OROSOMUCOID PROTEIN 1 regulation of sphingolipid synthesis is required for nodulation in Aeschynomene evenia.

Legumes establish symbiotic interactions with nitrogen-fixing rhizobia that are accommodated in root-derived organs known as nodules. Rhizobial recognition triggers a plant symbiotic signaling pathway that activates 2 coordinated processes: infection and nodule organogenesis. How these processes are orchestrated in legume species utilizing intercellular infection and lateral root base nodulation remains elusive. Here, we show that Aeschynomene evenia OROSOMUCOID PROTEIN 1 (AeORM1), a key regulator of sphingolipid biosynthesis, is required for nodule formation. Using A. evenia orm1 mutants, we demonstrate that alterations in AeORM1 function trigger numerous early aborted nodules, defense-like reactions, and shorter lateral roots. Accordingly, AeORM1 is expressed during lateral root initiation and elongation, including at lateral root bases where nodule primordium form in the presence of symbiotic bradyrhizobia. Sphingolipidomics revealed that mutations in AeORM1 lead to sphingolipid overaccumulation in roots relative to the wild type, particularly for very long-chain fatty acid-containing ceramides. Taken together, our findings reveal that AeORM1-regulated sphingolipid homeostasis is essential for rhizobial infection and nodule organogenesis, as well as for lateral root development in A. evenia.

Nodulating another way: what can we learn from lateral root base nodulation in legumes?

Certain legumes provide a special pathway for rhizobia to invade the root and develop nitrogen-fixing nodules, a process known as lateral root base (LRB) nodulation. This pathway involves intercellular infection at the junction of the lateral roots with the taproot, leading to nodule formation in the lateral root cortex. Remarkably, this LRB pathway serves as a backbone for various adaptative symbiotic processes. Here, we describe different aspects of LRB nodulation and highlight directions for future research to elucidate the mechanisms of this as yet little known but original pathway that will help in broadening our knowledge on the rhizobium-legume symbiosis.

A mutant-based analysis of the establishment of Nod-independent symbiosis in the legume Aeschynomene evenia.

Intensive research on nitrogen-fixing symbiosis in two model legumes has uncovered the molecular mechanisms, whereby rhizobial Nod factors activate a plant symbiotic signaling pathway that controls infection and nodule organogenesis. In contrast, the so-called Nod-independent symbiosis found between Aeschynomene evenia and photosynthetic bradyrhizobia, which does not involve Nod factor recognition nor infection thread formation, is less well known. To gain knowledge on how Nod-independent symbiosis is established, we conducted a phenotypic and molecular characterization of A. evenia lines carrying mutations in different nodulation genes. Besides investigating the effect of the mutations on rhizobial symbiosis, we examined their consequences on mycorrhizal symbiosis and in nonsymbiotic conditions. Analyzing allelic mutant series for AePOLLUX, Ca2+/calmodulin dependent kinase, AeCYCLOPS, nodulation signaling pathway 2 (AeNSP2), and nodule inception demonstrated that these genes intervene at several stages of intercellular infection and during bacterial accommodation. We provide evidence that AeNSP2 has an additional nitrogen-dependent regulatory function in the formation of axillary root hairs at lateral root bases, which are rhizobia-colonized infection sites. Our investigation of the recently discovered symbiotic actor cysteine-rich receptor-like kinase specified that it is not involved in mycorrhization; however, it is essential for both symbiotic signaling and early infection during nodulation. These findings provide important insights on the modus operandi of Nod-independent symbiosis and contribute to the general understanding of how rhizobial-legume symbioses are established by complementing the information acquired in model legumes.

The rhizobial type III effector ErnA confers the ability to form nodules in legumes.

Several Bradyrhizobium species nodulate the leguminous plant Aeschynomene indica in a type III secretion system-dependent manner, independently of Nod factors. To date, the underlying molecular determinants involved in this symbiotic process remain unknown. To identify the rhizobial effectors involved in nodulation, we mutated 23 out of the 27 effector genes predicted in Bradyrhizobium strain ORS3257. The mutation of nopAO increased nodulation and nitrogenase activity, whereas mutation of 5 other effector genes led to various symbiotic defects. The nopM1 and nopP1 mutants induced a reduced number of nodules, some of which displayed large necrotic zones. The nopT and nopAB mutants induced uninfected nodules, and a mutant in a yet-undescribed effector gene lost the capacity for nodule formation. This effector gene, widely conserved among bradyrhizobia, was named ernA for "effector required for nodulation-A." Remarkably, expressing ernA in a strain unable to nodulate A. indica conferred nodulation ability. Upon its delivery by Pseudomonas fluorescens into plant cells, ErnA was specifically targeted to the nucleus, and a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy approach supports the possibility that ErnA binds nucleic acids in the plant nuclei. Ectopic expression of ernA in A. indica roots activated organogenesis of root- and nodule-like structures. Collectively, this study unravels the symbiotic functions of rhizobial type III effectors playing distinct and complementary roles in suppression of host immune functions, infection, and nodule organogenesis, and suggests that ErnA triggers organ development in plants by a mechanism that remains to be elucidated.

RibBX of Bradyrhizobium ORS285 Plays an Important Role in Intracellular Persistence in Various Aeschynomene Host Plants.

Bradyrhizobium ORS285 forms a nitrogen-fixating symbiosis with both Nod factor (NF)-dependent and NF-independent Aeschynomene spp. The Bradyrhizobium ORS285 ribBA gene encodes for a putative bifunctional enzyme with 3,4-dihydroxybutanone phosphate (3,4-DHBP) synthase and guanosine triphosphate (GTP) cyclohydrolase II activities, catalyzing the initial steps in the riboflavin biosynthesis pathway. In this study, we show that inactivating the ribBA gene does not cause riboflavin auxotrophy under free-living conditions and that, as shown for RibBAs from other bacteria, the GTP cyclohydrolase II domain has no enzymatic activity. For this reason, we have renamed the annotated ribBA as ribBX. Because we were unable to identify other ribBA or ribA and ribB homologs in the genome of Bradyrhizobium ORS285, we hypothesize that the ORS285 strain can use unconventional enzymes or an alternative pathway for the initial steps of riboflavin biosynthesis. Inactivating ribBX has a drastic impact on the interaction of Bradyrhizobium ORS285 with many of the tested Aeschynomene spp. In these Aeschynomene spp., the ORS285 ribBX mutant is able to infect the plant host cells but the intracellular infection is not maintained and the nodules senesce early. This phenotype can be complemented by reintroduction of the 3,4-DHBP synthase domain alone. Our results indicate that, in Bradyrhizobium ORS285, the RibBX protein is not essential for riboflavin biosynthesis under free-living conditions and we hypothesize that its activity is needed to sustain riboflavin biosynthesis under certain symbiotic conditions.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Naturally occurring variations in the nod-independent model legume Aeschynomene evenia and relatives: a resource for nodulation genetics.

BACKGROUND: Among semi-aquatic species of the legume genus Aeschynomene, some have the unique property of being root and stem-nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the production of Nod factors. These species provide an excellent biological system with which to explore the evolution of nodulation in legumes. Among them, Aeschynomene evenia has emerged as a model legume to undertake the genetic dissection of the so-called Nod-independent symbiosis. In addition to the genetic analysis of nodulation on a reference line, natural variation in a germplasm collection could also be surveyed to uncover genetic determinants of nodulation. To this aim, we investigated the patterns of genetic diversity in a collection of 226 Nod-independent Aeschynomene accessions. RESULTS: A combination of phylogenetic analyses, comprising ITS and low-copy nuclear genes, along with cytogenetic experiments and artificial hybridizations revealed the richness of the Nod-independent Aeschynomene group with the identification of 13 diploid and 6 polyploid well-differentiated taxa. A set of 54 SSRs was used to further delineate taxon boundaries and to identify different genotypes. Patterns of microsatellite diversity also illuminated the genetic basis of the Aeschynomene taxa that were all found to be predominantly autogamous and with a predicted simple disomic inheritance, two attributes favorable for genetics. In addition, taxa displaying a pronounced genetic diversity, notably A. evenia, A. indica and A. sensitiva, were characterized by a clear geographically-based genetic structure and variations in root and stem nodulation. CONCLUSION: A well-characterized germplasm collection now exists as a major genetic resource to thoroughly explore the natural variation of nodulation in response to different bradyrhizobial strains. Symbiotic polymorphisms are expected to be found notably in the induction of nodulation, in nitrogen fixation and also in stem nodulation. Subsequent genetic analysis and locus mapping will pave the way for the identification of the underlying genes through forward or reverse genetics. Such discoveries will significantly contribute to our understanding of the molecular mechanisms underpinning how some Aeschynomene species can be efficiently nodulated in a Nod-independent fashion.

A phylogenetic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-dependent and Nod-independent symbioses.

BACKGROUND: Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. RESULTS: This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. CONCLUSIONS: The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses.

The evolutionary dynamics of ancient and recent polyploidy in the African semiaquatic species of the legume genus Aeschynomene.

The legume genus Aeschynomene is notable in the ability of certain semiaquatic species to develop nitrogen-fixing stem nodules. These species are distributed in two clades. In the first clade, all the species are characterized by the use of a unique Nod-independent symbiotic process. In the second clade, the species use a Nod-dependent symbiotic process and some of them display a profuse stem nodulation as exemplified in the African Aeschynomene afraspera. To facilitate the molecular analysis of the symbiotic characteristics of such legumes, we took an integrated molecular and cytogenetic approach to track occurrences of polyploidy events and to analyze their impact on the evolution of the African species of Aeschynomene. Our results revealed two rounds of polyploidy: a paleopolyploid event predating the African group and two neopolyploid speciations, along with significant chromosomal variations. Hence, we found that A. afraspera (8x) has inherited the contrasted genomic properties and the stem-nodulation habit of its parental lineages (4x). This study reveals a comprehensive picture of African Aeschynomene diversification. It notably evidences a history that is distinct from the diploid Nod-independent clade, providing clues for the identification of the specific determinants of the Nod-dependent and Nod-independent symbiotic processes, and for comparative analysis of stem nodulation.

Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling.

Arabidopsis (Arabidopsis thaliana) absorbs inorganic phosphate (Pi) from the soil through an active transport process mediated by the nine members of the PHOSPHATE TRANSPORTER1 (PHT1) family. These proteins share a high level of similarity (greater than 61%), with overlapping expression patterns. The resulting genetic and functional redundancy prevents the analysis of their specific roles. To overcome this difficulty, our approach combined several mutations with gene silencing to inactivate multiple members of the PHT1 family, including a cluster of genes localized on chromosome 5 (PHT1;1, PHT1;2, and PHT1;3). Physiological analyses of these lines established that these three genes, along with PHT1;4, are the main contributors to Pi uptake. Furthermore, PHT1;1 plays an important role in translocation from roots to leaves in high phosphate conditions. These genetic tools also revealed that some PHT1 transporters likely exhibit a dual affinity for phosphate, suggesting that their activity is posttranslationally controlled. These lines display significant phosphate deficiency-related phenotypes (e.g. biomass and yield) due to a massive (80%-96%) reduction in phosphate uptake activities. These defects limited the amount of internal Pi pool, inducing compensatory mechanisms triggered by the systemic Pi starvation response. Such reactions have been uncoupled from PHT1 activity, suggesting that systemic Pi sensing is most probably acting downstream of PHT1.

Nod Factor-Independent Nodulation in Aeschynomene evenia Required the Common Plant-Microbe Symbiotic Toolkit.

Nitrogen fixation in the legume-rhizobium symbiosis is a crucial area of research for more sustainable agriculture. Our knowledge of the plant cascade in response to the perception of bacterial Nod factors has increased in recent years. However, the discovery that Nod factors are not involved in the Aeschynomene-Bradyrhizobium spp. interaction suggests that alternative molecular dialogues may exist in the legume family. We evaluated the conservation of the signaling pathway common to other endosymbioses using three candidate genes: Ca(2+)/Calmodulin-Dependent Kinase (CCaMK), which plays a central role in cross signaling between nodule organogenesis and infection processes; and Symbiosis Receptor Kinase (SYMRK) and Histidine Kinase1 (HK1), which act upstream and downstream of CCaMK, respectively. We showed that CCaMK, SYMRK, and HK1 are required for efficient nodulation in Aeschynomene evenia. Our results demonstrate that CCaMK and SYMRK are recruited in Nod factor-independent symbiosis and, hence, may be conserved in all vascular plant endosymbioses described so far.

Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides.

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

Radiation of the Nod-independent Aeschynomene relies on multiple allopolyploid speciation events.

• The semi-aquatic legumes belonging to the genus Aeschynomene constitute a premium system for investigating the origin and evolution of unusual symbiotic features such as stem nodulation and the presence of a Nod-independent infection process. This latter apparently arose in a single Aeschynomene lineage. But how this unique Nod-independent group then radiated is not yet known. • We have investigated the role of polyploidy in Aeschynomene speciation via a case study of the pantropical A. indica and then extended the analysis to the other Nod-independent species. For this, we combined SSR genotyping, genome characterization through flow cytometry, chromosome counting, FISH and GISH experiments, molecular phylogenies using ITS and single nuclear gene sequences, and artificial hybridizations. • These analyses demonstrate the existence of an A. indica polyploid species complex comprising A. evenia (C. Wright) (2n = 2x = 20), A. indica L. s.s. (2n = 4x = 40) and a new hexaploid form (2n = 6x = 60). This latter contains the two genomes present in the tetraploid (A. evenia and A. scabra) and another unidentified genome. Two other species, A. pratensis and A. virginica, are also shown to be of allopolyploid origin. • This work reveals multiple hybridization/polyploidization events, thus highlighting a prominent role of allopolyploidy in the radiation of the Nod-independent Aeschynomene.

Arabidopsis thaliana high-affinity phosphate transporters exhibit multiple levels of posttranslational regulation.

In Arabidopsis thaliana, the PHOSPHATE TRANSPORTER1 (PHT1) family encodes the high-affinity phosphate transporters. They are transcriptionally induced by phosphate starvation and require PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR (PHF1) to exit the endoplasmic reticulum (ER), indicating intracellular traffic as an additional level of regulation of PHT1 activity. Our study revealed that PHF1 acts on PHT1, upstream of vesicle coat protein COPII formation, and that additional regulatory events occur during PHT1 trafficking and determine its ER exit and plasma membrane stability. Phosphoproteomic and mutagenesis analyses revealed modulation of PHT1;1 ER export by Ser-514 phosphorylation status. Confocal microscopy analysis of root tip cells showed that PHT1;1 is localized to the plasma membrane and is present in intracellular endocytic compartments. More precisely, PHT1;1 was localized to sorting endosomes associated with prevacuolar compartments. Kinetic analysis of PHT1;1 stability and targeting suggested a modulation of PHT1 internalization from the plasma membrane to the endosomes, followed by either subsequent recycling (in low Pi) or vacuolar degradation (in high Pi). For the latter condition, we identified a rapid mechanism that reduces the pool of PHT1 proteins present at the plasma membrane. This mechanism is regulated by the Pi concentration in the medium and appears to be independent of degradation mechanisms potentially regulated by the PHO2 ubiquitin conjugase. We propose a model for differential trafficking of PHT1 to the plasma membrane or vacuole as a function of phosphate concentration.

Occurrence and diversity of stem nodulation in Aeschynomene and Sesbania legumes from wetlands of Madagascar.

Legumes have the ability to establish a nitrogen-fixing symbiosis with soil rhizobia that they house in specific organs, the nodules. In most rhizobium-legume interactions, nodulation occurs on the root. However, certain tropical legumes growing in wetlands possess a unique trait: the capacity to form rhizobia-harbouring nodules on the stem. Despite the originality of the stem nodulation process, its occurrence and diversity in waterlogging-tolerant legumes remains underexplored, impeding a comprehensive analysis of its genetics and biology. Here, we aimed at filling this gap by surveying stem nodulation in legume species-rich wetlands of Madagascar. Stem nodulation was readily observed in eight hydrophytic species of the legume genera, Aeschynomene and Sesbania, for which significant variations in stem nodule density and morphology was documented. Among these species, A. evenia, which is used as genetic model to study the rhizobial symbiosis, was found to be frequently stem-nodulated. Two other Aeschynomene species, A. cristata and A. uniflora, were evidenced to display a profuse stem-nodulation as occurs in S. rostrata. These findings extend our knowledge on legumes species that are endowed with stem nodulation and further indicate that A. evenia, A. cristata, A. uniflora and S. rostrata are of special interest for the study of stem nodulation. As such, these legume species represent opportunities to investigate different modalities of the nitrogen-fixing symbiosis and this knowledge could provide cues for the engineering of nitrogen-fixation in non-legume crops.

Evolution of symbiosis in the legume genus Aeschynomene.

Legumes in the genus Aeschynomene form nitrogen-fixing root nodules in association with Bradyrhizobium strains. Several aquatic and subaquatic species have the additional capacity to form stem nodules, and some of them can symbiotically interact with specific strains that do not produce the common Nod factors synthesized by all other rhizobia. The question of the emergence and evolution of these nodulation characters has been the subject of recent debate. We conducted a molecular phylogenetic analysis of 38 different Aeschynomene species. The phylogeny was reconstructed with both the chloroplast DNA trnL intron and the nuclear ribosomal DNA ITS/5.8S region. We also tested 28 Aeschynomene species for their capacity to form root and stem nodules by inoculating different rhizobial strains, including nodABC-containing strains (ORS285, USDA110) and a nodABC-lacking strain (ORS278). Maximum likelihood analyses resolved four distinct phylogenetic groups of Aeschynomene. We found that stem nodulation may have evolved several times in the genus, and that all Aeschynomene species using a Nod-independent symbiotic process clustered in the same clade. The phylogenetic approach suggested that Nod-independent nodulation has evolved once in this genus, and should be considered as a derived character, and this result is discussed with regard to previous experimental studies.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

Aeschynomene evenia, a model plant for studying the molecular genetics of the nod-independent rhizobium-legume symbiosis.

Research on the nitrogen-fixing symbiosis has been focused, thus far, on two model legumes, Medicago truncatula and Lotus japonicus, which use a sophisticated infection process involving infection thread formation. However, in 25% of the legumes, the bacterial entry occurs more simply in an intercellular fashion. Among them, some Aeschynomene spp. are nodulated by photosynthetic Bradyrhizobium spp. that do not produce Nod factors. This interaction is believed to represent a living testimony of the ancestral state of the rhizobium-legume symbiosis. To decipher the mechanisms of this Nod-independent process, we propose Aeschynomene evenia as a model legume because it presents all the characteristics required for genetic and molecular analysis. It is a short-perennial and autogamous species, with a diploid and relatively small genome (2n=20; 460 Mb/1C). A. evenia 'IRFL6945' is nodulated by the well-characterized photosynthetic Bradyrhizobium sp. strain ORS278 and is efficiently transformed by Agrobacterium rhizogenes. Aeschynomene evenia is genetically homozygous but polymorphic accessions were found. A manual hybridization procedure has been set up, allowing directed crosses. Therefore, it should be relatively straightforward to unravel the molecular determinants of the Nod-independent process in A. evenia. This should shed new light on the evolution of rhizobium-legume symbiosis and could have important agronomic implications.

Molecular Mechanisms of Intercellular Rhizobial Infection: Novel Findings of an Ancient Process.

Establishment of the root-nodule symbiosis in legumes involves rhizobial infection of nodule primordia in the root cortex that is dependent on rhizobia crossing the root epidermal barrier. Two mechanisms have been described: either through root hair infection threads or through the intercellular passage of bacteria. Among the legume genera investigated, around 75% use root hair entry and around 25% the intercellular entry mode. Root-hair infection thread-mediated infection has been extensively studied in the model legumes Medicago truncatula and Lotus japonicus. In contrast, the molecular circuit recruited during intercellular infection, which is presumably an ancient and simpler pathway, remains poorly known. In recent years, important discoveries have been made to better understand the transcriptome response and the genetic components involved in legumes with obligate (Aeschynomene and Arachis spp.) and conditional (Lotus and Sesbania spp.) intercellular rhizobial infections. This review addresses these novel findings and briefly considers possible future research to shed light on the molecular players that orchestrate intercellular infection in legumes.

An integrated approach reveals how lipo-chitooligosaccharides interact with the lysin motif receptor-like kinase MtLYR3.

N-acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant-specific Lysin Motif Receptor-Like Kinases (LysM-RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo-chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen-fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM-RLK LYR3 (MtLYR3) as a specific LCO-binding protein. We also showed that the absence of LCO binding to LYR3 of the non-mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site-directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3-LysM3 by those of MtLYR3-LysM3 allowed the recovery of high-affinity LCO binding in experimental radioligand-binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.