Nod Factor Perception Research Articles

Medicago truncatula SOBIR1 controls pathogen immunity and specificity in the Rhizobium-legume symbiosis.

Medicago truncatula Nod Factor Perception (MtNFP)plays a role in both the Rhizobium-Legume (RL)symbiosis and plant immunity, and evidence suggests that the immune-related function of MtNFP is relevant for symbiosis. To better understand these roles of MtNFP, we sought to identify new interacting partners. We screened a yeast-2-hybrid cDNA library from Aphanomyces euteiches infected and noninfected M. truncatula roots. The M. truncatula leucine-rich repeat (LRR)receptor-like kinase SUPPRESSOR OF BIR1 (MtSOBIR1)was identified as an interactor of MtNFP and was characterised for kinase activity, and potential roles in symbiosis and plant immunity. We showed that the kinase domain of MtSOBIR1 is active and can transphosphorylate the pseudo-kinase domain of MtNFP. MtSOBIR1 could functionally complement Atsobir1 and Nbsobir1/sobir1-like mutants for defence activation, and Mtsobir1 mutants were defective in immune responses to A. euteiches. For symbiosis, we showed that Mtsobir1 mutant plants had both a strong, early infection defect and defects in the defence suppression in nodules, and both effects were plant genotype- and rhizobial strain-specific. This work highlights a conserved function for MtSOBIR1 in activating defence responses to pathogen attack, and potentially novel symbiotic functions of downregulating defence in association with the control of symbiotic specificity.

Chitin nanofibers promote rhizobial symbiotic nitrogen fixation in Lotus japonicus

Chitin, an N-acetyl-D-glucosamine polymer, has multiple functions in living organisms, including the induction of disease resistance and growth promotion in plants. In addition, chitin oligosaccharides (COs) are used as the backbone of the signaling molecule Nod factor secreted by soil bacteria rhizobia to establish a mutual symbiosis with leguminous plants. Nod factor perception triggers host plant responses for rhizobial symbiosis. In this study, the effects of chitins on rhizobial symbiosis were examined in the leguminous plants Lotus japonicus and soybean. Chitin nanofiber (CNF), retained with polymeric structures, and COs elicited calcium spiking in L. japonicus roots expressing a nuclear-localized cameleon reporter. Shoot growth and symbiotic nitrogen fixation were significantly increased by CNF but not COs in L.japonicus and soybean. However, treatments with chitin and cellulose nanofiber, structurally similar polymers to CNF, did not affect shoot growth and nitrogen fixation in L.japonicus. Transcriptome analysis also supported the specific effects of CNF on rhizobial symbiosis in L.japonicus. Although chitins comprise the same monosaccharides and nanofibers share similar physical properties, only CNF can promote rhizobial nitrogen fixation in leguminous plants. Taking the advantages on physical properties, CNF could be a promising material for improving legume yield by enhancing rhizobial symbiosis.

Putative molecular pathways of autoregulation of nodulation activated by CLE peptides in pea

Legume plants are important for ecosystems due to their ability to form root nodules in symbiosis with rhizobia, where nitrogen fixation takes place. The number of symbiotic nodules is regulated by the CLE peptides inhibiting excessive nodule formation. Previously, we have identified four genes encoding CLE peptides, activated in response to rhizobia inoculation in pea. Three of them, PsCLE13, PsCLE12 and PsNIC-like, were also activated by nitrate, and, therefore, they could mediate nitrate-dependent inhibition of nodulation [1]. Overexpression of PsCLE13 and PsCLE12 inhibited nodulation on transgenic roots: however, the role of PsNIC-like and PsCLE12-like have not been investigated.
 In this study, we constructed vectors for overexpression of the PsCLE12-like and PsNIC-like genes to study their possible role in nodulation, and also analyzed the expression levels of nodulation-related genes in transgenic roots overexpressing four PsCLEs genes. Moreover, vectors for CRISPR-Cas9-mediated gene editing of the PsCLE12 and PsCLE13 genes were constructed to further explore the role of these genes in nodulation. Overexpression of PsCLE12-like, PsCLE13 and PsCLE12 resulted in increased expression levels of TOO MUCH LOVE (PsTMLs) genes known as root-acting regulators of nodule number. In addition, in the roots overexpressing four PsCLEs genes, down regulation of the PsSYM37 gene (encoding the receptor for Nod-factors) was observed, suggesting that the CLE peptides might inhibit the development of symbiotic nodules at the earliest stages of symbiosis development upon Nod-factor perception.

RNA-Seq analysis of mung bean (Vigna radiata L.) roots shows differential gene expression and predicts regulatory pathways responding to taxonomically different rhizobia

Symbiotic interaction among legume and rhizobia is a complex phenomenon which results in the formation of nitrogen-fixing nodules. Mung bean is promiscuous host however expression profile of this important legume plant in response to rhizobial infection was particularly lacking and urgently needed. We have demonstrated the pattern of gene expression of mung bean roots inoculated with two symbionts Bradyrhizobium yuanmingense Vr50 and Sinorhizobium (Ensifer) aridi Vr33 and non-inoculated control (CK). The RNA-Seq data analyzed at two growth stages i.e., 1–3 h and 10–16 days post inoculation revealed significantly higher number of differentially expressed genes (DEGs) at nodulation stage. The DEGs encoding receptor kinases identified at early stage might be involved in perception of Nod factors produced by different rhizobia. At nodulation stage important genes involved in plant hormone signal transduction, nitrogen and sulfur metabolism were identified. KEGG pathway enrichment analysis showed that metabolic pathways were most prominent in both groups (Group 1: Vr33 vs CK; Group 2: Vr50 vs CK), followed by biosynthesis of secondary metabolites, plant hormone signal transduction and biosynthesis of amino acids. Furthermore, DEGs involved in cell communication and plant hormone signal transduction were found to be different among two symbiotic systems while DEGs involved in carbon, nitrogen and sulfur metabolism were similar but their expression varied in response to two rhizobial strains. This study provides the first insight into the mechanisms underlying interactions of mung bean host with two taxonomically different symbionts (Bradyrhizobium and Sinorhizobium) and the candidate genes for better understanding the mechanisms of symbiotic host-specificity.

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.

Photosynthetic Gains in Super-Nodulating Mutants of Medicago truncatula under Elevated Atmospheric CO2 Conditions.

Legumes are generally considered to be more responsive to elevated CO2 (eCO2) conditions due to the benefits provided by symbiotic nitrogen fixation. In response to high carbohydrate demand from nodules, legumes display autoregulation of nodulation (AON) to restrict nodules to the minimum number necessary to sustain nitrogen supply under current photosynthetic levels. AON mutants super-nodulate and typically grow smaller than wild-type plants under ambient CO2. Here, we show that AON super-nodulating mutants have substantially higher biomass under eCO2 conditions, which is sustained through increased photosynthetic investment. We examined photosynthetic and physiological traits across super-nodulating rdn1-1 (Root Determined Nodulation) and sunn4 (Super Numeric Nodules) and non-nodulating nfp1 (Nod Factor Perception) Medicago truncatula mutants. Under eCO2 conditions, super-nodulating plants exhibited increased rates of carboxylation (Vcmax) and electron transport (J) relative to wild-type and non-nodulating counterparts. The substantially higher rate of CO2 assimilation in eCO2-grown sunn4 super-nodulating plants was sustained through increased production of key photosynthetic enzymes, including Rieske FeS. We hypothesize that AON mutants are carbon-limited and can perform better at eCO2 through improved photosynthesis. Nodulating legumes, especially those with higher nitrogen fixation capability, are likely to out-perform non-nodulating plants under future CO2 conditions and will be important tools for understanding carbon and nitrogen partitioning under eCO2 conditions and future crop improvements.

Evolution of LysM-RLK Gene Family in Wild and Cultivated Peanut Species

In legumes, a LysM-RLK perception of rhizobial lipo-chitooligosaccharides (LCOs) known as Nod factors (NFs), triggers a signaling pathway related to the onset of symbiosis development. On the other hand, activation of LysM-RLKs upon recognition of chitin-derived short-chitooligosaccharides initiates defense responses. In this work, we identified the members of the LysM-RLK family in cultivated (Arachis hypogaea L.) and wild (A. duranensis and A. ipaensis) peanut genomes, and reconstructed the evolutionary history of the family. Phylogenetic analyses allowed the building of a framework to reinterpret the functional data reported on peanut LysM-RLKs. In addition, the potential involvement of two identified proteins in NF perception and immunity was assessed by gene expression analyses. Results indicated that peanut LysM-RLK is a highly diverse family. Digital expression analyses indicated that some A. hypogaea LysM-RLK receptors were upregulated during the early and late stages of symbiosis. In addition, expression profiles of selected LysM-RLKs proteins suggest participation in the receptor network mediating NF and/or chitosan perception. The analyses of LysM-RLK in the non-model legume peanut can contribute to gaining insight into the molecular basis of legume–microbe interactions and to the understanding of the evolutionary history of this gene family within the Fabaceae.

Genome-wide identification, expression, and characterization of CaLysM-RLKs in chickpea root nodule symbiosis

Legumes establish a nitrogen-fixing symbiosis with rhizobia which depends on the exchange of chemical signals between the two partners. Successful interaction between legumes and rhizobia depends on the perception of Nod factors (NFs) by the receptor-like kinases. A few cell surface receptors have been identified that perceive the rhizobial NFs and activate the downstream signaling pathway for nodulation in model legumes like Lotus japonicus and Medicago truncatula . However, crop legume such as chickpea lacks detailed analysis of LysM-RLKs genes for NFs perception. Here, we identified and characterized CaLysM-RLK genes and analyzed their gene structure, motif composition, chromosomal localization, phylogenetic relationship with Arabidopsis, Oryza sativa , and Medicago truncatula . To understand the spatiotemporal expression patterns, we performed transcript profiling of Ca LysM-RLKs genes at a different time interval of the chickpea-rhizobia symbiotic interaction that revealed CaNFP and CaLYK3 can act as candidate genes for NF perception. Down-regulation of CaNFP and CaLYK3 in chickpea by agrobacterium rhizogenes mediated hairy root sysytem leads to signifificant reduction in nodule numbers. This study will be useful for improving root nodule symbiosis in chickpea. • Seventeen CaLysM-RLKs genes were identified in chickpea. • Based on the structure CaLysM-RLKs family members were classified into three main groups LYS-I, LYS-II, and LYS-III. • Analysis of paralogous gene pair (PGP) leads to the identification of a total of 6 PGPs. • CaLYK3 , and CaNFP regulate root nodule symbiosis in chickpea.

Nod factor perception: an integrative view of molecular communication during legume symbiosis.

Compatible interaction between rhizobial Nod factors and host receptors enables initial recognition and signaling events during legume-rhizobia symbiosis. Molecular communication is a new paradigm of information relay, which uses chemical signals or molecules as dialogues for communication and has been witnessed in prokaryotes, plants as well as in animal kingdom. Understanding this fascinating relay of signals between plants and rhizobia during the establishment of a synergistic relationship for biological nitrogen fixation represents one of the hotspots in plant biology research. Predominantly, their interaction is initiated by flavonoids exuding from plant roots, which provokes changes in the expression profile of rhizobial genes. Compatible interactions promote the secretion of Nod factors (NFs) from rhizobia, which are recognised by cognate host receptors. Perception of NFs by host receptors initiates the symbiosis and ultimately leads to the accommodation of rhizobia within root nodules via a series of mutual exchange of signals. This review elucidates the bacterial and plant perspectives during the early stages of symbiosis, explicitly emphasizing the significance of NFs and their cognate NF receptors.

The importance of Rhizobium, Agrobacterium, Bradyrhizobium, Herbaspirillum, Sinorhizobium in sustainable agricultural production

Rhizobia which are soil bacteria capable of symbiosis with legume plants in the root or stem nodules and perform nitrogen fixation. Rhizobial genera include Agrobacterium, Allorhizobium, Aminobacter, Azorhizobium, Bradyrhizobium, Devosia, Mesorhizobium, Methylobacterium, Microvirga, Ochrobacterum, Phyllobacterium, Rhizobium, Shinella and Ensifer (Sinorhizobium). Review of the literature was carried out using the keywords Rhizobium, Agrobacterium, Bradyrhizobium, Herbaspirillum and Sinorhizobium. Rhizobial nodulation symbioses steps are included flavonoid signaling, Nod factor induction, and Nod factor perception, root hair responses, rhizobial infection, cell division and formation of nitrogen-fixing nodule. Rhizobium improves sustainable production by boosting organic nitrogen content.

Nod factor receptor complex phosphorylates GmGEF2 to stimulate ROP signaling during nodulation

The establishment of the symbiotic interaction between rhizobia and legumes involves the Nod factor signaling pathway. Nod factor recognition occurs through two plant receptors, NFR1 and NFR5. However, the signal transduction mechanisms downstream of NFR1-NFR5-mediated Nod factor perception remain largely unknown. Here, we report that a small guanosine triphosphatase (GTPase), GmROP9, and a guanine nucleotide exchange factor, GmGEF2, are involved in the soybean-rhizobium symbiosis. We show that GmNFR1α phosphorylates GmGEF2a at its N-terminal S86, which stimulates guanosine diphosphate (GDP)-to-GTP exchange to activate GmROP9 and that the active form of GmROP9 can associate with both GmNFR1α and GmNFR5α. We further show that a scaffold protein, GmRACK1, interacts with active GmROP9 and contributes to root nodule symbiosis. Collectively, our results highlight the symbiotic role of GmROP9-GmRACK1 and support the hypothesis that rhizobial signals promote the formation of a protein complex comprising GmNFR1, GmNFR5, GmROP9, and GmRACK1 for symbiotic signal transduction in soybean.

Auxin Response Factor 2 (ARF2), ARF3, and ARF4 Mediate Both Lateral Root and Nitrogen Fixing Nodule Development in Medicago truncatula.

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2, ARF3, and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/trans-acting small interference RNA 3 (TAS3) module through the action of TAS3-derived trans-acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a, and MtARF4b, in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2, MtARF3, and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a, and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a, and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a, and MtARF4b, as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules.

Rhizobia use a pathogenic-like effector to hijack leguminous nodulation signalling

Legume plants form a root-nodule symbiosis with rhizobia. This symbiosis establishment generally relies on rhizobium-produced Nod factors (NFs) and their perception by leguminous receptors (NFRs) that trigger nodulation. However, certain rhizobia hijack leguminous nodulation signalling via their type III secretion system, which functions in pathogenic bacteria to deliver effector proteins into host cells. Here, we report that rhizobia use pathogenic-like effectors to hijack legume nodulation signalling. The rhizobial effector Bel2-5 resembles the XopD effector of the plant pathogen Xanthomonas campestris and could induce nitrogen-fixing nodules on soybean nfr mutant. The soybean root transcriptome revealed that Bel2-5 induces expression of cytokinin-related genes, which are important for nodule organogenesis and represses ethylene- and defense-related genes that are deleterious to nodulation. Remarkably, Bel2-5 introduction into a strain unable to nodulate soybean mutant affected in NF perception conferred nodulation ability. Our findings show that rhizobia employ and have customized pathogenic effectors to promote leguminous nodulation signalling.

Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Functional characterization and physiological roles of the single Shaker outward K+ channel in Medicago truncatula.

The model legume Medicago truncatula possesses a single outward Shaker K+ channel, whereas Arabidopsis thaliana possesses two channels of this type, named AtSKOR and AtGORK, with AtSKOR having been shown to play a major role in K+ secretion into the xylem sap in the root vasculature and with AtGORK being shown to mediate the efflux of K+ across the guard cell membrane, leading to stomatal closure. Here we show that the expression pattern of the single M.truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch-clamp experiments on root hair protoplasts, besides the Shaker-type slowly activating outwardly rectifying K+ conductance encoded by MtGORK, a second K+ -permeable conductance, displaying fast activation and weak rectification, can be expressed by M.truncatula. A knock-out (KO) mutation resulting in an absence of MtGORK activity is shown to weakly reduce K+ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpirational water loss. In legumes, the early electrical signaling pathway triggered by Nod-factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In the absence of the functional expression of MtGORK, the rate of the membrane repolarization is found to be decreased by a factor of approximately two. This defect was without any consequence on infection thread development and nodule production in plants grown in vitro, but a decrease in nodule production was observed in plants grown in soil.

Expression of the Legume-Specific Nod Factor Receptor Proteins Alters Developmental and Immune Responses in Rice

Legumes form symbiosis with rhizobia, which fix nitrogen for the benefit of host plant in return for carbon resources. Development of this unique symbiosis in legumes is triggered by rhizobia-secreted nodulation (Nod) factors (NFs). NFs, upon perception, activate Nod signaling cascade, leading to reprogramming of host cell (root) developmental networks to pave way for accommodating rhizobial symbionts. A long-cherished goal of legume-rhizobia symbiosis research is to extend this symbiotic nitrogen-fixing capacity to cereal plants such as rice. As a part of achieving this ultimate goal, in this work, initially we expressed legume-specific Nod factor receptor protein (NFRP) genes, MtNFP, MtLYK3, and LjLNP, in rice and assessed their impact on NF perception and consequently triggered biological responses in roots. RNA-seq analysis revealed that roots of both control and NFRP-expressing plants perceive NFs, but NFs elicited contrasting impacts on gene expression patterns in roots of these plants. In contrast to suppressive role of NFs on expression of several genes involved in innate immune response in roots of control plants, in NFRP-expressing plants, NFs triggered massive upregulation of a vast array of genes associated with signaling, defense response, and secondary metabolism networks in roots. Expression of NFRPs in rice also conferred root hairs the ability to respond to NFs in terms of exhibiting deformations, albeit at low levels. Together, results of the study demonstrated that rice plants have inherent ability to perceive NFs, but the expression of legume NFRPs rendered rice roots hypersensitive to NFs.

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.

Mini-Review: Nod Factor Regulation of Phytohormone Signaling and Homeostasis During Rhizobia-Legume Symbiosis.

The rhizobia-legume symbiosis is a mutualistic association in which bacteria provide plants with nitrogen compounds and the plant provides bacteria with carbon sources. A successful symbiotic interaction relies on a molecular dialog between the plant and the bacteria, and generally involves rhizobial lipo-chitooligosaccharide signals called Nod factors (NFs). In most cases, specific NF perception is required for rhizobia to enter root cells through newly formed intracellular structures called infection threads (ITs). Concomitantly to IT formation in root hairs, root cortical cells start to divide to create a new root organ called the nodule, which will provide the bacteria with a specific micro-environment required for symbiotic nitrogen fixation. During all these steps of plant–bacteria interaction, new plant cellular compartments and developmental programs are activated. This interaction is costly for the plant that tightly controls symbiosis establishment and functioning. Phytohormones are key regulators of cellular and developmental plasticity in plants, and they are influential endogenous signals that rapidly control plant responses. Although early symbiotic responses were known for decades to be linked to phytohormone-related responses, new data reveal the molecular mechanisms involved and links between phytohormones and the control of early symbiotic events. Reciprocally, NF signaling also targets phytohormone signaling pathways. In this review, we will focus on the emerging notion of NF and phytohormone signaling crosstalk, and how it could contribute to the tight control of symbiosis establishment in legume host plants.

Epidermal auxin biosynthesis facilitates rhizobial infection in Lotus japonicus.

Symbiotic nitrogen fixation in legumes requires nodule organogenesis to be coordinated with infection by rhizobia. The plant hormone auxin influences symbiotic infection, but the precise timing of auxin accumulation and the genetic network governing it remain unclear. We used a Lotus japonicus optimised variant of the DII-based auxin accumulation sensor and identified a rapid accumulation of auxin in the epidermis, specifically in the root hair cells. This auxin accumulation occurs in the infected root hairs during rhizobia invasion, while Nod factor application induces this response across a broader range of root hairs. Using the DR5 auxin responsive promoter, we demonstrate that activation of auxin signalling also occurs specifically in infected root hairs. Analysis of root hair transcriptome data identified induction of an auxin biosynthesis gene of the Tryptophan Amino-transferase Related (LjTar1) family following both bacteria inoculation and Nod factor treatment. Genetic analysis showed that both expression of the LjTar1 biosynthesis gene and the auxin response requires Nod factor perception, while common symbiotic pathway transcription factors are only partially required or act redundantly to initiate auxin accumulation. Using a chemical genetics approach, we confirmed that auxin biosynthesis has a functional role in promoting symbiotic infection events in the epidermis.

Medicago Plants Control Nodulation by Regulating Proteolysis of the Receptor-Like Kinase DMI2.

Plants use receptor-like kinases to monitor environmental changes and transduce signals into plant cells. The Medicago truncatula (hereafter Mtruncatula) DOES NOT MAKE INFECTIONS2 (DMI2) protein functions as a coreceptor of rhizobial signals to initiate nodule development and rhizobial infection during nitrogen-fixing symbiosis, but the mechanisms regulating DMI2 protein level and folding are still unknown. Here, we report that DMI2 protein abundance changes during nitrogen-fixing symbiosis. DMI2 accumulates in the nodules and is induced by rhizobia treatment through a posttranscriptional process. However, DMI2 induction is independent of the perception of Nod factor, a group of lipochitooligosaccharides secreted by rhizobia. The stability of the DMI2 protein is controlled by the proteasome pathway: in rhizobia-free environments, DMI2 is degraded by the proteasome, but during rhizobial infection, DMI2 is protected from the proteasome, resulting in protein accumulation. Furthermore, proteasome inhibitor-promoted accumulation of DMI2 protein in Medicago roots induces the expression of two early nodulation marker genes, supporting the hypothesis that DMI2 accumulation activates downstream symbiosis signaling. The extracellular region of DMI2 contains two malectin-like domains (MLDs) and a leucine-rich repeat. When conserved amino acids in the MLDs are mutated, DMI2 fails to restore nodule development in dmi2 mutants, and point-mutated MLD proteins are degraded constitutively, suggesting that the MLD may be vital for the accumulation of DMI2. Our findings suggest that legumes control nodule development through modulating the protein level of DMI2, revealing a layer of regulation in the interaction between plants and rhizobia in nitrogen-fixing symbiosis.