Casandra Hernández-Reyes

Elisabeth Lichtenberg

Jean Keller

Pierre-Marc Delaux

Thomas Ott

Sebastian T Schenk
email: sebastian.schenk@biologie.uni-freiburg.de

NIN-Like Proteins; interesting Players in Rhizobia-Induced Nitrate Signaling Response during Interaction with Non-Legume Host Arabidopsis thaliana

Keywords: NIN-like Proteins (NLPs), NLP-pathway, nitrate-related signaling, Non-legume host Arabidopsis, rhizobia-interaction NIN-like Proteins (NLPs), NLP-pathway, nitrate-related signaling, Non-legume host Arabidopsis, rhizobia-interaction

Nitrogen (N) is an essential macronutrient and a key cellular messenger. Plants have evolved refined molecular systems to sense the cellular nitrogen status. This is exemplified by the root nodule symbiosis between legumes and symbiotic rhizobia, where nitrate availability inhibits this mutualistic interaction. Additionally, nitrate also functions as a metabolic messenger, resulting in nitrate signaling cascades which intensively cross-talk with other physiological pathways. (NODULE INCEPTION)-LIKE PROTEINS (NLPs) are key players in nitrate signaling and regulate nitrate-dependent transcription during legume-rhizobia interactions. Nevertheless, the coordinated interplay between nitrate signaling pathways and rhizobacteria-induced responses remains to be elucidated. In our study, we investigated rhizobia-induced changes in the root system architecture of the non-legume host Arabidopsis under different nitrate conditions. We demonstrate that rhizobium-induced lateral root growth and increased root hair length and density are regulated by a nitrate-related signaling pathway. Key players in this process are AtNLP4 and AtNLP5, since the corresponding mutants failed to respond to rhizobia. At the cellular level, AtNLP4 and AtNLP5 control a rhizobia-induced decrease in cell elongation rates, while additional cell divisions occurred independently of AtNLP4. In summary, our data suggest that root morphological responses to rhizobia are coordinated by a newly considered nitrate-related NLPpathway that is evolutionary linked to regulatory circuits described in legumes.

Introduction

Nitrogen, as a plant macronutrient and limiting factor for plant growth, is highly demanded in agriculture. Thus, organic and synthetic nitrogen fertilizers are essential for productive farming [START_REF] Vitousek | Agriculture. Nutrient imbalances in agricultural development[END_REF]. The advancement of fertilizer usage and the need for sustainable solutions drives research in two crucial perspectives (i) the molecular investigation of nitrate sensing/signaling of plants, to improve and understand nitrogen use -efficiency and -processing and (ii) the study of rhizospheric bacteria, that are important for the mineralization of nitrogen in the soil, but could also associate with plants and influence plant nitrogen consumption and uptake. In this context, nitrogen-fixing (diazotrophic) bacteria play a key role in making dinitrogen gas available for plants in the rhizosphere [START_REF] Carvalho | Nitrogen signalling in plant interactions with associative and endophytic diazotrophic bacteria[END_REF][START_REF] Rosenblueth | Nitrogen Fixation in Cereals[END_REF]. Besides the endosymbiotic nitrogen fixation with rhizobia species and their specialized legume host, there are several free-living, associated, and/or endophytic rhizobacteria that interact with non-legume plant hosts and promote plant growth [START_REF] Hardoim | Properties of bacterial endophytes and their proposed role in plant growth[END_REF][START_REF] Van Deynze | Nitrogen fixation in a landrace of maize is supported by a mucilageassociated diazotrophic microbiota[END_REF]. These mutualistic bacterial interactions facilitate biological nitrogen fixation (BNF), and further promote direct N uptake [START_REF] Carvalho | Nitrogen signalling in plant interactions with associative and endophytic diazotrophic bacteria[END_REF]. Within the group of soil-borne beneficial bacteria, several isolates of bacteria are collectively called Plant-Growth-Promoting Rhizobacteria (PGPR). Over the last decade a great number of PGPRs have been identified and studied in detail not only for their potential to promote plant growth, but also for their induction of biotic and abiotic stress tolerance [START_REF] Vacheron | Plant growth-promoting rhizobacteria and root system functioning[END_REF][START_REF] Gopalakrishnan | Plant growth promoting rhizobia: challenges and opportunities[END_REF][START_REF] Verbon | Beneficial Microbes Affect Endogenous Mechanisms Controlling Root Development[END_REF][START_REF] Etesami | Use of plant growth promoting rhizobacteria (PGPRs) with multiple plant growth promoting traits in stress agriculture: Action mechanisms and future prospects[END_REF]. The mode of action of these PGPRs are mechanistically diverse and far from being completely understood. However, plant growth hormone-signaling, and here specifically auxin and ethylene, are likely to be involved in driving rhizobacterial effects on plant physiology. Accordingly, plant hormones modulate root developmental changes, such as bacteriainduced inhibition of primary root length, positive effects on lateral root formation and root hair development, as well as inhibition of cell division and cell elongation within the root apical meristem and differentiation zone [START_REF] Zamioudis | Unraveling Root Developmental Programs Initiated by Beneficial Pseudomonas spp. Bacteria[END_REF][START_REF] Wintermans | Natural genetic variation in Arabidopsis for responsiveness to plant growth-promoting rhizobacteria[END_REF][START_REF] De Zelicourt | Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2-keto-4-methylthiobutyric acid production[END_REF]. These effects are often achieved by the secretion of plant hormone analogues directly produced by the rhizobacteria or by influencing endogenously plant growth hormone balance and signaling, causing plant developmental effects, as summarized previously [START_REF] Tsukanova | Effect of plant growthpromoting Rhizobacteria on plant hormone homeostasis[END_REF]. Moreover, these processes are often co-regulated by nutritional availability and nutritional signaling. For example, the PGPR Enterobacter radicincitans induced growth promotion and colonization increased under high-nitrogen conditions in tomato. Reciprocally, reduced N fertilization led to altered plant hormone signaling and consequently to an inhibition of these PGPR-effects [START_REF] Berger | Nitrogen supply influences plant growth and transcriptional responses induced by Enterobacter radicincitans in Solanum lycopersicum[END_REF]. In addition, inoculation of Arabidopsis thaliana (Arabidopsis) with a mixture of PGPR Bacillus spp. induced plant growth promotion effects by enhancing N uptake and chlorophyll content, which in turn influences nutritional signaling by a bacterial-induced transcriptional induction of nitrate and ammonium transporter genes [START_REF] Calvo | Plant growth-promoting rhizobacteria induce changes in Arabidopsis thaliana gene expression of nitrate and ammonium uptake genes[END_REF]. Over the last decade key players regulating N-homeostasis and signaling have been identified [START_REF] Wang | Nitrate Transport, Signaling, and Use Efficiency[END_REF][START_REF] Fredes | Nitrate signaling and the control of Arabidopsis growth and development[END_REF]. These include low-and high-affinity transporter protein families in roots such as the NITRATE-TRANSPORTER 1/PEPTIDE-TRANSPORTER FAMILY (NPF, also known as CHL1 and NRT1) and the NITRATE-TRANSPORTER 2 family (NRT2) [START_REF] Krapp | Nitrate transport and signalling in Arabidopsis[END_REF]O'Brien et al., 2016). Members of both protein families are transcriptionally regulated and functionally dependent on the plants' nutritional N-status. For example, the highaffinity-transporting-systems NRT2.1 and NRT2.2 are induced after nitrate uptake [START_REF] Cerezo | Major alterations of the regulation of root NO3-uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in arabidopsis[END_REF]. Examples for root-expressed high nitrate transporters are NRT1.1 (NPF6.3) (dual-affinity transporter) and NRT1.2 (NPF4.6) [START_REF] Wang | Nitrate Transport, Signaling, and Use Efficiency[END_REF]. Interestingly, it has been postulated that NRT1.1 is also able to transport auxin and consequently regulates lateral root growth in Arabidopsis [START_REF] Krouk | Nitrate-Regulated Auxin Transport by NRT1.1 Defines a Mechanism for Nutrient Sensing in Plants[END_REF][START_REF] Bouguyon | Nitrate Controls Root Development through Posttranscriptional Regulation of the NRT1.1/NPF6.3 Transporter/Sensor[END_REF][START_REF] Maghiaoui | The Arabidopsis NRT1.1 transceptor coordinately controls auxin biosynthesis and transport to regulate root branching in response to nitrate[END_REF]. Its auxin transporting activity depends on the availability of exogenous nitrate in a concentration-dependent manner. In nitrate-rich patches, auxin accumulates in lateral roots and induces root emergence and elongation, whereas auxin is depleted in sites with low nitrate availability resulting in reduced overall root growth [START_REF] Remans | The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches[END_REF][START_REF] Mounier | Auxin-mediated nitrate signalling by NRT1.1 participates in the adaptive response of Arabidopsis root architecture to the spatial heterogeneity of nitrate availability[END_REF]. Notably, root system architecture, and here primarily lateral-and primary-root length, are stimulated during moderate but homogenous nitrate availability, but inhibited under excess conditions [START_REF] Jia | Signaling pathways underlying nitrogen-dependent changes in root system architecture: from model to crop species[END_REF]. This indicates that different signaling cascades are activated in fluctuating nitrate conditions resulting in adaptive physiological and morphological processes. The most studied nitrogen-signaling cascade is the so called Primary Nitrate Response (PNR), which describes the response to nitrate resupply after depletion [START_REF] Medici | The Primary Nitrate Response: a multifaceted signalling pathway[END_REF]. This signaling pathway is rapidly activated upon nitrate perception and results in transcriptional changes within minutes. The PNR is directly regulated by transcription factors like the RWP-RK transcription factor NIN-like Protein7 (NLP7) [START_REF] Bellegarde | Signals and players in the transcriptional regulation of root responses by local and systemic N signaling in Arabidopsis thaliana[END_REF]. The Arabidopsis genome contains nine NIN-like Proteins (NLPs), with all of them comprising the RWP-RK motif, enabling DNA binding, and an additional PB-domain (Protein Binding) [START_REF] Chardin | The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development[END_REF]. Transcriptional activation of nitrate-related genes and binding capacity of RWP-RK motif to Nitrate-Responsive cis-Elements (NRE) have been shown for several NLPs of Arabidopsis [START_REF] Konishi | Arabidopsis NIN-like transcription factors have a central role in nitrate signalling[END_REF]. In addition, NLPs comprise a highly conserved nitrate-responsive domain (NRD), which determines nitrate perception and signal transmission [START_REF] Mu | Evolutionary analyses of NIN-like proteins in plants and their roles in nitrate signaling[END_REF]. In Arabidopsis, an NLP function is described for AtNLP8 and AtNLP6/AtNLP7 complex. AtNLP8 is a designated master regulator in nitrate-induced seed germination mediating catabolism of abscisic acid [START_REF] Yan | NIN-like protein 8 is a master regulator of nitrate-promoted seed germination in Arabidopsis[END_REF], while AtNLP7 functions as a key regulator in PNR [START_REF] Castaings | The nodule inception-like protein 7 modulates nitrate sensing and metabolism in Arabidopsis[END_REF]. Recently, AtNLP2 is postulated to have a redundant role in nitrate signaling and vegetative growth of Arabidopsis [START_REF] Konishi | Nitrate-responsive NIN-like protein transcription factors perform unique and redundant roles in Arabidopsis[END_REF]. Interestingly, NLP6 and NLP7 form heterodimers via the PB domain and are suggested to interact in complex with the transcription factor TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20 (TCP20) in the nucleus and regulate meristem growth under nitrate starvation condition [START_REF] Guan | Interacting TCP and NLP transcription factors control plant responses to nitrate availability[END_REF]. Also, other NLPs can form heterodimers that are proposed to play a role in the complexity of nitrate-related signaling Molecular Plant-Microbe Interactions® (MPMI) 6 pathways, as exemplified for AtNLP4 and AtNLP5 [START_REF] Konishi | The role of protein-protein interactions mediated by the PB1 domain of NLP transcription factors in nitrate-inducible gene expression[END_REF][START_REF] Hsin | Phylogenetic and Structural Analysis of NIN-Like Proteins With a Type I/II PB1 Domain That Regulates Oligomerization for Nitrate Response[END_REF]. Root nodule nitrogen-fixing symbiosis is evolutionary retained in different species of the Nitrogen-Fixing-(NFN) clade (Fabales, Rosales, Cucurbitales, Fagales), where it is established between several species and bacteria collectively named rhizobia (in Fabales and the Rosales species Parasponia andersonii) or Frankia, which enables them to access atmospheric dinitrogen after its fixation and conversion to ammonium by the symbiont [START_REF] Werner | Evolutionary signals of symbiotic persistence in the legume-rhizobia mutualism[END_REF][START_REF] Griesmann | Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis[END_REF][START_REF] Van Velzen | Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses[END_REF]. In this process, one of the central transcriptional regulators is the Nodule-INception protein (NIN), which was originally identified in Lotus japonicus [START_REF] Schauser | A plant regulator controlling development of symbiotic root nodules[END_REF]. Different to other NLPs, the Nitrate-responsive domain at the N-terminal region of legume NIN protein is only partially conserved, which explain the loss of direct nitrate responsiveness of NIN and thus may represent an evolutionary adaptation of legumes to maintain this mutualistic interaction [START_REF] Suzuki | The evolutionary events necessary for the emergence of symbiotic nitrogen fixation in legumes may involve a loss of nitrate responsiveness of the NIN transcription factor[END_REF][START_REF] Mu | Evolutionary analyses of NIN-like proteins in plants and their roles in nitrate signaling[END_REF]. While the binding capacities of NIN and NLPs to NRE sequences are similar [START_REF] Suzuki | The evolutionary events necessary for the emergence of symbiotic nitrogen fixation in legumes may involve a loss of nitrate responsiveness of the NIN transcription factor[END_REF], it should be noted that the functionality of rootnodule symbiosis is strictly regulated by the availability of nitrate. Furthermore, this mechanism is also regulated via NLP functions as exemplified by the Nitrate unResponsive SYMbiosis 1 (NRSYM1) and NRSYM2 in Lotus japonicus [START_REF] Nishida | A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus[END_REF][START_REF] Nishida | Different DNA-binding specificities of NLP and NIN transcription factors underlie nitrate-induced control of root nodulation. Plant Cell[END_REF]. Recently, these two LjNLPs were shown to have distinct roles in nitrate signaling, and, moreover, influence nitrate induced inhibition of nodulation on levels of symbiotic-gene regulation [START_REF] Nishida | Different DNA-binding specificities of NLP and NIN transcription factors underlie nitrate-induced control of root nodulation. Plant Cell[END_REF]. The LjNLP4 (NRSYM1)-LjNIN interaction complex accumulates in the nucleus in response to high nitrate conditions and regulates transcription of the CLE-RS2 peptide, which in turn negatively controls nodule numbers. Remarkably, expression of the Arabidopsis AtNLP6 and AtNLP7 rescued the nodulation phenotype of the nrsym1 mutant, suggesting a functional conservation of NLPs in legumes and non-legumes plants [START_REF] Nishida | A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus[END_REF]. In Medicago truncatula (Medicago), MtNLP1 was reported to mediate nitrate signaling and to re-localize from the cytosol to the nucleus under high nitrate conditions. Subsequently, complex formation between MtNLP and MtNIN proteins in the nucleus suppresses the activation of the root-nodule symbiosis marker genes CRE1 and NF-YA and thus induces the nitrate-dependent inhibition of nodulation [START_REF] Lin | NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula[END_REF]. Analyses of different natural soil samples indicates that the Arabidopsis rhizosphere also contains about 4% bacteria from the Rhizobiales order [START_REF] Garrido-Oter | Modular Traits of the Rhizobiales Root Microbiota and Their Evolutionary Relationship with Symbiotic Rhizobia[END_REF] and effects of rhizobia on Arabidopsis root development have already been described. For example, the nodulating Rhizobium sp. IRBG74 inhibits root development of Arabidopsis by impacting auxin signaling via a change in transcriptional activation of auxin responsive genes [START_REF] Zhao | Rhizobium sp. IRBG74 Alters Arabidopsis Root Development by Affecting Auxin Signaling[END_REF]. Similarly, Mesorhizobium loti (M. loti), the natural symbiont of L. japonicus, inhibits Arabidopsis primary root growth in an auxin-dependent manner, whereas an auxin-independent increase in root hair formation was observed [START_REF] Poitout | Local signalling pathways regulate the Arabidopsis root developmental response to Mesorhizobium loti inoculation[END_REF]. Furthermore, the Medicago symbiont Sinorhizobium meliloti (S. meliloti) induces rapid transcriptional reprogramming of defense genes in Arabidopsis cortex cells followed by late responses of stress-related genes in the pericycle, a possible stress adaptation response resulting in an increased number of lateral roots [START_REF] Walker | Changes in Gene Expression in Space and Time Orchestrate Environmentally Mediated Shaping of Root Architecture[END_REF]. Here, we investigated alterations of the Arabidopsis root system architecture upon inoculation with the widely used and well-characterized M. loti and S. meliloti with respect to nitrate availability and nitrate-related signaling. For our investigations we selected these two model rhizobia to adhere with the bacterial point of view as close as possible to symbiotic abilities on plant infection processes. We demonstrate that rhizobial effects on the Arabidopsis root architecture are initiated by a nitrate-related signaling pathway, specifically involving NIN-like proteins. Our study highlights the importance of nutrient-dependent signaling during bacterial-plant interactions and shed further light on the complex interplay between plant endogenous signaling and bacterial connectivity.

Results

Rhizobia associate with the root surface of Arabidopsis seedlings Following the findings that rhizobia and other diazotrophic bacteria are able to colonize the endosphere of non-legume roots by entering via natural openings such as sites of lateral root emergence [START_REF] De Zelicourt | Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2-keto-4-methylthiobutyric acid production[END_REF][START_REF] Schneijderberg | A genetically and functionally diverse group of non-diazotrophic Bradyrhizobium spp. colonizes the root endophytic compartment of Arabidopsis thaliana[END_REF], we assessed the colonization patterns of rhizobia on Arabidopsis roots. For this, Arabidopsis plants were grown vertically in vitro and inoculated with fluorescently labelled M. loti for three days. Bacterial biofilms formed along the root epidermis (Fig. 1a) with enrichments at the root cap and the root elongation zone (Fig. 1d). Similar colonization patterns of bacteria were observed on plants growing on high-and lownitrate medium (Fig. 1b,c). Furthermore, rhizobia accumulated at sites of lateral root emergence (Fig. 1e) and invaded the space of these natural epidermal openings (Fig. 1f). However, clear signs of intercellular colonization in further tissues were not observed. Thus, rhizobia associated with the non-legume Arabidopsis by establishing an interaction on the outside of the plant root, independent of nitrate-availability conditions.

Rhizobia affect plant root development

As several soil-borne bacteria are known to alter the plant root architecture, thus enabling plants to better access nutrients directly or indirectly, we tested whether M. loti and S. meliloti -effects are influenced by the availability of exogenous nitrate. For this, we transferred 10-days old Arabidopsis seedlings onto plant medium with either high nitrate (5mM KNO 3 ) or nitrate starvation (0.1mM KNO 3 ) conditions and additionally inoculated these plants with either S. meliloti or M. loti for 3 days. The number and length of lateral roots significantly increased when inoculating plants grown under high nitrate conditions with rhizobia compared to the non-inoculated control (Fig. 2ae) while the primary root length was unaffected by this treatment (Fig. 2f). Nitrate deprivation fully abolished the rhizobial effect on lateral root growth (Fig. 2d,e) and resulted in a moderate increase in the primary root length, which was independent of the presence of bacteria (Fig. 2f). These interaction responses are in contrast to observations in legumes, where a functional root-nodule symbiosis is strictly established only in limiting nitrate conditions. Among other cells, root hairs can serve as primary contact sites and can be altered by rhizobacteria [START_REF] Poitout | Local signalling pathways regulate the Arabidopsis root developmental response to Mesorhizobium loti inoculation[END_REF][START_REF] De Zelicourt | Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2-keto-4-methylthiobutyric acid production[END_REF]. Thus, we investigated root hair development along young segments of the primary root. Here, we decided to inoculate rhizobia as early as 7 days post germination, since root hairs develop faster than lateral roots. Furthermore, we included a low-nitrate condition (0.25 mM) to further assess nutrient-limiting conditions rather than nutrient stress responses. As expected, nitrate starvation led to a significant increase in root hair length (Fig. 3a-b, h) and number (Fig. 3g) even in the absence of rhizobia, as published earlier [START_REF] Vatter | Regulation of length and density of Arabidopsis root hairs by ammonium and nitrate[END_REF]. While the presence of S. meliloti moderately affected root hair patterns (Fig. 3c-d, g-h), a significant induction of root hair growth was observed upon inoculation with M. loti under high (5 mM) and low (0.25 mM) nitrate conditions. No rhizobial effects on root hairs were found under nitrate-starvation conditions (0.1mM nitrate) in comparison to non-treated plants (Fig. 3e-h). Therefore, we continued experiments on rhizobial effects on high-(5mM) and low-(0.25mM) nitrate conditions rather than observing nutrient stress conditions at 0.1mM nitrate. To further test the impact of rhizobia on root hair growth, we examined root hair-specific marker gene expression in roots treated with rhizobia under different nitrate conditions. Indeed, rhizobia tended to induce the morphogenetic root-hair genes RHD6-LIKE4 (RSL4), and EXTENSIN11 (EXT11), but not ROOT HAIR DEFECTIVE6 (RHD6) (Fig. 3). Overall, our results show that M. loti treatments were particularly pronounced under high-nitrate conditions. These data demonstrate that Arabidopsis root architecture and morphology are affected by rhizobia in a similar way as nitrate impacts root modifications.

Rhizobia influence nitrate-related gene expression

To continue analyzing rhizobia-induced root modifications and its impact on nitrate-dependent signaling responses, we investigated nitrate-related gene expression in roots treated with rhizobia. Due to the fact that nitrate conditions impact distinct set of genes, we studied NRT transceptors that have been reported to be up-regulated also in low-nitrate conditions and improve nitrate use efficiency [START_REF] Bouguyon | Nitrate Controls Root Development through Posttranscriptional Regulation of the NRT1.1/NPF6.3 Transporter/Sensor[END_REF][START_REF] Ye | A reevaluation of the contribution of NRT1.1 to nitrate uptake in Arabidopsis under low-nitrate supply[END_REF]. Although NRT2.1 and NRT1.1 were induced under low-nitrate conditions, their expression was further increased in samples treated with rhizobia, for NRT1.1 even in high-nitrate conditions (Fig 4a,b). By contrast, expression of the starvation marker gene NRT2.4 that was reported to be transcriptionally induced in low-nitrate conditions [START_REF] Kiba | The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants[END_REF] was not modified by rhizobia (Fig. 4c). Transcriptionally activated NIN-like proteins are known to play a role in nitrate signaling during rhizobia-legume interactions. These proteins suppress gene expression by either directly binding to NIN-proteins or by competitive binding to promotors of symbiosis-responsive genes [START_REF] Lin | NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula[END_REF][START_REF] Nishida | A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus[END_REF]. Large-scale phylogenetic analysis of the NLP family revealed their conservation in land plants [START_REF] Schauser | Evolution of NIN-Like proteins in Arabidopsis, rice, and Lotus japonicus[END_REF]. Furthermore, different duplication events led to three NLP-clades that evolved prior to the divergence of seed plants (Fig. S2 andS3). As NLP7 (NIN -like protein 7) was identified as key regulator during the primary nitrate response in Arabidopsis and accumulates as a heteromeric complex with NLP6 and TCP20 in the nucleus during nitrate-starvation responses [START_REF] Guan | Dancing with Hormones: A Current Perspective of Nitrate Signaling and Regulation in Arabidopsis[END_REF], we decided to analyze transcriptional activation of NLPs in our experimental conditions (Fig. 4d-f), (Fig. S4). While NLPs are not transcriptionally up-regulated during nutritional signaling per se [START_REF] Chardin | The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development[END_REF], we detected a significant induction of NLP4 and NLP5 expression in roots inoculated with M. loti in high-nitrate and S. meliloti in low-nitrate conditions (Fig. 4e,f). However, other NLPs were not influenced by the presence of rhizobia (Fig. S4). Additionally, genes involved in nitrate metabolism such as NIA1 and NIA2 (nitrate reductase), and NIR1 (nitrite reductase), which are regulated during nitrate responses, were also found to be induced upon rhizobial inoculation of plants grown under high-nitrate, but not under low nitrate conditions (Fig. 4g-i). These results suggest that rhizobia intervene nitrate-dependent signaling by differentially regulating nitrate-related marker genes in distinct nitrate responses.

M. loti-induced root morphology changes are controlled by NLP4 and NLP5

As expression patterns of nitrate-related genes in rhizobia treated root samples indicate a distinct nitrate-dependent signaling during rhizobium-induced root modifications, we further tested the genetic dependency of our observations on root development using different nitrate signaling mutants. Here, the most obvious M. loti -induced phenotype, the impact on root hair development at the primary root, was selected as experimental readout. Due to the importance of transceptors (NRTs) during nitrate -transport, -sensing and -signaling in roots, we studied the impact of M. loti inoculation on nrt1.1-5 (chl1-5) and nrt2.1 mutant backgrounds (Fig. S5). The nrt2.1 mutant exhibited wild-type responses towards the presence of rhizobia (Fig. S5c-d). This was different in the nrt1-1 mutant that already showed a nitrate-independent reduced root hair formation phenotype under control conditions that was not rescued by the application of rhizobia (Fig. S5ab). We then investigated downstream roles of the NLP master regulators during rhizobia-Arabidopsis interactions. For this, we obtained independent T-DNA insertion mutant alleles for NLP6 and NLP7 [START_REF] Castaings | The nodule inception-like protein 7 modulates nitrate sensing and metabolism in Arabidopsis[END_REF] and analyzed root hair development in these mutant plants. Under control conditions (uninoculated, high-nitrate condition) nlp6 and nlp7 mutants already developed more root hairs compared to the wild-type, while mutants responded to the presence of rhizobia in a wild-type-like and nitrate-independent manner (Fig. 5a-b). Due to the striking importance of the Nodule-Inception Protein NIN during root nodule symbioses and in line with the M. loti-induced expression of AtNLP4 and AtNLP5 expression in Arabidopsis (Fig. 4), we investigated the NLP-clade NLP1-NLP5 (closely related to the clade containing NIN from legumes such as Medicago) (Fig. S3). Mutants of this NIN-related gene family in Arabidopsis, specifically nlp1, nlp4 and nlp5, responded to nitrate limitation by increased root hair formation as observed for the wild-type control (Fig. S6, Fig. 5c-d, Fig. S7a-b). However, and in contrast to the nlp1 mutant, nlp4 and nlp5 mutants failed to induce pronounced root hair development upon application of M. loti (Fig. 5c-d, Fig. S7a-b). Furthermore, the rhizobial effects of induced lateral root emergence and overall lateral root length were also lost in nlp4 and nlp5 mutants (Fig. S7c-d). To further clarify the involvement of NLP4 and NLP5, we generated two constitutive over expression lines of genomic NLP4 and NLP5 into the respective mutant backgrounds. Two independent lines of genotype clearly showed rhizobia effects and were able to complement the nlp4 and nlp5 mutations (Fig. 5e, f, Fig. S7d). Following up on the significant phenotypes of nlp4 and nlp5 we further examined rhizobia-induced gene expression in these mutants (Fig. S8). Interestingly, the elevated transcription of nitrate-related genes in rhizobia treated samples as observed in wild-type plants (Fig. 4) was lost in nlp4 and nlp5 mutants (Fig. S8a-b). However, root hair development marker gene expression of RSL4 and EXT11 were still partially elevated in nlp4 and nlp5, respectively (Fig. S8c-d). In summary, the root-development response to nitrate is not impaired in these mutants, while nlp4 and nlp5 fail to induce distinct rhizobia-induced nitrate-related signaling responses. Thus, these data suggest the presence of an NLP4/5-dependent signaling pathway in Rhizobia-Arabidopsis interactions, similar to nitrate-dependent regulation of symbioses.

M. loti alters cell proliferation and elongation patterns in the root tip

Besides nitrate being a signaling component during root development it also influences cell divisions at the root apical meristem and subsequent cell cycle progression [START_REF] Guan | Interacting TCP and NLP transcription factors control plant responses to nitrate availability[END_REF][START_REF] Gigli-Bisceglia | Cell wall integrity modulates Arabidopsis thaliana cell cycle gene expression in a cytokinin-and nitrate reductasedependent manner[END_REF]. Similar effects were also described for treatments with PGPRs, which can influence root architecture via hormonal cross-talks [START_REF] Verbon | Beneficial Microbes Affect Endogenous Mechanisms Controlling Root Development[END_REF]. Since root hair development at the primary root is determined by the cell fate at the apical meristem [START_REF] Vissenberg | Hormonal regulation of root hair growth and responses to the environment in Arabidopsis[END_REF], we decided to test possible impacts of M. loti on Arabidopsis root cell patterning. For this, we transferred 7 days-old Arabidopsis seedlings on plant medium with highor low-nitrate, inoculated them with M. loti for 3 days and stained root cell membranes with the dye FM4-64 to visualize cell morphology and to quantify cell numbers (Fig. 6a-d). In wild-type Arabidopsis plants, the average length of the first five cortex cells above the root transition zone significantly decreased under high-but not under low-nitrate conditions in the presence of M. loti (Fig. 6e), whereas this effect was not observed in nlp4 and nlp5 mutant plants (Fig. 6f,g). This shorted elongation zone implies a bacteria-induced and NLP4/5-dependent early differentiation in high-nitrate conditions, and could explain the observed effects on root hair density on the primary root tip (Fig. 3h and5c, d). To quantify the meristem size, we counted the cell number from the quiescent center until the transition zone (first doubling of cell size) in the cortex cell layer along a longitudinal axis. Cortical cell numbers were found to be increased by M. loti in both wild-type and nlp4 mutant plants, while this rhizobial effect was lost in nlp5 mutants (Fig. 6h-j). This was accompanied by increased transcription of the cell cycle-related cyclin D3 (CYCD3;1) (Fig. 6h), but not of CYCB1;1 (Fig. 6i). Since CYCD3;1 is expressed during the meiotic G1/S phase of the cell cycle, while CYCB1;1 serves as a marker for a prolonged G2 phase, we conclude that cortical cell division rates are induced by M. loti. Overall, we demonstrate that rhizobium-induced signaling influences cell cycle progression and root cell elongation in a NLP5-dependent manner.

Discussion

Gene regulatory networks control plant responses to different nutrient conditions, leading to modifications of the root architecture. In natural soils, plant roots are additionally challenged by a great diversity of beneficial and pathogenic microbes and the plants' overall nutrient status determines the responsiveness to these microbes [START_REF] Shahzad | Food for thought: how nutrients regulate root system architecture[END_REF][START_REF] Singh | A novel function of Nsignaling in plants with special reference to Trichoderma interaction influencing plant growth, nitrogen use efficiency, and cross talk with plant hormones[END_REF]. Among them are rhizobia, which have been mostly investigated in frame of the root-nodule symbiosis, a process that ultimately results in the ability of these bacteria to fix atmospheric dinitrogen gas. This mutualistic interaction strictly depends on the unavailability of nitrate and is immediately terminated upon resupply of inorganic nitrate to the host plant [START_REF] Van Noorden | Molecular Signals Controlling the Inhibition of Nodulation by Nitrate in Medicago truncatula[END_REF]. While the root-nodule symbioses interaction mostly relies on a highly strain-specific interaction with a narrow host range, microbiome studies revealed the presence of rhizobia in the rhizosphere or as endophytes in roots of non-legume plants, as well as nitrate serving as a regulating factor mediating the microbial composition on Arabidopsis roots [START_REF] Konishi | Nitrate Supply-Dependent Shifts in Communities of Root-Associated Bacteria in Arabidopsis[END_REF][START_REF] Garrido-Oter | Modular Traits of the Rhizobiales Root Microbiota and Their Evolutionary Relationship with Symbiotic Rhizobia[END_REF]. In this manuscript, we investigated rhizobium-induced root morphology changes and their dependence on nitrate availability and nitrate-related signaling cascades in Arabidopsis. We demonstrate that rhizobia affect lateral root emergence and -elongation (Fig. 2) and increase root hair density and length (Fig. 3,5). Interestingly, these effects were found to be pronounced in high nitrate conditions and reduced or even abolished in low-nitrate conditions (Fig. 2,3). In contrast to the inhibitory effects of nitrate on root-nodule symbiosis, our findings imply a supporting nitratedependent signaling and suggest an underlying complex crosstalk between nutrient-related signaling and rhizobia-induced responses on non-legume plants. It is well documented that nitrate starvation is accompanied by stimulated lateral root emergence and primary root elongation, while excess nitrate leads to root growth inhibition [START_REF] Jia | Signaling pathways underlying nitrogen-dependent changes in root system architecture: from model to crop species[END_REF]. In line with this, the presence of PGPRs on Arabidopsis roots stimulates a low-nitrate response. This can be exemplified by the PGPR Phyllobacterium STM196 that positively influences root architecture under high-nitrate conditions. These rhizobacteria did not directly alter the N-status in leaves or root nitrate uptake, but induced the expression of nitrate starvation marker genes such as AtNRT2.5 and AtNRT2.6 [START_REF] Mantelin | Nitrate-dependent control of root architecture and N nutrition are altered by a plant growth-promoting Phyllobacterium sp[END_REF] that are also postulated to play a central role in bacterial-induced growth promotion [START_REF] Kechid | The NRT2.5 and NRT2.6 genes are involved in growth promotion of Arabidopsis by the plant growth-promoting rhizobacterium (PGPR) strain Phyllobacterium brassicacearum STM196[END_REF]. This is in agreement with our results on nitrate-influenced M. loti-induced root hair development (Fig. 3,5,S7). Indeed, one interesting aspect is the impact of rhizobia on nitrate starvation signaling (Fig. 4a-c), similarly to nitrate-induced inhibition circuits in legume symbiosis. However, our data indicate a rather specific rhizobium-induced high-nitrate signaling in plants, comprising NRT1.1, and nitrate metabolic gene regulations (Fig. 4a, g-i). Furthermore, the effectiveness of M. loti influenced root modifications (Fig. 3) can be explained by this specific rhizobium-induced high-nitrate transcriptional response, which specifically involves differential expression of the transcription factors NLP4 and NLP5 (Fig. 4e, f, Fig. 5e,f) and directs to a positive regulatory mechanism of an NLP-dependent pathway (Fig. 7). In line with this, the corresponding nlp4 and nlp5 mutants failed to morphologically respond to the presence of rhizobia at the primary root tip (Fig. 5, S7) and lose rhizobia-induced nitrate-related gene expression (Fig. S8a,b). These results are of particular interest, considering the evolutionary proximity of AtNLP4 and AtNLP5 to the legume gene NIN, and their co-orthologs legume genes MtNLP1, LjNLP4 and LjNLP1 that are essential for controlling nitrate-dependent root nodule symbiosis [START_REF] Schauser | Evolution of NIN-Like proteins in Arabidopsis, rice, and Lotus japonicus[END_REF][START_REF] Lin | NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula[END_REF][START_REF] Nishida | A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus[END_REF][START_REF] Mu | Evolutionary analyses of NIN-like proteins in plants and their roles in nitrate signaling[END_REF][START_REF] Nishida | Different DNA-binding specificities of NLP and NIN transcription factors underlie nitrate-induced control of root nodulation. Plant Cell[END_REF]. Thus, the response to rhizobia could be trait shared within the NLP clade (NLP1-5, angiosperms) and the Eudicots duplication leading to NLP4/5 and NLP1/2/3 could have later allowed the possible neofunctionalization of NIN for root nodule symbiosis (Fig. S2,S3). Additionally, we demonstrated a NLP4/NLP5-dependent and rhizobiuminduced stalling of cell elongation but increased cell division rates at the proximal part of the root elongation zone (Fig. 6), which suggests an NLP-dependent (nitrate related) -signaling in the root elongation zone and directs to an earlier differentiation of roots treated with rhizobia (Fig. 7). In line with this hypothesis, cell wall integrity and meristem cell progression are postulated to depend on nitrate reductase function and endogenous cytokinin homeostasis in roots tips [START_REF] Gigli-Bisceglia | Cell wall integrity modulates Arabidopsis thaliana cell cycle gene expression in a cytokinin-and nitrate reductasedependent manner[END_REF]. In addition, morphological changes also coincided with an enhanced activation of the cell-cycle marker gene CYCB1;1 [START_REF] Trinh | Evaluation of the plant growth-promoting activity of Pseudomonas nitroreducens in Arabidopsis thaliana and Lactuca sativa[END_REF]. Reciprocally, the application of Pseudomonas putida WCS417 on Arabidopsis resulted in an inhibition of root growth, which was likely caused by reduced cell elongation and driven by an observed activation of CYCB1;1 in inoculated samples [START_REF] Zamioudis | Unraveling Root Developmental Programs Initiated by Beneficial Pseudomonas spp. Bacteria[END_REF]. While M. loti induced similar effects on cell proliferation we observed an induced expression of CYCD1;3 but not of CYCB1;1 indicative for an accelerated cell progression into the G1 phase (Fig. 6). Such induction of D-like cyclins (here: CYCD1, D2 and D3) was also reported in rice seedlings when treated with S. meliloti [START_REF] Wu | Rhizobia promote the growth of rice shoots by targeting cell signaling, division and expansion[END_REF]. The importance of nitrate-regulated transcription factors during plant root development has been extensively described. For example, the bZIP transcription factors TGA1 and TGA4 control nitrateinduced lateral root formation (Alvarez et al., 2014) and root hair cell fate, latter by regulating the root hair cell-specific gene CAPRICE (CPC) [START_REF] Canales | Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana[END_REF]. Furthermore, a meta-analysis indicates nitrate regulation of root hair growth by bZIP and G2-transcription factors coordinated auxin signaling response [START_REF] Canales | Systems analysis of transcriptome data provides new hypotheses about Arabidopsis root response to nitrate treatments[END_REF]. Interestingly, the NRT1.1-TGA1/4 nitrate signaling complex additionally controls root hair density and trichoblast cell length [START_REF] Canales | Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana[END_REF]. Besides rhizobia-induced NRT1.1 regulation (Fig. 4a), we also assume that NRT1.1 controls rhizobia-induced changes in trichoblast patterning (Fig. S5). However, mutation in the NRT1.1 locus affects already root hair development in high and low nitrate conditions (control conditions) and may thus mask rhizobial responses. By contrast, nlp4 and/or nlp5 mutants are still responsive to nitrate limitation and lose the rhizobial impact on root morphogenesis (Fig. 5 and S7). Overall, these results reveal new promising NLP candidates and potential transcription factors regulating rhizobia-induced nitrate-related signaling responses in Arabidopsis. Still, specific functions of NLP4 and NLP5, including protein complex formation in the nucleus and the activation of target genes, within the crosstalk signaling of nitrate availability adaptation processes and rhizobia-induced responses need to be further investigated, however our results clearly point to a so far uncovered regulatory circuit. Taken together, we understand the investigated bacterial-plant interaction as a complex signaling pathway between nutritional sensing followed by metabolic progressing in crosstalk with bacteria interaction and stress-induced responses, which finally results in adaptive developmental processes. Being fully aware of the well-documented crosstalk between nitrate signaling and growth hormones [START_REF] Takei | AtIPT3 is a key determinant of nitrate-dependent cytokinin biosynthesis in Arabidopsis[END_REF][START_REF] Vidal | Nitrate-responsive miR393/AFB3 regulatory module controls root system architecture in Arabidopsis thaliana[END_REF][START_REF] Ma | Auxin biosynthetic gene TAR2 is involved in low nitrogen-mediated reprogramming of root architecture in Arabidopsis[END_REF][START_REF] Naulin | Nitrate Induction of Primary Root Growth Requires Cytokinin Signaling in Arabidopsis thaliana[END_REF], we consider the observed rhizobia-induced nitrate responses as early signaling events after bacterial perception, which finally lead to downstream fine-tuning of growth hormone effects (Fig. 7). Future research in this line will contribute to the development of bacterial inoculums as effective and sustainable solutions in agriculture.

Materials and Methods

Plant growth conditions and root observations

Arabidopsis thaliana wild type ecotype Columbia (Col-0), Wassilewskija (WS) and knockout mutant seeds were surface sterilized with 50% ethanol/0.5% Tritron X-100 and briefly washed with 95% ethanol. Seeds for mutant plants nlp4 (N563595), nlp5 (N527488), and nlp1 (N584809) were provided by The Nottingham Arabidopsis Stock Centre (NASC) and homozygosity for T-DNA insertion was validated by PCR with primers designed by SALK Institute Genomic Analysis Laboratory. Seeds were germinated on half-strength Murashige-Skoog-medium (1/2 MS) after stratification at 10°C for 3 days and grown horizontally for 7-days/10-days (as indicated) at 22°C with 150µmol/m 2 /s light in a 12/12-h day/night photoperiod. After germination seedlings were transferred and inoculated with bacteria on nitrate-variable plant medium containing 5mM/0.25mM/0.1mM (as indicated) KNO 3 or 5mM KCl (in low-nitrate conditions KCl was used to buffer ion concentrations), 1mM KH 2 PO 4 , 0.5 mM CaSO 4 , 0.5 mM MgCl 2 , 3 mM MES, pH 5.8, 0.25% (w/v) sucrose and 0,1% micronutrients [START_REF] Scheible | Genome-wide reprogramming of primary and secondary metabolism, protein synthesis, cellular growth processes, and the regulatory infrastructure of Arabidopsis in response to nitrogen[END_REF] including 1% ferric citrate, supplemented with 0.9% Phyto-Agar and grown vertically for 3 days. Root modifications were quantified on images photographed with scale bar and analyzed on Image J Software. For root hair quantifications pictures were taken using a stereomicroscope and root hairs were measured and counted in 5 mm from the start of the differentiation zone (first root hair appearance). Data were averaged from at least 7 root samples and experiments were performed at least 3 times independently.

Bacteria growth conditions

Sinorhizobium meliloti strain Sm2011, tetracycline resistant with mCherry as fluorescent marker, and Mesorhizobium loti strain MAFF, gentamycin resistant with dsRED as fluorophore, were used for bacterial treatments. For each experiment bacteria were freshly plated on TY-medium and incubated for 3 days at 28°C. Afterwards the inoculum culture was grown in 5 ml liquid TY-medium for 48h. For inoculation, bacteria were pelleted, resuspended in sterile H 2 O and diluted to a final OD 600 of 0.1. Ten µl of bacteria solution per root were used to treat Arabidopsis seedling and primary root length was marked after inoculation. For reproducibility the timing of bacterial preculture growth is essential for an adequate-equal inoculum preparation.

Quantitative RT-PCR All quantitative RT-PCR were performed with 7-day old seedlings grown for 3 days in high-(5mM) and low-(0.25mM) nitrate conditions. Total RNA was extracted from Arabidopsis root samples using an RNA extraction kit (Sigma Aldrich) and quality control was performed photometrically using a NanoDrop (Thermo Fisher Scientific) device and on agarose gel. To remove traces of genomic DNA, 1 g of total RNA from each sample was treated with DNase-I and genomic-DNA contamination was excluded via quantitative RT-PCR. Reverse transcription was accomplished from purified RNA with SuperScript cDNA Synthesis Kit (Quanta BioScience). Efficiency of cDNA synthesis was verified by semi-quantitative PCR on housekeeping actin2 transcripts. All primers used for qRT-PCR are listed in (Table S1). Quantitative RT-PCR was performed using an Applied Biosystems real time PCR system and a Sybr green Mix. Ct-values were normalized to the housekeeping gene EF1α4 or UBQ4, as indicated. Data were averaged from at least 3 independent experiments.

Characterization of mutants and over-expression lines

For genotyping of the T-DNA insertion mutants nlp4 and nlp5, the recommended PCR set up from Salk Institute Genomic Analysis Laboratory (SALK) with left-and right border primers and the LBb1.3 primer was used. Genomic DNA was extracted from soil grown wild type Col-0 and mutant plants and subjected to PCR analysis. Homozygous plants were then selected for further experiments. To proof that the respective T-DNA is inserted in an exonic region of the gene of interest, semi-quantitative PCR was carried out on cDNA samples (as described in "Quantitative RT-PCR") using a primer pair covering the T-DNA flanking sequence. For transcript quantification qRT-PCR was performed on cDNA extracted from plant roots of non-treated samples (Fig. S1). All primers used for characterization are listed in (Table S1).

Cloning method and selection of transgenic lines

The cloning procedure used for constructing complementation vectors was the Golden Gate cloning method [START_REF] Binder | A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants[END_REF]. Genomic DNA from Arabidopsis plants was extracted and the full-length genomic NLP4 and NLP5 was amplified with proofreading DNA polymerase PCR using corresponding Golden Gate primers. To keep Golden Gate compatibility silent mutations were performed on BpiI and BsaI restriction sites. All primers used for cloning are listed in Table S1. Level II constructs were assembled with vectors containing the promoter sequence of AtUBQ10, the fluorescent tag eGFP, and for transformant selection the herbicide resistance BASTA as well as a screenable marker FAST [START_REF] Shimada | A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana[END_REF]. The resulting constructs were quality controlled by full-length sequencing. For plant transformation Agrobacterium tumefaciens (AGL1) was transformed with described construct and flower buds of soil grown 5weeks old Arabidopsis mutant plants nlp4 and nlp5 mutant plants were infected with appropriate bacterial solution according the floral dip method [START_REF] Clough | Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[END_REF]. Infection was repeated after one week. T0 seeds were harvested and positive transformations were selected via FAST method [START_REF] Shimada | A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana[END_REF]. Positive seeds were germinated on 10mM BASTA ½ MS plates and plants were propagated until T3. For homozygosity of transformants segregation tests were proceed in T2 and resistant plant lines were selected in T3 for following experiments.

FM4-64 staining and image analyzes of root tip samples Treated seedlings were incubated in 5 µM FM TM 4-64 dissolved in liquid nitrate-variable plant medium for 15 min. After 5 min washing in liquid plant medium primary root tips were mounted on a microscopy slide. Images of 3880x3880 resolution were taken with SP8-Leica confocal microscope using a 25x water objective and a white laser, emission of 554 to 591 nm.

Phylogenetic analysis NLP sequences were retrieved by the tBLASTn algorithm v2.9.0+ [START_REF] Camacho | BLAST+: architecture and applications[END_REF] using the NIN protein from the model species Medicago truncatula as query, a database containing 124 land plants genomes from the SymDB database (http://www.polebio.lrsv.ups-tlse.fr/symdb/web/) and no e-value greater than 1 e-10 . Sequences were aligned using MAFFT v7.407 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF] with default parameters. Alignments were cleaned using trimAl v1.4 rev22 [START_REF] Capella-Gutierrez | trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[END_REF][START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF] to remove positions with more than 50% of gaps. Cleaned alignments were subjected to Maximum Likelihood analysis using IQ-TREE v1.6.7 [START_REF] Nguyen | IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[END_REF] after testing the best-fitting evolutionary model using ModelFinder [START_REF] Kalyaanamoorthy | ModelFinder: fast model selection for accurate phylogenetic estimates[END_REF]. Branch support was tested with 10,000 SH-aLRT [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF]. Trees were visualized and annotated using the iTOL platform v4.4.2 [START_REF] Letunic | Interactive Tree Of Life (iTOL) v4: recent updates and new developments[END_REF]. For readability reasons, presented phylogeny in (Fig. S3) has been reduced to 7 species with maintained topology of the 124 species-main tree (Fig. S2). Ten-days old seedlings were transferred on high-nitrate plant medium (5mM KNO 3 ) or nitratestarvation plant medium (0.1mM KNO 3 ) and roots were inoculated for 3 days with rhizobia (S. meliloti) or (M. loti). (a) control seedlings grown for 3 days on high-nitrate plant medium. (b) S. meliloti and (c) M. loti inoculated seedlings grown on high-nitrate plant medium scale bar presents 1 cm (starting root length, before inoculation, was marked with blue marker pen). ( d Seven-days old seedlings of wild-type (Col-0), mutant plants (as indicated), or over-expression lines were transferred on high-nitrate plant medium (5mM KNO 3 ) or low-nitrate plant medium (0.25mM KNO 3 ) and roots were inoculated for 3 days with M. loti. Quantification of root hair formation on primary root tip is presented. Down site representative close-up image of Col-0, and root differentiation zone treated with M. loti for 3 days of Col-0, nlp-mutant (nlp5). and nlp5/pAtUBQ10:NLP5 line (OENLP5). Statistical significance was determined by one-factorial ANOVA α=0.05 followed by Scheffé post-hoc test. Results obtained indicate rhizobia-induced local root developmental effects on the non-legume host Arabidopsis thaliana. We demonstrate an involvement of key NIN-like proteins and show an induction of nitrate signaling during rhizobia responses, which finally impacts on cell cycle progression and morphological modifications. Furthermore, described effects are pronounced in high nitrate conditions and suggest a complex crosstalk between bacterial-and nitrate-related signaling pathways. Fig. S5 Dual-affinity-transceptor NRT1.1 is needed for M. loti-induced impact on root morphology, but not NRT2.1. Seven-days old seedlings of wild-type (Col-0) and Wassilewskija (WS) or mutant plants (as indicated) were transferred on high-nitrate plant medium (5mM) or low-nitrate plant medium (0.25mM) and roots were inoculated for 3 days with M. loti. Quantification of root hair formation and root hair length on primary root tip (first 5 mm) is presented. Statistical significance was determined by one-factorial ANOVA α=0.05 followed by Scheffé post-hoc test.

Fig. S6 A NLP1 mutation is not influencing rhizobial effects on root hair development.

Seven-days old seedlings of wild-type (Col-0) or mutant plants (as indicated) were transferred on high-nitrate plant medium (5mM) or low-nitrate plant medium (0.25mM) and roots were inoculated for 3 days with M. loti. Quantification of root hair formation and root hair length on primary root tip (first 5 mm) is presented. Statistic significance was determined by one-factorial ANOVA α=0.05 followed by Scheffé post-hoc test Fig. S7 AtNLP4 and AtNLP5 are involved in M. loti induced root modifications. Seven-days old seedlings of wild-type (Col-0), mutant plants (as indicated) or over-expression lines were transferred on high-nitrate plant medium (5mM) or low-nitrate plant medium (0.25mM) and roots were inoculated for 3 days with M. loti. (a) and (b) quantification of root hair length on primary root tip (first 5 mm) is presented. Statistic significance was determined by one-factorial ANOVA α=0.05 followed by Scheffé post-hoc test. (c) and (d) quantifications of lateral root numbers and lateral root length are presented. Statistical significance was determined by students t-test *=p<0.05; ***=p<0.001. Fig. S8 Varying regulation of rhizobia-induced genes in nlp4 and nlp5 mutant roots. Transcript levels of indicated genes in 7-day old Arabidopsis root samples treated for 3-days with rhizobium M. loti or in 10-day old control samples grown on high-(5 mM) or low-(0.25 mM) nitrate plant medium conditions. Quantified by qRT-PCR and calculated relative to the housekeeping gene expression level of UBQ4.

Figure 1 .

 1 Figure 1.: Rhizobia attachment on roots of non-legume host plant Arabidopsis

Figure 2 .

 2 Figure 2.: Rhizobia-induced modifications on Arabidopsis root architecture depend

  )-(f) Quantifications of number of lateral roots, lateral root length, and primary root length on different nitrate conditions and indicated rhizobia treatment. Statistical significance was determined by students t-test *=p<0.05; **=p<0.01.

Figure 3 .

 3 Figure 3.: Rhizobia-induced root hair formation depends on high-nitrate availability.

Figure 4 .

 4 Figure 4.: Rhizobia-induced expression of nitrate-regulated genes.

Figure 5 .

 5 Figure 5.: Specific NIN-like Proteins are involved in M. loti induced root hair effect.

Figure 6 .

 6 Figure 6.: M. loti impacts cell -proliferation and -elongation.

Figure 7 .

 7 Figure 7.: Model of the rhizobium-induced NLP-pathway in Arabidopsis thaliana

Figure 1 .

 1 Figure 1.: Rhizobia attachment on roots of non-legume host plant Arabidopsis thaliana.Ten-days old Arabidopsis seedlings inoculated with transgenic rhizobia (M. loti) tagged with dsRed fluorophore grown on plant media with 5mM nitrate. (a-c) Bacterial biofilm formation on growing Arabidopsis primary root (a) white arrows indicate bacterial enrichments (hot spots) (b) inoculated plants grown in high-nitrate conditions (5mM) and (c) in low-nitrate conditions (0.25mM). (d) Bacteria accumulation on root tip and root elongation/differentiation zone. (e) and (f) M. loti accumulation at lateral root emergence sites and intercellular space invasion, validated by z-stack evaluation. Samples were observed under Zeiss stereo fluorescence microscope (a-e) and a confocal-microscope (f).190x275mm (600 x 600 DPI)

Figure 7 .

 7 Figure 7.: Model of the rhizobium-induced NLP-pathway in Arabidopsis thaliana roots.Results obtained indicate rhizobia-induced local root developmental effects on the non-legume host Arabidopsis thaliana. We demonstrate an involvement of key NIN-like proteins and show an induction of nitrate signaling during rhizobia responses, which finally impacts on cell cycle progression and morphological modifications. Furthermore, described effects are pronounced in high nitrate conditions and suggest a complex crosstalk between bacterial-and nitrate-related signaling pathways.190x275mm (600 x 600 DPI)

Fig. S2 Fig.

 S2 Fig. S2 Phylogenetic tree of NIN-like Proteins (main tree). Maximum Likelihood tree of the NLP family (model: GTR+F+R10; log-likelihood: -598245.4704). Tree is rooted on the non-vascular plants. Clades are colored and named according the Arabidopsis thaliana nomenclature. Only significant branch support values (>80%) are shown.

  Statistical significance was determined by students t-test *=p<0.05.

	qPCR Primer sequence Hernandez-Reyes et al. Mut_NLP5_BsaI_rev3 TAGAAGACAAtgaCacccatgtttgagc
	RSL4_F NLP5_BpiI_fwd4	AACCTTGTGCCAAACGGGAC TAGAAGACAAAacaatgaatgggttttg
	RSL4_R NLP5_BpiI_rev4	CCAGGCCGTTGTAAGCCAAT TAGAAGACAAtgtTttcatctaagtatttcagatcg
	EXT11_F	GGCAGTTTTTGGTTTATGTCGTG
	EXT11_R genotyping of nlp4 and nlp5 mutants used in Hernandez-Reyes et al. GATGGCTGGGAAAATTGTATTGTG
	RHD6_F LBb1.3-SALK	GCCCTAGATCCACCGAAACTCC ATTTTGCCGATTTCGGAAC
	RHD6_R NLP4-LP	TGGCTGCTAGGCTTTGTGG GATCTCATGAAGCTCGAAACG
	NRT1.1_F NLP4-RP	GCACATTGGCATTAGGCTTT CTCTAGCCAATGTTGCTCCAG
	NRT1.1_R NLP5-LP	CTCAATCCCCACCTCAGCTA AGCTCAAACATGGGTCTCATG
	NRT2.1_F NLP5-RP	AGTCGCTTGCACGTTACCTG TTGTAGGGATGATGCTTCTGG
	NRT2.1_R NLP4fwdEXON	ACCCTCTGACTTGGCGTTCTC GACGAGCAGCGAAATCGAGC
	NIA1_R NLP4revEXON	ACGGAGCATGGATGAGTT GCCATAAACTGTATTCAGTAGC
	NIA1_F NLP5fwdEXON	ATCGTCAAAGAAACCGAAGTC CACTGGCTCAGTTGATTTCG
	NIR1_F NLP5revEXON	TGCTGATGACGTTCTTCCACTCTGC CTTGGCAGCATCTTTGAGAC
	NIR1_R	CTGAGGGTTGACTCCGAAATAGTCTC
	CYCB1;1_F	TCAGCTCATGGACTGTGCAA
	CYCB1;1_R	GATCAAAGCCACAGCGAAGC
	CYCD3;1_F	CTTCAGCTCGTTTCTGTCGC
	CYCD3;1_R	TGTCTCCTCCACTTGAAAGTCT
	AtNLP4_F	TCA CAA CAA GCC AAG ACC AC
	AtNLP4_R	GGG TTT CAC TGG AGC AAC AT
	AtNLP5_F	ATT CAC CAC CTG CAA AGT CC
	AtNLP5_R	TAT CCG TGG GAT CTT CCT TG
	AtNLP1_F	TCA AGG AGC CTC TTG TTC TTG
	AtNLP1_R	ATG TCG CTT TCA CTT TGA TGG
	AtNLP2_F	TGG TAC TGG CAC TTC CTT CA
	AtNLP2_R	TGA TGG TGG TGA CTT TGG AG
	AtNLP3_F	TGT TTG GAG ATT CGA TGC TG
	AtNLP3_R	CGC TTT GCG ATC TCT CTT CT
	AtNLP6_F	CTC ACC AGA GTC GGT AAG CA
	AtNLP6_R	CGG AGA CCC ATT CTC TAT CG
	AtNLP7_F	CGA ACC ATC AGG CTC AGA A
	AtNLP7_R	CGT TCC TGC ATC AAC TTT CA
	AtNLP8_F	CAG CGA GAG TGG ATC AAC AA
	AtNLP8_R	CAA CTG ATG GCT CGA ACT TG
	AtNLP9_F	AAT CGA GCG AGG TAA TGG AA
	AtNLP9_R	GAA CTT CCC AGC AAA ACT GC
	cloning Primers used in Hernandez-Reyes et al.
	GG-NLP4-fwd1	ATGAAGACTTTACGGGTCTCACACCatgGTCCTATCACGACCTTCTGTTTG
	GG-NLP4-rev1	ATGAAGACTTCAGAGGTCTCTCCTTGTGCCATAAACTGTATTCAGTAGCT
	Mut_NLP4_BsaI_fwd2 TAGAAGACAAaCCAACTCAAGATACATCAGCTCT
	Mut_NLP4_BsaI_rev2 TAGAAGACAATGGtGACCACATGAAGGAAGT
	Mut_NLP4_BpiI_fwd3 TAGAAGACAAgAGACGAGCAGCGAAATCGAG
	Mut_NLP4_BpiI_rev3 TAGAAGACAATCTcCTTCAATGCACCTCCTAC
	NLP4_BpiI_fwd4	TAGAAGACAAaACAATGAATGGGTTTTGTTGACTTGC
	NLP4_BpiI_rev4	TAGAAGACAATGTtTTCGTCTAAGTACTTTAGATC
	GG-NLP5-fwd1	ATGAAGACTTTACGGGTCTCACACCatggaaaacaattctcttcctatgg
	GG-NLP5-rev1	ATGAAGACTTCAGAGGTCTCTCCTTtgaaagacatccactgacag
	Mut_NLP5_BsaI_fwd2 TAGAAGACAAGccaacgcaagacacatc
	Mut_NLP5_BsaI_rev2 TAGAAGACAAtggCgaccacatgaagg
	Mut_NLP5_BsaI_fwd3 TAGAAGACAAGtcatgtctcaaacaaagc

Acknowledgements

Authors thank Liên BACH for mutant plants chl1-5 (nrt1.1) and Anne Krapp for kindly providing seeds for nrt2.1-1, nlp6.2 and nlp7.1. The project was partially funded by the Deutsche Forschungsgemeinschaft (DFG) under Germany's Excellence Strategy grant CIBSS -EXC-2189 -Project ID 39093984 and central University funds. We thank the staff of the Life Imaging Center (LIC) in the Centre for Integrative Signalling Analysis (CISA) of the Albert-Ludwigs-University of Freiburg for help with their confocal microscopy resources, and the excellent support in image recording. The Leica SP8 confocal microscope was funded by the DFG project number 414136422. Especially, we thank all members of the OttLab team for the active discussions and supporting ideas that helped to develop the project.

Funding: This study was partially supported by the German Research Foundation (Deutsche Forschungsgemeinschaft) under Germany's Excellence Strategy (CIBSS-EXC-2189 -Project ID 39093984) and through a grant to the University of Cambridge by the Bill & Melinda Gates Foundation (OPP1172165) and UK government's Department for International Development (DFID). The Laboratoire de Recherche en Sciences Végétales (LRSV) laboratory belongs to the TULIP Laboratoire d'Excellence (ANR-10-LABX-41).

Accession numbers

Sequence data from this article can be found under the following Arabidopsis Genome Initiative accession numbers: NRT2.1 (AT1G08090), NRT1.1 (AT1G12110), NIA1 (AT1G77760), NIR1 (AT2G15620), RSL4 (AT1G27740), EXT11 (AT5G49080), RHD6 (AT1G66470), CYCD3;1 (AT4G34160), CYCB1;1 (AT4G37490), UBQ4 (AT5G25760), NLP1 (AT2G17150), NLP4 (AT1G20640), NLP5 (AT1G76350), NLP6 (AT1G64530), NLP7 (AT4G24020)

Author Contribution STS and TO conceived and designed the experiments; STS, CHR, EL, JK performed the experiments; STS, CHR, JK, P-MD, analyzed and interpreted the data; and STS, CHR and TO wrote the manuscript.

Literature Cited

Alvarez, J.M., Riveras, E., Vidal, E.A., Gras, D.E., Contreras-Lopez, O., Tamayo, K.P., Aceituno, F., Gomez, I., Ruffel, S., Lejay, L., Jordana, X., and Gutierrez, R.A. 2014. Systems

Supporting Information

Fig


Nicolas Lepage-Saucier

Juliette Schleich
email: juliette.schleich@sciences-po.fr

Etienne Wasmer

Cornelia Woll

Alexandre Biotteau

Iana Markevitch

Moving towards a single contract? Pros, cons and mixed feelings Bien que peu prisé des syndicats de salariés et des représentants patronaux, le contrat unique reste paradoxalement au coeur du débat public. Si le contrat unique semble à première vue être une réponse séduisante pour réduire le dualisme, il ne règle pas pour autant les problèmes liés à la précarité et à l'impact de la protection de l'emploi ; ceux-ci peuvent être traités par des politiques alternatives ciblées plus efficaces. De plus, bien que réels, les coûts du dualisme sont moins évidents et moins bien démontrés que ceux engendrés par la protection de l'emploi elle-même. Enfin, la suppression des CDD entrainerait, à protection inchangée, une perte d'emploi importante car tous ces emplois ne seraient pas remplacés par des CDI compte tenu de la prudence à l'embauche des entreprises. Un assouplissement fort de la protection de l'emploi du nouveau contrat unique serait alors nécessaire. Mais celui-ci peut intervenir dans le cadre existant et n'a pas de raison d'être lié à un nouveau contrat : d'autres contreparties en termes d'assurance-chômage ou de formation professionnelle sont possibles.

Although the single employment contract is not popular among both representatives of workers and company leaders, the proposal for a single labor contract, created with the goal of reducing dualism, has paradoxically remained at the forefront of debates on the need for reform of the labor market. At first glance, the single employment contract seems to be an attractive response to the issue of dualism. However, upon looking more closely, it appears that a single contract would not resolve problems linked to precariousness and the consequences of employment protection. Most of the time, alternative policies could be more efficient. Moreover, while there are costs to dualism, these are not as obvious and well established as the ones triggered by employment protection itself. Finally, suppressing temporary contracts would lead, at a constant level of employment protection, to important employment losses, given that not all temporary jobs would become permanent jobs. A drastic reform of employment protection would be needed. Such a reform does not need to be linked to the creation of a new employment contract: counterparts in terms of unemployment insurance or firm-sponsored training are possible.

1/ An overview of the actual situation in Europe

In Europe, dualism is pervasive: it mainly affects young people under temporary contracts and has led governments to initiate several labor market reforms. Some countries such as France, Germany and Spain focused their efforts on limiting the use of fixed-term contracts whereas countries such as the United Kingdom, Italy, Denmark and Finland have moved towards flexible employment protection regulations. Are these regulations really efficient in increasing the share of permanent contracts? Statistics suggest otherwise. Between 1985 and 2008, European countries (excepted Denmark) experienced an increase [1] Moving towards a single contract? Pros, cons and mixed feelings 2 LIEPP policy brief #8 -avril 2013 in the share of temporary contracts (12% on average in OECD countries). It is particularly high in Spain (24.9% in 2010, Eurostat) and in the Netherlands (18.5% in 2010, Eurostat), both countries where employment protection for regular contracts is high.

In order to reduce the negative consequences of dualism, exacerbated by a context of economic crisis and high unemployment, several policy experiments have been conducted with mixed results.

• In August 2005, the French government created the "Contrat Nouvelle Embauche" (CNE) for companies of less than 20 workers, a contract with a 2-year probation period. The French government tried to extend this contract to all companies for workers below the age of 26 (CPE). However, both reforms were overturned: the CNE for legal reasons (it was deemed contrary to International Termination of Employment Convention n°158) and the CPE after protests by employees' unions in March 2006.

• In February 2012, the Italian government launched a comprehensive reform to simplify its labor code: first, the forty different types of employment contracts were brought down to eight. A new contract with a probationary period of three years replaced the permanent one. Following these three years, employers were required to hire workers permanently. However, a large part of these modifications were abandoned during the ratification of the bill in July of 2012.

• In Spain, in March 2012, the government also proposed a drastic reform of its labor market, which mostly consisted in the introduction of a new open-ended contract for small companies (less than 50 workers) with a probationary period of one year. This reform gave tax breaks to employers who used this contract to hire workers below the age of 30 or that had faced a long-term unemployment period. It also clarified the notion of "termination for economic reasons" and limited the duration of temporary contracts to 24 months.

Among the proposals to increase flexibility and reduce dualism which arose through public debate, the single labor contract is one of the most popular. However, there are different ways to move towards such a contract. One, supported by some French unions, would be to remove all forms of temporary contracts. This solution would result in the loss of a margin of adjustment for firms. A second possibility would involve replacing all existing contracts by a unique single contract that would be somewhere in between the traditional permanent contract and the fixed term contract. This contract, more flexible than the current permanent one, could have an extended trial period or be subject to less dismissal requirements. Cahuc and Kramarz (2004) have notably proposed to remove all the collective regulations on layoffs and replace them with a termination tax. Generally speaking there are two forms of single employment contracts: contracts with Progressive Seniority Rights or contracts with Long Probationary Periods, and any combination of these two logics is possible. Changing the labor market in order to implement this type of single employment contracts requires a lot of work. The question we raise is whether it is really worth the trouble.

2/ Would the single contract reduce inequalities between workers?

The single employment contract is presented as a tool to reduce inequalities between workers caused by dualism in contracts. It also gives employers (but also landlords and banks) means to legally discriminate between different groups of workers based on demographics or skills. In particular, whereas workers under permanent contracts are highly protected in case of dismissals and therefore, can develop stable working habits, temporary workers face job insecurity.

and Kramarz (2004) argue that permanent workers are much more likely to be home owners because they benefit from an easier access to credit. Banks are less willing to grant loans to temporary workers, viewed as economically unstable. This makes it more difficult for them to purchase a house. More specifically they show that workers above the age of 30 in a permanent contract are 10 to 15 percentage points more likely to become home owners than workers in a temporary contract. However, in this context, introducing a single contract with a long probationary period is likely to be inefficient: workers in the probationary period would still be discriminated against by banks and the gap between workers would remain. Moreover, alternative reforms such as a partial deregulation of the housing market may be more effective.

Another consequence of the gap between temporary and permanent workers is the inequality in the access to training. Firms invest in training if it provides workers with skills that are profitable to them: hence, permanent workers are more likely to receive training than others. Yet again, a single contract with a long probationary period is unlikely to improve the current situation. The effects of a single contract with less dismissal requirements might be positive but have yet to be proven. However, more efficient solutions do exist. For instance, Lemoine and Wasmer (2010) [2] , proposed to introduce specific Pigouvian incentives for French firms by reducing their taxes or layoff costs depending on whether they train more or fewer workers, with more general training to internalize the externality created by general training, following upon the logic of Blanchard and Tirole of an experience rating system [3] .

Due to the difficulty for employers to dismiss their workers under a regular contract, they use temporary contracts as a screening device. However, temporary workers whose contracts have not been renewed are not necessarily presumed to be less motivated or less skillful since a bad economic situation could have made companies fearful of offering regular contracts. Instead, the termination of a worker under a Contract with Progressive Seniority Rights or with Long Probationary Periods, would serve as a much better signal on the worker: the termination of such a contract would necessarily be due to the workers' lack of skill or motivation, not to the firms' characteristics or to the formal impossibility to renew the contract. Furthermore, there is a risk that a single contract with a long probationary period could reinforce abuse: probationary periods, which are less regulated than the current temporary contracts, could be used in the same way while reducing the workers' protection to workers, since terminating a temporary contract is prohibitive in most countries.

3/ The coexistence of permanent and temporary contracts and the efficiency of the labor market

The cost differeence in terms of employment adjustement costs between temporary and permanent contracts leads firms to use temporary contracts as a cheaper adjustment variable to the economic conjuncture: in periods of economic booms, the high costs linked to permanent contracts lead firms to use temporary contracts (subject to weak regulations) to hire new employees. In periods of crisis, the existing regulations force firms to respond to economic fluctuations through labor turnover instead of using alternative methods such as changing the workplace organization. This results in an excessive turnover in the labor market (more hires in temporary contracts but also more contract terminations). Bentolila et alii. (2010). They find that the larger gap between the dismissal costs of workers with permanent and temporary contracts in Spain as compared to France is responsible for the much higher unemployment rate in Spain, as more temporary contracts to expire.

The model we built and quantified confirms what analyze

There is considerable heterogeneity in the labor market turnover rate even with a single labor contract. Removing temporary contracts would simply discourage firms to take risks by hiring new workers. Figure 1 represents the employment effect of varying layoff costs of regular contracts denoted by F. The solid line is equal to total employment under dualism (both permanent and temporary employment). The dashed line gives the level of employment in a world without temporary workers. Starting (from the right) from a large level of employment protection (say, F=0.06), the employment rate (averaging up the first and second employment period, which is interpreting the model as reflecting an overlapping generation of two-period firms) is approximately 76%. The model gives the following conclusions:

1. In this specific example, removing temporary contracts leads to a 7 percentage point drop in employment at a constant level of employment protection for regular contracts.

2. Reducing employment protection raises employment in both worlds;; it is faster in the absence of temporary contracts.

3. Without temporary contracts, one can restore the same level of employment as in the case of coexistence of permanent and temporary contract if layoffs costs in permanent contracts F are drastically reduced, namely a third of their initial value.

The essence of our argument is to focus more on the costs of employment protection than on the need to reduce dualism. In many countries, the prerequisites of "economic layoffs" are not well defined in the law and are thus a source of uncertainty: in France, 70% of litigation cases concern individual layoffs when in reality they are the result of a bad economic situation. These litigations often last more than one year. Employment protection is paradoxical: countries with the strongest employment protection are also those where employees perceive their job to be the less secure. Likewise, they are also those where stress is the highest among workers: for permanent workers, the risk of layoff is lower but can be associated with adverse social consequences. Costly economic layoffs lead firms to put more pressure on their workers to increase productivity or to obtain dismissals at lower costs. Employment protection has in addition an ambiguous effect on the allocation of productive units: on the one hand, it can favor less productive structures and slows the allocation process of workers on the most productive activities. A vast literature has shown that this results in distortive effects on capital accumulation and in misallocation of productive units leading to a lower productivity, even if the overall marginal effect of employment protection may sometimes be positive in low protection countries such as in the United States.

4/ Conclusion

A large part of the debate on how to favor employment and reduce dualism focuses on the idea of implementing a single labor contract. Advertising for labor market reforms through the single labor contract label is a way to argue for the convergence of different types of labor contracts and the removal of temporary contracts which appears as the least costly solution. But this focus undermines alternative reforms which could be more efficient in limiting the negative effects caused by dualism. Moreover, advocates of this proposal have conflicting views on the characteristics of such a contract. The 'single labor contract' is meant to be a quid pro quo (meaning an exchange of something against something else, understood by all parties). By single labor contract, proponents of this measure probably mean ambitious reforms of permanent contracts with less protection. This part may however be a quiproquo (a misunderstanding due to voluntary ambiguity on the concepts) if this amounts to the disappearance of temporary contracts in exchange for an extended probationary period. It is hard to believe that unions would favor this solution since fixed duration contracts are fairly well protected and would be replaced by contracts with long probationary periods, or an increase of individual layoffs even within permanent contracts.

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LIEPP policy brief

Figure 2 (

 2 Figure 2 (next page) further illustrate how changes in firing costs affects hiring decisions and termination of contracts. The green region is made of two areas: the light green region represents the number of workers initially hired as permanent

Figure 1 :

 1 Figure 1 : Impact of layoff costs for regular contracts