Sovanna Tan
email: sovanna.tan@ips2.universite-paris-saclay.fr

Myriam Sanchez
email: myriam.sanchez@inra.fr

Carole Laffont
email: carole.laffont@ips2.universite-paris-saclay.fr

Stéphane Boivin
email: stephane.boivin@inra.fr

Christine Le Signor
email: christine.le-signor@inra.fr

Richard Thompson
email: richard.thompson@inra.fr

Florian Frugier
email: florian.frugier@cnrs.fr

Mathias Brault
email: mathias.brault@ips2.universite-paris-saclay.fr

M B Wrote

A cytokinin signalling type-B response regulator transcription factor acting in early nodulation

Keywords: symbiotic nodule, type-B RR, NSP2, CCS52, endoreduplication

A Medicago truncatula cytokinin-signalling transcription factor is required for early symbiotic nodulation and

Introduction

Legumes species, such as Medicago truncatula, can form a symbiotic interaction with specific soil nitrogen-fixing bacteria commonly referred to as rhizobia, leading to the formation of new organs on the root system, the nodules [START_REF] Suzaki | Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria[END_REF]. Bacteria hosted in nodules find a favourable environment for fixing atmospheric nitrogen into ammonium that can then be assimilated by the host plant. In exchange, bacteria benefit from plant photoassimilates. The symbiotic interaction initiates with a molecular dialogue between the two symbionts: plant roots release specific flavonoids in the rhizosphere, which promote the production of bacterial lipochitooligosaccharidic signals called Nodulation Factors (NF; [START_REF] Lerouge | Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal[END_REF][START_REF] Suzaki | Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria[END_REF]. The NF perception in root-hair epidermal cells triggers microsymbiont recognition and infection thread (IT) formation. Concomitantly to these epidermal symbiotic events, cortical cells divide several cell layers away from the infection site, leading to the formation of a nodule primordium [START_REF] Kondorosi | Ubiquitin-mediated proteolysis. To be in the right place at the right moment during nodule development[END_REF][START_REF] Xiao | Fate map of Medicago truncatula root nodules[END_REF]. IT progression across the outer and middle cortex allows bacteria to reach the nodule organ primordium [START_REF] Fournier | Mechanism of Infection Thread Elongation in Root Hairs of Medicago truncatula and Dynamic Interplay with Associated Rhizobial Colonization[END_REF][START_REF] Oldroyd | Coordinating nodule morphogenesis with rhizobial infection in legumes[END_REF][START_REF] Suzaki | Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria[END_REF]. These NF-induced early symbiotic processes rely on a well described signalling cascade, involving notably the activation of a nucleocytoplasmic calcium spiking upstream of the transcriptional regulation of early nodulation genes [START_REF] Suzaki | Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria[END_REF]. Different transcription factors, such as Nodulation Signalling Pathway 1 and 2 (NSP1 and NSP2), are critical for the activation of the early symbiotic infection marker Early Nodulin 11 (ENOD11) and nodule formation [START_REF] Kaló | Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators[END_REF][START_REF] Smit | NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription[END_REF]. Accordingly, the nsp2 loss-offunction mutant does not nodulate. Nodule primordia then differentiate into nitrogen-fixing nodules, which in M. truncatula have an indeterminate growth due to a persistent apical meristem (or zone I ; [START_REF] Vasse | Correlation between ultrastructural differentiation of bacteriods and nitrogen fixation in alfalfa nodules[END_REF]. A rhizobial infection and differentiation zone (or zone II) is formed below this apical nodule meristem, where bacteria and plant cells codifferentiate to acquire the ability to fix and to assimilate atmospheric nitrogen, respectively, followed by a nitrogen fixation zone (or zone III). The differentiation of nodule symbiotic cells is tightly associated with an increase of cell endoreduplication, whose progression is notably controlled by the Anaphase-Promoting Complex (APC) activator Cell Cycle Switch 52A (CCS52A) in M. truncatula [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF]. MtCCS52A expression is induced during nodule formation prior to nodule differentiation, and reducing the MtCC52A transcript level decreases the endoreduplication rate and inhibits the formation of nitrogen-fixing nodules. In Lotus japonicus, endoreduplication was also suggested to be required for IT progression through the root cortex towards dividing cortical cells [START_REF] Suzaki | Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus[END_REF]. In addition, the two orthologs of MtCCS52A in L. japonicus are expressed in these cortical cells during early nodulation events.

Early nodulation is tightly regulated by plant hormones including cytokinins (CK; [START_REF] Frugier | Cytokinin: secret agent of symbiosis[END_REF][START_REF] Ferguson | Phytohormone Regulation of Legume-Rhizobia Interactions[END_REF]Boivin et al., 2016a;[START_REF] Gamas | Cytokinins in Symbiotic Nodulation: When, Where, What For?[END_REF]. This was notably highlighted in M. truncatula and L. japonicus by the identification of gainof-function and loss-of-function mutants affecting orthologs of the Arabidopsis thaliana Histidine Kinase 4/ Cytokinin Response 1 CK receptor (AHK4/CRE1), named respectively CRE1/ CHASE (Cyclases/Histidine kinases Associated Sensory Extracellular) Histidine Kinase 1 in M. truncatula (MtCRE1/MtCHK1) and Lotus Histidine Kinase in L. japonicus ( LHK1; [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Murray | A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis[END_REF][START_REF] Tirichine | A Gain-of-Function Mutation in a Root Nodule Organogenesis[END_REF][START_REF] Plet | MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF]Boivin et al., 2016b). Expression of a constitutively active LHK1 CK receptor is sufficient to induce cell divisions and "spontaneous" nodule organogenesis in the absence of rhizobia [START_REF] Tirichine | A Gain-of-Function Mutation in a Root Nodule Organogenesis[END_REF], while knockout of MtCRE1 or LHK1 dramatically decreases nodulation in both model legumes [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Murray | A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis[END_REF][START_REF] Plet | MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF]. Furthermore, consistent with these early nodulation phenotypes, CK regulates the expression of NF signalling genes, such as in M. truncatula, Ethylene Response factor required for Nodulation1 (MtERN1), Nodule INception (MtNIN) and MtNSP2, depending on the LHK1/MtCRE1 receptor [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Murray | A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis[END_REF][START_REF] Tirichine | A Gain-of-Function Mutation in a Root Nodule Organogenesis[END_REF][START_REF] Heckmann | Cytokinin Induction of Root Nodule Primordia in Lotus japonicus Is Regulated by a Mechanism Operating in the Root Cortex[END_REF][START_REF] Plet | MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF]. CKs have also been shown to negatively regulate rhizobial infections within the root epidermis. Indeed, the mutant affecting LHK1 was named hit1 for hyperinfected 1, as besides its low nodulation phenotype, it shows a higher number of rhizobial infections [START_REF] Murray | A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis[END_REF][START_REF] Held | Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation[END_REF][START_REF] Liu | Cytokinin responses counterpoint auxin signaling during rhizobial infection[END_REF].

In contrast in M. truncatula, the functionally related low-nodulating cre1 mutant does not show an increased number of infection events but forms ITs that are either blocked in outer layers of MtCRE1 RNA interference (RNAi) roots, or tend to have an exaggerated growth and to form sac-like structures in the cre1 mutant [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Plet | MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF].

More recently, an activation of CK responses in the root epidermis was linked in M. truncatula to a negative regulation on NF signalling [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]. Indeed, a shortterm CK treatment abolishes the NF induction of the MtENOD11 infection marker depending on the MtCRE1 receptor; and conversely, depletion of the CK pool specifically in the root epidermis by the ectopic expression of a CK oxidase ensuring CK degradation enhances the NF-induction of MtENOD11 expression as well as nodulation. Finally, other CK receptors than LHK1/MtCRE1 have a redundant activity in regulating early nodulation, and also participate in regulating nitrogen fixation at a later symbiotic stage [START_REF] Held | Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation[END_REF]Boivin et al., 2016b).

LHK1/MtCRE1 receptors are histidine kinases whose activation by CK triggers a phosphotransfer signalling pathway similar to bacterial two-component systems (TCS; [START_REF] Hwang | Two-component circuitry in Arabidopsis cytokinin signal transduction[END_REF][START_REF] Heyl | Cytokinin signal perception and transduction[END_REF]. This phosphotransfer involves histidine phosphotransfer proteins shuttling between the cytosol and the nucleus, to activate type-B response regulator (RRB) transcription factors in the nucleus [START_REF] Imamura | Compilation and characterization of Arabiopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction[END_REF][START_REF] Hwang | Two-component circuitry in Arabidopsis cytokinin signal transduction[END_REF][START_REF] Heyl | Nomenclature for members of the two-component signaling pathway of plants[END_REF][START_REF] Kabbara | CHASEing Cytokinin Receptors in Plants, Bacteria, Fungi, and Beyond[END_REF]. This ultimately regulates the transcription of CK primary response genes [START_REF] Sakai | Arabidopsis ARR1 and ARR2 response regulators operate as transcriptional activators[END_REF], such as type-A RRs (RRAs) and genes specific to various developmental contexts [START_REF] Kieber | Cytokinin signaling in plant development[END_REF]. During M. truncatula early nodulation, the NF signalling gene MtNSP2 was notably proposed to be a direct target of the MtCRE1-dependent CK signalling based on the identification in its promoter of MtRRB1 binding sites required for the activation of MtNSP2 expression by CK and during early nodulation [START_REF] Ariel | Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[END_REF]. However, no nodulation phenotype could be identified when silencing or overexpressing MtRRB1. In Arabidopsis, the ARR2 RRB transcription factor directly regulates in roots the expression of the APC activator subunitencoding gene AtCCS52A1 [START_REF] Takahashi | Cytokinins control endocycle onset by promoting the expression of an APC/C activator in Arabidopsis roots[END_REF]. A similar CK regulation of MtCCS52A and endoreduplication may thus occur during early nodulation [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF].

In this work, we aimed to identify a RRB transcription factor in M. truncatula relevant for symbiotic nodulation and to characterize potential target genes. Within the 31 genes encoding putative RRBs in the genome of M. truncatula, we selected the RRB most closely related to MtRRB1 that shows a structure typical of authentic RRB proteins acting in TCS signalling, MtRRB3, which is also the RRB gene most highly expressed in roots and nodules [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Tan | Diversification of cytokinin phosphotransfer signaling genes in Medicago truncatula and other legume genomes[END_REF]. Accordingly, in contrast to MtRRB1, MtRRB3 is able to activate a CK primary target RRA gene in a trans-activation assay. Downregulation of MtRRB3 expression by RNAi, or the identification of a mutant, revealed reduced nodulation and rhizobial infection phenotypes, associated with a reduced NF activation of the ENOD11 infection marker. Finally, by a combination of spatial expression and functional assays, we showed that the MtRRB3 transcription factor is able to bind and trans-activate the expression of two genes previously linked to early nodulation, MtNSP2 and MtCCS52A. These results indicate that MtRRB3 coordinates the CK-mediated regulation of key early nodulation genes, consistent with its positive function in regulating rhizobial infections and nodule initiation.

Results

The type-B response regulator MtRRB3 positively regulates nodulation and rhizobial infections in M. truncatula MtRRB1 binding sites required for the regulation of MtNSP2 in response to CK and in early nodulation were previously identified [START_REF] Ariel | Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[END_REF], suggesting that this protein could activate the transcription of this candidate target gene. A closer analysis of the MtRRB1 structure however revealed an atypical structure [START_REF] Tan | Diversification of cytokinin phosphotransfer signaling genes in Medicago truncatula and other legume genomes[END_REF] poorly compatible with a role in TCS signalling. Accordingly, MtRRB1 was unable to trans-activate the promoter of a typical CK primary response gene related to early nodulation (MtRRA11; Op den [START_REF] Op Den Camp | A Phylogenetic Strategy Based on a Legume-Specific Whole Genome Duplication Yields Symbiotic Cytokinin Type-A Response Regulators[END_REF]Supplemental Figure S1). A previously published phylogenetic analysis of the whole M. truncatula RRB family revealed that MtRRB3 is the protein having a canonical RRB structure that is the most closely related to MtRRB1 [START_REF] Tan | Diversification of cytokinin phosphotransfer signaling genes in Medicago truncatula and other legume genomes[END_REF]. In addition, MtRRB3 is the most highly expressed RRB gene which is additionally tightly co-expressed with the MtCRE1 CK receptor in roots and during nodulation. Accordingly, MtRRB3 can efficiently trans-activate the MtRRA11 CK primary response gene (Supplemental Figure S1). We therefore focused our studies on MtRRB3 with the aim of characterizing a canonical CKsignalling transcription factor involved in nodulation.

To establish the involvement of MtRRB3 in nodulation, two RNAi constructs specifically targeting MtRRB3 were first generated (RNAi#1 or RNAi#2; Figure 1A). Analysis of steady-state transcript levels of MtRRB3, as well as those of MtRRB1 and MtRRB2, its most closely related RRB genes [START_REF] Tan | Diversification of cytokinin phosphotransfer signaling genes in Medicago truncatula and other legume genomes[END_REF], revealed that both RNAi constructs efficiently decreased MtRRB3 transcript levels compared to a β-glucuronidase (GUS) RNAi control, while MtRRB1 and MtRRB2 expression was not affected. This therefore demonstrates the specificity of the RNAi silencing constructs (Figure 1B). MtRRB3-silenced roots inoculated with rhizobium had significantly reduced nodule numbers in both RNAi#1 and RNAi#2 roots (Figure 1C), suggesting a positive role of MtRRB3 during nodule initiation. To independently validate this result, a mutant was identified by TILLING (Targeting Induced Local Lesions IN Genomes) from an ethyl methane sulfonate (EMS) mutant population using the 3' region of MtRRB3, corresponding to the transactivation domain (Figure 1A; Le [START_REF] Signor | Optimizing TILLING populations for reverse genetics in Medicago truncatula[END_REF].

From the allelic series identified, one allele generating a premature stop codon at position 528 (W528*) was selected for further analyses, and referred to as rrb3 (Figure 1A). Analysis of root growth sensitivity to CKs showed that this rrb3 mutant has a reduced sensitivity to benzylaminopurine (BAP) compared to the wild type (WT; Supplemental Figure S2). In addition, the nodule number was significantly reduced in the rrb3 mutant (Figure 1D), consistent with the MtRRB3 RNAi nodulation phenotype. This nodulation phenotype was not indirectly due to a root growth defect since a similar decrease was observed when the nodule number was normalized by the total root system length (nodule density; Supplemental Figure S3A). Accordingly, no significant difference between the WT and the rrb3 mutant was identified regarding either root length, lateral root number, or lateral root density (lateral root number per cm of main root; Supplemental Figure S2 andS4), indicating again that the observed symbiotic phenotype was not indirectly caused by an altered root system architecture of the rrb3 mutant. In addition, the root and shoot dry weights of the rrb3 mutant were not significantly different from the WT under non-symbiotic conditions (Supplemental Figure S5A), even though a reduced growth of the rrb3 mutant was observed when nodulated, in agreement with its defective nodulation phenotype (Supplemental Figure S5B).

Importantly, the defective nodulation phenotype of rrb3 mutant roots was rescued by an ectopic expression of MtRRB3 (Supplemental Figure S6). To determine if this reduced nodulation phenotype was associated with an altered rhizobial infection capacity of the mutant, the number of ITs was determined in the rrb3 mutant using a rhizobium strain expressing a LacZ reporter. A decreased IT number was observed, which remained significant when the IT number was normalized by root length (Figure 1E, Supplemental Figure S3B). In agreement, the expression of the early symbiotic infection marker MtENOD11 was reduced in rrb3 mutant roots treated for 3 hours with NF compared to WT roots (Figure 1F). We further evaluated if MtRRB3 could also act at a later symbiotic stage, in relation to nodule tissue patterning, bacteria differentiation and/or viability, and/or the nitrogen fixation activity. Using a "live and dead" staining on rrb3 nodule sections, no major difference could be identified in the spatial organization of rrb3 mutant nodules, or in the differentiation status and survival of bacteria (Supplemental Figure S7A-D). Furthermore, the nitrogen fixation activity of rrb3 mutant nodules was evaluated using an acetylene reduction assay (ARA) allowing an indirect measure of bacterial nitrogenase activity. Whereas ARA activity per plant was significantly reduced in the rrb3 mutant (Supplemental Figure S7E), this difference was lost when ARA activity was normalized by nodule number (Supplemental Figure S7F). This suggests that the decreased ARA activity was caused by the reduced nodulation of the rrb3 mutant rather than by a direct effect on the nitrogen fixation capacity. Altogether, these results support a positive role for MtRRB3 in nodulation, notably on IT formation and on the NF induction of the MtENOD11 infection marker.

Rhizobial infections are associated with an increased nuclear size and the rrb3 mutant is altered in the cytokinin regulation of the MtCCS52A endoreduplication marker IT progression was previously associated in L. japonicus to enlarged nuclei and endoreduplication, consistent with the expression of LjCCS52A genes during early nodulation [START_REF] Suzaki | Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus[END_REF], but these features were not described in M. truncatula. We therefore quantified, using confocal microscopy, nuclear area in root regions infected or not with rhizobia, using a strain constitutively expressing a red fluorescent protein (RFP) reporter and DAPI staining of nuclei (Figure 2A-F). An increase of the nuclear area was indeed observed in infected root regions compared to uninfected root regions, in both root hairs (Figure 2E) and cortical cells (Figure 2F). No significant difference was however observed when comparing the nuclear area in the vicinity of developing ITs in WT and rrb3 mutant inoculated roots (Figure 2G). Similarly, flow cytometry analyses revealed no difference in endoreduplication levels either in response to rhizobial inoculation or between WT and rrb3 mutant roots (Supplemental Figure S8). In contrast, we observed that the regulation of MtCCS52A expression by a short-term CK treatment (BAP 10 -7 M, 30 min and 1 h) was decreased in the rrb3 mutant (Figure 3A). Interestingly, the NF regulation of this gene was previously shown to be dependent on the MtCRE1 CK signalling pathway (Supplemental Figure S9; [START_REF] Van Zeijl | Rhizobium Lipo-Chitooligosaccharide Signaling Triggers Accumulation of Cytokinins in Medicago Truncatula Roots[END_REF].

The MtRRB3 expression pattern overlaps with the expression domains of the MtNSP2 and MtCSS52A nodulation genes

As MtNSP2 was previously shown to be regulated by CKs depending on MtCRE1 and its promoter to be bound by MtRRB1 [START_REF] Ariel | Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[END_REF], we also analysed its regulation in response to a short-term CK treatment (BAP 10 -7 M, 30 min and 1 h) in WT or rrb3 mutant roots (Figure 3). The expression of MtRRA CK primary response genes previously linked to early nodulation [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Op Den Camp | A Phylogenetic Strategy Based on a Legume-Specific Whole Genome Duplication Yields Symbiotic Cytokinin Type-A Response Regulators[END_REF][START_REF] Saur | Crosstalk between the nodulation signaling pathway and the autoregulation of nodulation in Medicago truncatula[END_REF] was reduced in rrb3 mutant roots (Figure 3B; Supplemental Figure S10), indicating that CK signalling was indeed impaired in the rrb3 mutant. Similarly, the CK induction of MtNSP2 was also notably reduced after a 1-h BAP treatment (Figure 3C). These results demonstrate that, as previously shown for MtCCS52A, the expression of MtNSP2 is positively regulated by MtRRB3.

We then analysed in rhizobium-infected roots the spatial expression pattern of a MtRRB3 GUS transcriptional fusion in parallel with MtCCS52A GUS and MtNSP2 GUS fusions, to determine how these patterns overlapped (Figure 4). Histological analyses revealed that the MtRRB3 GUS transcriptional fusion was detected in the root apex (Figure 4A), in the root hair region susceptible to rhizobia (Figure 4B), and in nodule primordia (Figure 4C).

Interestingly, both MtNSP2 and MtCCS52A GUS fusions revealed an activity co-localizing spatially with MtRRB3 in roots, mostly in the elongation/differentiation zone (Figures 4D andG), in the differentiated root hair region (Figures 4E andH), and in nodule primordia (Figures 4F andI).

Overall, the MtRRB3 protein and the two early nodulation genes MtNSP2 and MtCCS52A have overlapping spatial expression domains in roots and nodules, which are compatible with a potential direct regulation of these genes by MtRRB3.

MtRRB3 interacts and trans-activates the promoter of MtNSP2 and MtCCS52A

Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) assays were then performed in roots transformed with an MtRRB3-HA construct to test a putative interaction between MtRRB3 and MtNSP2 or MtCCS52A promoters. Immunoprecipitations (IPs) with an anti-HA antibody revealed an enrichment for both MtNSP2 and MtCCS52A promoters compared to an anti-IgG antibody IP (Figure 5A), indicating that MtRRB3 can bind MtNSP2 and MtCCS52A promoters. In addition, promoter trans-activation assays were carried out in M. truncatula mesophyll protoplasts using the native MtRRB3 protein and a constitutively active form of MtRRB3 (MtRRB3-D64E). In this variant, the conserved aspartate involved in the activation of MtRRB3 by phosphotransfer was replaced by a glutamate to mimic the phosphorylation effect, leading therefore to its constitutive activation even in the absence of CK (Supplemental Figure S11). An activation of the expression of pMtNSP2::LUC and pMtCCS52A::LUC transcriptional fusions was detected (Figure 5B), showing the ability of MtRRB3 to transcriptionally activate these two promoters. Taken together, these results indicate that MtRRB3 can positively regulate the expression of MtNSP2 and MtCCS52A.

Discussion

The initiation of the symbiotic interaction between legumes and rhizobia requires a tight coordination between infection and nodule organogenesis, followed by a co-differentiation of bacteria and plant cells to allow nitrogen-fixation and assimilation [START_REF] Suzaki | Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria[END_REF]. All these processes are finely tuned in time and space by plant hormones that operate at different nodulation stages [START_REF] Buhian | Mini-Review: Nod Factor Regulation of Phytohormone Signaling and Homeostasis During Rhizobia-Legume Symbiosis[END_REF]. CKs notably play a major role in nodule initiation depending on the MtCRE1/LHK1 receptor, first by positively regulating root cortical cell divisions and nodule organogenesis as well as cortical infection, and second by negatively regulating NF signalling and/or rhizobial infections within the root epidermis [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][START_REF] Miri | Into the Root: How Cytokinin Controls Rhizobial Infection[END_REF][START_REF] Gamas | Cytokinins in Symbiotic Nodulation: When, Where, What For?[END_REF]. Beside the well described role of CK receptors, little is known about the involvement of downstream TCS elements which are recruited for symbiotic functions. We show in this study that MtRRB3-defective plants have a decreased ability to form nodules, as previously observed for MtCRE1 [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Plet | MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF]. In contrast, root length and lateral root number or density were not altered in the rrb3 mutant. The reduced nodule number phenotype of the rrb3 mutant is associated with a reduced number of infection events, whereas no significant difference was previously observed in MtCRE1 RNAi and cre1 mutant roots, even though IT growth in outer layers of the root seemed respectively blocked or altered. In agreement, the short-term NF induction of the MtENOD11 infection marker was not altered in the cre1 mutant [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], whereas it is decreased in the rrb3 mutant. As some symbiotic responses are still observed, this suggests that a functional redundancy with other RRBs may exist. In addition, the ectopic expression of MtRRB3 was not sufficient to rescue the cre1 reduced nodulation phenotype (Supplemental Figure S12), pointing to a potential functional specialization between RRBs. In addition, differential symbiotic phenotypes observed depending on the CK signalling mutant analyzed suggest that MtRRB3 mediates only a subset of the early symbiotic CK function. The greatly increased IT number detected in the L. japonicus lhk1/hit1 (hyperinfected 1) mutant [START_REF] Murray | A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis[END_REF][START_REF] Held | Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation[END_REF] further indicates that rhizobial infections are differentially regulated between L. japonicus and M. truncatula.

Concerning the response to NFs, whereas the NF induction of MtENOD11 was potentiated when the CK pool was decreased by ectopically expressing a CK oxidase gene in the root epidermis [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] [START_REF] Choi | The cytokininactivated transcription factor ARR2 promotes plant immunity via TGA3/NPR1dependent salicylic acid signaling in arabidopsis[END_REF].

We explored the hypothesis that some already identified early nodulation genes may be directly transcriptionally regulated by MtRRB3-dependent CK signalling. We focused on two genes: MtNSP2 previously shown to be regulated by CKs and to have a promoter bound by the atypical MtRRB1 protein [START_REF] Plet | MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF][START_REF] Ariel | Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[END_REF]; and MtCCS52A, whose closest Arabidopsis homolog was previously shown to be regulated by CK in the root apex through the binding of a RRB transcription factor on its promoter [START_REF] Fülöp | Arabidopsis anaphase-promoting complexes: Multiple activators and wide range of substrates might keep APC perpetually busy[END_REF][START_REF] Takahashi | Cytokinins control endocycle onset by promoting the expression of an APC/C activator in Arabidopsis roots[END_REF]. Based on a combination of genetic, reporter fusion, trans-activation, and ChIP analyses, we showed that the regulation of both MtNSP2 and MtCCS52A expression relies on an interaction with the MtRRB3 transcription factor.

MtNSP2 is a Gibberellic-acid insensitive (GAI), Repressor of gai (RGA) And Scarecrow (SCR) GRAS transcription factor critical for NF signalling and nodule establishment whose expression is regulated by CKs depending on the MtCRE1 receptor [START_REF] Kaló | Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators[END_REF][START_REF] Plet | MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF][START_REF] Ariel | Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[END_REF]van Zeil et al., 2015). Previous studies showed that the MtNSP2 promoter contains MtRRB1 binding sites (RRBS) required for its induction by CK and for its expression in nodule primordia [START_REF] Ariel | Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[END_REF]. MtRRB1 is however unable to regulate a

MtRRA promoter in a trans-activation assay, consistent with its atypical structure lacking a Cterminal trans-activation domain [START_REF] Tan | Diversification of cytokinin phosphotransfer signaling genes in Medicago truncatula and other legume genomes[END_REF]. In contrast, MtRRB3 acts as a bona fide TCS RRB including on the MtNSP2 promoter. This MtRRB3-dependent regulation of the MtNSP2 transcription factor essential for NF signalling and rhizobial infections may explain the reduced NF induction of MtENOD11 expression and of the number of ITs observed in the rrb3 mutant. In addition, the CK-mediated regulation of MtNSP2 expression might be one of the evolutionary innovations that are associated with the capacity of legume plants to nodulate.

Another gene recruited during nodulation and that is a target of MtRRB3 is the APC activator, MtCCS52A, promoting endoreduplication cycles [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF]. In Arabidopsis, two CCS52A genes exist, AtCCS52A1 and AtCCS52A2, which display a complementary expression pattern in the root apical meristem (RAM; [START_REF] Vanstraelen | APC/C CCS52A complexes control meristem maintenance in the Arabidopsis root[END_REF]. In contrast to the [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF] study, we observed that MtCCS52A was expressed not only in nodules but also in roots and at early stages of the interaction with rhizobium, in agreement with data obtained with CCS52A genes in L. japonicus [START_REF] Suzaki | Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus[END_REF]. Interestingly, the expression of the M. truncatula CCS52A gene in the RAM recapitulates the combined expression of AtCC52A1 and AtCCS52A2 Arabidopsis genes. As observed in M. truncatula, AtCCS52A1 is regulated by CK depending on the ARR2 transcription factor, to positively regulate the progression of endoreduplication cycles in the elongation zone of the RAM [START_REF] Takahashi | Cytokinins control endocycle onset by promoting the expression of an APC/C activator in Arabidopsis roots[END_REF]. In legume roots infected by symbionts, an increase in ploidy levels correlates with an expanded nuclear area and the induction of CCS52A genes, either in relation to the progression of rhizobial ITs in L. japonicus or of fungal hyphae from the arbuscular mycorrhizal endosymbiotic fungus Gigaspora margarita in M. truncatula [START_REF] Suzaki | Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus[END_REF][START_REF] Carotenuto | Local endoreduplication as a feature of intracellular fungal accommodation in arbuscular mycorrhizas[END_REF]. We now also showed in M.

truncatula that the nuclear area is increased in roots infected by rhizobia, despite that no change in endoreduplication levels could be identified, potentially because of a lack of sensitivity to detect discrete endoreduplication events associated to a limited number of et al., 2008) or by TILLING in the same EMS mutant collection as for RRB3, did not contain the predicted retrotransposon insertion or did not affect any conserved residue that would be anticipated to generate a nodulation phenotype, respectively. In addition no efficient CCS52A

RNAi construct could be generated, overall impeding the analysis of any potential IT progression and/or nuclear enlargement phenotype. Despite the fact that no difference in nuclear area could be identified in infected or non-infected rrb3 mutant roots compared to the WT, the analysis of MtCCS52A expression was however sensitive enough to reveal an alteration of its regulation in the rrb3 mutant, indicating that the MtRRB3 transcription factor positively regulates MtCCS52A expression. Overall, data obtained suggest that the MtRRB3dependent CK regulation of MtCCS52A was recruited at the onset of nodulation potentially in relation with the activation of endoreduplication cycles in root cells associated with IT progression. It would be interesting to determine if the MtRRB3-dependent CK regulation of CCS52A expression is also associated to mycorrhizal infections.

Conclusion

In this study, we showed that the CK-signalling transcription factor MtRRB3 is required for nodulation, rhizobial infection and NF signalling, and that these functions correlate with the regulation of diverse CK-regulated genes associated to early nodulation such as MtNSP2 and MtCCS52A. Rhizobial infection and NF signalling response phenotypes are strikingly different in the rrb3 mutant from those of cre1 or lhk1 receptor mutants [START_REF] Held | Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation[END_REF]Boivin et al., 2016b). This suggests that CK seems to regulate rhizobial infections differently depending on plant species and that other RRB(s) are functionally redundant with MtRRB3 to regulate nodulation initiation.

Materials and Methods

Materials, plant growth conditions and treatments

The Medicago truncatula Jemalong A17 genotype was used in this study. A rrb3 mutant was identified using a TILLING screen on an EMS mutagenized population (Le [START_REF] Signor | Optimizing TILLING populations for reverse genetics in Medicago truncatula[END_REF][START_REF] Sanchez | Targeting Induced Local Lesions IN Genomes (TILLING) in Medicago truncatula[END_REF]Supplemental were genotyped by PCR and subsequent sequencing of amplicons (primers used are given in Supplemental Table S1). Seeds were scarified by immersion in pure sulfuric acid for 3 min, rinsed six times with water, and sterilized for 20 min in Chlorofix (8.25 mg/L. Bayrol, France). After three washes with sterilized water, seeds were sown on 1% w/v bacto-agar (Gibco) plates, and stratified for 3 days at 4°C in the dark. Germination was triggered by an overnight incubation at 24°C in the dark. Germinated seeds were grown either in vitro on 1.5% w/v agar plates or in pots containing a mixture of perlite:sand (3:1), and placed in growth chambers with the following conditions:16h light at 150 µE intensity, 24°C, 60% relative air humidity.

For nodulation phenotyping experiments, a Sinorhizobium medicae WSM419 strain [START_REF] Terpolilli | The model legume Medicago truncatula A17 is poorly matched for N2fixation with the sequenced microsymbiont Sinorhizobium meliloti 1021[END_REF] was used, as well as corresponding strains expressing either the lacZ reporter (WSM419 phemA-lacZ; [START_REF] Domonkos | NAD1 controls defense-like responses in Medicago truncatula symbiotic nitrogen fixing nodules following rhizobial colonization in a BacAindependent manner[END_REF], or the pBHR-mRFP construct described in [START_REF] Smit | NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription[END_REF]WSM419-RFP BRC3915 strain). Bacteria were grown overnight at 30°C on a yeast extract broth (YEB) medium [START_REF] Vervliet | Characterization of different plaque-forming and defective temperate phages in Agrobacterium[END_REF], plants were grown in vitro on a Fahraeus medium without nitrogen [START_REF] Truchet | Identification of a Rhizobium meliloti pSym2011 region controlling the host specificity of root hair curling and nodulation[END_REF], and roots were inoculated for 1h with a bacterial suspension (OD 600nm =0.05). Nodules were counted after 6 and 14 days, and root length was measured using Fiji (https://fiji.sc/; [START_REF] Schindelin | Fiji: An open source platform for biological image analysis[END_REF].

For root architecture phenotyping experiments, germinated seedlings were grown on a low nitrogen "i" medium [START_REF] Blondon | Contribution à l'étude du développement des graminées fourragères raygrass et dactyle[END_REF]. Lateral roots were counted 10 days after germination and primary root lengths were measured using Fiji (https://fiji.sc/; [START_REF] Schindelin | Fiji: An open source platform for biological image analysis[END_REF].

For CK short-term treatments, germinated seedlings were placed on a grid in a Magenta box containing the low nitrogen "i" liquid medium [START_REF] Blondon | Contribution à l'étude du développement des graminées fourragères raygrass et dactyle[END_REF] and grown for five days under a gentle agitation in the same growth chamber as described above. Seedlings were treated either with CKs (10 -7 M BAP, Sigma) or with NFs (10 -8 M) for different incubation times (indicated in figure legends). Roots were collected and immediately frozen in liquid nitrogen for RNA extraction.

For CK sensitivity root growth assays, plants were grown on growth papers (Mega International, https://mega-international.com/) placed on the low nitrogen 'i' medium [START_REF] Blondon | Contribution à l'étude du développement des graminées fourragères raygrass et dactyle[END_REF]. After four days, plants on growth papers were transferred onto a fresh 'i' medium supplemented or not with 10 -7 M BAP. The position of root tips was marked at the time of transfer, and root growth from this point was measured after 3 days using Fiji (https://fiji.sc/; [START_REF] Schindelin | Fiji: An open source platform for biological image analysis[END_REF].

For shoot and root dry weight analyses, germinated seedlings were grown in pots watered with a liquid Fahraeus medium [START_REF] Truchet | Identification of a Rhizobium meliloti pSym2011 region controlling the host specificity of root hair curling and nodulation[END_REF] supplemented or not with nitrogen (NH 4 NO 3 1 mM). Nitrogen-starved plants were inoculated with rhizobium seven days after germination, as described above. Roots and shoots were collected 35 days after germination and dried during seven days in an oven at 60°C before weighing.

Cloning Procedures and Agrobacterium rhizogenes Root Transformation

For the pMtRRB3-GUS transcriptional fusion, a sequence of 2.55 kb upstream of the PCR from genomic DNA using the Phusion High-Fidelity DNA polymerase (Thermo Scientific) and forward and reverse primers flanked, respectively, with KpnI and AatII restriction sites (Supplemental Table S1). The PCR product was then cloned using these enzymes into the pFRN-GUS vector (Boivin et al., 2016b). Transcriptional GUS fusions with MtNSP2 (Medtr3g072710 and MtrunA17_Chr3g0114841 in M. truncatula genome v4.0 and v5.0, respectively) and MtCCS52A (Medtr4g102510 and MtrunA17_Chr4g0057131 in M.

truncatula genome v4.0 and v5.0, respectively) promoters (2542 bp and 2460 bp, respectively) were previously described, respectively, in [START_REF] Ariel | Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[END_REF] and [START_REF] Vinardell | Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules[END_REF].

For RNAi constructs, specific sequences targeting the MtRRB3 transactivation domain that is the most divergent region at the nucleotide level between M. truncatula RRB genes, were amplified by PCR using the Phusion High-Fidelity DNA polymerase (Thermo Scientific) and primers indicated in Supplemental Table S1, and subsequently cloned by Gateway recombination into the pDONR207 plasmid (Invitrogen). This sequence was introduced in the pFRN-RNAi destination vector (deriving from pFGC5941; NCBI accession number AY310901, [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF]. The GUS-RNAi control vector was previously described [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF].

For transcriptional promoter-luciferase fusions used in protoplast assays, the ARR6 promoter was removed from the pARR6-luciferase plasmid (NCBI accession number primers (Supplemental Table S1) and cloned in the pHBT plasmid [START_REF] Sheen | Protein phosphatase activity is required for light-inducible gene expression in maize[END_REF]; NCBI accession number EF090408) downstream of a 35S-CaMV promoter. p35S:MtRRB3-D64E

was obtained by site-directed mutagenesis of the p35S:MtRRB3 plasmid using the QuikChange XL Site-Directed Mutagenesis Kit (Agilent) and the appropriate primers (Supplemental Table S1).

For the 35S-CaMV:MtRRB3-HA construct, 35S promoter and MtRRB3 coding sequences were PCR amplified respectively from the pFRN-RNAi vector and from M. truncatula root cDNAs using the Phusion High-Fidelity DNA polymerase (Thermo Scientific)

and forward and reverse primers flanked respectively with KpnI and BamHI restriction sites for the promoter, and BamHI and StuI restriction sites for the MtRRB3 coding sequence (Supplemental Table S1). PCR products were then cloned using these restriction enzymes into the pFRN-203 vector containing a C-terminal HA tag (Boivin et al., 2016b). The p35S:MtRRB3-D64E-HA construct was prepared by subcloning the MtRRB3-D64E-HA region from the pHBT vector into the pFRN-203 vector using BamHI and StuI restriction enzymes.

All plasmids were verified by sequencing.

For in planta experiments, constructs were introduced into the Agrobacterium rhizogenes ARqua1 strain to transform M. truncatula roots and generate composite plants as described in [START_REF] Boisson-Dernier A | Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations[END_REF]. Transformed roots, selected on kanamycin (50 µg/ml, Sigma), were then transferred onto growth papers (Mega International) on a Fahraeus medium without nitrogen [START_REF] Truchet | Identification of a Rhizobium meliloti pSym2011 region controlling the host specificity of root hair curling and nodulation[END_REF]. After one week, plants were then inoculated with rhizobia as described above.

Live and dead staining, nuclear staining and confocal imaging

For the live and dead staining, nodules from in vitro cultivated plants were harvested at 21 days post inoculation (dpi), embedded in agarose (Eurobio) 4% (w/v), and prepared for "live-dead" staining (using the "Live/Dead BacLight" kit, ref. L7007, Invitrogen) containing Syto-9 to label living bacteria and propidium iodide (PI) to label dead bacteria and plant cell genomic DNA [START_REF] Haag | Protection of sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF]. Nodule sections (70 μm) were obtained with a vibratome (VT1200S, Leica) and incubated for 20 min in the "live-dead" staining solution supplemented with 0.01% w/v calcofluor white M2R (Sigma). Sections were then rinsed in a 50 mM Tris-HCl pH 7.2 buffer before observation using a LSM880 Zeiss confocal microscope with a 488 nm laser for Syto-9 excitation, a 561 nm laser for PI excitation and a 405 nm laser for calcofluor excitation.

For the nuclear area quantification, roots inoculated with the S. medicae RFP strain were fixed, embedded in 3% w/v agarose (Eurobio), sectioned with a vibratome (100 µm thick; VT1200S, Leica), and di-aminido-phenyl-indol (DAPI) stained as described in [START_REF] Carotenuto | Local endoreduplication as a feature of intracellular fungal accommodation in arbuscular mycorrhizas[END_REF]. Imaging was performed using a LSM880 Zeiss confocal microscope with the 405 nm diode for DAPI excitation and 561 nm laser for RFP excitation. Fluorescence was collected between 425 nm-485 nm for DAPI emission and 595 nm-640 nm for RFP emission. Nuclear areas were segmented and measured using Fiji [START_REF] Schindelin | Fiji: An open source platform for biological image analysis[END_REF] with a threshold/watershade-based segmentation on maximal z-projection. At least 10 independent sections (from 5 independent roots) per experimental condition and from two independent experiments were analysed.

GUS staining assays

Roots of composite plants expressing a reporter construct were stained for GUS activity as described in [START_REF] Pichon | Rhizobium meliloti Elicits Transient Expression of the Early Nodulin Gene ENOD12 in the Differentiating Root Epidermis of Transgenic Alfalfa[END_REF]. Samples were incubated overnight at 37°C.

Tissues were then rinsed with water and cleared in 20% v/v sodium hypochlorite for 1 h.

Samples were observed with a bright-field light microscope (Olympus BX53) and images were taken with an Olympus DP73 camera. Pictures included in figures are representative of GUS patterns observed in at least 70% of the transgenic roots (n > 15 roots per replicate; two independent biological experiments).

Flow cytometry

DNA content of root cell nuclei was assessed as described in [START_REF] Carotenuto | Local endoreduplication as a feature of intracellular fungal accommodation in arbuscular mycorrhizas[END_REF].

To release nuclei, roots were chopped with a razor blade in a buffer containing 45 mM MgCl 2 , 30 mM sodium citrate, 60 mM MOPS pH 7.2, and 0.1% v/v Triton. Nuclei suspensions were then filtered through 50 μm Celltrics filters (Partec, Germany), stained with 50 μg.mL -1 PI, and at least 20,000 nuclei per sample were analysed using a CyFlow cytometer (Partec, Germany) with a 488-nm laser for scattering and a 532-nm laser for PI excitation.

Acetylene Reduction Assay (ARA)

Acetylene reduction assays were performed on individual plants 25 days postrhizobium inoculation following a protocol modified from [START_REF] Koch | Reduction of acetylene to ethylene by soybean root nodules[END_REF]. Whole plants were placed in 21 mL glass vials sealed with rubber septa. Acetylene gas (200 µL) was injected into each vial. After a 2-h incubation, ethylene production was measured by injecting 1 mL of vial gas into a gas chromatographer (Agilent Technologies, https://www.agilent.com/) equipped with a GS-Alumina column. Column temperature and gas flow were adjusted at 120°C and 7.5 mL min -1 , respectively.

Gene expression analysis

For reverse transcription quantitative PCR (RT-qPCR), total RNAs were extracted from frozen roots using the RNeasy plant mini kit (Qiagen). The first-strand cDNA was synthesized from 1.5 μg of total RNA using the Superscript II first strand synthesis system (Invitrogen). Primer design was performed using the OligoPerfect Designer software (https://tools.thermofisher.com/content.cfm?pageid=9716&icid=fr-oligo-6?CID=floligoperfect). Primer combinations showing a minimum amplification efficiency of 90% were retained (Supplemental Table S1), and qPCR reactions were performed using the Light Cycler Fast Start DNA Master SYBR Green I kit on a Light Cycler 480 apparatus according to manufacturer's instructions (Roche). Cycling conditions were as follows: 95°C for 10 min, and then 40 cycles at 95°C for 15 sec, 60°C for 15 sec, and 72°C for 15 sec. PCR amplification specificity was verified using a dissociation curve and sequencing of amplicons.

MtRBP1 and MtACTIN11 were previously selected as reference genes using the Genorm software (http://medgen.ugent.be/~jvdesomp/genorm/ ; [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF].

ChIP-qPCR

Roots expressing the 35S-CaMV:MtRRB3-HA construct (~3 g of fresh weight) were used for chromatin purification and immunoprecipitation (IP) as described in [START_REF] Ariel | Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[END_REF] except that an anti-IgG IP (Abcam172730, dilution 1:1000) was performed as a negative control in parallel to the anti-HA IP (Abcam9110, dilution 1:400). Primers used for the ChIP-qPCR analysis are listed in Supplemental Table S1. qPCR was performed on the input genomic DNA and after IP using the same experimental conditions as for RT-qPCR, but with 45 cycles. For each gene, the input data were used to normalize results obtained after IP.

Protoplast transactivation assays

Arabidopsis thaliana protoplasts were prepared and transfected as described by [START_REF] Yoo | Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[END_REF]. For M. truncatula protoplasts, a similar protocol was followed except that 2% w/v Cellulase Onozuka R10 (Yakult Pharmaceutical Industry CO LTD), 1% w/v Macerozyme R10 (Yakult Pharmaceutical Industry CO LTD) and 4% v/v Viscozyme (Sigma) enzymes were used to digest cell walls. Arabidopsis and M. truncatula protoplasts (1.6.10 5 ) were transfected with 20 µg of a DNA mixture consisting of 9 µg of the luciferase (LUC) reporter construct (pMtRRA11:LUC, pMtNSP2:LUC or pMtCCS52A:LUC), 10 µg of a plasmid expressing a RRB (p35S:MtRRB1, p35S:MtRRB3 or p35S:MtRRB3-D64E), and of 1 µg of the pUBQ10:GUS vector generated in [START_REF] Yoo | Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[END_REF] and used as an internal control to normalize the transfection efficiency. After transfection, protoplasts were incubated overnight at room temperature in darkness. Protoplasts were then harvested by centrifugation (100 g for 5 min), lysed in a protoplast lysis buffer [START_REF] Yoo | Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[END_REF]

  rhizobial infections, in contrast to the more extensive mycorrhizal root colonization. Potential ccs52A mutants, identified either from the Tobacco (Nicotiana tabacum) Tnt1 retrotransposon insertional mutant collection (NF9775 and NF9308; http://bioinfo4.noble.org/mutant/; Tadege

  to amplify two different regions of the MtNSP2 (left graph) and MtCCS52A (right graph) promoters as shown on the schemes above each graph. IP values were normalized for each promoter as a ratio against the genomic DNA input (in %). Ratios with anti-IgG control were calculated to visualize MtRRB3 binding fold enrichment. Results are the mean of two technical replicates with n>15 independent transgenic roots per construct, from one representative experiment out of three. Error bars represent standard deviations and a Student's t-test was performed to assess significant differences (p<0.05) compared to the control IP, indicated with asterisks. B. Trans-activation assays. M. truncatula mesophyll protoplasts were transformed with pMtNSP2:LUC (left graph) or pMtCCS52A:LUC (right graph) transcriptional fusions and a vector containing a MtRRB3 native version (MtRRB3), a constitutively active MtRRB3 variant (MtRRB3-D64E), or an empty vector used as a negative control. Results are the mean of two technical replicates from one biological experiment out of four. Error bars represent standard deviations and a Student's ttest was performed to assess significant differences (p<0.05) compared to the control, indicated with asterisks.

  , indicating a negative role of CK in the regulation of the NF signalling, it was reduced in the rrb3 mutant. This suggests that depending on the strategy used, targeting different CK metabolism or signalling genes, and/or the root tissue considered, antagonistic roles of CK on the regulation of NF signalling can be identified. Overall, these results suggest a positive function of MtRRB3 in NF signalling and rhizobial infections, and that differences with infection phenotypes previously identified for CK receptor mutants pinpoint first that diverse CK regulations of rhizobial infections exist depending on plants and/or root tissues, and second that various RRB(s) likely contribute to the regulation of early nodulation. More generally, CKs can have variable and sometimes opposite effects on plantmicrobe interactions, and notably promote either plant defence or susceptibility depending on pathogens, plant species, and even plant organs[START_REF] Choi | Cytokinins and plant immunity: old foes or new friends?[END_REF] Boivin et al., 2016a;

	Shigenaga and Argueso, 2016). It would be therefore interesting to determine, on the one
	hand, if the positive function of MtRRB3 on rhizobial infection and NF signalling is
	conserved in other nodulating legumes such as L. japonicus where a very different role of
	CKs in regulating these processes is observed; and on the other hand, if MtRRB3 could
	participate in determining resistance/susceptibility to pathogens, as demonstrated for the
	Arabidopsis RRB encoding gene ARR2 that promotes resistance against Pseudomonas
	syringae

Table 1 )

 1 , and backcrossed to the WT.

	The region used for TILLING was selected using the CODDLE (Codons Optimized to
	Discover	Deleterious	Lesions)	software

(http://wwwuser.cnb.csic.es/~tiller/sequenceToAnalyse.html) as the most likely to generate stop codons. Homozygous rrb3 mutants introducing a stop codon at position 528 (W528*)

  https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/,[START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF] was amplified by

	MtRRB3	start	codon	(Medtr3g106220;	M.	truncatula	genome	v4.0;
	http://www.medicagogenome.org/	;	MtrunA17Chr3g0137101;

  and protein extracts were clarified by centrifugation (2,000 g for 5 min). For GUS assays, protein extracts were incubated 30 min at 37°C in a 4-methylumbelliferyl-ß-D-glucuronide (Duchefa) substrate mix and fluorescence (excitation: 365 nm, emission: 455 nm) was measured with a TECAN Infinite200 microplate reader. For LUC assays, protein extracts were mixed with a LUC assay buffer (20 mM tricine pH 7.8, 5 mM MgCl 2 , 0.1 mM EDTA, 3.3 mM DTT, 270 mM Coenzyme A, 500 µM luciferin (Duchefa) and 500 µM ATP (Sigma), and luminescence was immediately integrated for 10 s using a TECAN Infinite200 microplate reader.

	Statistical Analyses
	Statistical	analyses	were	performed	with	the	Xlstat	software

(https://www.xlstat.com/fr/). For RT-qPCR, ChIP-qPCR, and transactivation assays, we used a Student's t-test; and for phenotyping experiments, a Mann-Whitney test when n = 2 independent conditions are compared, and a Kruskal-Wallis test when n > 2 independent conditions are compared.

Accession Numbers

All accession numbers of genes mentioned are indicated in Supplemental Table

1

.

Acknowledgements

We thank Fabienne Maillet (LIPM, Toulouse, France), Rene Geurts (WUR, Wageningen, The Netherlands), Gabriella Endre (BRC, Szeged, Hungary), Leo Serra (IJPB, Versailles, France), Peter Mergaert (I2BC, Gif sur Yvette, France) and Pascal Ratet (IPS2, Gif-sur-Yvette, France), for providing respectively Nod factors, the pKGW-RRGG-243-pRRA11 plasmid, the WSM419-lacZ and WSM419-RFP rhizobium strains, a Fiji macro to quantify nuclear area, and the pPR97 pCCS52A:GUS plasmid. We also thank students from the Paris-Diderot University for generating additional biological replicates of trans-activation experiments.

Funding from ANR, the Labex "Saclay Plant Science" (SPS), and the Idex "Plant Phenotyping Pipeline" (3P) are acknowledged. S.T. was the recipient of a PhD fellowship from the Paris-Sud/Paris-Saclay University.

Supplemental

Supplemental Table

 1. List of primers. Arrowheads indicate enlarged nuclei associated to infection events. In A-F, the colour scale represents the nuclear area (in µm 2 ). Each picture is representative of the nuclear area pattern observed in n>15 sections from 5 independent roots per genotype from two independent experiments. Scale bar: 50 µm.

G. Quantification of the nuclear area through a z-stack confocal projection (n>15 sections from 5 independent roots per genotype from two independent experiments). A Kruskal-Wallis test was performed to assess significant differences (α<0.05), indicated with letters. A control immunoprecipitation was performed with an anti-IgG antibody. Specific primers were used