Marie-Françoise Jardinaud

Justine Fromentin

Marie-Christine Auriac

Sandra Moreau

Yann Pecrix

Ludivine Taconnat

Ludovic Cottret

Grégoire Aubert

Sandrine Balzergue

Judith Burstin

Pascal Gamas
email: pascal.gamas@inrae.fr

Sébastien Carrère

MtEFD and MtEFD2: Two transcription factors with distinct neofunctionalization in symbiotic nodule development

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The rhizobium-legume symbiosis provides agro-ecosystems with considerable amounts of assimilable nitrogen (N), a critical factor for plant growth, thanks to the capacity of the bacterial nitrogenase to fix atmospheric nitrogen. The symbiotic N fixation (SNF) takes place in dedicated root organs, called nodules, that provide rhizobia with carbon sources and a micro-oxic environment, essential for the nitrogenase to be functional. Nodules can be of indeterminate (e.g., in pea (Pisum sativum) and Medicago sp.) or determinate (e.g., in soybean (Glycine max) and Lotus sp.) type, i.e., with or without a permanent (apical) meristem, respectively. Two main steps can be distinguished in nodule organogenesis: the first one is nodule inception in root tissues, which follows a signaling process triggered by the perception by the plant of specific rhizobial chito-oligosaccharidic molecules, the Nod factors. This leads to cell divisions in the root cortex and rhizobial infections in the root epidermis. Both processes involve a series of symbiotic transcription factors (TFs), with NODULE INCEPTION (NIN) as a master regulator [START_REF] Schauser | A plant regulator controlling development of symbiotic root nodules[END_REF][START_REF] Marsh | Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase[END_REF], as well as phytohormones, among which cytokinins (CK) and auxins are major players [START_REF] Lin | No Home without Hormones: How Plant Hormones Control Legume Nodule Organogenesis[END_REF]. CK signaling in the root cortex leads to NIN induction, itself activating NF-Y and LATERAL ORGAN BOUNDARIES-DOMAIN PROTEIN 16 (LBD16) TF expression, contributing to local auxin accumulation and consequently cell division [START_REF] Schiessl | NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula[END_REF][START_REF] Soyano | A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus[END_REF]. LBD16 being also involved in lateral root organogenesis, this reveals an important overlap between the genetic programs respectively leading to nodule and lateral root inception, also evidenced by the importance of the SHORTROOT-SCARECROW module in both developmental programs [START_REF] Dong | An SHR-SCR module specifies legume cortical cell fate to enable nodulation[END_REF]. The second step is cell differentiation associated with nodule development, which allows symbiotic traits to be acquired by plant and bacterial cells. At this stage, a large number of genes are strongly and specifically up-regulated in developing nodules compared to the roots [START_REF] Mergaert | Gene Expression in Nitrogen-Fixing Symbiotic Nodule Cells in Medicago truncatula and Other Nodulating Plants[END_REF]. In Medicago truncatula, the symbiotic differentiation process first takes place in cells generated by divisions of inner (C4-C5) root cortical cells in the nodule primordium, and later in cells produced by an apical nodule meristem established from the C3 cortical cell layer [START_REF] Xiao | Fate map of Medicago truncatula root nodules[END_REF].

The meristematic region of a M. truncatula nodule, termed Zone I (ZI), involves distinct division centers, associated with peripheral vascular bundles (VB) and central tissues respectively. The nodule vasculature is ontologically related to roots, based on the ontogeny of the VB meristems and the expression of key regulators of the root meristem [WUSCHEL-RELATED HOMEOBOX (MtWOX5) and PLETHORA (PLT1 and PLT2) TFs] [START_REF] Osipova | Wuschel-related homeobox5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF][START_REF] Magne | MtNODULE ROOT1 and MtNODULE ROOT2 Are Essential for Indeterminate Nodule Identity[END_REF]. Two genes, with distinct but overlapping expression profiles in the nodule meristem, are essential to regulate the meristematic subdomains and control nodule identity: MtNODULE ROOT 1 (MtNOOT1) and MtNOOT2 [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Magne | MtNODULE ROOT1 and MtNODULE ROOT2 Are Essential for Indeterminate Nodule Identity[END_REF]. A Mtnoot1 Mtnoot2 double mutant shows a complete nodule to root conversion, with ectopic roots developing from VB meristems.

The symbiotic differentiation of indeterminate nodules involves cells generated by the central nodule meristem, along a longitudinal gradient from apical to proximal nodule zones. Once meristematic cells exit the division cycles in the sub-meristematic zone (termed distal ZII), they rapidly enlarge and begin to endoreduplicate. This endoreduplication is controlled by MtCCS52A, a cell cycle switch gene activated at the final stage of nodule primordium formation [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF]. Knocked down lines of MtCCS52A exhibit a reduction of the endoreduplication level (predominantly 8C, vs 32C nuclei in control nodules) and produce small fix-nodules, thereby demonstrating the importance of endoreduplication for nodule development. MtCCS52A is expressed in the whole nodule ZII (or infection zone) [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF], where rhizobia are released from infection threads (tubular structures of plant origin). Rhizobia are surrounded by a plant membrane showing specific identity markers [START_REF] Limpens | Medicago N2-fixing symbiosomes acquire the endocytic identity marker Rab7 but delay the acquisition of vacuolar identity[END_REF], thereby forming the so-called symbiosome. In the same time, hundreds of plant genes are massively up-regulated in successive waves [START_REF] Maunoury | Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF]. This allows the cell environment required for SNF to be gradually set up, leading to the formation of the interzone II-III (IZ) and nitrogen-fixing ZIII.

A hallmark of nodule differentiation is the accumulation of leghemoglobin, essential to keep a low free oxygen concentration while maintaining respiration. Differentiation also involves the upregulation of various transporters and important modifications of the cytoskeleton organization and membrane trafficking in the plant cell [e.g. [START_REF] Gavrin | Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago[END_REF][START_REF] Zhang | The host actin cytoskeleton channels rhizobia release and facilitates symbiosome accommodation during nodulation in Medicago truncatula[END_REF]]. A C2H2 TF termed RSD (REGULATOR OF SYMBIOTIC DIFFERENTIATION) is thought to promote symbiosome development by regulating the plant secretory pathway [START_REF] Sinharoy | The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula[END_REF]. Furthermore, in Medicago and other legumes of the Inverted Repeat Lacking Clade, a gene family strongly upregulated in the infection / differentiation zones encodes so-called Nodule-specific Cysteine-Rich peptides (NCRs) [START_REF] Pan | Nodule cysteine-rich peptides maintain a working balance during nitrogenfixing symbiosis[END_REF][START_REF] Mergaert | Role of antimicrobial peptides in controlling symbiotic bacterial populations[END_REF][START_REF] Stonoha-Arther | Tough love: accommodating intracellular bacteria through directed secretion of antimicrobial peptides during the nitrogen-fixing symbiosis[END_REF], processed and targeted to the symbiosomes by a signal peptidase complex (SPC) including DOES NOT FIX NITROGEN 1 (DNF1) [START_REF] Wang | A nodulespecific protein secretory pathway required for nitrogen-fixing symbiosis[END_REF]. More than 600 NCR genes have been identified in M. truncatula [START_REF] De Bang | Genome-Wide Identification of Medicago Peptides Involved in Macronutrient Responses and Nodulation[END_REF][START_REF] Montiel | Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides[END_REF]. While the role of most of them remains elusive, several have been demonstrated to be essential factors of bacteroid differentiation and / or survival [START_REF] Van De Velde | Plant peptides govern terminal differentiation of bacteria in symbiosis[END_REF][START_REF] Farkas | Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms[END_REF][START_REF] Horváth | Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant[END_REF][START_REF] Kim | An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis[END_REF].

Interestingly, the DNF1 SPC was recently shown to process NIN, to produce a smaller NIN form that controls late nodule development and numerous associated genes [START_REF] Feng | Processing of NODULE INCEPTION controls the transition to nitrogen fixation in root nodules[END_REF].

As with plant cells, rhizobia undergo several rounds of endoreduplication, along with strong morphological changes and modifications in gene expression triggered by the micro-oxic environment and NCR peptides, resulting in terminally differentiated bacteroids. Importantly, in spite of their presence in huge numbers within plant cells, rhizobia do not trigger plant immune responses, unless the interaction is impaired by mutations. Thus, several fix-M. truncatula mutants exhibit defense reactions which affect the maintenance of rhizobia: dnf2, nad1 (nodules with activated defense 1), symcrk (symbiotic cys-rich receptor kinase), npd1 (nodule-specific Plat-domain 1) or npd1, 2, 3, 4, 5 [START_REF] Bourcy | Medicago truncatula DNF2 is a PI-PLC-XD-containing protein required for bacteroid persistence and prevention of nodule early senescence and defense-like reactions[END_REF][START_REF] Berrabah | Multiple steps control immunity during the intracellular accommodation of rhizobia[END_REF][START_REF] Wang | NODULES WITH ACTIVATED DEFENSE 1 is required for maintenance of rhizobial endosymbiosis in Medicago truncatula[END_REF][START_REF] Yu | Suppression of innate immunity mediated by the CDPK-Rboh complex is required for rhizobial colonization in Medicago truncatula nodules[END_REF][START_REF] Pislariu | The Nodule-Specific PLAT Domain Protein NPD1 Is Required for Nitrogen-Fixing Symbiosis[END_REF].

Not many transcriptional regulators controlling symbiotic differentiation per se have been demonstrated in M. truncatula, besides MtRSD and MtNIN. MtNF-YA1, MtNOOT1 and MtNOOT2 are essential for the establishment and the maintenance of the central nodule meristem respectively, but their mutation does not prevent the symbiotic differentiation [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Xiao | Fate map of Medicago truncatula root nodules[END_REF][START_REF] Magne | MtNODULE ROOT1 and MtNODULE ROOT2 Are Essential for Indeterminate Nodule Identity[END_REF]. Not considering TFs expressed in nodule VBs, three TFs appear to be more [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF], under the direct or indirect control of INTERACTING PROTEIN OF DMI3 (IPD3) transcriptional regulator [START_REF] Ovchinnikova | IPD3 controls the formation of nitrogen-fixing symbiosomes in pea and Medicago Spp[END_REF]. The two others are NIN [START_REF] Feng | Processing of NODULE INCEPTION controls the transition to nitrogen fixation in root nodules[END_REF][START_REF] Liu | NIN is essential for development of symbiosomes, suppression of defence and premature senescence in Medicago truncatula nodules[END_REF] and MtNLP2, a NIN-like protein expressed in the IZ and the ZIII, where it notably controls the expression of leghemoglobins, together with NIN [START_REF] Jiang | NIN-like protein transcription factors regulate leghemoglobin genes in legume nodules[END_REF]. Furthermore, epigenetic regulators are also important, likely by modulating the access of TFs to chromatin regions. Thus a knocked-down mutant of DEMETER (DME), a DNA demethylase strongly up-regulated in proximal ZII and IZ, is fix-and impaired in nodule differentiation [START_REF] Satgé | Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula[END_REF]. In addition, gene expression in the nodule differentiation zone often coincides with reduced levels of repressive histone marks (e.g., H3K27me3) and increased levels of active histone marks (H3K9ac) [START_REF] Nagymihaly | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF][START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF].

directly involved in nodule differentiation / development. MtEFD (ETHYLENE RESPONSE FACTOR INVOLVED IN NODULE DIFFERENTIATION) is expressed in ZII
MtEFD belongs to the large family of AP2/ERF TFs, more specifically the group V, as MtERN1 (ERF REQUIRED FOR NODULATION 1) and MtERN2, two key regulators of NF signaling, nodule inception and rhizobium infection [START_REF] Andriankaja | AP2-ERF transcription factors mediate Nod factor dependent Mt ENOD11 activation in root hairs via a novel cis-regulatory motif[END_REF][START_REF] Middleton | An ERF Transcription Factor in Medicago truncatula That Is Essential for Nod Factor Signal Transduction[END_REF][START_REF] Cerri | The Symbiosis-Related ERN Transcription Factors Act in Concert to Coordinate Rhizobial Host Root Infection[END_REF]. MtEFD, however, differs from MtERN1 and MtERN2, both in its AP2/ERF DNA binding domain and in its expression profile, since MtEFD is neither induced by NF nor associated with infection threads, in contrast to MtERN1 and MtERN2 [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF]. Mtefd-1, a deletion mutant of MtEFD, is fixand exhibits strong alterations in the nodule ZII and III [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF]. In addition, Mtefd-1 exhibits a hypernodulation phenotype and frequent multilobed nodules. Transcriptomic analyses supported by a transactivation assay indicated that MtEFD positively controls a negative regulator of the CK pathway, the type A response regulator MtRR4 gene. Because CKs play a key role in nodule inception, it was hypothesized that the downregulation of MtRR4 might explain the hypernodulation phenotype of Mtefd-1. It was also speculated that MtEFD might regulate a gradient of CK activity within the nodule and thereby impact nodule differentiation [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF]. The importance of CKs for nodule development / differentiation has later been established by the nodule phenotype of a Mtcre1/chk1 CK receptor mutant, which exhibits multiple lobes and incomplete differentiation [START_REF] Plet | MtCRE1dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF]. A multilobed nodule phenotype corresponding with a decreased MtEFD and MtRR4 expression was also observed in M. truncatula roots where three KNOX homeodomain TF genes, termed MtKNAT3/4/5-like (or MtKNOX3, MtKNOX5 and MtKNOX9), were simultaneously knockeddown [START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF]. These TFs, expressed in nodule primordia and different nodule zones, belong to a KNOX homeodomain subclass promoting the differentiation of aerial organs in Arabidopsis (Arabidopsis thaliana).

Here we revisited the impact of the Mtefd-1 mutation on nodule development, with analyses of the endoreduplication levels and affected genes, notably MtRR4. We also investigated MtEFD evolution, by testing the capacity of a close MtEFD paralog, MtEFD2, and of the A. thaliana EFD orthologue, AtERF003, to complement the Mtefd-1 mutation. Finally, we assessed whether MtEFD and MtEFD2, which exhibit distinct but overlapping expression profiles, could both be involved in the control of nodulation.

Results

The Mtefd-1 mutant exhibits an endoreduplication phenotype To further investigate the impact of the Mtefd-1 mutation on nodule differentiation, we analyzed nodule endoreduplication. In wild type (wt) 13-day-old nodules, the DNA content of plant cells ranged from 2C to 64C (five cycles of endoreduplication), with 32C and 64C nuclei representing on average 9.4% and 0.35% of total nuclei respectively (in attapulgite growth condition, supplemental Table S1). In Mtefd-1 nodules, the relative abundance of 32C and 64C nuclei was strongly decreased (0.8% and 0.08% of the population on average, respectively), whereas the frequency of 16C nuclei was not statistically different from wt (3.5% vs. 4.2%) (Fig1. A; supplemental Table S1). This was observed in different growth conditions (aeroponic or attapulgite condition) and at different time points, from 13 to 30 days post inoculation (supplemental Table S1), therefore ruling out a mere delay of endoreduplication. This phenotype differed from the one observed with five other fix-M. truncatula mutants, namely TR3, TR36, TRV36, TR183 and TRV43 [START_REF] Maunoury | Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches[END_REF], for which corresponding genes have not yet been identified, except for TR3, allelic to ipd3 [START_REF] Ovchinnikova | IPD3 controls the formation of nitrogen-fixing symbiosomes in pea and Medicago Spp[END_REF]. These five mutants showed either a low proportion (TRV36 and TRV43) or a total absence (TR3, TR183 and TR36) of both 16C and 32C cells (Fig. 1 C-F; supplemental Table S1), as previously reported [START_REF] Maunoury | Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches[END_REF]. While the rsd-1 [START_REF] Sinharoy | The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula[END_REF] and dme [START_REF] Satgé | Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula[END_REF] fix-mutants are also affected in the level of both 16C and 32C cells, Mtefd-1 appears to be a plant fixmutant specifically affected at the 16C-32C transition.

The endoreduplication of bacterial cells (Sinorhizobium meliloti 2011) was also clearly reduced in Mtefd-1 nodules (Fig. 1B). In addition, another hallmark of bacteroid differentiation, the expression of NifH, a nitrogenase subunit, was severely affected, as seen with a NifH:GFP fusion. While a strong NifH:GFP signal was observed in the IZ/ZIII of wt nodules, only a weak signal was observed in a few scattered cells of Mtefd-1 nodules at 21 dpi (Fig. 1G-H). The weakness of the signal was not due to cell death, which was found by a live/dead assay to be very limited (Fig. 1I-J). The impact of Mtefd-1 on nodule differentiation is therefore not due to a deregulated plant immune response, in contrast to several other fix-plant mutants (dnf2, symcrk, nad-1, npd1), which was further confirmed by transcriptomic data (see below).

The Mtefd-1 mutation impacts a large proportion of genes expressed during late nodule development One of the first generations of M. truncatula microarrays (Mt16KOLIPlus) was used for the original transcriptomic analyses of the Mtefd-1 mutant, which revealed 223 and 34 genes respectively downand up-regulated at least two fold (adj. p-value <0.05) vs wt nodules at 10 dpi, and none at 4 dpi [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF]. Here, to get a more complete view of genes affected by Mtefd-1, we successively used whole-genome Nimblegen microarrays [START_REF] Verdier | A regulatory network-based approach dissects late maturation processes related to the acquisition of desiccation tolerance and longevity of Medicago truncatula seeds[END_REF] and RNAseq to compare wt and Mtefd-1 non-inoculated roots, immature (4-day-old) and mature (10-day-old) isolated nodules. The two approaches gave consistent results (supplemental Table S2; supplemental Table S3), but we focus here on RNAseq results (supplemental Fig. S1), which are more sensitive and take advantage of the last (Mt5.0) M. truncatula genome release [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF].

The analysis of wt samples showed a large set of genes strongly differentially regulated between nodules and roots, with 2,140 and 4,311 genes >4-fold up-regulated (FDR<0.01) in 4 dpi and 10 dpi nodules respectively (supplemental Fig. S2A-B). Furthermore, the comparison of mature vs immature wt nodules revealed 4,189 genes up-regulated during late nodule development (fold change= FC>4, FDR<0.01) (supplemental Fig. S2C). To determine where they are expressed in the nodule, we made use of the 16 expression patterns previously defined from RNAseq analysis of laser-dissected nodule zones [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF] (see legend of Fig. 2A). As expected, they were mostly expressed in the differentiation and nitrogen-fixation nodule zones (expression patterns 6 to 11 and 15-16) (Fig. 2A-C).

The analysis of Mtefd-1 transcriptomes revealed 246 and 1,719 strongly down-regulated genes compared to wt in 4 and 10 dpi nodules respectively (FC<-4, FDR<0.01) (Fig. 2D; supplemental Fig. S2D). This represented 8.6% and 37.6% of the genes up-regulated in wt nodules vs roots, respectively. By contrast, only 14 genes were down-regulated in Mtefd-1 roots vs wt roots (supplemental Table S3). The analysis of 4 dpi nodules was particularly interesting, because those are less impacted by indirect, developmental effects of Mtefd-1. At this stage, the induction of several genes was completely abolished in Mtefd-1, including notably MtRR4 (Table 1). A >4-fold reduction of expression was detected for 18 early (patterns 4 to 6) NCR genes, with notably 9 out of 12 NCR genes in pattern 4 and 5 affected, while the nodule-specific thioredoxin MtTRX S1, regulating NCR activity and required for bacteroid differentiation [START_REF] Ribeiro | Regulation of Differentiation of Nitrogen-Fixing Bacteria by Microsymbiont Targeting of Plant Thioredoxin s1[END_REF] was 65-fold down-regulated in Mtefd-1 vs wt 4 dpi nodules. Two other families proposed to be involved in nodule differentiation were also affected, namely nodule-specific Glycine-rich proteins [START_REF] Alunni | Genomic organization and evolutionary insights on GRP and NCR genes, two large nodule-specific gene families in Medicago truncatula[END_REF] and the Medicago lineage-specific SNARP/LEED..PEED family [START_REF] Trujillo | Genomic characterization of the LEED..PEEDs, a gene family unique to the medicago lineage[END_REF], with MtNODGRP36 and MtLP9/MtLP10 down-regulated (153-, 31-and 405-fold respectively) in 4 dpi nodules and additional members of both families affected at 10 dpi (Table 1; supplemental Table S3). In addition, the expression of various genes encoding other secreted Cys-rich peptides of unknown function was severely impaired (50-to 220-fold downregulation), including a cluster of eight leginsulin genes (de [START_REF] De Bang | Genome-Wide Identification of Medicago Peptides Involved in Macronutrient Responses and Nodulation[END_REF] and three knottin-domain nodulins (MtN1, MtN1b and MtN15) (Table 1 and supplemental Table S3). Of note, MtCCS52a was moderately but significantly down-regulated (FC=-2.3 and -1.6 at 4 and 10 dpi, respectively), which is interesting considering the importance of this gene for nodule endoreduplication. Finally, the GO analysis revealed a downregulation of defenserelated genes in Mtefd-1 nodules (GO:0006952; p-value = 7.5E-04; supplemental Table S3).

At 10 dpi, many genes down-regulated in Mtefd-1 vs wt nodules corresponded to the late differentiation and nitrogen-fixing zones (Fig. 2E), with 48.1% of pattern 6-11 genes down-regulated in Mtefd-1 (1,890 genes FC<-2, FDR<0.01). 455 NCR genes were down-regulated in Mtefd-1 (FC<-4), along with a series of leghemoglobin (GO:0015671) and transporter genes (GO:0006810) (Table 1 and supplemental Table S3). This reflects a major developmental impact of the Mtefd-1 mutation, with likely many indirect effects. As an illustration of possible cascade effects, the MtRSD (pattern 7) and MtNCR169 (corresponding to the dnf7 mutant; pattern 9) genes were down-regulated 4-and 9fold, respectively, in 10 dpi Mtefd-1 nodules (Table 1). Transcriptomic analyses of rsd and dnf7 mutant nodules have previously identified 163 and 93 down-regulated genes (FC≤-2) compared to wt controls, respectively [START_REF] Sinharoy | The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula[END_REF][START_REF] Horváth | Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant[END_REF], all expressed in the late nodule differentiation zone (pattern 7-11 and 15-16; supplemental Fig. S2F-G). We found that a majority of them were down-regulated in Mtefd-1 (78.4% for dnf7 and 79.5% for rsd-1; supplemental Fig. S2E), while only 12 genes were shared between dnf7 and rsd-1 affected genes. Likewise, MtDME was down-regulated 3.4-fold in Mtefd-1, similar to the downregulation level leading to fix-nodules in MtDMEi knock-down mutants [START_REF] Satgé | Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula[END_REF], while MtNLP2 [START_REF] Jiang | NIN-like protein transcription factors regulate leghemoglobin genes in legume nodules[END_REF] was 10-fold down-regulated. In summary, Mtefd-1 affects several key regulators of late nodule development.

The dnf7 mutant nodules exhibit early senescence, with a high induction of MtCP2 and MtCP6 cysteine protease genes, which are markers of nodule developmental senescence [START_REF] Horváth | Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant[END_REF]. By contrast, MtCP2 and MtCP6 were not differentially expressed in Mtefd-1 nodules as compared to wt nodules (Table 1), possibly because of the residual level of NCR169 expression in Mtefd-1. Furthermore, genes preventing plant immune responses against rhizobium in nodules (MtDNF2, MtSYMCRK, MtNAD1) were only modestly impacted by the efd-1 mutation (around 5-fold down-regulation), by comparison with their considerable level of induction in wt nodules (1760-, 756-and 477-fold respectively in 10 dpi nodules vs roots) (Table 1 and supplemental Table S3).

Consistently, the defense response genes highly activated in dnf2, symcrk or nad1 mutants were either not [MtVSP (VACUOLAR SORTING PROTEIN); MtBGL (BETA-1,3-GLUCANASE); MtNDR1 (NON-RACE SPECIFIC DISEASE RESISTANCE PROTEIN 1); MtPR3 (PATHOGENESIS RELATED PROTEIN 3); MtPAL (PHENYLALANINE AMMONIA LYASE); or only moderately up-regulated (MtPR10-6; MtVPE (VACUOLE PROCESSING ENZYME)] in 10 dpi Mtefd-1 vs wt nodules.

Consistent with the live/dead assay (Fig. 1J), these transcriptomic data thus confirmed that MtEFD controls the differentiation process and not immune responses.

In addition to down-regulated genes, we found a number of up-regulated genes in the Mtefd-1 mutant compared to wt samples (42 in non-inoculated roots, 718 in 4 dpi nodules and 380 in 10 dpi nodules; FC>4, FDR<0.01; Fig. 2D, supplemental Table S3, supplemental Fig. S2D). At 10 dpi, a fraction of up-regulated genes likely reflects the absence of ZIII development in efd-1 nodules, mechanically leading to a relative enrichment in ZI and II transcripts compared to wt nodules (Fig. 2A-C). We also observed the upregulation at 4 dpi of auxin-related genes, including MtLBD11 [START_REF] Schiessl | NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula[END_REF] and MtARF13 (AUXIN RESPONSE FACTOR 13) transcriptional activator genes, as well as MtGH3 (GRETCHEN HAGEN 3) and MtSAUR1 (SMALL AUXIN UP RNA 1) auxin-responsive genes, potentially related to increased cortical cell divisions (ccd) that we observed in Mtefd-1 (see below).

The 10-fold upregulation at 4 dpi of MtCEP11 (C-TERMINALLY ENCODED PEPTIDE 11) (Table 1), related to the positive regulator of nodulation MtCEP1 [START_REF] Gautrat | Compact Root Architecture 2 Promotes Root Competence for Nodulation through the miR2111 Systemic Effector[END_REF], might also be linked to the Mtefd-1 hypernodulation phenotype. More intriguing was the observation that 44 genes normally expressed in the late differentiation zone (patterns 8-11, 15), were up-regulated at 4 dpi while down-regulated at 10 dpi (FC>4, FDR<0.01) (Table 1 andsupplemental Table S3). Those included 21 late NCR genes (notably MtNCR169), a cluster of nodule-specific calmodulin-like genes, whose products are targeted to the symbiosome [START_REF] Liu | Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula[END_REF], as well as a redox control gene, MtRbohA (RESPIRATORY BURST OXIDASE HOMOLOG A), normally expressed in the zone III [START_REF] Marino | A Medicago truncatula NADPH oxidase is involved in symbiotic nodule functioning[END_REF]. These genes are thus prematurely induced in Mtefd-1 nodules. In other words, MtEFD seems to both positively regulate differentiation genes and prevent early expression of a set of late symbiotic genes.

MtRR4 can complement the hypernodulation but not the nodule differentiation phenotype of the Mtefd-1 mutant

In line with our original observations indicating that MtEFD positively controls MtRR4 expression [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF], we found here that MtRR4 and MtEFD have a similar expression profile in nodules (Fig. 2A bottom) and that MtRR4 induction is abolished in Mtefd-1 nodules (Table 1 and supplemental Fig. S3A for a validation by RT-qPCR analysis). MtRR4 is by far the most highly expressed member of the MtRRA gene family (ten genes) in mature wt nodules (supplemental Table S3). Using RNAseq data from an independent time course analysis [START_REF] Schiessl | NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula[END_REF], we observed two waves of MtRR4 induction taking place during nodulation, the first one in the first 24h following rhizobium infection, and the second one about three days later, i.e., at the stage of nodule primordium growth and young nodule emergence from the root (supplemental Fig. S3B). Since MtEFD is not induced before about 2 dpi [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF][START_REF] Schiessl | NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula[END_REF], it is likely that only the second wave of MtRR4 induction is impaired in Mtefd-1.

Since MtRR4 is thought to negatively regulate cytokinin signaling, we decided to assess the impact of the Mtefd-1 mutation on the expression of a reporter of CK signaling, TCSn:GUS [START_REF] Zürcher | A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta[END_REF][START_REF] Jardinaud | A Laser Dissection-RNAseq Analysis Highlights the Activation of Cytokinin Pathways by Nod Factors in the Medicago truncatula Root Epidermis[END_REF], using hairy root transformation. In wt and Mtefd-1 S. meliloti-inoculated roots, TCSn:GUS was expressed in nodule primordia at all developmental stages, from stage I to stage VI as defined by [START_REF] Xiao | Fate map of Medicago truncatula root nodules[END_REF] (Fig. 3A-D). In the Mtefd-1 mutant, we observed a more intense GUS signal in individual cells within nodule primordia (Fig. 3B andD) as well as frequent unorganized cortical cell divisions, associated or not with infection threads (Fig. 3E). In mature (10 dpi) wt nodules, the TCSn:GUS signal was observed in a narrow, meristematic or submeristematic region (Fig. 3F, supplemental Fig. S4). By contrast, in Mtefd-1 nodules, TCSn:GUS was expressed in a broader region including the infection / differentiation zones (Fig. 3G, supplemental Fig. S4). Yet, a marker of the central and vascular nodule meristems, the pWOX5 (WUSCHEL-RELATED HOMEOBOX-5):GUS construct [START_REF] Osipova | Wuschel-related homeobox5 gene expression and interaction of CLE peptides with components of the systemic control add two pieces to the puzzle of autoregulation of nodulation[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF], remained expressed in highly localized apical cells in Mtefd-1, as in wt nodules (Fig. 3H andI). Of note, Agrobacterium rhizogenes-mediated transformation may change the hormonal balance but this would also be the case in the control wt background. Overall, this suggested an alteration of CK signaling in the infection/differentiation zones of Mtefd-1 nodules, while the nodule meristematic centers are not affected. By contrast, auxin signaling appeared to be minimally affected in mature Mtefd-1 nodules, based on the expression profile of a DR5:GUS auxin-responsive reporter (Ulmasov et al, 1995) (supplemental Fig. S4).

Bearing in mind the importance of CK for both early and late stages of nodule development [START_REF] Plet | MtCRE1dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF], we then asked whether MtRR4 could complement the different nodulation phenotypes of Mtefd-1. MtRR4 was expressed under the control of MtEFD promoter to ensure a proper localization of its transcripts and transformed in Mtefd-1 using A. rhizogenes. To assess the complementation efficiency in a sensitive way, nodules were induced using S. meliloti carrying the NifH:GFP construct.

As expected, transformation of Mtefd-1 with pMtEFD:MtEFD produced pink nodules, with numerous cells expressing NifH:GFP, as in wt control nodules (Fig. 4A-C). By contrast, nodules produced in pMtEFD:MtRR4-transformed roots were very similar to empty vector-transformed nodules, i.e., small, white and with very few GFP-expressing cells (Fig. 4D) even though MtRR4 was well expressed (as verified by RT-qPCR; supplemental Fig. S5). Furthermore, an analysis of nodule cell endoreduplication showed that the pMtEFD:MtRR4 construct was unable to restore the 32C peak in Mtefd-1 nodules, in contrast to pMtEFD:MtEFD (Fig. 4E). We then tested whether pMtEFD:MtRR4 could complement the Mtefd-1 hypernodulation phenotype. In contrast to the nodule development phenotype, we found that pMtEFD:MtRR4 was as efficient as pMtEFD:MtEFD in restoring a normal level of nodulation (Fig. 4F). Altogether, this suggested that MtRR4 downregulation is a major (and possibly the only) component of the Mtefd-1 hypernodulation phenotype, in contrast to its nodule differentiation phenotype, thus likely to involve other players.

The Arabidopsis orthologue of MtEFD, AtERF003, complements the nodule differentiation phenotype of Mtefd-1, in contrast to a close MtEFD paralogue, MtEFD2

We identified proteins closely related to MtEFD in a variety of legume and non-legume plant species.

Those included a MtEFD paralogue in M. truncatula dubbed MtEFD2 (MtrunA17_Chr3g0137174; 67.7% amino acid identity). The top 30 hits of Blastp searches in the ncbi non-redundant (nr) protein database with MtEFD were only legume ERFs (expect values <1e-82). By contrast, 14 non-legume ERFs were found amongst the 30 top hits for MtEFD2 (expect values <1e-92). Multiple alignment analyses indicated that the AP2/ERF DNA binding domain of MtEFD/MtEFD2 subgroups and AtERF003, the only Arabidopsis thaliana ERF closely related to MtEFD, are highly similar, with a perfect conservation of amino acids involved in the direct interaction with DNA (Fig. 5A, supplemental Fig. S6). A phylogenetic tree was generated with the protein fraction following the AP2/ERF domain, using AtERF003, the 15 best hits for MtEFD and MtEFD2, and MtERN1 as an outgroup (subgroup Vb, vs subgroup Va for MtEFD). It showed that MtEFD2 and AtERF003 belong to a same subgroup, clearly distinct from the MtEFD subgroup (supplemental Fig. S7), with a further separation among MtEFD2-related proteins between legume and non-legume proteins. In the protein fraction following the AP2/ERF domain, two motifs described for the group V ERFs, termed CMV-1 and CMV-2 [START_REF] Nakano | Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice[END_REF][START_REF] Cerri | AP2/ERF transcription factors and root nodulation[END_REF] are also conserved amongst EFD/EFD2related proteins, albeit with some differences between the two subgroups (Fig. 5A; supplemental Fig. S6). The remaining region of about sixty amino acids, located between CMV-1 and the C-terminal CMV-2, is more variable and poorly structured, according to AlphaFold modeling (supplemental Fig. S8). Yet an analysis of a set of 39 accessions of Pea (a species genetically closely related to M. truncatula), representing a wide range of diversity [START_REF] Siol | Patterns of Genetic Structure and Linkage Disequilibrium in a Large Collection of Pea Germplasm[END_REF], showed that this region is well conserved in PsEFD (supplemental Fig. S9), suggesting a possible role in the structure and /or function of EFD.

We found that pMtEFD2:GUS had a different expression pattern than pMtEFD:GUS in A. rhizogenestransformed roots and nodules. In non-inoculated roots, pMtEFD2:GUS was expressed in the meristematic zone of the root tip, while pMtEFD:GUS was expressed in both zones flanking the root meristem, notably in the transition zone (where root cells elongate and begin to differentiate) (Fig. 5B-C). Consistently, independent transcriptome analyses revealed that MtEFD2 expression is positively regulated by auxin, which is not the case for MtEFD [START_REF] Herrbach | Nod factors potentiate auxin signaling for transcriptional regulation and lateral root formation in Medicago truncatula[END_REF]. Following S. meliloti inoculation, the expression of both genes was detected in nodule primordia (Fig. 5D-E), including at very early stages, consistent with RNAseq analyses of spot-inoculated roots [START_REF] Schiessl | NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula[END_REF] (supplemental Fig. S3B). However, from the stage III primordia (establishment of the nodule meristem [START_REF] Xiao | Fate map of Medicago truncatula root nodules[END_REF]), pMtEFD:GUS seemed more expressed at the base of the primordium (Fig. 5D), possibly corresponding to cells undergoing a differentiation process, which was not the case with pMtEFD2:GUS. In 4 dpi nodules, MtEFD and MtEFD2 were up-regulated 133-fold and seven-fold vs roots, respectively, with MtEFD transcripts being about nine times more abundant than MtEFD2 transcripts (supplemental Table S3). In mature wt nodules, the maximal expression of MtEFD and MtEFD2 was in ZII and in ZI, respectively (laser capture microdissection-RNAseq and promoter:GUS analyses; Fig. 2A and Fig. 5F-G), with some overlapping expression in distal ZII. In the Mtefd-1 mutant, pMtEFD2:GUS was weakly expressed except for a few scattered cells in the nodule primordia (Fig. 5H), consistent with a 2.6-fold decrease of MtEFD2 expression detected by RNAseq in Mtefd-1 vs wt 4 dpi nodules (supplemental Table S3).

Since MtEFD and MtEFD2 proteins are closely related, we then wondered whether MtEFD2 could complement the Mtefd-1 nodule phenotype if expressed from the MtEFD promoter. To further explore possible neofunctionalization processes, we also tested a non-legume protein from the MtEFD2 subgroup, AtERF003. Following hairy root transformation of Mtefd-1 and inoculation with S. meliloti NifH:GFP, we found that pMtEFD:MtEFD2-transformed roots were unable to develop pink nodules at three weeks post-inoculation (wpi), in contrast to pMtEFD:MtEFD -transformed roots, while a few pink nodules were observed at six wpi (Table 2, Fig. 5I). In contrast, surprisingly, pMtEFD:AtERF003 efficiently complemented Mtefd-1 at three wpi, with the production of pink nodules harboring numerous pnifH::GFP expressing cells, and plant and bacteroid endoreduplication levels similar to the pMtEFD:MtEFD control (Table 2, Fig. 5I). Furthermore, pMtEFD:AtERF003 also complemented the hypernodulated phenotype of Mtefd-1, in contrast to pMtEFD:MtEFD2 (Fig. 5J).

We identified one non-conservative modification (P57 replaced by T57) in the AP2/ERF DNA binding site of MtEFD2, not found in MtEFD nor AtERF003 (Fig. 5A). However, the reciprocal substitution of this amino acid residue in MtEFD and MtEFD2 did not modify the outcome of the complementation assay (Table 2). We then performed a reciprocal domain swap between MtEFD and MtEFD2, joining the N-terminal part, including the AP2/ERF DNA binding site, of one protein (amino acids 1-64) to the remaining C-terminal part of the other. We found that a MtEFD2:MtEFD chimeric protein (MtEFD2 Nterminal part joined to MtEFD C-terminal part) was fully able to complement Mtefd-1, whereas the reciprocal MtEFD:MtEFD2 chimeric protein was totally inefficient (Table 2). This confirmed that the AP2/ERF domains of MtEFD and MtEFD2 are functionally identical, and showed the importance and neofunctionalization of the remaining part of the proteins.

Mtefd2 mutations decrease the nodule number in Mtefd-1

Since MtEFD2 is up-regulated seven-fold in wt 4 dpi nodules, we tested whether Mtefd2 mutations could impact nodulation. The analysis of a mutant with a TnT1 insertion in the DNA binding site (Noble Foundation population, R108 background, line NF-9808) revealed no nodulation phenotype.

Then, to investigate possible interactions between Mtefd-1 and Mtefd2 mutations, we set up a multiguide CRISPR-CAS9 mutagenesis approach based on A. rhizogenes-mediated transformation, as previously described for MtNLP2 [START_REF] Jiang | NIN-like protein transcription factors regulate leghemoglobin genes in legume nodules[END_REF]. The four-guide construct that we used had a very high editing rate of MtEFD2 (>70%) (supplemental Fig. S10), while no off-target edits were detected in MtEFD (n=36). As for insertion mutants, no significant difference (P-value=0.26, Kruskal-Wallis test) was observed in nodule number for these Mtefd2 mutants in a wt A17 background. In contrast, in the Mtefd-1 background, a significant decrease in nodule number was observed for biallelic mutants of Mtefd2 at 17 dpi (Fig. 5K). The nodules formed in the double mutant were similar in terms of development to those formed in Mtefd-1 (supplemental Fig. S11).

RNAseq analysis of nodulated root fragments at 4 dpi revealed a set of 122 genes that were downregulated in the Mtefd2 mutants, in both the wt A17 and Mtefd-1 backgrounds, whereas they were up-regulated in the Mtefd-1 single mutant (Table 3; supplemental table S4). A majority of them (70.5%) were induced in wt nodules 4 dpi (FC>4). These genes represent possible candidates for mediating the impact of Mtefd2 on nodulation. They included notably five MtNodGRP [START_REF] Alunni | Genomic organization and evolutionary insights on GRP and NCR genes, two large nodule-specific gene families in Medicago truncatula[END_REF] (mean downregulation of ~30-fold in Mtefd2), the symbiotic remorin gene MtSYMREM1 [START_REF] Lefebvre | A remorin protein interacts with symbiotic receptors and regulates bacterial infection[END_REF] (downregulated ~20-fold), as well as MtNF-YA1 and MtNF-YB16, whose products interact in the nodule [START_REF] Baudin | A phylogenetically conserved group of NF-Y transcription factors interact to control nodulation in legumes[END_REF] (downregulated >4-fold and 2.5-fold in Mtefd2, respectively). Consistently, the six STY (SHORT INTERNODES/STYLISH) genes and one YUCCA gene belonging to the NF-YA1 regulation cascade involved in auxin signaling and nodule emergence [START_REF] Shrestha | Lotus japonicus Nuclear Factor YA1, a nodule emergence stage-specific regulator of auxin signalling[END_REF] were also downregulated in Mtefd-2 at 4 dpi (table 3 and supplemental table S4).

Discussion

While substantial progress has been made in recent years in understanding nodule initiation, the control of later stages of nodule development remains relatively undocumented. Our further characterization of the EFD transcription factor supports its key role in the control of symbiotic differentiation of M. truncatula nodules. Thus, we found that endoreduplication of both rhizobium and plant cells, a hallmark of differentiation, is impaired in the Mtefd-1 KO mutant, with a pattern not yet described in other fixmutants, namely the absence of 32C plant nuclei (64C nuclei being barely detectable in wt nodules in our conditions). Importantly, no concomitant cell death and strong defense reactions were observed in Mtefd-1 nodules, supporting the primary defect of Mtefd-1 being differentiation and not the control of the plant immune response toward rhizobium, in contrast to other reported plant fixmutants. The absence of 32C nuclei in Mtefd-1 might be related to the down-regulation of MtCCS52a expression. Indeed MtCCS52a antisense lines with a ~2.5 fold decrease in MtCCS52a transcript accumulation (similar to the decrease of MtCCS52a expression in 4 dpi Mtefd-1 nodules) also exhibit a strong reduction of 32C nuclei [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF]. Furthermore, since transcription waves coincide with growing ploidy levels [START_REF] Nagymihaly | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF], there might be an EFD-dependent developmental transition corresponding to the last cycle(s) of endoreduplication, possibly taking place in the nodule proximal zone II, where MtCCS52a is maximally expressed [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF]. In agreement with this, our RNAseq analysis indicated that the expression of a strong proportion of genes specifically expressed in wt IZ is impaired in Mtefd-1 nodules (notably 46.7% of pattern 8 and 9 genes, i.e., 616 genes, with a >4-fold down-regulation compared to wt nodules). Those include two important symbiotic regulator genes, namely MtRSD [START_REF] Sinharoy | The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula[END_REF] and MtNCR169 [START_REF] Horváth | Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant[END_REF], expressed at a later stage of development than MtEFD (Fig. 2A). Consistent with MtRSD and MtNCR169 acting downstream MtEFD, we found that a large fraction (~75%) of genes affected in rsd-1 and ncr169 mutants were also down-regulated in Mtefd-1.

Another advance provided by this work was the identification of hundreds of genes affected in very young (4 dpi) Mtefd-1 nodules, less prone to indirect developmental effects. In addition to the previously identified EFD target gene, MtRR4 [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF], we found a series of genes whose upregulation is abolished or highly impaired in Mtefd-1. Those include a set of genes encoding small secreted, Cys-rich peptides, that is 24 early NCR, 8 leginsulin genes, MtN1-related and MtN15 nodulin genes, 4 LTP and defensin genes, all massively induced during wt nodulation. Some of them may contribute to rhizobium differentiation, as NCR peptides or nodule-specific Glycine-rich proteins [START_REF] Alunni | Genomic organization and evolutionary insights on GRP and NCR genes, two large nodule-specific gene families in Medicago truncatula[END_REF] and SNARP/LEED..PEED peptides [START_REF] Laporte | A novel RNA-binding peptide regulates the establishment of the Medicago truncatula-Sinorhizobium meliloti nitrogen-fixing symbiosis[END_REF][START_REF] Trujillo | Genomic characterization of the LEED..PEEDs, a gene family unique to the medicago lineage[END_REF], also affected in Mtefd-1. Expression of the nodule-specific, symbiosome-localized, redox regulator MtTRX S1 [START_REF] Ribeiro | Regulation of Differentiation of Nitrogen-Fixing Bacteria by Microsymbiont Targeting of Plant Thioredoxin s1[END_REF] was also strongly inhibited in Mtefd-1 (>60-fold at both 4 and 10 dpi), suggesting that not only the expression but also the activity of symbiosome-targeted peptides is likely affected. Of note, RNAi-mediated downregulation of MtTRX S1 is sufficient to alter bacteroid differentiation and nodule activity [START_REF] Ribeiro | Regulation of Differentiation of Nitrogen-Fixing Bacteria by Microsymbiont Targeting of Plant Thioredoxin s1[END_REF].

We also found out that many (718) genes are up-regulated in 4dpi Mtefd-1 nodules, compared to wt nodules. In addition to auxin-related genes, hypothesized to relate to increased Rhizobium-induced ccd in Mtefd-1 (see below), 44 genes, normally expressed in the late differentiation zone of wt nodules (patterns 8, 9, 10, 11, 15), were up-regulated at 4 dpi but down-regulated in 10 dpi Mtefd-1 nodules. This suggested an altered timing of transcriptional activation, with MtEFD controlling both the repression (in young nodules) and activation (in mature nodules) of genes associated with late stages of nodule development, notably encoding: NCR peptides [including NCR169 [START_REF] Horváth | Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant[END_REF]], nodule-specific symbiosome-targeted calmodulin-like proteins [START_REF] Liu | Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula[END_REF], and a zone III-associated redox regulator, MtRbohA [START_REF] Marino | A Medicago truncatula NADPH oxidase is involved in symbiotic nodule functioning[END_REF]. Since the expression of these genes is normally finely regulated in successive waves [START_REF] Maunoury | Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches[END_REF][START_REF] Guefrachi | Extreme specificity of NCR gene expression in Medicago truncatula[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF], it is possible that their premature expression is deleterious to the differentiation process.

Thus, NCR peptides are diverse in terms of biochemical properties, localization in the symbiosome, and probably interactors and functions [START_REF] Mergaert | Role of antimicrobial peptides in controlling symbiotic bacterial populations[END_REF]. Modifying the timing of expression and the balance between different types of NCRs may therefore strongly modify the outcome of their action.

In summary, both the down and upregulation of multiple nodule-associated genes in Mtefd-1 could be responsible for Mtefd-1 defects in plant and bacteroid differentiation. From a mechanistic point of view, EFD and all related proteins carry a so-called conserved motif V-2, highly enriched in acidic and apolar amino-acid residues. This motif is strongly reminiscent of the EDLL motif found in the subclass IX of ERFs and demonstrated to be a transcriptional activation domain [START_REF] Tiwari | The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors[END_REF]. By contrast, EFD does not seem to carry a repressive domain, such as the EAR motif found in several ERFs [START_REF] Nakano | Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice[END_REF]. Consequently, the EFD repressive activity is probably either indirect or mediated by interactions with repressive factors. This was observed with AP2 proteins which act mainly as activators, but are able to interact with the TOPLESS repressor in other protein complexes [START_REF] Horstman | AINTEGUMENTA-LIKE proteins: hubs in a plethora of networks[END_REF].

Among the set of target genes (direct or indirect) of MtEFD, we paid particular attention to MtRR4 because of the key role played by CK for nodule inception and development. MtRR4 is the most highly expressed type A response regulator gene in mature wt nodules, and its expression is abolished in Mtefd-1 nodules. The CK signaling reporter, TCSn:GUS, was found to be expressed in a narrow submeristematic region in wt nodules, but in a much larger zone in Mtefd-1 nodules (Fig. 3F-G). This might be due to the absence of MtRR4, thought to be a negative regulator of CK signaling.

Considering the links between CK and the cell cycle control [START_REF] Schaller | Cytokinin and the cell cycle[END_REF], including endoreduplication, the Mtefd-1 mutant would probably be a useful tool to investigate this issue in nodules. Our attempts to complement Mtefd-1 using MtRR4 expressed from the MtEFD promoter were successful for the hypernodulation but not the nodule differentiation phenotype. This suggests that MtRR4 plays a major role in a MtEFD-dependent negative feedback loop for nodulation, while additional or alternative players are involved in the control of nodule differentiation. Examination of RNAseq data from a detailed time course study [START_REF] Schiessl | NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula[END_REF] revealed two waves of MtRR4 transcriptional activation during early nodulation stages (supplemental Fig. S3). We propose that the first wave is induced by the burst of CK signaling in the cortex leading to ccd and nodule primordium formation, since MtRR4 is a primary CK response gene [START_REF] Plet | MtCRE1dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF]. The second one might be consecutive to MtEFD transcriptional activation, and contribute to the negative feedback regulation of the CK pathway (see model in Fig. 6). Considering the numerous additional and disorganized ccd that we observed in Mtefd-1 roots (Fig. 3E and Fig. 5H), it is tempting to hypothesize that MtEFD may act locally via MtRR4 to restrict ccd and the number of nodules. In the same time, MtEFD would contribute to the differentiation of cells resulting from ccd and nodule meristem activity (most likely once infected). Whether the MtEFD-dependent control of nodulation relates to the systemic autoregulation of nodulation (AON) mechanisms remains an open question. It was proposed that MtEFD expression could be activated by the CLE/SUNN (CLAVATA3/EMBRYO-SURROUNDING REGION / SUPERNUMERIC NODULES) AON pathway because MtCLE12 overexpression leads both to MtEFD induction (albeit moderate: 2.3 fold) and repression of nodulation, in a SUNN-dependent way [START_REF] Saur | Crosstalk between the nodulation signaling pathway and the autoregulation of nodulation in Medicago truncatula[END_REF]. However, the impact of MtCLE12 overexpression on nodulation could not be tested in the Mtefd-1 mutant [START_REF] Saur | Crosstalk between the nodulation signaling pathway and the autoregulation of nodulation in Medicago truncatula[END_REF], preventing evaluation of the role of MtEFD in the CLE/SUNN pathway.

During nodule evolution, MtEFD has probably been recruited from a root developmental program, because it is only expressed in roots and nodules, in both cases in the differentiation zone. We could not however identify a robust root phenotype co-segregating with the Mtefd-1 mutation, possibly because of functional redundancy issues or technical difficulties due to root plasticity. As a first approach to investigate the evolution of MtEFD, we asked whether two MtEFD closely related genes, from M. truncatula (MtEFD2) and from A. thaliana (AtERF003), expressed under the control of the MtEFD promoter, could complement the nodule differentiation phenotype of Mtefd-1.

Unexpectedly, we found that AtERF003 but not MtEFD2 efficiently complemented Mtefd-1. This strongly suggests that the main innovation for the symbiotic function of MtEFD resides in its promoter, even though MtEFD regulates a large set of nodule-specific genes that do not exist in A. thaliana (which is unable to establish endosymbiotic interactions, whether with nitrogen-fixing bacteria or mycorrhizal fungi). This is in line with recent studies which revealed that a major mechanism for the emergence of nitrogen-fixing symbioses was the evolution of cis regulatory elements in promoter regions [START_REF] Liu | Evolution of NIN and NIN-like Genes in Relation to Nodule Symbiosis[END_REF][START_REF] Mergaert | Gene Expression in Nitrogen-Fixing Symbiotic Nodule Cells in Medicago truncatula and Other Nodulating Plants[END_REF][START_REF] Dong | An SHR-SCR module specifies legume cortical cell fate to enable nodulation[END_REF].

Still, how AtERF003 can activate nodule-specific genes is an intriguing question. A first element of response is that the DNA binding sites (DBS) of MtEFD and AtERF003 are highly conserved. Furthermore, MtEFD does not activate its nodule-specific target genes in the root, nor following its induction by the bacterial pathogen Ralstonia solanacearum [START_REF] Moreau | The symbiotic transcription factor MtEFD and cytokinins are positively acting in the Medicago truncatula and Ralstonia solanacearum pathogenic interaction[END_REF]. Therefore, another layer of regulation must exist in the nodule, to which AtERF003 is also responsive. This could be the presence of nodule-specific interactors in transcriptional complexes and / or the presence of a peculiar epigenetic landscape. Indeed many nodule differentiation genes are found in genomic regions (notably the so-called symbiotic islands) strongly enriched in epigenetic marks which are highly differential in nodules vs roots or between nodule zones [START_REF] Satgé | Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula[END_REF][START_REF] Nagymihaly | Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression[END_REF][START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF]. DNA demethylation and replacement of repressive by active histone marks could thus be important factors to give MtEFD/AtERF003 access to a set of promoter regions. In support of this hypothesis, DNA affinity purification sequencing (DAP-seq) showed that the binding to DNA of many AP2/ERFs is sensitive to cytosine methylation [START_REF] O'malley | Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape[END_REF].

The DBS of MtEFD2 is also very similar to MtEFD DBS, which it could efficiently replace in a chimeric EFD2:EFD protein, in contrast to the rest (C-terminal part) of MtEFD2 protein. This suggests a neofunctionalization of the MtEFD2 protein involving modifications in its C-terminal part, in addition to a distinct expression pattern (MtEFD2 being maximally expressed in/very close to the root and nodule division zones). Precise information is lacking regarding the role of the C-terminal part of MtEFD-related proteins, but it is likely involved in protein-protein interactions. Although we were unable to detect a nodulation phenotype of single Mtefd2 mutants, Mtefd-1 Mtefd2 double mutants were affected in the number of nodules produced. This suggests that MtEFD2 plays a role in nodule inception, possibly partially overlapping with MtEFD role, or perhaps only in the particular environment resulting from the Mtefd-1 mutation. It is possible, for example, that the downregulation of MtNF-YA1 and MtNF-B16 resulting from the Mtefd2 mutations does not affect the normal level of nodulation but becomes a limiting factor for hypernodulation in the Mtefd-1 background. Transcriptome analyses suggested that MtEFD2 contributes to the activation of MtNF-YA1/MtNF-B16 and their regulatory cascade involved in auxin biosynthesis six STY genes and one YUCCA gene. Since STY genes are required for nodule emergence [START_REF] Shrestha | Lotus japonicus Nuclear Factor YA1, a nodule emergence stage-specific regulator of auxin signalling[END_REF], it is tempting to speculate that this contributes to decreased nodule formation in Mtefd-1 Mtefd2. In any case, the MtEFD/MtEFD2 case is reminiscent of several other examples of paralogous genes playing overlapping or synergistic roles during nodulation, such as MtNOOT1/MtNOOT2 [START_REF] Magne | MtNODULE ROOT1 and MtNODULE ROOT2 Are Essential for Indeterminate Nodule Identity[END_REF] MtERN1/MtERN2 [START_REF] Cerri | The Symbiosis-Related ERN Transcription Factors Act in Concert to Coordinate Rhizobial Host Root Infection[END_REF], MtNF-YA1/MtNF-YA2 [START_REF] Laloum | Two CCAAT-box-binding transcription factors redundantly regulate early steps of the legume-rhizobia endosymbiosis[END_REF].

Conclusions

Our study revealed the recruitment to the nitrogen-fixing symbiosis of two proteins probably originally operating in roots, MtEFD and MtEFD2, expressed in the differentiation and meristematic zones of both roots and nodules, respectively, and with distinct neofunctionalization processes for each of them. We propose that, following the evolution of its promoter region leading to a strong induction in nodules, MtEFD has become a major regulator of nodule symbiotic differentiation, contributing directly or indirectly to the activation of almost half of the genes specifically expressed in the late differentiation zone, while preventing the premature activation of some of them. In addition, MtEFD is involved in the control of root cortical cell divisions and nodule number, probably through MtRR4 action on CK signaling. For a better understanding of the MtEFD-associated upstream and downstream regulation network, it would now be necessary to identify the cis and trans elements that control MtEFD expression, and to identify the DNA sites bound by MtEFD, keeping in mind that these may be affected by DNA methylation. Identification of protein interactors and possible post-translational modifications would also be valuable to better understand the differences in the mode of action of MtEFD and MtEFD2, and more generally to document the functional importance of the C-terminal part of AP2/ERF proteins.

Materials and Methods

Plant genotypes

The Medicago truncatula A17 was used as the wt reference. The Mtefd-1 null deletion mutant (backcrossed twice) was previously described [START_REF] Vernié | EFD Is an ERF Transcription Factor Involved in the Control of Nodule Number and Differentiation in Medicago truncatula[END_REF]. The TR36, TRV36, TRV43, TR183 and TR3 mutant seeds [START_REF] Maunoury | Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches[END_REF] were provided by Peter Mergaert (I2BC, Gif-sur-Yvette). The Mtefd2 mutant was generated by multi-guide CRISPR-Cas9 mutagenesis of MtEFD2 using M. truncatula A17 hairy root transformation. It was therefore a mix of mutations, with >95% of large deletions or frame-shift mutations, as described in supplemental Fig. S9. The Mtefd-1 Mtefd2 double mutant was similarly generated by CRISPR-Cas9 mutagenesis of MtEFD2 using Mtefd-1 hairy roots.

Plant growth and hairy root transformation

Seeds were sterilized and placed at 4°C during 5 days then germinated one night at 25°C. Germinated seeds were grown on agar plates containing Fahraeus medium supplemented with 0.5 mM NH4NO3 and covered with a filter paper (at 25°C; light/dark photoperiod of 16h/8h), in attapulgite (at 25°C) or in aeroponic caissons (at 22°C; 75% hygrometry; light intensity: 200 E.m -2 .s -1 ; light-dark photoperiod: 16 h-8 h). In aeroponic caissons, plants were grown for about seven days in aeroponic medium supplemented with 5 mM NH4NO3 [START_REF] Barker | Growing Medicago truncatula: choice of substrates and growth conditions[END_REF] and then in nitrogen-free aeroponic medium three days before inoculation with Sinorhizobium meliloti 2011.

Hairy root transformations (analyses of all GUS constructs, Mtefd-1 complementation experiments and CRISPR-Cas9 mutagenesis of MtEFD2) were performed using ARqua1 Agrobacterium rhizogenes as described [START_REF] Boisson-Dernier | Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations[END_REF]. Composite plants were selected on kanamycin 25 mg/L (20°C; day/night, 12h) and systematically checked for the expression of the DsRED marker present on the T-DNA construct (stereomicroscope MZFLIII, Leica). They were transferred in aeroponic caissons following two weeks of growth on plates at 20°C (Fahraeus medium with 5 mM NH4NO3) and grown for seven days in the presence of 5 mM NH4NO3 before nitrogen starvation and inoculation with S.

meliloti.

Under aeroponic conditions, nodules started fixing nitrogen at around 7 dpi and 10 dpi for nontransformed plants and transformed composite plants, respectively.

Light and fluorescence microscopy

Bacterial viability (LIVE/DEAD® BacLight Viability Kit -Thermoscientific) and NifH:GFP expression were analyzed using 80 µm sections (microtome LEICA VT 1000S) of nodules embedded in 8% low-melting agarose (NuSieve TM GTG TM Agarose Lonza). They observed with an Axioplan 2 microscope (Zeiss).

For GFP and SYTO TM 9, the fluorescence observations were made using an excitation wavelength of 475nm, a barrier filter of 394 nm and an emission filter of 397nm. For propidium iodide and DsRed, the set up was an excitation wavelength of 546 nm, a barrier filter of 580 nm and an emission filter of 590 nm. Images were taken with an Axiocam MRc camera (Zeiss). In complementation assays, sections were stained with 0.5µg/ml DAPI (4′, 6-diamidino-2-phenylindole) (excitation wavelength of 365nm, barrier filter 395nm and emission filter 397 nm).

For analysis of promoter:GUS fusions, roots or nodules were pre fixed in paraformaldehyde 0.4% (v/v) for 1h, stained for β-glucuronidase (GUS) activity for 4h at 37°C and fixed in glutaraldehyde 2% (v/v). Longitudinal sections were made from roots embedded in 8% (w/v) low-melting agarose, using a vibrating microtome (Leica VT 1000S), and observed with a Zeiss Axiophot light microscope.

For analyses of thinner sections (TCSn:GUS-transformed S.meliloti-inoculated roots), roots fragments were fixed under vacuum in 0.1 M sodium phosphate buffer containing 0.3% (v/v) paraformaldehyde, rinsed in Phosphate buffer (three times) and incubated in GUS staining buffer [0.1 M NaPO4 pH7.4, 400 µM K3 Fe(CN)6, 400 µM K4Fe(CN)6, 0.625% (w/v) X-Gluc] for 6 hours at 37°C. Samples were rinsed and fixed again in 0.1 M Phosphate buffer containing 2% (v/v) glutaraldehyde. Roots were then dehydrated by sequential treatments in 30, 50, 70, 90 and 100% (v/v) ethanol and embedded in glycolmethacrylate (Technovit 7100; Haereus-Kulzer) according to the manufacturer's instructions. Images of 10 µm sections were acquired using a Nanozoomer HT (Hamamatsu).

RNA extraction and transcriptome analyses

Total RNA was extracted from ~1cm-long root tips, isolated root nodules or nodulated root fragments, using the Qiagen rNeasy Plant kit according to the supplier's instructions. These root tips represent good reference samples as they contain the region of MtEFD and MtEFD2 expression and part of the nodulation competence zone. Microarray analyses were carried out at URGV (Evry, France), using microarrays based on the Roche-NimbleGen technology. High density microarray slides contained twelve chambers, each with 249,087 oligonucleotides representing 83,029 M. truncatula A17 loci (each oligonucleotide in triplicate). Four independent biological replicates were analyzed, with ≥ 12 aeroponically grown plants per replicate. For each comparison, one technical replicate per biological replicate with fluorochrome reversal was performed (i.e., four dye-switch hybridizations per comparison). The labeling of cRNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products) and the hybridization to the slides were performed as described in [START_REF] Lurin | Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis[END_REF]. Two micron scanning was performed with InnoScan900 scanner (Innopsys R , Carbonne, FRANCE) and raw data were extracted using Mapix R software (Innopsys R , Carbonne, FRANCE). For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths 635 nm (red) and 532 nm (green); a global intensity-dependent normalization using the Loess procedure [START_REF] Yang | Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation[END_REF] was performed to correct the dye bias. Differential analysis was based on log-ratios averaged over duplicate probes and technical replicates. Hence the number of available data for each gene equaled the number of biological replicates and was used to calculate the moderated t-test [START_REF] Smyth | Linear models and empirical bayes methods for assessing differential expression in microarray experiments[END_REF]. Since no evidence that specific variances vary between probes was found by Limma, moderated t-statistic was assumed to follow a standard normal distribution.

Adjusted p-values were calculated using the optimized FDR approach [START_REF] Storey | Statistical significance for genomewide studies[END_REF] with R software (R development Core Team, 2005). SqueezeVar function of the library Limma was used to smooth the specific variances by computing empirical Bayes posterior means. The library kerfdr was used to calculate the adjusted p-values.

RNAseq analyses were performed with three biological replicates, each representing pools of >12 independent plants or transformed roots. Oriented RNAseq was carried out at the GeT-PlaGe core facility, INRAE Toulouse, France (http://www.get.genotoul.fr). RNAseq libraries were prepared according to Illumina's protocols using the TruSeq Stranded mRNA sample prep kit (Illumina) to analyze mRNA. Briefly, mRNA was selected using poly-T beads. Then, RNA was fragmented to generate double-stranded cDNA, and adaptors were ligated to be sequenced. Libraries were amplified by 11 cycles of PCR. Library quality was assessed using a Fragment Analyzer (Advanced Analytical Technologies, Inc.) and libraries were quantified by qPCR using a Library Quantification Kit (Kapa). RNA-seq experiments were performed using a NovaSeq technology, with a paired-end read length of 2 × 150 bp.

Expression measure was performed using M. truncatula annotation version 5.1.8. We used nfcore/rnaseq pipeline version 3.0 (doi:10.5281/zenodo.4323183) with the following parameters " --skip_alignment --pseudo_aligner salmon " that performs adapter and quality trimming with Trim Galore software version 0.6.6. followed by transcript assignation and quantification with salmon tool (version 1.4.0). DEGs were detected with EdgeR Bioconductor package version 3.34.0 [START_REF] Robinson | Small-sample estimation of negative binomial dispersion, with applications to SAGE data[END_REF]. Genes with no counts across all libraries were discarded prior to further analysis. Normalization was performed using trimmed mean of M values method [START_REF] Robinson | A scaling normalization method for differential expression analysis of RNA-seq data[END_REF]. Heatmaps showing correlation between replicates were generated using the package pheatmap version 1.0.12 (https://CRAN.R-project.org/package=pheatmap) with sample-to-sample Euclidean distances. DEGs were called using the GLM (Fitted generalized linear models) likelihood ratio test, with an FDR adjusted p-value [START_REF] Benjamini | The control of the false discovery rate in multiple testing under dependency[END_REF]. Analyses of GO term enrichment were performed using the topGO package version 2.44.0 [START_REF] Rahnenfuhrer | Improved scoring of functional groups from gene expression data by decorrelating GO graph structure[END_REF] Correspondences between gene identifiers (Nimblegen, Affymetrix, Mt20120830, Mt4.0 and Mt5.0), as well as gene names/acronyms were from Legoo [START_REF] Carrère | LeGOO: An Expertized Knowledge Database for the Model Legume Medicago truncatula[END_REF][START_REF] Carrere | MtExpress, a Comprehensive and Curated RNAseq-based Gene Expression Atlas for the Model Legume Medicago truncatula[END_REF] and downloaded from the downloads section of Mt5.0 genome browser (https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/), as of 20211025. NCR genes were annotated using [START_REF] Montiel | Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides[END_REF][START_REF] De Bang | Genome-Wide Identification of Medicago Peptides Involved in Macronutrient Responses and Nodulation[END_REF], with correspondences to Mt5.0 annotated proteins established using blastP. The published NCR number was used for hits with > 90% identity over >90% query. Additional NCR numbers (MtNCR-new1…) were used for 86 Mt5.0 proteins with the Interpro domain IPR009810:Late nodulin and not corresponding to published NCRs. In total 691 NCR genes were annotated in Mt5.0.

Phylogenetic analysis

Sequences were aligned with MAFFT (v7.310) [START_REF] Katoh | Recent developments in the MAFFT multiple sequence alignment program[END_REF]. Columns with more than 50% of gaps were pruned. The phylogenetic tree was built with IQ-TREE (v1.5.5) [START_REF] Nguyen | IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF]. IQtree used ModelFinder [START_REF] Kalyaanamoorthy | ModelFinder: fast model selection for accurate phylogenetic estimates[END_REF] to identify JTT+G4 as the best-fit evolutionary model according to Bayesian information criterion (BIC). Branches were tested by SH-like aLRT [START_REF] Anisimova | Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative[END_REF]. Finally, the tree was drawn with Itol [START_REF] Letunic | Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation[END_REF].

Statistical analysis

Endoreduplication levels were compared using one-way Anova followed by post hoc Tukey test.

Normality (Shapiro's test) and homoscedasticity (Bartlett's test) were previously verified (P-value>0.05). Transcriptomic and phylogenetic data were analyzed as described above. Nodule numbers were statistically analyzed with the Kruskal-Wallis multiple comparison test or the Wilcoxon two-sample method using R software (version 4.1.0) (two-sided tests) and the "agricolae" package (version 1.3-5). Boxplots were drown with the 'ggplot2' package (version 3.3.5).

Construct production

The TCSn:GUS construct was previously described [START_REF] Jardinaud | A Laser Dissection-RNAseq Analysis Highlights the Activation of Cytokinin Pathways by Nod Factors in the Medicago truncatula Root Epidermis[END_REF]. For pMtWOX5:GUS, a 2108 bp fragment upstream the ATG was PCR amplified (PrimeSTAR® HS DNA Polymerase, TaKaRa) using MtWOX5 promoter Forward and Reverse (supplemental Table S5). The amplicon was then cloned into a modified pCAMBIA2200 vector (Fliegman et al., 2013) using the BsaI sites to generate the pMtWOX5:GUS binary vector. For the transcriptional fusion between the MtEFD and MtEFD2 promoters and the GUS gene, 960bp and 2457bp regions were amplified respectively, using MtEFD and MtEFD2 promoter Forward and Reverse primers (supplemental Table S5). The GUS fusion was achieved using BsaI digest-T4 DNA ligase ligation protocol with a mix of amplicon and level 0 plasmids EC47822 (pL1V-R2), EC75111 (pL0M-SC-GUS) and EC41414 (pL0M-T-T35S-1) [START_REF] Schiessl | NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula[END_REF]. Level2 assembly was performed using BpiI digest-T4 DNA ligase ligation protocol and the following level1 blocs: the previously generated level1 assembly, EC15529 (pL1M-R1-p35S-Kana-TNOS); pUbiq (EC15062-pAtUbi10; EC15073 -DSRed; EC41432-TOCS-1;EC47822), End Linker (EC41766 -pL1M-ELE-3) and the binary vector EC50506 (pL2V-1-50506).

For the complementation experiments, MtEFD, MtEFD2, MtRR4 and AtERF003 wild type coding sequences were amplified with MtEFD, MtEFD2, MtRR4, AtERF003 Forward and Reverse primers respectively (supplemental Table S5). To introduce P57/T57 point mutations in MtEFD and MtEFD2, fragments were amplified using MtEFD and MtEFD2 mut Forward and Reverse (supplemental Table S5). For the chimeric MtEFD:MtEFD2 and MtEFD2:MtEFD constructs (domain swaps), fragments were amplified with MtEFD:MtEFD2 chim Forward and Reverse, and MtEFD2:MtEFD1 chim Forward and Reverse, respectively (supplemental Table S5). Level1 (pMtEFD:coding sequence: T35S) and Level2 assemblies were generated as previously described.

For CRISPR-Cas9 mutagenesis, guide RNAs (gRNA; listed in supplemental table 5) were designed with CRISPOR (version 4.8, http://crispor.tefor.net/) program, with the INRA A17r5.0 r1.6 M. truncatula genome release [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF] and the "20bp-NGG-Sp Cas9, SpCas9-HF1, eSpCas9 1.1" option.

The S. pyogenes Cas9 coding DNA sequence was used, with a SV40 NLS sequence added at the Cterminus. Four gRNAs, interspaced by tRNAs, were preassembled as a synthetic polycistronic gene, as described [START_REF] Xie | Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system[END_REF]. They were expressed from M. truncatula U6.1 (MtrunA17_Chr3g0136831) and U6.6 (MtrunA17_Chr7g0251721) promoters (each for two guides). Genotyping was performed by nested PCR and systematic sequencing of PCR products, using individual transformed root systems, on DNA from root segments adjacent to nodules.

Accession numbers

GSE138899 (Nimblegen analyses); SRP349933 (RNAseq of CRISPR-Cas9 mutant and control samples); SRP349926 (RNAseq of non-transformed wt and Mtefd-1 samples) Tables Table 1. The Mtefd-1 mutation leads to downregulation of a set of genes expressed in immature nodules and early differentiation zone, as well as premature activation of a set of genes expressed in the late differentiation zone. Genes associated with nodule senescence and immune response are weakly affected in Mtefd-1. Gene expression patterns are from [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF]. FC = fold change. A, Top: 16 expression patterns defined in wt nodules [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF] et al., 2013[START_REF] Feng | Processing of NODULE INCEPTION controls the transition to nitrogen fixation in root nodules[END_REF][START_REF] Jiang | NIN-like protein transcription factors regulate leghemoglobin genes in legume nodules[END_REF] regarding MtRSD, NIN and NLP2 in nodule development and activity. In the primordium cells, MtEFD would be first involved in the differentiation of nodule, following the Nod Factor signaling pathway leading to induction of root cortical cell divisions by cytokinins (CK) and auxin. MtEFD would also contribute to the transcriptional activation of MtRR4, a negative regulator of CK signaling, thereby contributing to negative feedback regulation of ccd and nodulation. In the nodule, MtEFD would be involved in the differentiation of cells generated by the nodule meristem, including late endoreduplication cycles (C16 to C32 and C64). Based on their respective expression profile (Fig. 2), MtEFD would act before two other regulators of nodule development, namely MtRSD and MtNLP2. MtNIN is only weakly downregulated in the Mtefd-1 mutant at 10 dpi. MtEFD also contributes to the regulation of CK signaling in the nodule (Fig. 3G), possibly through MtRR4, but MtRR4 cannot complement the Mtefd-1 differentiation nodule phenotype, whereas it complements the Mtefd-1 hypernodulation phenotype. MtEFD2 is proposed to contribute to MtNF-YA1 expression and action, through its regulation cascade comprising MtSTY genes. NF = Nod Factors; CK = cytokinins; ccd = cortical cell divisions; P= Pericycle; E= endodermis; C3, C4, C5 = cortical cell layers 3, 4, 5 respectively.

RT-qPCR

MtEFD

  They were cloned using Golden Gate cloning technology into backbone plasmids provided by the ENSA project (Schiessl et al., 2019) (Engineering Nitrogen Symbiosis for Africa ; https://www.ensa.ac.uk ). T-DNAs included a kanamycin resistance module (p35S:KanR:TNos) and a DsRed fluorescent reporter module (pAtUbi10:DsRed:TOcs), located respectively close to the right and left borders of the T DNA.

Figure 1 .

 1 Figure 1. Mtefd-1 nodules exhibits an unusual plant cell endoreduplication phenotype, and a poor S. meliloti nifH expression level, without significant cell death. A, B, Comparison of plant (A) and bacterial (B) cell endoreduplication in wt and Mtefd-1 nodules (n=19). C, D, E, F, Levels of plant cell endoreduplication in wt (C), TRV43 (D), Mtefd-1 (E), and TR183 (F); each graph is representative of observations from two biological repeats (n=22, 9, 19, 16 respectively). G, H, Expression of S. meliloti pnifH:GFP in wild-type (G) and Mtefd-1 (H) nodules. DNA was stained with DAPI (4′,6-diamidino-2-phenylindole; blue fluorescence). I, J Live-dead assay of wild-type (I) and Mtefd-1 (J) nodules. Cells showing a cyan signal (SYTO TM 9) are alive while the magenta signal (propidium iodide) correspond to dead cells as well as plant nuclei (particularly abundant in the nodule meristem). Asterisks indicate the nodule meristematic region. Nodules were analyzed at 21 days postinoculation. Bars= 250 µm. Plants were grown under aeroponic condition for A, B, G, H, I, J, and attapulgite condition for C, D, E, F.

Figure 2 .

 2 Figure 2. Expression pattern of MtEFD and other regulators of nodule development, and downregulation by the Mtefd-1 mutation of many genes expressed during late nodule development.A, Top: 16 expression patterns defined in wt nodules[START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF], based on the average relative expression levels (in percentage of total expression) in five laser-dissected nodule zones, namely zone I (ZI), distal zone II (dZII), proximal zone II (pZII), interzone II-III (IZ) and fixation zone III (ZIII) (RNAseq data from[START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF], mapped on the MtV5.0 genome release and reanalyzed). P1 to P11 are mostly expressed in a single zone, while P12 to P15 are expressed in all zones with a preferential one. A, Bottom: expression profile (with pattern number in parentheses) of MtEFD and its paralogue MtEFD2, as well as MtRR4 and three TF genes thought to regulate nodule differentiation, shown in our final model (Fig.6). B, Distribution of all nodule-expressed genes amongst the 16 expression patterns (wild-type nodules). C, Number of genes upregulated in mature (10 dpi) vs immature (4 dpi) wild-type nodules (fold change FC>4; FDR<0.01), per expression pattern. D, Cumulative number of genes up-and down-regulated at least 2-fold in Mtefd-1 vs wt 10 dpi nodules. E, Number of genes down-regulated in Mtefd-1 vs wild-type 10 dpi nodules (FC<-4; FDR<0.01), per expression pattern.

Figure 3 .

 3 Figure 3. Cortical cell divisions and cytokinin signaling are modified in Mtefd-1 S.meliloti-inoculated roots and nodules. A-D, Expression pattern of the CK signaling reporter TCSn:GUS in young (stage III: A, B) and older (stage IV-V: C, D) nodule primordia, in wild-type (A, C) and Mtefd-1 (B, D) backgrounds, at 4 dpi with S. meliloti phemA:lacZ, using A. rhizogenes-transformed plants (10 µm sections), following revelation of GUS and LacZ activity (blue and violet signal, respectively). Note the stronger GUS signal in Mtefd-1.E, Section (10 µm) of TCSn:GUS Mtefd-1 roots at 4 dpi following GUS staining. Note the presence of numerous cortical cell divisions (asterisks) within and between nodule primordia. F-G, Expression of TCSn:GUS in mature wild-type (F) and Mtefd-1 (G) nodules (50 µm sections) H-I, Expression of pWOX5:GUS in mature wild-type (H) and Mtefd-1 (I) nodules (50 µm sections) F to I nodules were analyzed at 21 dpi. Bars =100 µm (A-E) or 250 µm (F-I).

Figure 4 .

 4 Figure 4. The type A response regulator MtRR4 complements the hypernodulation phenotype of Mtefd-1 but not its nodule differentiation phenotype. A-D, Mtefd-1 complementation assays, using S. meliloti NifH:GFP-induced nodules and Agrobacterium rhizogenes-mediated root transformation. A, wild-type mature nodules; B, Mtefd-1 nodules are efficiently complemented by pMtEFD:MtEFD; C, pMtEFD:MtRR4 fails to restore full nodule development and NifH:GFP expression in Mtefd-1; D, Mtefd-1 nodule transformed with an empty vector. Blue and green fluorescence: DAPI-staining of DNA and GFP expression, respectively (50 µm sections). DsRed expression (transformation marker) was checked for all nodules. E, Analysis by flow cytometry of the endoreduplication level of wt, efd-1, and efd-1 transformed by MtEFD or MtRR4; MtRR4 fails to restore the 32C level of plant cell endoreduplication.

  Figure 1

  Figure 2

  indicate the BsaI restriction site and spacer used for Golden Gate cloning; letters in bold correspond to point mutations Supplemental Figure S1. Clustering analysis of RNAseq data produced using non-inoculated N-starved root tips and isolated S.meliloti 2011-induced nodules at 4 and 10 days post-inoculation, in wt (A17) and Mtefd-1 backgrounds. Three biological replicates (R1, R2, R3) per sample. Color scale : euclidian distances between samples. Supplemental Figure S2. Genes up-and down-regulated in wt nodules, and comparison of genes downregulated in the Mtefd-1, rsd-1 and dnf-7 mutants. A, B, C, range of up-and down-regulation in wt nodules (cumulative values) D, range of up-and down-regulation in Mtefd-1 nodules 4 dpi (cumulative values) E, Venn diagram showing the overlap between genes downregulated in the Mtefd-1 (10 dpi), rsd-1 (8 dpi) and dnf7 (15 dpi) mutant nodules (≥2-fold downregulation; based on available Mt5.1.8 corresponding loci) E, Venn diagram showing the overlap between genes downregulated in the Mtefd-1 (10 dpi), rsd-1 (8 dpi) and dnf7 (15 dpi) mutant nodules (≥2-fold downregulation; based on available Mt5.1.8 corresponding loci) F, G: Distribution of down-regulated genes in rsd-1 and dnf7 mutant nodules, amongst the 16 expression patterns defined in Pecrix et al. 2018 (see Fig. 2A) Time course analysis of MtEFD, MtRR4 and MtEFD2 expression. A, Q-RT-PCR analysis of expression in wild-type siblings (SWT) or Mtefd-1 mutant. N0 = non-inoculated roots; N4, N6, N11= isolated nodules at 4, 6 and 11 days post-inoculation (dpi), Respectively. Mean of four biological replicates, with 20 plants per replicate. Bars = ± SE. B, RNAseq analysis of spot-inoculated roots vs mock treatment; data extracted from (Schiessl et al., 2019) . Macroscopic development of nodules in the Mtefd-1 mutant and the Mtefd-1 Mtefd2 double mutant. Nodules harvested at 17 days post inoculation with S.meliloti 2011. Scale bar = 2mm

Table 3 .

 3 Examples of genes downregulated in Mtefd2 mutant 4 dpi nodules. The three last columns correspond to CRISPR-Cas9 experiments in wt M.truncatula A17 or Mtefd-1 background. MtEFD2 correspond to non-edited MtEFD2 gene, as determined by genotyping and sequencing. FC= fold change. Except where specified, all FC values are significant (FDR<0.01)

	Gene Id (Mt5.0 r1.8)	Gene name	FC wt 4 dpi nodules	FC Mtefd-1	FC Mtefd-2	FC Mtefd-2
			vs root tips (non-	vs wt 4 dpi	vs MtEFD2	vs MtEFD2
			transformed)	nodules	(wt A17	(Mtefd-1
					background)	background)
	MtrunA17_Chr1g0177091 MtNF-YA1	1964.6	2.5	-4.3	-5.3
	MtrunA17_Chr4g0067091 MtNF-YB16	240.5	2.3	-2.5	-4.1
	MtrunA17_Chr2g0296321 MtNF-YB18	344.9	2.2	-2.2	-3.4
	MtrunA17_Chr2g0301491 MtNodGRP12	588.1	3.2	-6.9	-33.8
	MtrunA17_Chr4g0033101 MtNodGRP23	6427.3	2.8	-56.9	-45.6
	MtrunA17_Chr3g0094011 MtNodGRP4;MtN25	2896.3	2.6	-41.1	-31.8
	MtrunA17_Chr8g0386521 MtSYMREM1	3350.1	2.6	-19.6	-15.9
	MtrunA17_Chr5g0422861 MtNCR025	1234.7	2.3	-23.4	-35.5
	MtrunA17_Chr6g0474261 MtNCR109	1105.1	3.1	-8.6	-17.6
	MtrunA17_Chr8g0372461 MtSTY2	35.8	-1.6	-2.4	-2.2
				(FDR=0.99)
	MtrunA17_Chr8g0353111 MtSTY9	51.6	-1.7	-3.3	-2.3
				(FDR=0.99)
	MtrunA17_Chr7g0262591 MtYUC8	1.9	-1.8	-1.8	-2.2
				(FDR=0.06)

  , based on the average relative expression levels (in percentage of total expression) in five laser-dissected nodule zones, namely zone I (ZI), distal zone II (dZII), proximal zone II (pZII), interzone II-III (IZ) and fixation zone III (ZIII) (RNAseq data from[START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF], mapped on the MtV5.0 genome release and reanalyzed).

	P1 to P11 are mostly expressed in a single zone, while P12 to P15 are expressed in all zones with a
	preferential one. A, Bottom: expression profile (with pattern number in parentheses) of MtEFD and
	its paralogue MtEFD2, as well as MtRR4 and three TF genes thought to regulate nodule
	differentiation, shown in our final model (Fig. 6).
	B, Distribution of all nodule-expressed genes amongst the 16 expression patterns (wild-type
	nodules).
	C, Number of genes upregulated in mature (10 dpi) vs immature (4 dpi) wild-type nodules (fold
	change FC>4; FDR<0.01), per expression pattern.
	D, Cumulative number of genes up-and down-regulated at least 2-fold in Mtefd-1 vs wt 10 dpi
	nodules.
	E, Number of genes down-regulated in Mtefd-1 vs wild-type 10 dpi nodules (FC<-4; FDR<0.01), per
	expression pattern.

  F, Analysis of the average nodule number produced in wt, efd-1, and efd-1 transformed by MtEFD or MtRR4; pMtEFD:MtRR4 complements the hypernodulation phenotype of Mtefd-1 Statistical significance was analyzed by Kruskal-Wallis multiple comparison test. Samples with different letters (a and b) show a statistical difference for P ≤ 0.001. Root fresh weight, measured after nodule removal. Center lines, box range, whiskers, black points and red points indicate the median, lower to upper quartile, 1.5× interquartile, outliers, and individual values respectively. All nodules were analyzed at 21 dpi. Bars = 250 µm. MtEFD2, a MtEFD paralogue with a distinct expression pattern, is unable to complement the Mtefd-1 nodule phenotypes, in contrast to AtERF003, and positively regulates the nodule number in the Mtefd-1 mutant background. A, Multiple alignment analysis of MtEFD-like proteins in M. truncatula (MtEFD2; 79.1% similarity) and A. thaliana (AtERF003; 77.9% similarity). Amino acids involved in the direct interaction with DNA[START_REF] Nakano | Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice[END_REF] are indicated by an asterisk (AP2/ERF domain). CMV-1 and CMV-2 (= conserved motifs 1 and 2 of group V ERF proteins) are as previously defined[START_REF] Cerri | AP2/ERF transcription factors and root nodulation[END_REF]. Red and blue fonts: high and low consensus value (>90% and >50%, respectively). AtERF003, MtEFD and MtEFD2 expressed from MtEFD promoter. K, Average number of nodules produced at 17 dpi in the Mtefd-1 mutant and the Mtefd-1 Mtefd2 Model of MtEFD and MtEFD2 involvement in M. truncatula nodule development, integrated with other known regulators. Inspired by[START_REF] Xiao | Fate map of Medicago truncatula root nodules[END_REF] Shresta et al., 2020 for primordium and nodule meristem formation, by[START_REF] Schiessl | NODULE INCEPTION Recruits the Lateral Root Developmental Program for Symbiotic Nodule Organogenesis in Medicago truncatula[END_REF] Soyano et al., 2019 regarding NIN and auxin biosynthesis, and by Sinharoy

	Figure 5. double mutant.
	b: P-value<0.001 (Kruskal-Wallis test). Root fresh weight, measured after nodule removal.
	Bars = 250 µm (B, C, F, G, I) or 100 µm (D, E, H).
	Agrobacterium rhizogenes-mediated root transformation for B to K.
	Figure 6.

B, C, Expression of pMtEFD:GUS (B) and pMtEFD2:GUS (C) in wild-type root tips (cleared roots) D, E, Expression of pMtEFD:GUS (D) and pMtEFD2:GUS (E) in wild-type nodule primordia (10 µm sections) F, G, Expression of pMtEFD:GUS (F) and pMtEFD2:GUS (G) in wild-type mature nodules (50 µm sections) H, Expression of pMtEFD2:GUS in a Mtefd-1 mutant roots inoculated with S. meliloti 2011 (10 µm section). Asterisks indicate unorganized cortical cell divisions adjacent to nodule primordia. B to H: observations following revelation of GUS activity. I, Sections (50 µm) of S.meliloti NifH:GFP-induced nodules (17 dpi) produced on wt M. truncatula A17 transformed with an empty vector, and Mtefd-1 mutant transformed with an empty vector, MtEFD2 and AtERF003 expressed from MtEFD promoter; note the presence of differentiated bacteroids around a central vacuole in wt nodules and pEFD:AtERF003 transformed nodules only. Blue and green fluorescence: DAPI-staining of DNA and GFP expression, respectively. DsRed expression (transformation marker) was checked for all nodules. J, Average number of nodules produced at 21 dpi by Mtefd-1 transformed by an empty vector,

Table S1 .

 S1 Values with different letters are significantly different (P-val<0.05; Anova Tukey post hoc test); distinct groups were identified in function of the percentage of nuclei in 16C, 32C and 64C. TR36 produced pink (TR36p) and white (TR36w) nodules with different levels of endoreduplication. Groups I, II, III and IV are as defined in[START_REF] Maunoury | Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches[END_REF] Percentage of 16C, 32C and 64C nuclei in nodules of wt A17, Mtefd-1 and other fix minus mutants Supplemental

	Supplemental
	8 a	4.7 a	4.2 a	3.8 ab	2.7 bc	2.4 bcd	3.5 ab	1.7 d	2 cd	1.4 d	2.3 a	2 a
	32C (%) 9.9 a	1.2 b	9.4 a	7.9 a	2.9 b	3.1 b	0.8 c	1 c	0.5 c	0,5 c	10.5 a	0.6 b
	64C (%) 1.2 a	0.2 b	0.4 ab	0.5 a	0.2 bc	0.1 cd	0.1 d	0.1 d	0.1 d	0 d
	group		I	I	II	II	III	IV	IV	IV

Table S5 .

 S5 Primers and guides

		FORWARD	REVERSE
	CLONING
	MtEFD promoter	ggtctcaggagGGTACCCACCCCGAACCC	ggtctcacattCAGCTTTGCACATATTTATAT
	MtEFD2 promoter	ggtctcaggagATAAGACTTAAACATATATATAACT	ggtctcacattACCTTGTTTTTCTTTATGTAT
	MtWOX5 promoter	ggtctccaaatCTTAAATTCCTATCATCCAATTTTTGG	ggtctcccaatATTTCAATTCTAGATGTTTTTACTCTGAACTC
	MtEFD	ggtctcaaatgCTTCACCTTCACTTCACTTCACTTAAGTCAC	ggtctcaaagcTAAGATGAACCAACAGAACAAAGCTCAATTGAACC
	MtEFD2	ggtctcaaatgATGGCTAGACCACAACAACGTTACCGAGGC	ggtctcaaagcGTCTTTTGACAAAACTCCCTTACATTAC
	MtEFD mut	ggtctccggaACAAAAGCACGTACCAATTTCCCATACAACCCAAATGG ggtctccTTCCACACATTAATCTTGCTGCTTCATCATATGCTCTTGC
	MtEFD2 mut	ggtctccggaCCAAGGGCAAGAACAAATTTCCCTTAC	ggtctccGTCCACACATTAATCTTGCTGCTTCATCG
	MtEFD:MtEFD2 chim	ggtctcctgggAAATTGGTACGTGCTTTTGGTCCACACATT	ggtgtgggggaTACAACCCCAATGTGTCACAATCATCATCTT
	MtEFD2:MtEFD chim	ggtctccccctTACAACCCAAATGGACCACAATCTTCTTCAT	ggtctccagggAAATTTGTTCTTGCCCTTGTTCCACACA
	AtERF003 (At5G25190)	ggtctcaaatgAGGTCTCGATTGTCCTTCACTCTCATCATTCTTTC	ggtctcaaagcAGGTCTCGCGTAATACATAACATAGCATATGATC
	MtRR4	ggtctcaaatgATGGAGACAAACAGTGTTGT	ggtctcaaagcTTCGGTGCCGGTCATTTTAAG
	NF9808 analysis
	MtEFD2 primers	GACCACAACAACGTTACC	GAGGACACTAGCTGAATG
	CRISPR GUIDES
	mgMtEFD2a-F	TAGGTCTCGACAAAGGCATTGGTTTTAGAGCTAGAA	CGGGTCTCTTTGTCGGACGCCTGCACCAGCCGGG
	mgMtEFD2b-F	TAGGTCTCACAACGTTACCGGTTTTAGAGCTAGAA	CGGGTCTCCGTTGTTGTGGTCTTGCACCAGCCGGG
	mgMtEFD2c-F	TAGGTCTCCTAGAATATGGCTGTTTTAGAGCTAGAA	CGGGTCTCTTCTAGTTTTTCTTGCACCAGCCGGG
	mgMtEFD2d-F	TAGGTCTCATGTGTGGAACAAGTTTTAGAGCTAGAA	CGGGTCTCACACATTAATCTTTGCACCAGCCGGG
	MtEFD2 genotyping
	MtEFD2	TCTACAACTAGGAATGCAAATTGC	CCCCAACTTGCAATTGACCCAA
	MtEFD2 nested	AAATCCTTTGTGGGACCCTC	GAGGACACTAGCTGAATG

Acknowledgments

We are grateful to Peter Mergaert (ISV CNRS, Gif-sur-Yvette) for providing us with Fix-Medicago mutants, Ludovic Legrand (LIPME, Toulouse) for helping us with Alphafold, Rene Geurts and Eric Limpens for providing us with the S. meliloti NifH:GFP strain, and Jean-Marie Prospéri (INRA, Montpellier) for supplying M. truncatula A17 seeds. We thank the ENSA project for providing plasmids for Golden Gate cloning, and the FRAIB-TRI Imaging Platform (Université de Toulouse, CNRS) for its excellent support. RT-qPCR and RNAseq experiments were carried out at the Toulouse Genopole GeT-PlaGe platform.

Funding information

This work was supported by the Agence Nationale de la Recherche (grant "GENOPEA" ANR-09-GENM-026), by INRAE (CJS fellowship for J.F.; SPE grant "EFFOR") and the "Laboratoire d'Excellence (LABEX) "TULIP" (ANR-10-LABX-41). The POPS platform benefited from the support of the LABEX Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).

Supplementary Data

Supplemental Table S1. Percentage of 16C, 32C and 64C nuclei in nodules of wt A17, Mtefd-1 and other fix minus mutants. S2. Differentially expressed genes detected using Nimblegen gene chips. S3. RNAseq analysis of nitrogen-starved root tips, 4 dpi and 10 dpi nodules in wt A17 and Mtefd-1 backgrounds. Tab 2: RNAseq data of the nine MtRRA genes with RNAseq reads. Tab 3: Venn diagrams showing the overlap between differentially regulated genes detected using Nimblegen chips and RNAseq data. Tab 4-7: GO term enrichment analysis for genes down-and upregulated in Mtefd-1 vs wt 4 and 10 dpi nodules. S4. RNAseq analysis of 4 dpi nodulated root fragments of Mtefd2 mutants generated by CRISPR-Cas9 in wt A17 and Mtefd-1 backgrounds. Tab2: GO term enrichment analysis for genes downregulated in efd2 mutants in both wt A7 and Mtefd-1 mutant, while upregulated in Mtefd-1 at 4 dpi. RNA from 40 dpi nodules harvested from 8 (EV, MtEFD), 11 (MtRR4) and 13 (MtEFD2) independently transformed roots (2 biological replicates). Transcript levels were normalized using the mean of two housekeeping genes.

Supplemental Table

Supplemental Table

Supplemental Table

Supplemental Table S5. Primers and guides

Supplemental

Error bars = ± SE.

Relative expression

Relative expression

Relative expression [START_REF] Sehnal | Mol* Viewer: modern web app for 3D visualization and analysis of large biomolecular structures[END_REF]. The AP2/ERF domain comprises three β-sheets and two α-helices, as indicated in [START_REF] Nakano | Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice[END_REF]; the conserved CMV-1 comprises a small β-sheet and one α-helix; the CMV-2 motifs comprises one α-helix. Only the positions of the predicted structural motifs in CMV-1 and CMV-2 are shown.

MtEFD