protein (PDLP3-like; Table II) also wasup-regulated at 4 h, suggesting early modifications ofcell-to-cell communication, potentially involved in thepreparation of neighboring outer cortical cells for in-fection.

Annexins are calcium-binding proteins involved inmembrane organization, vesicle trafficking, and sig-naling (Clark et al., 2012). Two tandem annexin genes,MtAnn1 and MtAnn2, are known to be involved insymbiotic interactions in M. truncatula, with MtAnn1up-regulated by NF (de Carvalho Niebel et al., 1998,

Figure 1. Multiple comparisons (Venn diagrams) of NF up-regulatedM. truncatula genes detected in recent transcriptomics experiments.Top, 1- to 6-h NF treatments; bottom, 24-h NF treatment and S. meliloti-inoculatedRHs5dpost inoculation (RH-5dpi) in a hyperinfected sklmutantbackground. ART-1h, 1028

M NF-treated (1 h) whole roots (Rose et al.,2012); ART-3h, 1029

M NF-treated (3 h) root segments in the presence of1mM aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis(van Zeijl et al., 2015); ART-6h and ART-24h, 1028

M NF-treated root seg-ments (6 and 24 h; Czaja et al., 2012); LCM-4h/-24h, laser-dissected epi-dermal cells (this study) with 4 or 24 h of 1028

MNF treatment, respectively;RH-24h, 1026

M NF-treated RHs (24 h; Breakspear et al., 2014). MtV4.0identifierswere used in all cases. Thresholds chosen to identify up-regulatedgeneswere defined in the corresponding publications. Venn diagramsweregenerated with the tool provided by Bardou et al. (2014).


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2002) and MtAnn2 expressed in nodule primordiaand vasculature (Manthey et al., 2004). We found herethat MtAnn2 also is strongly, but transiently,up-regulated 4 h after NF treatment in epidermal cells(Table II). Two small GTPase Ras-related proteins, po-tentially involved in polarized membrane trafficking(Vernoud et al., 2003), and three patellins (Sec14 andGOLD domain proteins, also implicated in membranetrafficking; Peterman et al., 2004), were additionallyidentified as NF-induced genes. Arabidopsis (Arabi-dopsis thaliana) PATELLIN1 is thought to bindphosphatidylinositol and its phosphorylated deriv-atives, themselves known to regulate membranetrafficking (Peterman et al., 2004). Noteworthy, twogenes (Medtr1g105930 and Medtr3g073100) likelyinvolved in the production of phosphoinositideswere found to be NF induced (Table II; SupplementalTable S3).Transporter and channel genes represented one of

the largest classes of NF up- and down-regulated genesin epidermal cells (53 and 29 genes, respectively), with avariety of gene families (Supplemental Tables S3 andS4). Many of the transport genes are transcriptionallyactivated both strongly and early (4 h) by NF, eventhough some (e.g. the sugar transporter SWEET13)were described previously as only activated at the laterrhizobium infection stage (Breakspear et al., 2014). Sixcorresponding proteins were reported to be phos-phorylated 1 h after NF addition (Rose et al., 2012; TableII; Supplemental Table S3). Those included the previ-ously mentioned MtHA1 ATPase, while a paralogousgene, MtAHA5, also encoding a protein rapidly phos-phorylated uponNF addition (Nguyen et al., 2015), wasdown-regulated. Three NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (PTR) proteins are encodedby NF-induced genes and likely transport oligopep-tides, similar to other PTR1- and PTR3-like proteins(Léran et al., 2014; von Wittgenstein et al., 2014). In-terestingly, the M. truncatula Gene Atlas (MtGEA;http://mtgea.noble.org/v3/) indicates that the NF-in-duced PTR3-like gene (Mtr.31737.1.S1_s_at) is al-most nodule specific.Other components of NF-induced epidermal cell

dynamics included genes involved in the S-phase of the

cell cycle (coding for cyclin D1 and three mini-chromosome maintenance proteins, MCM2, MCM5,and MCM6, consistent with Breakspear et al., 2014;Table II; Supplemental Table S3). MetExplore (Cottretet al., 2010) was used as an unbiased approach topredict bioinformatically which metabolic pathwayswere NF regulated (Supplemental Table S5). Thisanalysis notably revealed the NF induction (at 24 h)of genes involved in GA (Bonferroni = 1.1E207;Supplemental Fig. S6) and trans-zeatin CK biosynthesis(Bonferroni = 7.7E203; Supplemental Fig. S7). Down-regulation of the indole-3-acetyl-amide conjugatebiosynthesis pathway was additionally identifiedtransiently for the 4-h NF treatment (Bonferroni = 1.0E205)and of the CK degradation pathway after 24 h of NFtreatment (Bonferroni = 2.1E206). Overall, this suggeststhat an increase in GA, auxin, and CK biosynthesis/accumulation occurs in the epidermis in response toNFs.

Genes Encoding Proteins Involved in Perception andSignaling Pathways

Numerous receptor-like and protein kinase genes(33 and 29, respectively) were found to be induced byNF. Expression of the NF receptor gene NFP was ap-parently not affected by the NF treatment, in contrast towhat was reported by Breakspear et al. (2014), while theclosely related LYK2 and LYK3 genes were down-regulated (Supplemental Table S4). Four other LYSM-RLK genes from three distinct subgroups (Arrighi et al.,2006) were up-regulated (Table III; Supplemental TableS3): LYR3, which encodes a high-affinity LCO-bindingprotein (Fliegmann et al., 2013; Fliegmann and Bono,2015); LYR6, of unknown function, a chitin recep-tor CERK1-like gene; and, more strikingly, LYK10,the ortholog of LjEPR3 recently described as encod-ing a receptor of Rhizobium spp. exopolysaccharide(Kawaharada et al., 2015), required for rhizobial infec-tion in L. japonicus. LjEPR3 is expressed in epidermalcells exclusively in response to NF or Rhizobium spp.(Kawaharada et al., 2015), consistent with dataobtained forMtLYK10 in our study. Other up-regulatedgenes encoded different types of RKs, including

Figure 2. Functional classificationofM. truncatulaNF-regulated genesin the root epidermis. Categorieswere manually defined, based uponbest BLASTP hits using predictedencoded proteins against SWIS-SPROT and The Arabidopsis Infor-mation Resource databases. Thenumber of genes is indicated foreach category, with black andwhitebars corresponding to up- anddown-regulated genes, respectively.









http://mtgea.noble.org/v3/









10 lectin domain receptors, 16 LRR-RLKs, two wall-associated RKs, and five Cys-rich RKs (CRKs). Inter-estingly, those included MtSymCRK (Table III),required at late stages of nodule development andproposed to be involved in the control of symbioticimmunity (Berrabah et al., 2014). A second gene

thought to control immunity in nodules, MtDNF2(Bourcy et al., 2013), also was identified as NF inducedin the root epidermis (Table III). MtSymCRK and nineother NF up-regulated RKs exhibit a non-RD kinasemotif, characteristic of kinases involved in innate im-mune signaling (Schwessinger and Ronald, 2012; Table

Table II. Transcriptomic and protein phosphorylation data for selected NF-induced genes involved in cell dynamics

The eight right columns indicate detected NF-induced phosphorylation (P) sites and the regulation levels (fold change) following NF treatment forthe indicated times or S. meliloti inoculation at 5 d post inoculation (RH-5dpi; skl mutant background) determined in this study versus previousstudies: a, Rose et al. (2012); b, van Zeijl et al. (2015); c, Czaja et al. (2012); and d, Breakspear et al. (2014). For entries in parentheses, differenceswere not statistically significant (adjusted P. 0.05). ART, All root tissues from whole root systems (a) or 1- to 2-cm-long root segments (b and c); ND,not detected; NF, RNAseq reads detected only in the NF-treated samples.

Mt20120830 MtV4.0Functional

CategoryAnnotation

P Sites

(a)LCM-4h LCM-24h

ART-3h

(b)

ART-6h

(c)

ART-24h

(c)

RH-24h

(d)

RH-5

dpi (d)

Mt0017_10221 Medtr3g010330 Cell wall Cellulase3-like 15.3 19.6 1.4 1.9 33.3Mt0006_00323 Medtr3g082440 Cell wall KOR2

endoglucanase10.1 1.8 3.3 5.5 6.1 4.0

Mt0008_01038 Medtr0118s0070 Cell wall Expansin EXPB14-like

740.3 152.5 177.1 259.6 118.4 93.5 795.4

Mt0004_11139 Medtr1g073320 Cell wall Plasmodesmata-locatedprotein3-like

20.9 0.2

Mt0015_10081 Medtr8g006790 Transport Plasma membraneproton pumpMtAHA1

NF NF 268.8 11.6 149.6

Mt0009_00137 Medtr8g087710 Transport MtNip1 (NOD26) 384.0 187.2 206.3 8.1 5.1 56.0 688.2Mt0005_10852 Medtr5g023240 Transport Lys-His transporter S27 49.2 31.6 3.4 10.8 5.7 35.6 164.2Mt0040_00155 Medtr3g112460 Transport Peptide transporter

PTR1-like163.1 121.3 433.9 3.3 5.6

Mt0023_10223 Medtr2g011570 Transport Equilibrativenucleotidetransporter

NF NF

Mt0007_00517 Medtr3g098930 Transport Sugar transporterSWEET13

NF ND 652.5 23.2

Mt0006_10110 Medtr3g087730 Transport Sulfate transporter 1,335.9 337.0 105.4Mt0009_00487 Medtr8g095360 Membrane

traffickingPatellin 7.9 366.4 4.3 2.6 1.9 5.1 31.3

Mt0037_10126 Medtr8g038220 Membranetrafficking

MtAnn2 annexin 447.6 0.2 149.5

Mt0057_00074 Medtr1g105930 Membranetrafficking

Inositol-1,3,4-trisphosphate5/6-kinase

6.8 10.2 4.8 2.4 2.6 3.6 26.0

Mt0036_00256 Medtr7g116710 Cytoskeleton ABIL (ABI-like) S255 3.9 66.1 4.5 1.4 1.7 20.3Mt0006_10532 Medtr3g078623 Cytoskeleton Actin-binding

formin-likeprotein8

579.1 (NF) 755.6

Mt0013_00466 Medtr3g060900 Cytoskeleton Kinesin-4microtubulebinding

136.1 1.3 4.3 20.2

Mt0036_00045 Medtr7g112420 Cytoskeleton Kinesin-4microtubulebinding

S277 4.7 6.2 3.1 2.2

Mt0054_10146 Medtr6g061690 Cytoskeleton Microtubule-associatedprotein MAP65

NF (22.1) 1,699.6

Mt0001_10069 Medtr4g133890 Cytoskeleton Microtubule-associatedprotein MAP70

10.7 37.7 11.2 4.7 3.4 8.0 15.0

Mt0031_00057 Medtr7g099290 Cytoskeleton MYOSIN2 S1117 2.0 10.9Mt0040_10068 Medtr3g110720 Cytoskeleton Tubulin a-4 chain S439 7.1 2.7Mt0001_10832 Medtr4g116870 Cell cycle DNA replication

licensing factor316.1 2.6 8.1


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III; Supplemental Table S3). Noteworthy, 41 defense-related genes were NF up-regulated, including 16 clas-sified in the defense response category GO:0006952,whereas 28 defense-related genes, including six genes

from the GO:0006952 class, were NF down-regulated(Fig. 2; Supplemental Table S4). Genes from the PR-1and PR-10 families showed either up- or down-regulation depending on the members.

Table III. Transcriptomic data for selected NF-induced genes involved in perception, signaling, and gene expression

The seven right columns indicate the regulation levels (fold change) following NF treatment for the indicated times or S. meliloti inoculation at 5 dpost inoculation (RH-5dpi; skl mutant background) determined in this study versus previous studies: a, van Zeijl et al. (2015); b, Czaja et al. (2012);and c, Breakspear et al. (2014). ART, All root tissues from 1- to 2-cm-long root segments; ND = not detected; NF = RNAseq reads detected only in theNF-treated samples.

Mt20120830 MtV4.0Functional

CategoryAnnotation LCM-4h LCM-24h

ART-3h

(a)

ART-6h

(b)

ART-24h

(b)

RH-24h

(c)

RH-5

dpi (c)

Mt0012_10650 Medtr5g033490 Receptor-like(RD) kinase

MtLYK10/EPR3 NF NF 10.8 8.7 7.8 13.9 50.8

Mt0057_10185 Medtr1g104890 Receptor-like(RD) kinase

Cys-rich RLK 160.8 3.6 8.0 1.3 2.7

Mt0006_00443 Receptor-like(non-RD)kinase

MtSymCRK 101.7 NF 2.8 1.6 8.3 40.0

Mt0039_00125 Medtr1g088930 Receptor-like(non-RD)kinase

LRR RLK 51.5 NF 10.6

Mt0077_10096 Medtr2g068650 Receptor-like(non-RD)kinase

G-type lectin S RLK 66.7 15.8 19.1

Mt0036_00102 Medtr7g113490 Protein kinase MAPKKK1 ANP1-like

16.6 3.5 2.7

Mt0068_00014 Medtr2g023890 Protein kinase MAPKKK15-like 17.8 19.8 4.2 6.9 3.4 12.0Mt0016_00257 Medtr2g100290 Protein kinase Protein kinase 9.0 NF 31.8 1.8 1.6Mt0020_00194 Medtr8g074920 Protein kinase MtSPK2 protein

kinase2NF 988.0 236.6 38.6 20.8 20.2 118.7

Mt0015_10342 Medtr8g012795 Defense Defensin-like protein 1,025.5 559.3 1,292.0 21.5 63.7 46.7 25.5Mt0003_11544 Medtr4g085800 Immunity

controlMtDNF2 87.9 NF 4.1

Mt0101_10077 Medtr2g437800 Signaling CLE-related peptide 55.6 NDMt0020_10418 Signaling MtCEP7 peptide 162.0 13.6Mt0004_01114 Medtr1g075730 Signaling Protein

phosphatase2C27.8 7.9 15.3

Mt0026_00412 Medtr7g075900 Signaling Subtilase, AtSBT1.1ortholog

NF NF

Mt0003_00135 Medtr4g053630 Signaling Subtilase, LjSBTS/AtAIR3-like

3.6 13.7 7.4 17.8 4.8 3.2 25.6

Mt0001_11381 Medtr4g102400 Signaling Subtilase, P69C-like 39.4 1,373.4 17.1 75.9 41.1 36.7 53.4Mt0036_10297 Medtr7g117415 Proteasome E3 ubiquitin-protein

ligase7.6 1.8 4.7 1.5 1.7 5.6

Mt0008_00403 Medtr5g061290 Proteasome Seven-in-absentia(SINA) protein

NF NF 134.6 2.8 3.4 72.6

Mt0011_00804 Medtr7g078150 Proteasome Zinc finger, RING/FYVE/PHD-type

136.5 77.7 4.3 2.5 2.1 10.2 33.6

Mt0009_00840 Medtr8g103227 Proteasome MtLIN-like E3 ligase NF NF 21.0Mt0042_00049 Medtr7g106340 Proteasome Plant U-box22

(PUB22)1.6 3.6 2.8 2.3 2.1

Mt0012_00744 Medtr5g031880 Transcriptionalregulation

MtPLT3 ethyleneresponse factor

NF NF 13.2 1.1 1.7


GRASSCARECROW-likeTF

40.7 NF 11.8 2.4 7.5


Lateral organboundariesdomain TF

53.6 285.3 4.6


No apical meristemTF (ANAC048-like)

9.5 3.4 3.5 2.2







Twenty NF up-regulated protein kinases were iden-tified, including three calcium-dependent protein ki-nases and two orthologs of Arabidopsis MAPKKkinases (MAPKKK), namely MAPKKK15 and Arabi-dopsis NPK1-RELATED KINASE1 (ANP1; Table III;Supplemental Table S3). Five of these are phosphory-lated upon a 1-h NF treatment, including the ANP1-likeprotein (Rose et al., 2012). SYMBIOTIC PROTEIN KI-NASE1 (MtSPK1; Table I) was shown previously to bealso induced by exogenous hydrogen peroxide (H2O2)and relevant for nodulation as well as for NIN andMtNF-YA1 expression (Andrio et al., 2013). Three pro-tein phosphatases (PP) also were identified as earlyinduced by NF (Table III; Supplemental Table S3). Theyall belong to the PP2C family that emerged as a majorplayer in abiotic and biotic stress signaling (Fuchs et al.,2013). One of them (Medtr5g080680) is the predictedortholog of Arabidopsis PP2CA, a major negative reg-ulator of abscisic acid signaling (Fuchs et al., 2013).

As mentioned previously, various secondary signalsare generated in response to NF treatment, notablycalcium fluxes and ROS production, as well as modifi-cations in phytohormone accumulation and/or re-sponses. Five calcium-binding protein genes, includinga calcineurin B and MtCAML4 (a nodule-specificcalmodulin-like protein; Liu et al., 2006), were rapidlyinduced by NF, along with genes suggesting the pro-duction of CK, auxin, ethylene, GA, strigolactones, andbrassinosteroids (Supplemental Table S3), consistentwith results from Breakspear et al. (2014). In addition,two genes encoding signaling peptides were stronglyinduced, encoding a CLE and the C-TERMINALLYENCODED PEPTIDE7 (CEP7; Table III). These twogenes have not been characterized so far but belong tofamilies known to regulate nodulation (Mortier et al.,2012b; Imin et al., 2013).

Subtilases (Ser peptidases) were classified here assignaling proteins, since they belong to a large family ofproteins among which some play important roles insignaling/developmental processes (Schaller et al.,2012). Seven subtilases were found to be NF induced(Table III; Supplemental Table S3). Two belong to asubgroup closely related to AUXIN-INDUCED ROOTPROTEIN3 (AtAIR3), expressed at sites of lateral rootemergence (Neuteboom et al., 1999), and LjSbtS (sub-tilase S), induced by mycorrhizal fungi and NF (Kistneret al., 2005). The other five belong to the so-called SBT1subgroup (Schaller et al., 2012), including one closehomolog of the tomato (Solanum lycopersicum) PR-P69Cprotein (up-regulated by biotic stresses; Jordá et al.,1999) and the predicted ortholog of AtSBT1.1, requiredfor the processing of phytosulfokine precursors(Schaller et al., 2012). Phytosulfokines are small sulfatedpeptide hormones that activate cell proliferation(Matsubayashi and Sakagami, 2006), and some of themseem to positively regulate nodulation in L. japonicus(Wang et al., 2015). The seven subtilase genes exhibitvarious expression patterns based on MtGEA data, buttwo of them (SBT1 subgroup) are clearly symbiosisspecific, with one (Mtr.45771.1.S1_at) expressed only in

nodules and the other (Mtr.13963.1.S1_at) induced bothby rhizobium and mycorrhizal fungi or their signals.

The ubiquitin proteasome machinery comprisesmany genes involved in the regulation of signaling/developmental processes, including response to phy-tohormones and positive or negative roles for rhizo-bium infection and nodule development (Vinardellet al., 2003; Shimomura et al., 2006; Kiss et al., 2009;Mbengue et al., 2010; Den Herder et al., 2012; Yuanet al., 2012). Nineteen putative ubiquitin proteasomegenes were scored as NF up-regulated, among whichtwo genes encoding, respectively, a close homolog ofLUMPY INFECTIONS (MtLIN), primarily required forinfection thread growth in RH (Kiss et al., 2009), and anodulation-specific SEVEN-IN-ABSENTIA (SINA) do-main protein, very distantly related to LjSINA4, in-volved in SYMRK turnover (Den Herder et al., 2012;Table III; Supplemental Table S3). The ortholog ofAtPUB22, encoding a negative regulator of immunity(Trujillo et al., 2008), also was found to be moderatelybut significantly induced by NF at 24 h (Table III).

A large number (38) of NF-induced TFs were iden-tified (Table III; Supplemental Table S3). Six of themwere expected: NSP1, NIN, ERN1, ERN2, NF-YA1, aswell as NF-YC2, recently shown to interact withNF-YA1 (Baudin et al., 2015). Only one of the remainingNF-induced TFs, a noncharacterized SCARECROW-like GRAS factor (Table III), was found to beexpressed specifically in symbiotic samples (mycorrhi-zal and nodulated roots), based on MtGEA data. ThreeNF-induced TFs belong to the ERF family in addition toERN1 and ERN2, namely PLETHORA3 (MtPLT3), as-sociated with both nodule and root meristems togetherwith three other PLT proteins (Franssen et al., 2015),and two ERFs belonging to subgroup VIII, which car-ries an ERF-associated amphiphilic repression motif(Nakano et al., 2006). The expression of PLT1, PLT2, andPLT4 was not detected in the epidermis, suggesting aspecific role of PLT3 at this early stage. Other NF-induced TFs notably included the ortholog ofANAC042, a NAC TF induced by H2O2 (Wu et al.,2012), two auxin-response factors (ARFs), five basichelix-loop-helix, one MADS box, four MYB-like, andtwo lateral organ boundaries domain (LBD) TFs.Among the latter was the predicted ortholog ofAtLBD16, which regulates lateral root formation viadirect activation by ARFs (Okushima et al., 2007). In-terestingly, six homeodomain protein genes also wereNF up-regulated, including MtKNOX3, a gene up-regulated during nodulation and recently shown to actdepending on CK pathways (Azarakhsh et al., 2015).

CK Pathways Are Activated during NF Signaling inthe Epidermis

The role of CK during nodule initiation in the rootcortex is well demonstrated, whereas it remains poorlydocumented in epidermal cells. In this study, severalgenes involved in CK biosynthesis (SupplementalFig. S7), perception (MtCHK1/CRE1), and response


Jardinaud et al.











(MtRRA2, MtRRA8, and MtRRA9) were found to beup-regulated by NF in the epidermis, mostly at 4 h forCK perception and signaling genes. In mock-treatedepidermal cells, MtCHK1/CRE1 was expressed at amuch higher level thanMtCHK2 and at a similar level toMtCHK3 and MtCHK4, while in NF-treated samples(4 h), MtCHK1 was the only (transiently) induced pu-tative CK receptor gene (becoming expressed about10-fold more thanMtCHK3 andMtCHK4; SupplementalTable S2).Knowing the essential role of CK in nodulation, we

conducted promoter:GUS analyses to support theLCM-RNAseq data. We first analyzed the expression ofa pMtCRE1:GUS transcriptional fusion treated or notfor 4 h with NFs at 1028 and 1029

M. The pMtCRE1:GUSfusion showed a strong expression in the root stele andthe apex, as described previously (Lohar et al., 2006),which was increased by the NF treatment (Fig. 3). Aprolonged staining revealed, in contrast to the previousstudy, a weaker pMtCRE1:GUS signal in RHs, detectedonly in response to NF. This CRE1 spatial expressionpattern is in agreement with the RH and LCM tran-scriptome data sets (Breakspear et al., 2014; this study).We then used TCSn:GUS, a newly improved version

of the TCS reporter consisting of repeated cis-elementsfrom the promoter of an RRA CK primary responsegene, and therefore used as a proxy to monitor the ac-tivation of CK signaling pathways (Zürcher et al., 2013).We also used in parallel pENOD11:GUS as a positivecontrol for the NF response. To validate the TCSn:GUSreporter, never described in M. truncatula, we first ex-amined its expression pattern in nontreated roots ver-sus roots treated with 1027

M 6-benzylaminopurine(BAP; a CK; Fig. 4, A and B). In nontreated roots, TCSn:GUS was mostly expressed in the columella and thedifferentiation region of the root meristem, while it wasstrongly induced by the BAP treatment, as expected fora CK response reporter. Roots treated with 1028

M NF(4 h) exhibited a strong induction of GUS activity inRHs of the region competent for nodulation in about40% of the roots (Fig. 4C), consistent with the LCM-RNAseq data. The NF induction of TCSn:GUS was notdetected in an M. truncatula nfp mutant (Fig. 4D). Rootsections indicated that the TCSn:GUS induction takesplace in epidermal cells as well as subtending cells fromthe outer cortex (Fig. 4E), while no induction was ob-served in the inner cortex. The NF-induced pENOD11:GUS expression seemed to be more restricted to epi-dermal cells (Fig. 4F). Roots inoculated with S. melilotisimilarly revealed a strong activation of GUS expres-sion in the nodulation-competent root region 24 to 48 hpost inoculation (Supplemental Fig. S8). No TCSn:GUSor pENOD11:GUS activation was observed using anodA S. meliloti mutant (Supplemental Fig. S8), indi-cating that the inductions observed were strictly de-pendent on NF. To define in which cell layers TCSn:GUS was activated first, we conducted a kinetic anal-ysis from 4 to 72 h following S. meliloti inoculation. TheTCSn:GUS up-regulation was detected at 4 and 8 h postinoculation, with maximal expression in epidermal and

outer cortical cells (Fig. 4, G and H). The TCSn:GUSactivation became strong in inner cortical cells only at2 d post inoculation (Fig. 4I).

In conclusion, the TCSn:GUS expression patternsuggests an activation of the CK signaling pathway inresponse to NF treatment or S. meliloti inoculation, firstin the outer root tissues (epidermis and outer cortex)and then in inner root tissues.

The Epidermal MtCRE1 Signaling Pathway NegativelyRegulates the NF Induction of ENOD11 Expression and theNumber of Nodules

To determine the functional relevance of the ob-served NF induction of CK signaling in the epidermis,we first analyzed by quantitative reverse transcription-PCR the impact of a root pretreatment with exogenousCK on the level of ENOD11 induction by NF. ENOD11was selected as a marker because it is strongly and rap-idly induced in the epidermis byNFs (Fig. 4F).Wild-type

Figure 3. NFs induce pMtCRE1:GUS in M. truncatula RHs. A and B,GUS staining of untreated roots transformed with a pMtCRE1:GUS fu-sion. C and D, GUS staining of roots transformed with a pMtCRE1:GUSfusion, treated with NFs (1029

M) for 4 h. Similar results were obtainedwith 1028

M NFs. Bars = 300 mm.









and cre1 mutant plants were pretreated or not with CK(1027

M BAP for 1 or 3 h) and then treated or not withNFs(1029

M for 3 h). While ENOD11 induction in response toNFs was detected in the wild type as expected, this in-duction was strongly reduced after a 1- or 3-h CK pre-treatment (Fig. 5A; Supplemental Fig. S9A). In the cre1mutant, the NF induction of ENOD11 expression wassimilar in the absence of CK and after a 1-h CK pre-treatment, indicating that the CRE1 signaling pathway isrequired for the inhibition of ENOD11NF induction (Fig.5B; Supplemental Fig. S9B). A reduction of the NF in-duction ofENOD11 expressionwas observed in cre1 aftera 3-h CK pretreatment, suggesting that the other M.truncatula CK CHK receptors, the expression of which isdetected in the epidermis and RHs (Supplemental TableS3; Breakspear et al., 2014), are likely functionally re-dundant with CRE1.

To independently evaluate the role of CK in regu-lating NF signaling in the epidermis, we expressed theArabidopsis cytokinin oxidase/dehydrogenase gene(AtCKX3) from the pLeEXT1 promoter specifically act-ing in the epidermis of M. truncatula roots (Mirabella

et al., 2004; Rival et al., 2012) and referred to henceforthas pEPI. Expression of the pEPI:AtCKX3 construct waschecked in transformed roots by quantitative reversetranscription-PCR analysis (Supplemental Fig. S10). Todirectly assess the impact of the pEPI:AtCKX3 constructon CK accumulation, root CKs were profiled by liquidchromatography-tandem mass spectrometry (LC-MS/MS), using root fragments corresponding to thenodulation-competent region analyzed by RNAseq,with or without a 1028

M NF (4-h) treatment. Threedifferent nucleotide CK types were detected, namelytrans-zeatin nucleotide (tZNT), cis-zeatin nucleo-tide, and isopentenyl adenine nucleotide (iPNT;Supplemental Table S6). Similar CK levels were detec-ted for the NF- and mock-treated roots, but all pEPI:CKX3 samples presented significantly lower levels (P#0.05) of iPNT and tZNT (Supplemental Fig. S11), whileno differences were observed for cis-zeatin nucleotidelevels. This indicates that the pEPI:CKX3 construct ef-ficiently reduced CK accumulation in the transformedroots, thereby demonstrating that a portion of the rootCK pool locates in the epidermis.

Figure 4. CKs, NFs, and S. meliloti induce TCSn:GUS in M. truncatula root tissues. A to D, GUSstaining of whole roots transformed with TCSn:GUS fusion viaAgrobacterium rhizogenes. Shownare mock-treated roots (A), roots treated with CKs(1027

M BAP for 4 h; B), and wild type (C) and nfp(D) roots treated with NFs (1028

M for 4 h). Bars =500 mm. E to I, GUS staining of longitudinal25-mm sections (E, F, H, and I) or a transverse10-mm section (G) of roots transformed via A.rhizogenes with TCSn:GUS (E, G, H, and I) orpENOD11:GUS (F) and treated with NFs (1028

M

for 4 h; E and F) or inoculatedwith S. meliloti at 8 h(G and H) and 72 h (H) post inoculation, respec-tively. Bars = 50 mm.


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We then assessed the impact of the pEPI:AtCKX3construct on the induction of ENOD11 by NF. WhileAtCKX3 expressed from the strong constitutive 35Spromoter previously revealed a positive role of CK innodulation (Lohar et al., 2004), roots transformed with

pEPI:AtCKX3 exhibited a stronger ENOD11up-regulation by a 3-h NF treatment compared withcontrol roots (Fig. 5C; Supplemental Fig. S9C). Thissuggested that the NF response is enhancedwhen iPNTand/or tZNT are depleted in the epidermis.

Finally, we compared rhizobium infection and nod-ulation in pEPI:AtCKX3 versus control roots at 6 and14 d post inoculation with S. meliloti, respectively. Al-though there was a trend suggesting possible increasedinfection in pEPI:AtCKX3 versus control roots and de-creased infection when expressing AtCKX3 from acortex-specific promoter (pCO; Rival et al., 2012), thedifferences were not statistically significant (Fig. 6, Aand B). However, using a larger population of trans-formed roots, we observed enhanced nodulation ofpEPI:AtCKX3 versus control roots (P = 0.008, one-sidedWelch’s test; Fig. 6C). Increased nodulation also wasobserved with pEPI:AtCKX3 roots inoculated with S.medicae (Supplemental Fig. S12). By contrast, whenAtCKX3 was expressed from the pCO, nodulation wasdecreased (P = 0.0001; Fig. 6D), consistent with dataobtained with the 35S promoter (Lohar et al., 2004).Altogether, these results suggest a possible negativerole of the epidermal CK/CRE1 pathway in the NFinduction of ENOD11 and nodulation.

DISCUSSION

In past years, transcriptomics approaches have en-abled the identification of genes playing major roles inthe control of nodulation, such as NF-YA or NCR, andtherefore are complementary to forward genetics, es-pecially in the case of functional redundancies or whenused to identify genes for which mutation leads to le-thal phenotypes. One challenge in transcriptomics nowconsists of analyzing single tissues rather than a mix-ture of tissues, in order to uncover the roles of regula-tory and metabolic pathways in specific tissues. In thisstudy, NF-induced gene expression reprogrammingwas analyzed by laser microdissection of epidermalcells from the root region that is competent for nodu-lation and responds to NF. This was combined withhigh-depth RNAseq and mapping of the RNAseqreads on the most recent M. truncatula genome se-quence, which thereby enabled a sensitive genome-wide analysis.

More than 300 genes showed a greater than 10-foldlevel of NF activation, reflecting both dramatic modi-fications in gene expression and increased sensitivity ofsingle-tissue studies. Forty-four of the NF-inducedgenes were found to be symbiosis specific based onMtGEA data (Supplemental Table S3) and corre-sponding to a variety of functions (receptors, kinases,TFs, proteases and proteasome elements, transporters,defense-related proteins, etc.), including 18 genes alsoup-regulated during mycorrhizal interactions. Surpris-ingly, these 44 genes include three NCR genes (NCR117,NCR150, andNCR160, the last two also being induced inmycorrhizal roots) not yet listed among the very fewNCR genes that are known to be not exclusively

Figure 5. The NF induction of ENOD11 is rapidly repressed by CKs viaCRE1 and can be increased by expressingAtCKX3 in the root epidermis.A, Real-time reverse transcription-PCR analysis of ENOD11 relativeexpression in response to a 1027

M BAP treatment (1 or 3 h) followed bya 1028

M NF treatment (3 h) in wild-type M. truncatula roots. B, Real-time reverse transcription-PCR analysis of ENOD11 relative expressionin response to a 1027

M BAP treatment (1 or 3 h) followed by a 1028MNF

treatment (3 h) in a cre1mutant. C, Real-time reverse transcription-PCRanalysis of ENOD11 relative expression in pEPI:AtCKX3 roots treated ornotwith 1028

MNFs for 3 h.One biological replicate is shown out of twoindependent experiments (for the other biological replicate, seeSupplemental Fig. S9). Error bars indicate SD of two technical replicates.









expressed in the nodule (five out of 334 analyzed in267 experimental conditions; Guefrachi et al., 2014).NCR genes are currently only known for their key rolesin later nodulation stages for bacteroid differentiation,so this symbiotic induction in the absence of rhizobiasuggests a role independent of bacterial infections. AsNCRs are defensin-like proteins with antimicrobialproperties demonstrated for some of them (Marótiet al., 2015), this early induction might be related totransient defense responses induced during symbioticinteractions (see below).

The complexity of NF-induced responses just withina few hours and a single tissue may seem, at first sight,somewhat surprising. However, the dissection of an-other plant-microbe signaling pathway, associatedwith the perception of microbe-associated molecularpatterns, also shows great complexity, with a variety ofplayers involved in positive or negative regulation(Macho and Zipfel, 2014). It is very likely that NF

signaling is just as complex, leading to the productionof secondary signals of various natures (ion fluxes,ROS, flavonoids, phytohormones, and peptides). Anumber of genes identified are certainly associatedwithgene networks required for their production, transport,perception, signaling, and action. This probably ex-plains the many (145) NF-induced signaling genesidentified, notably encoding receptors, protein kinases,and phosphatases, as well as regulators of gene ex-pression at the transcriptional or posttranscriptionallevel. For example, several genes are good candidatesfor being part of a possible ROS pathway, such as theortholog of ANAC042, a NAC TF gene induced byH2O2 (Wu et al., 2012), and a gene encoding aMAPKKKclosely related to ANP1 kinase (Table III). ANP1 isknown to be activated by exogenous H2O2, while con-stitutively active ANP1 mimics the H2O2 effect, withactivation of the MAPK cascade and up-regulation ofspecific genes (Kovtun et al., 2000). This potential linkbetween NF signaling and ROS is supported by the factthat 102 NF up-regulated genes (Supplemental TableS3) were shown previously to be affected (directly orindirectly) by the inhibition of H2O2 production inS. meliloti-inoculated roots (Andrio et al., 2013). More-over, up-regulation by an exogenous H2O2 treatmentwas shown for five NF-induced genes: MtSPK1,MtSPK2, MtSRL1, MtABlL, and MtRIP1 (Ramu et al.,2002; Andrio et al., 2013).

Biological functions associatedwith theNF signaling/preinfection stage include both cell-autonomous andnon-cell-autonomous processes, with the preparation ofRHs for rhizobial infection involving the activation ofcell cycle genes, the regulation of defense responses, andthe induction of cell-cell communication with inner rootcell layers. The non-cell-autonomous effects range fromthe preparation of the outer cortical cell to enable in-fection thread progression, to the production of anunknown signal leading to the initiation of nodule or-ganogenesis by cell divisions in the inner cortex andpericycle cells, to a negative control in epidermal cells toprevent overinfection and control the number of nod-ules formed. A number of NF-regulated candidategenes that have potential to take part in these differentprocesses were described in “Results.” For example,numerous genes, coding for proteins involved in thecell wall and cytoskeleton structure or dynamics, aswell as membrane or vesicular trafficking, were iden-tified in line with the major subcellular remodeling as-sociated with infection thread formation (Fournieret al., 2015). The strong NF up-regulation of MtLYK10,for which the expression is not detectable in con-trol epidermal cells, probably also contributes tothe preparation for infection, since the orthologousL. japonicus EPR3 gene encodes a receptor for Rhizo-bium spp. exopolysaccharidic signals, a key factor forsuccessful infections (Kawaharada et al., 2015).Down-regulation of MtLYK3, also observed usingisolated RHs (Breakspear et al., 2014), is more sur-prising, since MtLYK3 is known to be involved ininfection. MtLYK3 expression might concentrate in

Figure 6. Expression of the AtCKX3 gene leads to an increased nodu-lation in the root epidermis and to a decreased nodulation in the rootcortex. Infection threads and nodules were counted on individual rootsat 6 and 14 d post inoculation with S. meliloti, respectively, using rootstransformed by A. rhizogenes with pEPI:CKX3 specifically expressingAtCKX3 in the root epidermis, or the corresponding control vector (Aand C), or with pCO:CKX3 specifically expressing AtCKX3 in the rootcortex, or the corresponding control vector (B and D). P = 0.1592 and0.1658 in A and B respectively, and 0.00786 and 0.00010 in C and D,respectively (one-sided Welch’s test). Values shown are means of threebiological replicates 6 SE.


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certain epidermal cells of the nodulation-competentroot region, in preparation for infection, and decreasein others. Alternatively MtLYK3 down-regulation maybe part of a negative feedback to prevent overinfection.Successful rhizobial infection requires a tight regu-

lation of plant immune responses in cells that getinfected by the symbiont. The transient induction ofdefense responses following Rhizobium spp. inoculationhas been reported often (for review, see Gourion et al.,2015; Limpens et al., 2015), and it was suggested thatNF may actively suppress plant immunity, based ondecreased ROS production and PR2-like (TC 78899 =Medtr4g076430) expression upon NF addition in M.truncatula (Shaw and Long, 2003; Mitra and Long,2004). However, whileMedtr4g076430 is indeed down-regulated by NF, as described previously, a paralogousgene (Medtr4g076470), located close to Medtr4g076430in the genome, is up-regulated by NF, along with sev-eral other defense-related genes (PR1, PR3, PR10,MLO8, WRKY, etc; Supplemental Table S3), in agree-ment with several other transcriptomic studies(Nakagawa et al., 2011; Rose et al., 2012; Breakspearet al., 2014). Recent data additionally showed a dualfunction for the LCO receptors LjNFR1/MtLYK3 andLjNFR5/MtNFP and for orthologous genes in rice(Oryza sativa) in symbiosis and in defense responses (forreview, see Limpens et al., 2015). This dual functioningmay involve distinct LYSM RLK receptor complexes,and the fact that several LYSMRLK genes are expressedat different levels in the epidermis, with three of themNF induced in addition to LYK10 (MtLYR3, MtLYR6,and CERK1-like), suggests that several pathways couldbe activated. It also has been proposed that immuneresponses (e.g. ROS production) were recruited to fa-cilitate and regulate symbiotic infections. We observedthe activation of several non-RD kinases, which arethought to recognize conserved microbial signaturesand to be involved in immunity (Schwessinger andRonald, 2012). This raises the additional possibility thatNF might activate a set of genes, including receptors ofmicrobe-associated molecular patterns, that are usefulfor preventing nonrhizobium microbes from penetrat-ing into root tissues during the infection stage. Thiscould be important under natural conditions, whererhizobia are competing with numerous microorgan-isms. Intriguingly, we found out that two genesthought to down-regulate the plant immune responsein functional nodules,MtDNF2 andMtSYMRK (Bourcyet al., 2013; Berrabah et al., 2014), are induced by NF at4 and 24 h. This implies that MtDNF2 and MtSYMRKhave an early symbiotic function in addition to theirrole at later nodulation stages, and this might contrib-ute to the control of the transient NF-induced defenseresponses in the root. The predicted ortholog of PUB22,a gene known to negatively control the plant immuneresponse (Trujillo et al., 2008), is NF induced and alsocould contribute to the control of defense reactions.NF signaling induces several plant secondary signals,

such as phytohormones, which can be involved in cell-cell communication. Our study generated evidence for

the NF regulation of classic hormone pathways such asthose for auxins, GAs, strigolactones, and CK (consis-tent with Breakspear et al., 2014), as well as peptidehormones such as CLE and CEP (Table III). We wereparticularly interested in CKs, known for their criticalrole in the initiation of nodule organogenesis andshown to accumulate rapidly in response to NF (vanZeijl et al., 2015; Reid et al., 2016) but not considered asearly epidermal signals. Indeed, in Rhizobiumspp.-inoculated L. japonicus roots, TCS:GUS expressionis detected first in the cortex (Held et al., 2014). Yet, ourRNAseq data coupled to metabolic network modelingsuggest that CK biosynthesis is activated rapidly by NFin the epidermis. In addition, by LC-MS/MS quantita-tive analysis, we established that significantly loweramounts of two root CKs (tZNT and iPNT) are found inpEPI:AtCKX3-transformed roots that express a CK-degrading enzyme specifically in the root epidermis.This indicates that a portion of the root CK, in particulartZNT, is located in the epidermis. LC-MS/MS analysisrevealed the presence of only the nucleotide forms ofCK, which are known to be the precursors of active CK.The production of bioactive free base CK then requires areaction catalyzed by phosphoribohydrolase of the LOGfamily (Sakakibara, 2006; Kamada-Nobusada andSakakibara, 2009). Our RNAseq data indicate that sev-eral LOG-like genes (Medtr3g113710, Medtr1g015830,Medtr4g058740, andMedtr1g057020) are expressed in theroot epidermis, suggesting that active CKs can be pro-duced in this tissue even if not detected by LC-MS/MS.As a matter of fact, the activation of CK signaling path-ways by NF was indicated by the transient induction ofMtCRE1 and, more importantly, three RRA genes(MtRRA2, MtRRA8, and MtRRA9), in agreement withOp den Camp et al. (2011) for MtRRA9 (formerlyMtRR9), based on GUS transcriptional fusions and othertranscriptomic analyses performed on isolated RHs un-der symbiotic conditions (Breakspear et al., 2014; Liuet al., 2015). This symbiotic activation of CK signaling inthe root epidermis was further validated by the use of apCRE1:GUS fusion and a new version of the CK-responsive reporter, TCSn:GUS (Zürcher et al., 2013),which both gave signals in the epidermis of thenodulation-competent zone in response to NF and S.meliloti. The discrepancy with the pattern of the TCS:GUS reporter described by van Zeijl et al. (2015) likelyrelates to the increased CK sensitivity of the TCSn:GUSvariant. A detailed kinetic analysis revealed that, in M.truncatula, the TCSn:GUS reporter expression is acti-vated by Rhizobium spp. first in the epidermis andsubtending cortical cells and becomes strong in the in-ner cortex only at later time points, in striking contrastto the TCS:GUS pattern in L. japonicus (Held et al., 2014).Once more, this could reflect differences between theCK sensitivity of the TCS and TCSn reporters (Zürcheret al., 2013), but alternatively, CK signaling activationcould differ between M. truncatula and L. japonicus (i.e.indeterminate versus determinate nodulation types).

In spite of evidence for the activation of CK signalingpathways, we were not able to detect by LC-MS/MS an






NF-induced accumulation of CK. This might be due tohigher global CK concentration in A. rhizogenes-transformed roots as compared with nontransformedroots, which could mask a moderate NF-induced CKaccumulation (only a 2-fold CK increase was repo-rted by van Zeijl et al. [2015] in response to NF innontransformed roots in the absence of amino-ethoxyvinylglycine). A possible candidate for the director indirect regulation of CK pathways is MtKNOX3,which we found here to be NF induced in the epider-mis, in addition to being up-regulated in nodule pri-mordia (Azarakhsh et al., 2015). KNOX genes areindeed known to control CK metabolism in the shootapical meristem, and MtKNOX3-RNAi lines exhibitreduced expression of IPT, LOG and RRA genes in-volved in CK biosynthesis, activation and response re-spectively (Azarakhsh et al., 2015).

It was demonstrated recently that CRE1 is activated inthe root cortex in response to an elusive epidermalsymbiotic signal, leading to NIN expression itself am-plifying CRE1 expression via a positive feedback loopand eventually leading to nodule primordium formation(Vernié et al., 2015). The presence of CK in the epidermisand the activation of CK signaling in epidermal andsubtending cortical cells at very early symbiotic stagesmake CKs attractive candidate molecules for the mobilesignal that activates CRE1 in the cortex. In this regard, itis interesting that several genes belonging to familiesproposed to be involved in CK translocation (encodingATP-binding cassette transporters and equilibrativenucleoside transporters; Sakakibara, 2006; Ko et al.,2014) are strongly up-regulated in NF-treated epidermalcells (e.g. Medtr2g011570; Table II).

A possible positive role of epidermal CK as a mobilesignal activating cortical responses may seem difficultto reconcile with the increased nodulation observedwhen AtCKX3 is expressed in the epidermis. However,our study also suggested a distinct, cell-autonomous,and possibly antagonistic function of CK in the epi-dermis. A negative role of CK in the NF induction ofMtENOD11 expression was identified, depending onthe CRE1 CK receptor, either by performing a CK pre-treatment before the NF treatment or by expressing thepEPI:AtCKX3 construct (Fig. 5). Bearing inmind that anexogenous CK treatment induces ERN1, NIN, andNSP2 (Plet et al., 2011) and that CRE1 is required for theNF induction of many genes (van Zeijl et al., 2015), CKmight have both positive and negative roles in the root.This would be similar to NIN, which is required forsymbiotic gene induction in the epidermis and cortex,as well as Rhizobium spp. infection and nodule initia-tion, while restricting ENOD11 expression on the onehand and nodulation on the other hand, via CLE pep-tides (Marsh et al., 2007; Soyano et al., 2013, 2014;Laloum et al., 2014; Yoro et al., 2014; Fournier et al.,2015; Vernié et al., 2015). Consequently, the impact ofthe pEPI:AtCKX3 construct might depend on the rela-tive efficiency of different processes (e.g. speed of CKtranslocation to the cortex versus degradation byAtCKX3 in the epidermis). In L. japonicus, the mutation

of a CKX3 gene expressed in nodule primordia but notin the epidermis leads to a reduction of both nodulationand infection thread formation (Reid et al., 2016), sug-gesting that CK overproduction in the cortex induces anegative feedback on infection and that CK may havetemporally and spatially restricted antagonistic func-tions. In M. truncatula, the opposite impact on nodula-tion observed for the epidermal pEPI:AtCKX3 andcortical pCO:AtCKX3 constructs suggests distinct pre-dominant roles for CK in outer and inner root tissuesduring NF signaling and nodulation, with a strongernegative component in the epidermis.

It remains an open questionwhichdownstream factorsmediate the various effects of CK in different root tissues.Among possible candidates, beyond the NIN transcrip-tional regulator itself, are the flavonoid biosynthesis andauxin-responsive genes, as demonstrated previously forthe activation of nodule organogenesis depending onMtCRE1 (Mathesius et al., 2000; Plet et al., 2011; Ng et al.,2015) as well as genes of the ethylene and GA pathways,activated both in response to a CK treatment (Ariel et al.,2012) and to NF (van Zeijl et al., 2015), notably in theepidermis (Breakspear et al., 2014; this study).

CONCLUSION

Investigations of tisue-specific responses have onlyjust begun at the genome-wide scale. This represents amajor challenge, now made accessible by recent tre-mendous technical progress, such as new-generationsequencing coupled to laser dissection approaches.Here, NF signaling could be investigated in the rootepidermis, revealing the extent and complexity of geneexpression reprogramming,with numerous NF-inducedgenes never identified before. This notably revealed theearly activation of CKmetabolic and signaling pathwaysin this tissue, thereby suggesting possible epidermis-specific cell-autonomous and non-cell-autonomousfunctions during early rhizobium-legume interactions.Interestingly, the activation of the CK-signaling pathwayin the epidermis was correlated to a negative regulation ofENOD11 gene expression and nodulation, whereas thesame pathway acts positively in the cortex for the stimu-lation of cell divisions. The possible dual (positive andnegative) control of nodulation and infection by the samesignal could contribute to the fine regulation and coordi-nation of these two key symbiotic processes. In any case,our study emphasizes the critical importance of tissue-specific analyses needed to decipher the complex regula-tions that occur during the rhizobium-legume symbiosisand, more generally, in host-microbe interactions.

MATERIALS AND METHODS

Plant Growth and Treatment

To generate root samples for laser dissection,Medicago truncatula ‘JemalongA17’ plants were grown in aeroponic caissons for 3 d in nitrogen-free aeroponicnutrient medium (Barker et al., 2007) prior the NF treatment (chamber condi-tions: temperature, 22°C; 75% hygrometry; light intensity, 200 mE m22 s21; and


Jardinaud et al.



light/dark photoperiod, 16/8 h). Plantlets were then immersed for either 4 or24 h in 1028

M NFs (in Fahraeus medium, without aminoethoxyvinylglycine) orin Fahraeus medium only (mock treatment), using five plantlets per 50-mLFalcon tube. Twenty-five plants per time point and per biological repetitionwere treated and used for laser dissection. Each biological repetition (more than50 plants) corresponded to an independent caisson. The cre1-1 and nfp-1 mu-tants are described by Plet et al. (2011) and Ben Amor et al. (2003), respectively.Plant growth conditions for promoter:GUS analyses and CK quantitativeanalysis are described below.

Laser Microdissection, RNAseq, and RNAseqData Analysis

One-centimeter-long root fragments (without root tips) were collected andtreated as described previously (Roux et al., 2014; for details, see SupplementalMaterials and Methods S1). Pooled RNAs were ethanol precipitated, ribosomalRNA was eliminated by oligocapture, and remaining RNA was amplified byin vitro transcription as described (Roux et al., 2014). One microgram of am-plified RNAper samplewas used for oriented paired-endRNA sequencing (2350 bases) by Fasteris using an Illumina HiSeq 2000 platform. Three biologicalrepetitions were used per sample except for the 24-h NF treatment, for whichonly two repetitions were retained, based on quality criteria (r . 0.90).

Expressedgenesweredefinedusingdata-basedglobalfiltering (Rauet al., 2013),giving a threshold of 28,255 normalized reads for one library. Differential expres-sion analysis of RNAseq data was performed with the DESeq software version1.20.0 (Anders and Huber, 2010). Dispersions were estimated using the pooledmethod with the fit-only criteria. P values were adjusted for multiple testing usingthe Benjamini and Hochberg false discovery rate (Benjamini and Hochberg, 1995).

The LEGoo knowledge base (https://www.legoo.org) Nicknames tool wasused to find correspondences between various gene, transcript, andmicroarrayoligonucleotide identifiers. Orthologous genes were predicted based on recip-rocal best hits (BLASTP).

Metabolic Network Analyses

M. truncatula metabolic reactions and pathways were built from transcriptsobtained in previous experiments (Roux et al., 2014). The relation betweentranscripts and biochemical reactions was established by comparing predictedprotein sequences with the protein sequences found in the pathway genomedatabases (PGDBs) available in Pathway Tools (http://bioinformatics.ai.sri.com/ptools/; Karp et al., 2002). The resultingM. truncatula PGDB is available athttps://pathway-tools.toulouse.inra.fr/MEDICAGO2 (login/password, guest/guest). From the PGDB and thanks to ad hoc bioinformatics tools, we built ametabolic graph that links reactions by their substrates and their products, notconsidering any a priori classification into pathways (Lacroix et al., 2008). Westored metabolic graphs in the MetExplore Web server (Cottret et al., 2010).Metabolic networks are directly available at http://metexplore.toulouse.inra.fr/metexplore2/index.html?idBioSource=3423. Thanks to the MetExploremapping functions, we established a list of reactions for which correspondinggenes are down-regulated or up-regulated by NF at 4 and 24 h. Pathway re-action enrichment significance was computed for each condition in MetExploreusing the Bonferroni test. Metabolic networks composed of whole sets of re-actions corresponding to up- or down-regulated genes were displayed usingCytoscape (Shannon et al., 2003). Then, we visually located interesting sub-networks crossing pathways of interest.

Transcriptional Fusion Constructs

For the transcriptional fusion between the MtCRE1 promoter, defined as2,500 nucleotides upstream of the ATG, and the uidA reporter gene, thepMtCRE1 region was amplified by PCR using the following primers: pCRE1_F,59-ggtaccTAACATAAGGACCTAGAACCAATATAAAGA-39, and pCRE1_R,59-gacgtcTACAACACCAACTAACACCAAATCTC-39 (lowercase letters indi-cate KpnI and AatII restriction sites used for cloning). The PCR product wascloned into the pFRN-GUS vector (derived from the pFGC5941 plasmid; Na-tional Center for Biotechnology Information accession no. AY310901) using theabove-mentioned restriction sites.

For epidermis-specific expression, the pFRN-RNAi vector (Gonzalez-Rizzoet al., 2006) was cut with EcoRI and SmaI to remove the RNAi cassette andreligated with a linker containing EcoRI and KpnI sites. The pLeEXT1 promoterwas excised from a pGEM-T vector (Rival et al., 2012) and cloned into these sitesto generate the pFRN-pEpi vector. An AtCKX3 PCR fragment was amplified

from a pUC19 plasmid described by Werner et al. (2001) with primersAtCKX3_F, 59-ggatcATGGCGAGTTATAATCTTCGTTC-39, and AtCKX3_R,59-aggcctACTCGAGTTTATTTTTTGAAATATATTTTG-39 (lowercase lettersindicate BamHI and StuI restriction sites used for cloning) and inserted into thepFRN-pEpi vector to generate the pEPI:AtCKX3 construct.

The TCSn reporter was amplified by PCR from the TCSn:GFP vector(Zürcher et al., 2013) using the following primers: TCSn F, 59-aggtctccaaat-CAAAGATCTTTAAAAGATTTTGAAAG-39, and TCSn R, 59-aggtctcc-caatTGTTATATCTCCTTGGATCGATCCCC-39 (lowercase letters indicate BsaIrestriction site and spacer used for cloning). The PCR product was Golden Gatecloned (Engler and Marillonnet, 2011) into a modified pCAMBIA2200 vector(Fliegmann et al., 2013) using BsaI sites to generate the TCSn:GUS binary vector.

Plant Transformation, Promoter:GUS Analyses, CKQuantitative Analysis, and Nodulation Assays

M. truncatula roots were transformed using Agrobacterium rhizogenes as de-scribed (Boisson-Dernier et al., 2001). All transformations were performed in atleast three biological repetitions. Composite plants were selected for 2 to3 weeks on 25 mg L21 kanamycin (25°C; day/night, 12/12 h) and then trans-ferred into slanted square petri dishes (123 12 or 243 24 cm) on Fahraeus agarmedium without nitrogen covered with autoclaved seed germination paper(38# Seed Germination Paper 2M/CTN; Anchor Paper).

For composite plants expressing the TCSn:GUS or the pENOD11:GUSconstruct, analyses were conducted following a 4-h treatment with 1028

M NF,1027

M BAP, or Fahraeus medium without nitrogen (mock), or Sinorhizobiummeliloti 2011 inoculation (pXLGD4 phemA:lacZ [GMI6526]; Ardourel et al., 1994)or nodA::Tn5#2208 at optical density at 600 nm = 0.01. Roots were collected andprefixed in 0.4% paraformaldehyde for 1 h, stained for GUS activity 4 h at 37°C,and fixed in 2% glutaraldehyde. Longitudinal sections were made from rootsembedded in 8% agarose, using a vibrating microtome (Leica VT 1000S), andobserved with a Zeiss Axiophot light microscope. For CK LC-MS/MS analysis,approximately 1-cm-long root fragments were collected before or after 4 h oftreatment with 1028

M NF versus mock, after cutting off root tips (approxi-mately 1–2 mm long) to remove a potential source of non-NF-regulated CK.Samples of 180 mg fresh weight (about 50 root systems per sample) were ly-ophilized and then processed as described by Kisiala et al. (2013); for details seeSupplemental Materials and Methods S1.

In nodulation assays, composite plants expressing the pEPI:AtCKX3 or pCO:AtCKX3 construct were grown in vitro on paper-covered Fahraeus mediumwithout nitrogen and inoculatedwith S. meliloti 2011 as before; infection threadsand nodules were counted at 6 and 14 d post inoculation, respectively, fol-lowing lacZ staining performed as described previously (Vernié et al., 2008). Inaddition, plants grown in vitro on i medium (Gonzalez-Rizzo et al., 2006) wereinoculated with S. medicae WSM419 (Terpolilli et al., 2008).

The Sequence Read Archive project accession number for original RNAseqdata is SRP058185.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. LCM of root epidermal cells.

Supplemental Figure S2. Pairwise comparisons of NF-regulated genesdetected in recent transcriptomic studies.

Supplemental Figure S3. Multiple comparisons (Venn diagrams) of NFdown-regulated M. truncatula genes detected in recent transcriptomicsexperiments.

Supplemental Figure S4. topGo analysis of NF up- and down-regulatedM. truncatula genes (4-h treatment).

Supplemental Figure S5. topGo analysis of NF up- and down-regulatedM. truncatula genes (24-h treatment).

Supplemental Figure S6. Expression of the GA12 metabolic network inresponse to NFs (24 h).

Supplemental Figure S7. Expression of the trans-zeatin CK metabolic net-work in response to NFs (24 h).

Supplemental Figure S8. TCSn:GUS and pENOD11:GUS are up-regulatedby wild-type S. meliloti in an NF-dependent way.






https://www.legoo.org

http://bioinformatics.ai.sri.com/ptools/

http://bioinformatics.ai.sri.com/ptools/

https://pathway-tools.toulouse.inra.fr/MEDICAGO2

http://metexplore.toulouse.inra.fr/metexplore2/index.html?idBioSource=3423

http://metexplore.toulouse.inra.fr/metexplore2/index.html?idBioSource=3423











Supplemental Figure S9. Biological repetition of experiments shown inFigure 5.

Supplemental Figure S10. AtCKX3 is strongly expressed in M. truncatularoots transformed with the pEPI:AtCKX3 construct.

Supplemental Figure S11. Statistical analysis of CK quantitative analysisin root fragments, transformed with the pEPI:AtCKX3 construct or acontrol plasmid, and treated or not with 1028

M NF for 4 h.

Supplemental Figure S12. Nodulation assays following S. medicae inocu-lation of roots transformed by A. rhizogenes with the pEPI:CKX3 con-struct.

Supplemental Table S1. Number of total and unambiguously mappedRNAseq read pairs.

Supplemental Table S2. Complete RNAseq and annotation data for mock-or NF-treated laser-dissected root epidermis samples.

Supplemental Table S3. List of genes (and annotation data) up-regulatedin M. truncatula root epidermis in response to 1028

M NFs, identified bylaser microdissection coupled with RNAseq analysis.

Supplemental Table S4. List of genes (and annotation data) down-regulated in M. truncatula root epidermis in response to 1028

M NFs,identified by laser microdissection coupled with RNAseq analysis.

Supplemental Table S5. List of metabolic networks regulated in M. trun-catula root epidermis in response to 1028

M NFs, identified using MetEx-plore.

Supplemental Table S6. Levels of CKs in roots transformed via A. rhizo-genes with a control plasmid or a plasmid carrying the pEPI:CKX3 con-struct.

Supplemental Materials and Methods S1. Laser microdissection, RNAsequencing, and CK extraction and quantification.

ACKNOWLEDGMENTS

We thank Bruno Müller (University of Zurich) for the TCSn:GFP construct,Thomas Schmuelling (Free University Berlin) for the AtCKX3 clone, FabienneMaillet (LIPM) for providing NFs, Sandra Bensmihen (LIPM) for the pGEM-T:pLeEXT1 vector, Fernanda de Carvalho Niebel (LIPM) for valuable advice andsupervising the NF treatments, Christine Hervé and Andreas Niebel (LIPM) forhelp in NF treatments, Sébastien Carrere (LIPM) for the SYMbiMICS Web site,Alain Jauneau and the Fédération de Recherche Agrobiosciences, Interactions,en Biodiversité microscopy platform for the laser microdissection equipment,and the IMAGIF cell biology platform.

Received May 6, 2016; accepted May 18, 2016; published May 23, 2016.

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