Julie Cullimore

Judith Fliegmann

Virginie Gasciolli

Chrystel Gibelin-Viala

Noémie Carles

Thi-Bich Luu

Ariane Girardin

Marie Cumener

Fabienne Maillet

Stéphanie Pradeau

Thi- Bich Luu

Sébastien Fort

Jean-Jacques Bono

Clare Gough

Benoit Lefebvre
email: benoit.lefebvre@inrae.fr

B L Wrote

Evolution of Lipochitooligosaccharide Binding to a LysM-RLK for Nodulation in Medicago truncatula

Keywords: Lysin motif receptor like kinases, plant symbiosis, lipochitooligosaccharide perception

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

INTRODUCTION

N-acetyl glucosamine containing oligosaccharides are important signalling molecules in plants and can elicit defence and symbiotic responses to microorganisms, depending on their structure [START_REF] Chiu | Receptor-Like Kinases sustain symbiotic scrutiny[END_REF][START_REF] Khokhani | Deciphering the Chitin Code in Plant Symbiosis, Defense, and Microbial Networks[END_REF][START_REF] Zipfel | Plant signalling in symbiosis and immunity[END_REF]. Short chain chitooligosaccharides (CO4-CO5) are produced by symbiotic arbuscular mycorrhizal (AM) fungi and elicit symbiotic responses in a variety of plants, whereas longer chain COs (CO7-CO8) elicit defence responses [START_REF] Carotenuto | The rice LysM receptor-like kinase OsCERK1 is required for the perception of short-chain chitin oligomers in arbuscular mycorrhizal signaling[END_REF][START_REF] Feng | A combination of chitooligosaccharide and lipochitooligosaccharide recognition promotes arbuscular mycorrhizal associations in Medicago truncatula[END_REF][START_REF] Kaku | Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor[END_REF]. Lipochitooligosaccharides (LCOs), in which the terminal non-reducing sugar is N-acylated generally with a C16 -C20 fatty acid, are produced by a variety of fungi, including AM fungi [START_REF] Cope | The Ectomycorrhizal fungus Laccaria bicolor produces lipochitooligosaccharides and uses the common symbiosis pathway to colonize Populus roots[END_REF][START_REF] Maillet | Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza[END_REF][START_REF] Rush | Lipochitooligosaccharides as regulatory signals of fungal growth and development[END_REF], and also by rhizobial bacteria [START_REF] D'haeze | Nod factor structures, responses, and perception during initiation of nodule development[END_REF]. The production of these rhizobial LCOs, called Nod factors (NFs), is essential for most rhizobia to establish the nitrogen-fixing, root-nodule symbiosis with legume plants, and species-specific chemical substitutions enable interaction with the correct symbiotic host. For example, Sinorhizobium meliloti typically produces tetrameric NFs, that are O-sulphated on the reducing sugar, N-acylated with predominantly a C16:2 acyl chain on the terminal non-reducing sugar (termed LCO-IV(C16:2,S); Fig. S1), and partially Oacetylated on this sugar. These decorations allow nodulation of Medicago [START_REF] Lerouge | Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal[END_REF]). NFs are very potent signals, eliciting symbiotic activities at pM to nM concentrations on roots of the legume host.

The discrimination of CO and LCO structures by plants is largely determined by lysin motif (LysM) containing proteins [START_REF] Chiu | Receptor-Like Kinases sustain symbiotic scrutiny[END_REF][START_REF] Khokhani | Deciphering the Chitin Code in Plant Symbiosis, Defense, and Microbial Networks[END_REF][START_REF] Zipfel | Plant signalling in symbiosis and immunity[END_REF]. The LysM is a small (less than 65 amino acid) protein domain, which occurs in Bacteria and many Eukaryotes and generally has a conserved  fold. In plants three divergent tandem LysMs (LysM1, LysM2, LysM3) are associated in the extracellular region (ECR) with a transmembrane spanning helix and an intracellular region (ICR) containing a kinase domain to form plasmamembrane (PM) located LysM receptor like kinases (LysM-RLKs). Phylogenetically these LysM-RLKs can be divided into two major classes, the LYRs (generally with an inactive, pseudokinase domain) and the LYKs (in which the kinase domain is predicted to be active). These two classes can be further divided into sub-classes [START_REF] Buendia | LysM receptor-like kinase and LysM receptor-like protein families: An update on phylogeny and functional characterization[END_REF].

Studies on CO perception, particularly in rice (Oryza sativa (Os)) and Arabidopsis thaliana (At), have identified a gene of the LYK-I subclass, CERK1, which is essential for CO-elicited responses [START_REF] Yang | CERK1, more than a co-receptor in plant-microbe interactions[END_REF]. In rice, OsCERK1 interacts with a LysM protein, OsCEBIP, for defence responses whereas it interacts with a LYR-IA subclass protein, OsMYR1 (also called OsNFR5) for AM [START_REF] Zhang | Discriminating symbiosis and immunity signals by receptor competition in rice[END_REF]. Crystal structures of the AtCERK1 and OsCEBIP ECRs have identified that COs bind in a groove in LysM2 [START_REF] Liu | Molecular Mechanism for fungal cell wall recognition by rice chitin receptor OsCEBiP[END_REF][START_REF] Liu | Chitin-induced dimerization activates a plant immune receptor[END_REF]. In dicots, such as tomato and Petunia hybrida, the LYR-IA protein, SlLYK10 and PhLYK10 respectively, is important for establishing AM and binds LCOs, rather than COs [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]). Thus plants have adopted a variety of ways in which to recognise CO/LCO signals via LysM proteins.

Legumes contain a highly expanded LysM-RLK family due to both segmental duplication to form clusters and by a whole genome duplication (WGD) event, which occurred in the Papilionoid legumes [START_REF] Young | The Medicago genome provides insight into the evolution of rhizobial symbioses[END_REF]. Thus there is more scope for variation, particularly to accommodate NF signalling. Both a LYK protein belonging to the LYK-I subclass (termed MtLYK3 in M. truncatula (Mt), LjNFR1 in Lotus japonicus (Lj), and PsSYM37, in Pisum sativum (Ps)) and a LYR protein belonging to the LYR-IA subclass (termed MtNFP, LjNFR5 and PsSYM10) are implicated in NF recognition and are essential for rhizobial nodulation [START_REF] Krönauer | Understanding Nod factor signalling paves the way for targeted engineering in legumes and non-legumes[END_REF]. By domain swapping and mutagenesis, the LysM1 of MtLYK3/LjNFR1 [START_REF] Bozsoki | Ligandrecognizing motifs in plant LysM receptors are major determinants of specificity[END_REF]) and the LysM2 of MtNFP/LjNFR5 [START_REF] Bensmihen | Contribution of NFP LysM domains to the recognition of Nod factors during the Medicago truncatula/Sinorhizobium meliloti symbiosis[END_REF][START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF] have been shown to be particularly important for nodulation. Structural and biochemical studies on the ECRs, expressed in insect-cells, show that the proteins adopt a similar conformation to CO-binding LysM proteins and bind specific NFs with Kds in the M range [START_REF] Bozsoki | Ligandrecognizing motifs in plant LysM receptors are major determinants of specificity[END_REF][START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF]. The slow kinetics of LCO binding, compared to fast kinetics of CO binding, has been suggested to be more important than affinity for receptor activation and signalling specificity, through a kinetic proofreading mechanism [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF].

We have worked on the perception of LCOs for many years, initially through using a radiolabelled ligand corresponding to a major NF of S. meliloti (LCO-IV(C16:2, 35 S)). We identified a high affinity (nM range) LCO binding protein in the PM fraction of cell cultures as the LYR-IIIA protein, MtLYR3 [START_REF] Fliegmann | Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula[END_REF][START_REF] Gressent | Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures[END_REF]. MtLYR3 is located next to and in inverse orientation to MtNFP [START_REF] Buendia | LysM receptor-like kinase and LysM receptor-like protein families: An update on phylogeny and functional characterization[END_REF][START_REF] Gough | Evolutionary history of plant LysM Receptor proteins related to root endosymbiosis[END_REF], and legume orthologs of MtLYR3 bind LCOs [START_REF] Bouchiba | An integrated approach reveals how lipo-chitooligosaccharides interact with the lysin motif receptor-like kinase MtLYR3[END_REF][START_REF] Malkov | Molecular basis of lipo-chitooligosaccharide recognition by the lysin motif receptor-like kinase LYR3 in legumes[END_REF]. We also used a radiolabelled LCO-V ligand (LCO-V(C18:1,NMe, 35 S)), which has more similarity to a Myc-LCO than the S. meliloti NFs, to characterise LCO binding to the tomato and P. hybrida LYR-IA protein, and showed the relevance of these proteins for AM [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]. In view of the physiological importance of LYR-IA, in this article we have investigated the binding properties of this subclass of protein from the Poaceae and M. truncatula, leading to a hypothesis on its ancestral role in plants. We also show that the two LYR-IA paralogs in M. truncatula have different LCO binding properties, which are unexpected in view of the physiological roles of these proteins.

RESULTS

MtLYR1 is a high affinity LCO binding protein with selectivity for LCOs versus short-chain COs

In order to analyse the LCO binding capability of the M. truncatula LYR-IA proteins, (MtNFP and MtLYR1), we made fusions of their ECRs with the transmembrane (TM) and ICR of MtNFP tagged with YFP, as previously done for MtLYR3 and tomato SlLYK10 [START_REF] Fliegmann | Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula[END_REF][START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF].

The aim was to ensure good expression of the proteins when expressed in Nicotiana benthamiana leaves, whilst leaving the ECRs intact. Membrane fractions of the leaves expressing the LysM-RLKs and of the negative control, mock-treated leaves (MOCK), were then prepared and analysed by Western blotting using anti-YFP antibodies. These membrane fractions were analysed for highaffinity binding to the S. meliloti NF ligand (LCO-IV(C16:2, 35 S); Fig. S1) using about 1 nM of the labelled ligand. Specific LCO binding was deduced by subtracting the non-specific binding (not competed by high (2 µM) concentrations of unlabelled ligand) from the total binding. Only MtLYR3 extracts, and not those of MtNFP or MtLYR1, showed specific binding to this NF ligand compared to binding to MOCK (Fig. 1A). This lack of specific binding is particularly unexpected for MtNFP in view of its essential role in the perception of NFs during symbiosis [START_REF] Arrighi | The Medicago truncatula lysin motif-receptor-like kinase gene family includes NFP and new noduleexpressed genes[END_REF] and the recent binding studies on MtNFP [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF]. In terms of expression in this system, MtNFP localises to the PM as expected [START_REF] Lefebvre | Role of N-Glycosylation sites and CXC motifs in trafficking of Medicago truncatula Nod Factor perception protein to plasma membrane[END_REF]) and its expression level is better than MtLYR3 (Fig 1A).

We then tested the remaining 8 LYR proteins (MtLYR2, MtLYR4-MtLYR10) and although there was considerable variation in the level of expression, all proteins, except the MtLYR6 fusion, were expressed at least as well as MtLYR3 (Fig. S2), but in comparison to binding to MOCK, only MtLYR3 extracts showed specific binding to this NF ligand (Fig. S2).

In tomato and Petunia, LYR-IA are represented by a single ortholog (called SlLYK10 and PhLYK10), which binds LCO-V(C18:1,NMe, 35 S) (Fig. S1), an LCO structure different from the NF ligand, and able to reveal the Myc-LCO binding site [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]. We thus analysed binding to MtNFP and to its LYR-IA paralog, MtLYR1, with this ligand. The MtLYR1 extracts showed binding to the ligand (Fig. 1B), whereas despite similar expression level of the MtNFP protein, no specific LCO binding to the MtNFP extracts was detected.

To determine the parameters of LCO binding to MtLYR1, a ligand saturation experiment using LCO-V(C18:1,NMe,S) was subjected to Scatchard analysis (Fig. 1C). The results are consistent with the presence of a single class of binding site, exhibiting a high affinity for the ligand (Kd = 6.5 nM). The LCO-V(C18:1,NMe,S) binding could be competed with the unlabelled NF LCO-IV(C16:2,S) (Fig. 1D) but with an IC 50 greater than 500 nM, suggesting at least an 80-fold lower affinity than for LCO-V(C18:1,NMe,S). This observation could explain why binding to MtLYR1 of the radiolabelled S. meliloti NF could not be detected (Fig. 1A). Competition experiments showed that an LCO mixture of LCO-V(C18:1,Fuc)/LCO-V(C18:1,MeFuc) recently-described as abundant Myc factor structures [START_REF] Rush | Lipochitooligosaccharides as regulatory signals of fungal growth and development[END_REF], could efficiently compete for binding while both CO4 and CO8 could only partially compete the LCO-V(C18:1,NMe, 35 S) binding to MtLYR1 (Fig. 1E). To test whether MtLYR1 has affinity for short-chain COs on a binding site independent of LCOs, we developed a crosslinkable biotinylated CO (thereafter called CO5-biot, Fig. S1), which can form a covalent bond with proteins in its vicinity. It allowed us to measure affinity of proteins for short-chain COs, following expression in N. benthamiana, incubation of membrane extracts with CO5-biot and detection by Western blotting using Streptavidin. A saturation experiment showed MtLYR1 labelling with CO5-biot, but only at high concentration of the ligand and not saturated between 500 nM and 5 µM, showing that the affinity for CO5 is in the µM range (Fig. 1F). A similar result was found for OsCEBIP, whereas no CO5-biot binding was detected to MtNFP (Fig. S3). Stronger competition of CO5-biot binding to MtLYR1 by LCOs compared to COs (Fig. 1G), shows that CO5-biot binds to the LCO binding site, which has higher affinity for LCOs than COs.

We previously found that Solanaceae LYR-IA genes could complement a Mtnfp mutant for nodulation but with a lower efficiency compared to MtNFP [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]. MtLYR1 showed a similar low ability to complement Mtnfp, which could not be increased significantly by replacing the TM+ICR with that of NFP. Both the efficiency of nodulation and the nodule number were much lower with the MtLYR1 constructs than with MtNFP (Fig. 1H).

Transcriptomic data show that MtLYR1 is induced in roots during AM and expressed particularly in arbuscule-containing cells, whereas it is not induced during nodulation (Fig. S4; [START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF]). To study the potential symbiotic role(s) of MtLYR1, we tested Mtlyr1 mutants for both AM and nodulation. Two independent knockout alleles, were obtained; Mtlyr1-1, an EMS mutant in M. truncatula A17 (C58T, introducing an early stop codon), and Mtlyr1-2, a Tnt1 insertion in M. truncatula R108 (before the region encoding LysM1) (Fig. 2A).

When these mutants were tested with spores of Rhizophagus irregularis, no differences between mutant and WT plants were observed either in proportions of colonized roots at 6 wpi (Fig. 2B) or in arbuscule development (Fig. S5). As Mtnfp mutants show no observable AM phenotype [START_REF] Ben Amor | The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation[END_REF], we tested the possibility of redundancy between MtLYR1 and MtNFP, and generated a double Mtlyr1-1/Mtnfp-2 mutant. No differences were observed between the R. irregularis colonization levels of these plants compared to plants of the single mutants or WT (Fig. 2B).

We then quantified the number of nodules in Mtlyr1 mutant plants inoculated by WT S. meliloti. No differences in nodule number between mutants and their corresponding WT plants were observed (Fig. 2C). Since MtLYR1 has higher affinity for LCO-V(C18:1,NMe,S) than LCO-IV(C16:2,S) we also tested whether MtLYR1 plays a role in nodulation through perception of NFs bearing a C18:1 fatty acid rather than a C16:2 fatty acid, as produced by the S. meliloti nodFE mutant [START_REF] Ardourel | Rhizobium Meliloti Lipooligosaccharide nodulation factors -different structural requirements for bacterial entry into target root hair-cells and induction of plant symbiotic developmental responses[END_REF]. Again, no differences in nodule number were observed (Fig. 2D). With either rhizobia strain, the Mtnfp mutant was devoid of nodules (Nod -), as expected [START_REF] Ben Amor | The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation[END_REF].

Our results show that, despite an induction of its expression during AM, MtLYR1 is dispensable both for AM and for nodulation.

Poaceae LYR-IA proteins have similar LCO/CO binding properties as dicot LYR-IA proteins.

We previously showed that Solanaceae LYR-IA proteins have high affinity for LCO-V(C18:1,NMe,S) and selectivity for LCOs versus COs [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]. To determine whether these properties are conserved among plant species, we characterized LYR-IA from monocotyledons, Brachypodium distachyon (Bd), Oryza sativa (Os) and Triticum aestivum (Ta). A single gene was found in the diploid species B. distachyon and O. sativa (BdLYR1 and OsMYR1/OsNFR5 respectively), while three genes were found in the hexaploid T. aestivum (TaLYR1, TaLYR1B and TaLYR1D; Fig. S6). We tested extracts of all 5 proteins, expressed in N. benthamiana leaves, and found specific LCO-V(C18:1,NMe, 35 S) binding to all of them (Fig. 3A). Scatchard analysis of the BdLYR1, OsMYR1 and TaLYR1D binding, which in each case correlated to a single class of high affinity binding sites (Fig. S7), yielded affinities (Kd) from 9 to 19 nM (Fig. 3B), which are in the same range as those found for MtLYR1, SlLYK10 and PhLYK10. We also tested the selectivity of these LCO binding sites for COs and found that CO4 and CO8 are poor competitors of the LCO-V(C18:1,NMe, 35 S) binding (Fig. 3C) for each of the proteins.

Using CO5-biot, absence of high affinity for short-chain COs was confirmed for OsMYR1 as, like MtLYR1, OsMYR1 was labelled only at a high concentration of the ligand (Fig. 3D). We characterized BdLYR1 in more detail. We found that synthetic LCO-V(C18:1,Fuc/MeFuc) (Fig. S1), but not LCO-IV(C16:2,S), can fully compete LCO-V(C18:1,NMe, 35 S) binding to BdLYR1 (Fig. 3E). High affinity for LCO-V(C18:1,NMe,S), but not for Sm LCO-IV(C16:2,S) and selectivity for LCOs versus CO4 was confirmed by using a radiolabelled crosslinkable LCO derivative (Fig. S1) on BdLYR1 purified from N. benthamiana leaves (Fig. 3F, Fig. S8). This LCO can form a covalent bond with proteins in its vicinity and allowed us to measure affinity of proteins for LCO, following detection by Phosphorimaging.

Finally, we also tested the ability of BdLYR1 to complement the Mtnfp mutant for nodulation. As for MtLYR1, BdLYR1 allowed the formation of a few nodules with much lower efficiency than MtNFP (Fig. 3G). The nodules were well colonized by S. meliloti (Fig. S9).

LysM domain swaps reveal the critical role of LysM2 in the functions of MtLYR1 and MtNFP

We have shown that MtLYR1 has high-affinity LCO-binding properties and lower ability to complement Mtnfp for nodulation than MtNFP. To pinpoint the regions responsible for the differences, we made domain swaps between the three LysMs of the MtNFP (NNN) and MtLYR1 (LLL) ECRs in fusion proteins to MtNFP-TM+ICR/YFP (Fig. 4A).

We tested the constructs for their ability to complement the Mtnfp mutant for nodulation.

The results show that the introduction of MtNFP LysM2 in MtLYR1 (LNL), but not of LysM1 (NLL) or LysM3 (LLN), led to a gain in nodulation efficiency to a level similar to that of NNN while conversely introducing MtLYR1 LysM2 into MtNFP (NLN), led to a loss of function in nodulation (Fig. 4B). To further explore the structure-function of the LysM2 of the MtLYR1 protein, three mutants were designed in which MtLYR1 residues differing from MtNFP were converted to the corresponding MtNFP residue(s) in the LLL construct (Fig. 4D). Analysis of the constructs for nodulation revealed that the LLL-S114N and LLL-K147P mutants, but not the double LLL-N142S/Y144T mutant, gained some ability to complement the Mtnfp mutant (Fig. 4B), thus suggesting that these MtNFP residues in LysM2 are important for nodulation.

We then tested LCO-V(C18:1,NMe, 35 S) binding to membrane extracts of the various chimeras/mutated proteins, after expression in N. benthamiana. The LysM2 (LNL) but not the LysM3 (LLN) domain swap, showed almost complete loss of binding to LCO-V(C18:1,NMe,S), whereas replacing the LysM2 of MtNFP with that of MtLYR1 (NLN) led to gain of LCO-V(C18:1,NMe,S) binding.

Binding assays of the 3 LLL proteins mutated in LysM2 to corresponding MtNFP residues, showed that LLL-S114N and the double mutant LLL-N142S/Y144T, and to a lesser extent LLL-K147P, have reduced LCO-V(C18:1,NMe,S) binding, supporting the importance of these LysM2 residues for LCO-V(C18:1,NMe,S) binding. Although the NLL construct showed reduced binding, this construct showed poor plasma membrane localization of the protein (Fig. S10), suggesting that it may not be folded correctly [START_REF] Lefebvre | Role of N-Glycosylation sites and CXC motifs in trafficking of Medicago truncatula Nod Factor perception protein to plasma membrane[END_REF].

These results show that residues of the LysM2 are very important for the role of MtLYR1 in LCO binding and of MtNFP in nodulation.

MtLYR1 ECR modelling suggests that residues critical for LCO binding lie on the putative CO binding groove and near the hydrophobic patch

A model of the MtLYR1-ECR has previously been produced, based on the MtNFP-ECR crystal structure, with placement of a CO in a groove [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF], similar to the one found by AtCERK1-ECR/CO co-crystallization [START_REF] Liu | Chitin-induced dimerization activates a plant immune receptor[END_REF]. Due to the higher similarity of MtLYR1-ECR with the LjLYS11-ECR, we made models of MtLYR1 ECR, based on the available crystal structure of LjLYS11-ECR in order to place the key binding residues found here in relation to the proposed binding site.

The model of MtLYR1-ECR based on LjLYS11 (Fig. S11) is very similar to the one based on MtNFP [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF]. The key LCO binding residues discussed in the previous section, S114 and N142/Y144 lie on opposite sides of the proposed CO-binding groove in LysM2, while K147 lies at the end of the groove closest to the proposed non-reducing end of the CO chain (Fig. 4E, [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF]). The hydrophobic patch, described by [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF] is in a position to accommodate the acyl chain of the LCO, if the CO moiety is located in the same orientation and location as the CO in AtCERK1/OsCEBIP. Although we have not shown whether both the N142S and Y144T mutations are responsible for the decrease in MtLYR1 binding in the double mutant, N142 is close to the hydrophobic patch, formed by L145, L146 and possibly I148. Superposition of the crystal structure of the MtNFP-ECR on the model of the MtLYR1-ECR reveals differences in the structure of the two proteins in the key residues that we identified to be required for the LLL mutant proteins to complement for nodulation (Fig. 4F). The two Leu residues, identified by [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF] in which a double mutation L147D/L154D reduces the ability of MtNFP to complement the Mtnfp mutant for nodulation, form part of the hydrophobic patch, and are conserved in MtLYR1 (L141, I148).

In conclusion, residue positions, critical for high affinity LCO-V(C18:1,NMe,S) binding to MtLYR1, lie in a groove, conserved in CO binding proteins, and near a hydrophobic patch, described by [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF]. The corresponding residues of MtNFP change the structure of the groove and are critical for permitting the MtLYR1 ECR construct to complement for nodulation of Mtnfp.

DISCUSSION

An ancestral role of LYR-IA as high affinity LCO binding proteins involved in AM?

We previously found that two Solanaceae LYR-IA proteins have high affinity for LCOs and selectivity for LCOs versus COs. Here we extended our analysis to Poaceae LYR-IA proteins (rice, wheat and Brachypodium) and found that these proteins have similar binding properties to those of the Solanaceae LYR-IA, with a Kd for LCOs in the nM range (Fig. 3). Our binding results suggest that the LYR-IA ancestor was an LCO binding protein, with a lower ability to bind COs.

Our data on the rice LYR-IA protein, OsMYR1 (also called OsNFR5), differ from those of [START_REF] He | A LysM receptor heteromer mediates perception of arbuscular mycorrhizal symbiotic signal in rice[END_REF], who measured a high affinity for CO4 (Kd of 90 nM) and did not detect LCO binding, using the S. meliloti NF as LCOs. We found that a major S. meliloti NF, LCO-IV(C16:2,S) binds poorly to OsMYR1 compared to LCO-V(C18:1,NMe,S), which gives a possible explanation of how they may have missed high-affinity LCO binding to this protein. Moreover, we have shown that short chain COs bind with lower affinity than LCOs to OsMYR1, by both direct binding of the CO5-biot ligand and by poor CO competition of LCO binding. Our results also suggest that the LCOs and COs bind at the same site.

The lack of high affinity CO binding is consistent with the observation of Carotenuto et al (2017) that CO4-induced Ca 2+ spiking responses are not affected in a Osmyr1 (Osnfr5) mutant, although conflicting results were found by [START_REF] He | A LysM receptor heteromer mediates perception of arbuscular mycorrhizal symbiotic signal in rice[END_REF].

The LYR-IA gene has generally been lost in non-mycorrhizal plants suggesting a role in AM [START_REF] Gough | Evolutionary history of plant LysM Receptor proteins related to root endosymbiosis[END_REF]. Indeed, analysis of the expression of LYR-IA genes and phenotyping of mutants in tomato, Petunia and rice [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF][START_REF] He | A LysM receptor heteromer mediates perception of arbuscular mycorrhizal symbiotic signal in rice[END_REF][START_REF] Miyata | Evaluation of the role of the LysM receptor-like kinase, OsNFR5/OsRLK2 for AM symbiosis in rice[END_REF] suggest that Myc-LCO binding LYR-IA proteins play an important and widespread role in AM, which in view of the antiquity of this symbiosis (established over 400 MYA) is probably an ancestral role. However, we cannot exclude that they may also be involved in interaction with other LCO-producing fungi or other microorganisms [START_REF] Rush | Lipochitooligosaccharides as regulatory signals of fungal growth and development[END_REF].

Duplication and divergence of LYR-IA in M. truncatula

In dicotyledonous plants, the evolution of LYR-IA genes is associated with the evolution of nodulation and nitrogen fixation [START_REF] Rutten | Duplication of symbiotic Lysin motif receptors predates the evolution of nitrogen-fixing nodule symbiosis[END_REF] The MtNFP/LjNFR5 orthogroup has clearly become specialized for an essential and paramount role in rhizobial NF perception and nodulation [START_REF] Krönauer | Understanding Nod factor signalling paves the way for targeted engineering in legumes and non-legumes[END_REF], whereas it was speculated that MtLYR1/LjLYS11 retained an analogous role in AM [START_REF] Young | The Medicago genome provides insight into the evolution of rhizobial symbioses[END_REF].

Transcriptomic data support this hypothesis as both MtLYR1 and LjLYS11 are induced during AM and in arbuscule-containing cells (Fig. S4; [START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harboring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Rasmussen | Intraradical colonization by arbuscular mycorrhizal fungi triggers induction of a lipochitooligosaccharide receptor[END_REF], like the tomato LYR-IA gene [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]. However, analysis of Mtlyr1 mutants from two divergent M. truncatula genotypes, A17 and R108, did not reveal a symbiotic role for this protein either in AM or rhizobial nodulation (Fig. 2). This lack of AM phenotype is not due to redundancy with MtNFP as the double Mtlyr1/Mtnfp mutant also had no observable AM phenotype (Fig. 2). Similar results have been obtained in L. japonicus, using Ljlys11 and Ljnfr5 mutants [START_REF] Rasmussen | Intraradical colonization by arbuscular mycorrhizal fungi triggers induction of a lipochitooligosaccharide receptor[END_REF].

The role of LYR-IA in AM might be masked by redundancy with other LysM-RLKs, particularly in legumes where this family is considerably expanded (about 22 genes in M. truncatula versus 10 or 11 in rice, depending on the genotype). This hypothesis is supported by the AM phenotype of Mtlyk9 mutants (LYK-I subclass; Gibelin-Viala et al. 2019), which is enhanced in a Mtlyk9/Mtnfp double mutant [START_REF] Feng | A combination of chitooligosaccharide and lipochitooligosaccharide recognition promotes arbuscular mycorrhizal associations in Medicago truncatula[END_REF]. Other contenders for a role in AM in M. truncatula are MtLYR3 (LYR-IIIA subclass) since loss of LCO binding in MtLYR3 orthologs can be associated with loss of AM [START_REF] Bouchiba | An integrated approach reveals how lipo-chitooligosaccharides interact with the lysin motif receptor-like kinase MtLYR3[END_REF][START_REF] Malkov | Molecular basis of lipo-chitooligosaccharide recognition by the lysin motif receptor-like kinase LYR3 in legumes[END_REF]) and MtLYR8 (LYR-IB subclass) since, like LYR-IA, this subclass is absent in the non-AM species A. thaliana [START_REF] Buendia | LysM receptor-like kinase and LysM receptor-like protein families: An update on phylogeny and functional characterization[END_REF]) and shown to be involved in AM in barley [START_REF] Li | Nutrient regulation of lipochitooligosaccharide recognition in plants via NSP1 and NSP2[END_REF]. Clearly, analysis of double, triple and higher order mutants are required to unravel possible LysM-RLK redundancy in AM.

Using a LCO-V(C18:1,NMe,S) ligand, we found high affinity binding to MtLYR1, but not to MtNFP (Fig. 1). Binding to MtLYR1 showed similar affinity and specificity as the non-legume proteins (Fig. 3; [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]. Neither protein directly bound the S. meliloti NF ligand (LCO-IV(C16:2,S)) to high affinity although this LCO can compete for LCO-V(C18:1,NMe,S) binding to MtLYR1 at high concentrations, as shown for non-legume LYR-IA proteins (Fig. 3; [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]).

Together, our and other results suggest that the MtLYR1 gene has retained many characteristics, in terms of enhanced expression in arbuscules and LCO binding of the encoded protein, to an ancestral gene, although its role in AM remains enigmatic and is probably redundant with other LysM proteins. A redundant role for MtLYR1 in the rhizobia symbiosis cannot be excluded, although there is no indication of this from the expression pattern.

Has MtNFP a high affinity for S. meliloti LCO?

In our assays, no evidence was found for high affinity LCO or CO binding to MtNFP, using either the LCO-IV(C16:2,S) S. meliloti NF ligand, the LCO-V(C18:1,NMe,S) ligand, or indeed the CO5-biot ligand (Fig. 1). Our results on absence of high affinity NF binding to MtNFP are robust as they have been confirmed in many experiments over a long period of time, using different MtNFP constructs and expression systems. Moreover, a clear difference is seen, using the same techniques, to the previously described high affinity LCO binding protein MtLYR3 (Fliegmann et al. 2013, positive control) whereas extracts of MtNFP, other LYR proteins and MOCK (negative control) showed no high-affinity LCO-IV(C16:2,S) NF binding (Fig. 1, Fig. S2). It should be noted that our work used a non-O-acetylated S. meliloti NF as ligand, whereas at least part of the NFs from this species is Oacetylated on the terminal non-reducing sugar (specified by the NodL gene; [START_REF] Ardourel | Rhizobium Meliloti Lipooligosaccharide nodulation factors -different structural requirements for bacterial entry into target root hair-cells and induction of plant symbiotic developmental responses[END_REF]). This decoration is not essential for MtNFP-dependent signalling as a S. meliloti nodL mutant is able to nodulate M. truncatula, although with a slightly lower efficiency than the WT [START_REF] Smit | Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling[END_REF]) and nodL NFs are active on Medicago roots down to pM concentrations in biological assays [START_REF] Ardourel | Rhizobium Meliloti Lipooligosaccharide nodulation factors -different structural requirements for bacterial entry into target root hair-cells and induction of plant symbiotic developmental responses[END_REF][START_REF] Journet | Rhizobium meliloti Nod factors elicit cell-specific transcription of the ENOD12 gene in transgenic alfalfa[END_REF][START_REF] Wais | Structure-function analysis of nod factor-induced root hair calcium spiking in Rhizobium-legume symbiosis[END_REF]. Even if such NFs have a 10-fold lower affinity for MtNFP compared to O-acetylated NFs, as suggested by [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF], we should still be able to detect affinities in the 100 nM range, as shown in previous studies on other proteins [START_REF] Fliegmann | Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula[END_REF][START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]. We cannot however measure low affinity binding in the µM range.

Using biolayer interferometry assays, [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF] have reported that the soluble MtNFP-ECR, expressed and purified from insect cells, binds to LCO chips with a specificity expected from genetic studies, but with an affinity in the µM range. They suggest that the slow kinetics of LCO binding to MtNFP, rather than affinity, is important for receptor activation and signalling specificity.

It is thus possible that MtNFP has a lower affinity for Sm LCOs compared to other LCO binding proteins such as MtLYR3, and that this is sufficient for mediating NF perception.

Evolution in the LCO binding site in MtNFP allowed higher efficiency for nodulation

All tested non-legume LYR-IA genes showed ability to complement the Mtnfp mutant for nodulation and rhizobial colonization (This study and [START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF]), but with low efficiency since the proportion of nodulating plants and the nodule number were much lower than by complementation with MtNFP. Similarly, MtLYR1 showed poor complementation of Mtnfp for nodulation, consistent with the protein retaining properties inherited from the ancestral gene.

By analysing LysM domain swaps of the MtLYR1 (LLL) and MtNFP (NNN) proteins we have confirmed a critical role of the MtNFP LysM2 for a role in nodulation (Fig. 4; [START_REF] Bensmihen | Contribution of NFP LysM domains to the recognition of Nod factors during the Medicago truncatula/Sinorhizobium meliloti symbiosis[END_REF][START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF]). This was found by both gain of function in the LLL construct (LNL) and loss of function in NNN (NLN). Moreover replacing some of the MtLYR1 LysM2 residues in LLL by those of MtNFP has highlighted the critical role of changing MtLYR1/MtNFP residues S114/N122 and K147/P153 in conferring nodulation ability. In MtLYR1, these residues are important for LCO binding (Fig. 4), as are N142 (replaced by S) and/or Y144 (replaced by T). Through homology modelling to the crystal structure of the LjLYS11-ECR (Fig. 4), our work is consistent with the CO moiety of the LCO binding in a groove in LysM2, similar to the one where CO binds in the crystal structure of Arabidopsis AtCERK1 and rice OsCEBIP [START_REF] Liu | Molecular Mechanism for fungal cell wall recognition by rice chitin receptor OsCEBiP[END_REF][START_REF] Liu | Chitin-induced dimerization activates a plant immune receptor[END_REF]. Our work is also compatible with the acyl chain of the LCO being accommodated in a hydrophobic patch, identified on certain LYR proteins [START_REF] Gysel | Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors[END_REF]). However, because there is no crystal structure of a LCO ligand in a LysM protein, this location and orientation of the LCO remain speculative. It should be noted that the residues identified in this work are important for the difference between MtLYR1 and MtNFP in terms of high affinity LCO binding (and nodulation) but are not fully conserved between the LYR-IA LCO binding proteins of different species (Fig. S12). This suggests that substitutions in this groove and the associated hydrophobic patch render the protein still capable of accommodating the notoriously flexible lipid chain and oligosaccharide of the LCO [START_REF] Groves | The relative orientation of the lipid and carbohydrate moieties of lipochitooligosaccharides related to nodulation factors depends on lipid chain saturation[END_REF].

Model of NF perception and nodulation

As MtNFP, in addition to its essential role in rhizobial nodulation, plays a role in immunity to pathogens and in Myc-LCO responses, it has been suggested that it may participate in different protein complexes to lead to different responses [START_REF] Gough | Nod factor perception protein carries weight in biotic interactions[END_REF]. For nodulation, its essential role in mediating sulfated S. meliloti NF perception at pM to nM concentrations, suggests that MtNFP is a key component of a NF receptor, although, as shown here, not as a high affinity NF binding protein. We propose that it could either share NF binding with another LysM protein [START_REF] Moling | Nod factor receptors form heteromeric complexes and are essential for intracellular infection in Medicago nodules[END_REF][START_REF] Igolkina | Structural insight into the role of mutual polymorphism and conservatism in the contact zone of the NFR5-K1 heterodimer with the Nod Factor[END_REF][START_REF] Solovev | Towards understanding afghanistan pea symbiotic phenotype through the molecular modeling of the interaction between LykX-Sym10 receptor heterodimer and Nod Factors[END_REF] or be more involved in the signalling role of the NF receptor. Because of the exquisite sensitivity to sulfated NFs, it is possible that NF perception in Medicago shows some differences compared to other legumes. A precedent for species-specific differences in LysM-RLK function exists from work on CO perception in rice/Arabidopsis [START_REF] Yang | CERK1, more than a co-receptor in plant-microbe interactions[END_REF]).

It should be noted, that during nodulation in the natural environment the role of high affinity LCO binding proteins, such as MtLYR1 and MtLYR3, could be to prevent infection by non-compatible rhizobia or other LCO producing microorganisms.

In conclusion, our results on different plant families suggest that the ancestor of the LYR-IA subclass of LysM-RLKs was an LCO binding protein, with a role in AM, properties that have been retained throughout evolution. Duplication of the gene in legumes led to specialisation of one paralog (corresponding to MtNFP) for interacting with rhizobia. The other paralog (MtLYR1) retained many characteristics of the original protein in terms of LCO binding and a potential, but probably redundant, role in AM. In M. truncatula, changes in the expression pattern of MtNFP coupled with evolution of the putative ancestral LCO binding site have been important for the function of MtNFP in nodulation with S. meliloti, and our results suggest that the latter has been through a decrease in LCO binding, compared to MtLYR1.

MATERIALS AND METHODS

Plant material

Medicago truncatula Gaertn. A17 was used as the gene source and for phenotyping experiments, unless otherwise stated. Brachypodium distachyon Bd21.3 and wheat Chinese spring were used as the gene source.

Mutants and phenotyping

The A17 Mtnfp mutant (Mtnfp-2), carrying the ProMtENOD11:GUS construct, has been previously described [START_REF] Arrighi | The Medicago truncatula lysin motif-receptor-like kinase gene family includes NFP and new noduleexpressed genes[END_REF]. Mutant Mtlyr1-1 (C58/T that leads to a stop codon), was derived from EMS mutagenesis of A17 (Le [START_REF] Signor | Optimizing TILLING populations for reverse genetics in Medicago truncatula[END_REF]. Mutant Mtlyr1-2 (NF3951) is a Tnt1 insertional mutant of M. truncatula ssp. tricycla R108-1 [START_REF] Tadege | Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula[END_REF]. Putative alternative translation would lead to proteins without signal-peptide and thus not functional.

Phenotyping of the mutants for nodulation was done in test tubes on Fahraeus agar slants [START_REF] Luu | A newly evolved chimeric lysin motif receptor-like kinase in Medicago truncatula spp. tricycla R108 extends its Rhizobia symbiotic partnership[END_REF]) using about 10 6 bacteria/plant of Sinorhizobium meliloti 2011 (GMI6526) or the nodFE mutant (GMI6528), containing a constitutive LacZ fusion on plasmid pXLGD4 [START_REF] Ardourel | Rhizobium Meliloti Lipooligosaccharide nodulation factors -different structural requirements for bacterial entry into target root hair-cells and induction of plant symbiotic developmental responses[END_REF].

Mycorrhiza analysis was performed on mycorrhizal roots using the gridline intersect method after plants were grown for 6 weeks, as described [START_REF] Gibelin-Viala | The Medicago truncatula LysM receptor-like kinase LYK9 plays a dual role in immunity and the arbuscular mycorrhizal symbiosis[END_REF] in the presence of 20 spores Rhizophagus irregularis (DAOM197198, Agronutrition, Toulouse, France) / plant, with 7-20 plants in total per genotype.

Complementation assays

Using Agrobacterium rhizogenes-mediated transformation [START_REF] Boisson-Dernier | Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations[END_REF], seedlings of the Mtnfp-2 or Mtnfp-2/ProMtENOD11:GUS lines were transformed using strains containing either empty vector (EV) or the appropriate construct, and transformants were selected on media containing 25 µg ml -1 kanamycin. After two-weeks growth, the plants were transferred to pots [START_REF] Luu | A newly evolved chimeric lysin motif receptor-like kinase in Medicago truncatula spp. tricycla R108 extends its Rhizobia symbiotic partnership[END_REF]) and analysed for nodulation following inoculation with 10 6 bacteria/plant of S. meliloti 2011.

Constructs for in planta protein expression

The predicted extracellular region (ECR) of the M. truncatula LYR genes [START_REF] Lefebvre | Role of N-Glycosylation sites and CXC motifs in trafficking of Medicago truncatula Nod Factor perception protein to plasma membrane[END_REF]) was amplified from genomic DNA using the primers listed in Fig. S13 and cloned behind either the Pro35S or the ProLjUb and in front of the TM+KIN domains of MtNFP, followed by YFP, in a pcambia vector modified for Golden Gate cloning [START_REF] Fliegmann | LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor[END_REF]. The MtLYR3 and MtLYR4 constructs are described in [START_REF] Fliegmann | Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula[END_REF]. The complete coding sequences (CDS) of BdLYR1 (Bradi1g69290),

TaLYR1A (TraesCS4A03G0122100), TaLYR1B (TraesCS4B03G0646400)
and TaLYR1D

(TraesCS4D03G0574900) were similarly cloned in a pbin vector modified for Gateway cloning [START_REF] Lefebvre | A remorin protein interacts with symbiotic receptors and regulates bacterial infection[END_REF], in fusion with YFP, using the primers listed in Fig. S13. The OsMYR1/OsNFR5 (LOC_Os03G13080) sequence was optimized with the N. benthamiana codon usage (Fig. S14) by gene synthesis and cloned in the pcambia vector modified for Golden Gate cloning. For the domain swapping experiments the ECR of MtNFP and MtLYR1 was each divided into three domains at the conserved CXC sequences (between the LysMs) to form LysM1, LysM2 and LysM3 modules. Each module was amplified by PCR using the primers listed in Fig. S13 and cloned. Full length proteins were reconstituted by Golden Gate cloning using the same modules described above. Mutations were introduced in the LysM2 of MtLYR1 by site-directed mutagenesis. All constructs were verified by sequencing.

Expression in N. benthamiana leaves and preparation of protein extracts.

Agrobacterium tumefaciens LBA4404 strains containing expression cassettes for the fusion constructs and/or the silencing inhibitor P19 were used to agro-infiltrate the three to four oldest leaves of N.

benthamiana plants, such that P19 was co-expressed with the LysM-RLKs. Leaves expressing only P19

were used as control (MOCK). Three days post infiltration, the protein expression in leaves was assessed by confocal microscopy and the leaves were harvested, frozen in liquid nitrogen and stored at -70 °C until extraction. Membrane extracts of the MOCK or fusion-expressing leaves (sedimenting between 3 000 -80 000 g) were prepared as described [START_REF] Fliegmann | Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula[END_REF][START_REF] Girardin | LCO receptors involved in arbuscular mycorrhiza are functional for rhizobia perception in legumes[END_REF] and then stored at -70 °C in 30 % glycerol in binding buffer until use.

BdLYR1-YFP was solubilized from membrane fraction in IP buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 % glycerol with protease and phosphatase inhibitors) with 0.2 % dodecylmaltoside (DDM, ratio detergent:proteins 1:4) and purified with magnetic anti-GFP beads (Chromotek,.

CO5-biot synthesis

The CO5-biot was derived from the CO5-propargyl [START_REF] Masselin | Unprecedented affinity labeling of carbohydrate-binding proteins with s-Triazinyl Glycosides[END_REF]). CO5-propargyl (5 mg, 4 µmol) and azide-Peg3-biotin conjugate (Aldrich) (2.7 mg, 6 µmol) were mixed in 0.1 M phosphate buffer (600 µL, pH 7.4), and copper sulfate (100 µL of an aqueous solution at 10 mg/mL, 4 µmol) and sodium ascorbate (100 µL of an aqueous solution at 10 mg/mL, 5 µmol) were successively added.

LCO and CO binding assays

Synthetic LCO-IV(C16:22E9Z), LCO-V(C18:111Z,NMe) purified from the supernatant of a Rhizobium tropici nodH mutant and synthetic azido photoactivatable LCO derivative (crosslinkable LCO; [START_REF] Fliegmann | Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula[END_REF], were enzymatically sulfated with 35 S and then purified by HPLC to separate out the non-sulfated fraction (see [START_REF] Gressent | Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures[END_REF]) for details). After sulfation the ligand generally has a specific radioactivity of 100 to 200 Ci/mmol. Binding assays generally used 5-60 g protein of membrane fraction and 0.5 to 1 nM labelled ligand in binding buffer (25 mM sodium cacodylate pH 6, 1 mM MgCl 2 , 1 mM CaCl 2 , 250 mM saccharose, 1 mg/L antipain, 1 mg/L leupeptin, 1 mg/L aprotinin, 1 mg/L pepstatin, 1 mg/L chymostatin and 100 µM AEBSF). Non-specific binding was assessed by competition with 2 M unlabelled ligand. Bound ligand was separated from un-bound by filtration [START_REF] Gressent | Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures[END_REF]. Specific binding (non-specific subtracted from total) was calculated.

Each LCO binding assay consisted of 3 technical replicates. Kd and Ki were determined in cold ligand saturation and competitive inhibition experiments respectively, with concentrations of the molecules ranging from 10 pM to 2 µM ligand, and calculated using the RadLig software. Competition experiments with LCOs and COs used 0.5 to 1 nM of labelled ligand and 1 to 2 M of competitor.

Crosslink of the azido photoactivatable LCO was performed on purified proteins attached to the anti-GFP beads, incubated with 3 nM radioactive photoactivatable ligand in binding buffer upon UV irradiation as described in [START_REF] Fliegmann | Lipo-chitooligosaccharidic symbiotic signals are recognized by LysM receptor-like kinase LYR3 in the legume Medicago truncatula[END_REF]. Samples were irradiated for 10 min at 254 nm.

For CO5-biot crosslink 50 µg of membrane protein was incubated in binding buffer with 5 µM CO5biot.

Bioinformatic analysis and structural modelling

The percentages of sequence identity (% ID) between proteins was calculated by EMBOSS Needle and protein sequence alignments used Multalin. Homology modelling used SWISS-MODEL and the crystal structure of LjLYS11 ECD (PDB: 7BAX) or MtNFP ECD (7AU7) as templates. Electrostatic surfaces were calculated using PDB2PQR and APBS webservers. The structures were visualised in PyMOL.

Data, Materials and Software Availability

All plant lines, microbial strains and constructs are available upon request. All study data are included in the article and/or SI Appendix.

Figure legends

  . A duplication of LYR-IA associated with the origin of the socalled nitrogen fixing clade of plants (orders Fabales, Fagales, Cucurbitales and Rosales) led to two ancestral paralogs, termed NFP-I and NFP-II. LCO-driven nodulation is correlated with a functional NFP-II type gene (the orthogroup of MtNFP/MtLYR1) and in legume plants the NFP-I-type gene has been lost (Rutten et al. 2020). In Papilionoid legumes, a WGD event occurred more recently (about 58 MYA), leading to two NFP-II genes. While MtNFP/LjNFR5 is present in the majority of Papilionoids, the paralogous MtLYR1/LjLYS11 is found in Galegoid and Robinoid legume species, but has apparently been lost in others, including in most of the related Millettioids, which include Glycine and Phaseolus spp. (Fig. S6; Rutten et al. 2020).

Fig. 1 .

 1 Fig. 1. MtLYR1 has a high affinity for Myc-LCOs but not for COs. A) Binding of the radiolabelled LCO-IV(C16:2,S) ligand to membrane extracts of N. benthamiana leaves expressing the indicated M. truncatula LYR proteins (upper panel). Proteins correspond to ECR of the MtLYR proteins fused to the TM and ICR of MtNFP and YFP. MOCK corresponds to membrane extracts of leaves expressing only the silencing inhibitor P19. Bars represent the specific LCO binding/mg membrane protein (means and SD between at least 5 independent experiments). Statistical differences were calculated using a Kruskal-Wallis test. Different letters indicate significant difference (P < 0.05). Western blot of 10 µg total protein of the membrane extracts using anti-GFP antibodies (lower panel). B) Binding of the radiolabelled LCO-V(C18:1,NMe,S) ligand to membrane extracts of N. benthamiana leaves expressing the same fusion proteins as in (A). Bars represent the specific LCO binding/mg membrane protein (means and SD between at least 5 independent experiments). Statistical differences were calculated using a Kruskal-Wallis test. Different letters indicate significant difference (P < 0.05). C) Affinity of MtLYR1 for LCO-V(C18:1,NMe,S). Scatchard plot analysis of a cold saturation experiment using a membrane extract expressing MtLYR1, the radiolabelled LCO-V(C18:1,NMe,S) ligand and a range of concentrations of unlabeled LCO-V(C18:1,NMe,S). D) Affinity of MtLYR1 for LCO-IV(C16:2,S). Competitive inhibition of the radiolabelled LCO-V(C18:1,NMe,S) binding using a range of concentration of unlabeled LCO-IV(C16:2,S) as competitor. E) Specificity of the MtLYR1 LCO binding site for LCOs versus COs. Bars represent the percentage competition of specific binding of radiolabelled LCO-V(C18:1,NMe,S) in presence of 2 µM of the indicated competitor (means and SD between 2 independent experiments, each with 3 technical replicates). Statistical differences were calculated using pairwise Student tests. Different letters indicate significant difference (P < 0.05). F) Affinity of MtLYR1 for short-chain COs. Saturation experiment using a range of concentration of a crosslinkable biotinylated CO5 (CO5-biot). Western blots using sequentially anti-GFP antibodies and Streptadivin were performed on the same membrane. The arrowhead indicates the position of MtLYR1, whereas * indicates an endogenously (biotinylated) protein in the extracts. G) Specificity of the MtLYR1 CO binding site for LCOs versus COs. CO5-biot binding was competed using CO4 or LCO-V(C18:1,NMe,S). Western blots using sequentially anti-GFP antibodies and Streptadivin were performed on the same membrane. The arrowhead indicates the position of MtLYR1 and the * an endogenously (biotinylated) protein. H) Number of nodulated plants and mean number of nodules per plant on the Mtnfp mutant complemented with the indicated constructs. Data from 2 independent experiments. Statistical differences were calculated using a Chi2 test for the number of nodulated plants and a Kruskal-Wallis test for the mean number of nodules. Different letters in each column indicate significant difference (P < 0.05).

Fig. 2 .

 2 Fig. 2. MtLYR1 is dispensable for AM and nodulation. A) Position of point mutation and transposon (Tnt1) insertion in Mtlyr1-1 (A17) and Mtlyr1-2 (R108) mutants, respectively, relative to the encoded MtLYR1 protein structure. Grey: LysM, Black: transmembrane domain (TM), White: kinase domain. B) Root-length colonization at 6 wpi with Rhizophagus irregularis in Mtlyr1-1 (A17), Mtlyr1-2 (R108), Mtnfp (A17) and Mtlyr1-1/Mtnfp (A17) mutants and their respective controls (A17 or R108). Boxplots represent the distribution between 13-25 individuals from 2 independent experiments. No significant differences were found between each Mtlyr1 mutant and its corresponding WT line using a Van der Waerden test (P > 0.05). C) Number of nodules at 28 dpi with Sinorhizobium meliloti in Mtlyr1-1 (A17), Mtlyr1-2 (R108) and Mtnfp (A17) mutants and their respective wild-type controls (A17 or R108). Boxplots represent the distribution between 10 individuals. Different letters indicate significant difference using a Van der Waerden test (P < 0.05). D) Same as in C) except that the plants were inoculated with S. meliloti nodFE mutant instead of WT S. meliloti.

Fig. 3 .

 3 Fig. 3. Poaceae LYR-IA have similar biochemical properties as MtLYR1. A) Binding of the radiolabelled LCO-V(C18:1,NMe,S) to membrane extracts of N. benthamiana leaves expressing the indicated fusion proteins (upper panel, means and SD between at least 2 independent experiments, each with 3 technical replicates). MOCK corresponds to membrane extracts of leaves expressing only the silencing inhibitor P19. Statistical differences were calculated using a Kruskal-Wallis test. Different letters indicate significant difference (P < 0.05). Proteins correspond to full length proteins fused to YFP. Western blot of 10 µg total protein of the membrane extracts using anti-GFP antibodies (lower panel). B) Affinities of the indicated proteins for radiolabelled LCO-V(C18:1,NMe,S) deduced from Scatchard plot analysis (Fig. S7). C) Selectivity of the indicated protein LCO binding sites to COs. Bars represent the percentage competition of specific radiolabelled LCO-V(C18:1,NMe,S) binding by 1 µM of the indicated competitors (mean and SD between 3 technical replicates). Statistical differences were calculated using pairwise Student tests. Different letters indicate significant difference (P < 0.05). D) Affinity of OsMYR1 for short-chain COs. Saturation experiment using a range of concentrations of a crosslinkable biotinylated CO5 (CO5-biot). Western blots using sequentially anti-GFP antibodies and Streptadivin were performed on the same membrane. The arrowhead indicates the position of OsMYR1 and the * an endogenously (biotinylated) protein. E) Specificity of the BdLYR1 LCO binding site for LCOs. Bars represent the percentage competition of specific LCO-V(C18:1,NMe,S) binding in presence of 2 µM of the indicated competitors (means and SD between 3 independent experiments). Statistical difference was calculated using a Student test. * indicates a significant difference (P < 0.05). F) Competition experiments on purified BdLYR1-YFP using a radiolabelled-and crosslinkable -LCO derivative and a range of concentrations of unlabeled LCO-V(C18:1,NMe,S) or CO4 as competitor. Phosphorimaging was performed on the same nitrocellulose

Fig. 4 .

 4 Fig. 4. The structure of MtLYR1 LysM2 is critical for high affinity LCO binding and for poor efficacy to complement Mtnfp mutant for nodulation. A) Cartoon of domain and AA swaps between MtLYR1 (green) and MtNFP (cyan). B) Number of nodules per plant on the Mtnfp mutant complemented with constructs of the indicated fusion proteins. EV corresponds to the empty vector control. Boxplots represent the distribution between 21-30 individuals from 2 independent experiments. Different letters indicate significant difference using a Van der Waerden test (P < 0.05). C) Binding of radiolabelled LCO-V(C18:1,NMe,S) to membrane extracts of N. benthamiana leaves expressing the indicated fusion proteins. Error bars show SD of the means of 2 independent experiments, each with 3 technical replicates. MOCK corresponds to membrane extracts of leaves expressing only the silencing inhibitor P19. Statistical differences were calculated using pairwise Student tests. Different letters indicate significant difference (P < 0.05). D) Sequence of LysM2 of MtLYR1 (green) and MtNFP (cyan) showing position of mutations. AA highlighted in yellow are conserved between the proteins. E) Structural model of the MtLYR1 LysM2 coloured for surface electrostatic potential to show the hydrophobic patch described in Gysel et al. (2021) (dotted line), the putative CO-binding groove and the position of the mutated residues. F) Superposition of the MtLYR1 LysM2 (green) on the MtNFP LysM2 (cyan) showing residues in MtLYR1 (red) and MtNFP (blue) important for LCO binding or nodulation activity respectively. The importance of MtNFP L147/L154 is described by Gysel et al (2021). Corresponding residues with unknown/unclear roles are shown in the same colour as the main protein.

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  Fig. 1.

ACKNOWLEDGEMENTS

We thank Cécile Pouzet (FRAIB-TRI Imaging Platform Facilities) for confocal imaging, Ton Timmers for nodule sectioning and imaging and Sandra Bensmihen for critical reading of the manuscript. For the Mtlyr1 mutants we are very grateful to Christine Le Signor (Agroécologie, Dijon), Pascal Ratet (Paris Saclay), and the FP6 EU Project GLIP Grain Legumes FOOD-CT-2004-506223. For the Tnt1 mutants, the Medicago truncatula plants utilized in this research project, which are jointly owned by the Centre National de la Recherche Scientifique, were obtained from Noble Research Institute, LLC and were created through research funded, in part, by grants National Science Foundation NSF-0703285 and IOS-1127155. This work was supported by the Agence Nationale de la Recherche (ANR) projects NodBindsLysM (ANR-05-BLAN-0243-01, coordinator J. Cullimore/C. Gough), NICE CROPS (ANR-14-CE18-0008-01, coordinator G. Bécard), SYMNALING (ANR-12-BSV7-01, coordinator J. Cullimore), MycSignalling (ANR-09-BLAN-0241, coordinator C. Gough), DUALITY (ANR-20-CE20-0017-01, coordinator C. Gough) and WHEATSYM (ANR-16-CE20-0025-01, coordinator B. Lefebvre). This study is set within the framework of the "Laboratoires d'Excellences (LABEX)" TULIP (ANR 10 LABX 41) and of the "École Universitaire de Recherche (EUR)" TULIP GS (ANR 18 EURE 0019). TBL gratefully acknowledges receipt of a Bourse d'Excellence from the Ambassade de France au Vietnam. SF acknowledges Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003) for partial support and NanoBio ICMG (UAR 2607) for providing facilities for mass spectrometry (A. Durand, L. Fort, R. Gueret) and NMR analyses (I. Jeacomine).