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

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.     

The DMI1 and DMI2 early symbiotic genes of medicago truncatula are required for a high-affinity nodulation factor-binding site associated to a particulate fraction of roots

The establishment of the legume-rhizobia symbiosis between Medicago spp. and Sinorhizobium meliloti is dependent on the production of sulfated lipo-chitooligosaccharidic nodulation (Nod) factors by the bacterial partner. In this article, using a biochemical approach to characterize putative Nod factor receptors in the plant host, we describe a high-affinity binding site (Kd = 0.45 nm) for the major Nod factor produced by S. meliloti. This site is termed Nod factor-binding site 3 (NFBS3). NFBS3 is associated to a high-density fraction prepared from roots of Medicago truncatula and shows binding specificity for lipo-chitooligosaccharidic structures. As for the previously characterized binding sites (NFBS1 and NFBS2), NFBS3 does not recognize the sulfate group on the S. meliloti Nod factor. Studies of Nod factor binding in root extracts of early symbiotic mutants of M. truncatula reveals that the new site is present in Nod factor perception and does not make infections 3 (dmi3) mutants but is absent in dmi1 and dmi2 mutants. Roots and cell cultures of all these mutants still contain sites similar to NFBS1 and NFBS2, respectively. These results suggest that NFBS3 is different from NFBS2 and NFBS1 and is dependent on the common symbiotic genes DMI1 and DMI2 required for establishment of symbioses with both rhizobia and arbuscular mycorrhizal fungi. The potential role of this site in the establishment of root endosymbioses is discussed.     

豆科植物早期共生信号转导的研究进展

豆科植物与根瘤菌建立特异的共生关系,在寄主根部产生固氮根瘤。此过程包含了共生信号识别与传递、根瘤菌侵染、根瘤形成以及固氮功能实现等生物学事件。研究人员已经从2种豆科模式植物蒺藜苜蓿（Medicago truncatula）和百脉根（Lotus japonicus）的共生固氮体系中,筛选到许多与根瘤菌共生相关的突变体及其相对应的功能基因,建立起包含结瘤因子识别、共生信号传递和转录响应在内的早期共生信号途径。该文对豆科植物早期共生信号途径的新进展进行了综述。     

The receptor kinase CERK1 has dual functions in symbiosis and immunity signalling

The establishment of symbiotic interactions between mycorrhizal fungi, rhizobial bacteria and their legume hosts involves a common symbiosis signalling pathway. This signalling pathway is activated by Nod factors produced by rhizobia and these are recognised by the Nod factor receptors NFR1/LYK3 and NFR5/NFP. Mycorrhizal fungi produce lipochitooligosaccharides (LCOs) similar to Nod factors, as well as short‐chain chitin oligomers (CO4/5), implying commonalities in signalling during mycorrhizal and rhizobial associations. Here we show that NFR1/LYK3, but not NFR5/NFP, is required for the establishment of the mycorrhizal interaction in legumes. NFR1/LYK3 is necessary for the recognition of mycorrhizal fungi and the activation of the symbiosis signalling pathway leading to induction of calcium oscillations and gene expression. Chitin oligosaccharides also act as microbe associated molecular patterns that promote plant immunity via similar LysM receptor‐like kinases. CERK1 in rice has the highest homology to NFR1 and we show that this gene is also necessary for the establishment of the mycorrhizal interaction as well as for resistance to the rice blast fungus. Our results demonstrate that NFR1/LYK3/OsCERK1 represents a common receptor for chitooligosaccharide‐based signals produced by mycorrhizal fungi, rhizobial bacteria (in legumes) and fungal pathogens. It would appear that mycorrhizal recognition has been conserved in multiple receptors across plant species, but additional diversification in certain plant species has defined other signals that this class of receptors can perceive.     

Peptide signalling in the rhizobium-legume symbiosis

For two decades, signalling research in the rhizobium-legume symbiosis field has been dominated by oligosaccharide signals (mainly Nod factors and, to a lesser extent, surface polysaccharides made by the microsymbionts) and phytohormones. Recently, plant peptides have emerged as another major class of signalling molecules in the rhizobium-legume symbioses contributing to the control of nodulation, infection and bacteroid differentiation. Here we focus on three examples of symbiotically relevant peptides, namely Enod40, CLE and NCR peptides. The number of genes encoding these peptides, as well as the recent discovery of additional peptide players in the context of symbiosis, suggests that we might be seeing only the tip of the peptide iceberg in the sea of symbiotic regulations.     

The Medicago truncatula E3 ubiquitin ligase PUB1 interacts with the LYK3 symbiotic receptor and negatively regulates infection and nodulation

LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogen-fixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box-dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant-microbe interactions and development through interacting with receptor-like kinases is discussed.     

Evidence for structurally specific negative feedback in the Nod factor signal transduction pathway

Nod factor is a critical signalling molecule in the establishment of the legume/rhizobial symbiosis. The Nod factor of Sinorhizobium meliloti carries O-sulphate, O-acetate and C16:2 N-acyl attachments that define its activity and host specificity. Here we assess the relative importance of these modifications for the induction of calcium spiking in Medicago truncatula. We find that Nod factor structures lacking the O-sulphate, structures lacking the O-acetate and N-acyl groups, and structures lacking the O-acetate combined with a C18:1 N-acyl group all show calcium spiking when applied at high concentrations. These calcium responses are blocked in dmi1 and dmi2 mutants, suggesting that they function through the Nod factor signal transduction pathway. The dmi3 mutant, which is proposed to function in the Nod factor signal transduction pathway downstream of calcium spiking, shows increased sensitivity to Nod factor. This increased sensitivity is only active with wild-type Nod factor and was not present when the plants were treated with mutant Nod factor structures. We propose that the Nod factor signal transduction pathway is under negative feedback regulation that is activated at or downstream of DMI3 and requires structural components of the Nod factor molecule for activity.     

Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway

Legumes form two different types of intracellular root symbioses, with fungi and bacteria, resulting in arbuscular mycorrhiza and nitrogen-fixing nodules, respectively. Rhizobial signalling molecules, called Nod factors, play a key role in establishing the rhizobium-legume association and genes have been identified in Medicago truncatula that control a Nod factor signalling pathway leading to nodulation. Three of these genes, the so-called DMI1, DMI2 and DMI3 genes, are also required for formation of mycorrhiza, indicating that the symbiotic pathways activated by both the bacterial and the fungal symbionts share common steps. To analyse possible cross-talk between these pathways we have studied the effect of treatment with Nod factors on mycorrhization in M. truncatula. We show that Nod factors increase mycorrhizal colonization and stimulate lateral root formation. The stimulation of lateral root formation by Nod factors requires both the same structural features of Nod factors and the same plant genes (NFP, DMI1, DMI2, DMI3 and NSP1) that are required for other Nod factor-induced symbiotic responses such as early nodulin gene induction and cortical cell division. A diffusible factor from arbuscular mycorrhizal fungi was also found to stimulate lateral root formation, while three root pathogens did not have the same effect. Lateral root formation induced by fungal signal(s) was found to require the DMI1 and DMI2 genes, but not DMI3. The idea that this diffusible fungal factor might correspond to a previously hypothesized mycorrhizal signal, the 'Myc factor', is discussed.     

Identification and characterization of nodulation-signaling pathway 2, a gene of Medicago truncatula involved in Nod actor signaling

Bacterially derived Nod factor is critical in the establishment of the legume/rhizobia symbiosis. Understanding the mechanisms of Nod factor perception and signal transduction in the plant will greatly advance our understanding of this complex interaction. Here, we describe the identification of a new locus, nodulation-signaling pathway 2 (NSP2), of Medicago truncatula that is involved in Nod factor signaling. Mutants at this locus are blocked for Nod factor-induced gene expression and show a reduced root hair deformation response. nsp2 plants also show a complete absence of infection and cortical cell division following Sinorhizobium meliloti inoculation. Nod factor-induced calcium spiking, one of the earliest responses tested, is still functional in these mutant plants. We conclude that the gene NSP2 is a component of the Nod factor signal transduction pathway that lies downstream of the calcium-spiking response.     

Perception of Bradyrhizobium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions

Suboptimal growth conditions, such as low rhizosphere temperature, high salinity, and low pH can negatively affect the rhizobia-legume symbioses, resulting in poor nodulation and lower amounts of nitrogen fixed. Early stages of the Bradyrhizobium japonicum-soybean [Glycine max (L.) Merr.] symbiosis, such as excretion of genistein (the plant-to-bacteria signal) and infection initiation can be inhibited by abiotic stresses; however, the effect on early events modulated by Nod factors (bacteria-to-plant signalling), particularly root hair deformations is unknown. Thus, the objective of this study was to evaluate the perception of Nod factor by soybean root hairs under three stress conditions: low temperature, low pH, and high salinity. Three experiments were conducted using a 1:1 ratio of Nod Bj-V (C(18:1), MeFuc) and Nod Bj-V (Ac, C(16:0), MeFuc). Nod factor induced four types of root hair deformation (HAD), wiggling, bulging, curling, and branching. Under optimal experimental conditions root hair response to the three levels of Nod factor tested (10(-6), 10(-8), and 10(-10) M) was dose-dependent. The highest frequency of root hair deformations was elicited by the 10(-6) M level. Root hair deformation decreased with temperature (25, 17, and 15 degrees C), low pH, and high salinity. Nod factor concentration did not interact with either low temperature or pH. However, salinity strongly inhibited HAD responses to increases in Nod factor concentration. Thus, the addition of higher levels of Nod factor is able to overcome the effects of low pH and temperature stress, but not salinity.     

Metabolomic profiling reveals suppression of oxylipin biosynthesis during the early stages of legume-rhizobia symbiosis

The establishment of symbiosis between leguminous plants and rhizobial bacteria requires rapid metabolic changes in both partners. We utilized untargeted quantitative mass spectrometry to perform metabolomic profiling of small molecules in extracts of the model legume Medicago truncatula treated with rhizobial Nod factors. One metabolite closely resembling the 9(R)-HODE class of oxylipins reproducibly showed a decrease in concentration within the first hour of in planta nod factor treatment. Oxylipins are precursors of the jasmonic acid biosynthetic pathway and we showed that both this metabolite and jasmonic acid inhibit Nod factor signaling. Since, oxylipins have been implicated as antimicrobial compounds produced by plants, these observations suggest that the oxylipin pathway may play multiple roles in facilitating Nod factor signaling during the early stages of symbiosis.     

Rhizobial lipo-oligosaccharide nodulation factors: multidimensional chromatographic analysis of symbiotic signals involved in the development of legume root nodules

Nod factors are a group of biologically active oligosaccharidesignals that are secreted by symbiotically competent bacteriaof the family Rhizobiaceae. Their biosynthesis is determinedby rhizobial nodulation (nod) genes, and is specifically inducedin response to flavonoids secreted from the roots of host leguminousplants. The biological activity of Nod factors on these hostlegumes dramatically mimics the early developmental symptomsof the Rhizobium-legame symbiosis including, amongst other effects,root hair deformations and nodule initiation. Structurally,all Nod factors are short oligomers of ß-1,4-linkedN-acetylglucos-amine residues [usually degree of polymerization(dp) 4 or 5] that are N-acylated on the distal glucosarnine.This common ‘core’ structure may be modified bya number of species-specific substituents on the distal or reducingsugars. These modifications are governed by rhizobial host specificitynod genes. The biological activity of purified Nod factors mirrorsthis host specificity, indicating that the symbiotic host rangeof individual Rhizobium species is, at least partially, determinedby the variety of Nod factors they are able to produce. Herewe describe techniques that are universally applicable to theextraction, chromatographic separation and identification ofNod factors. We have applied these techniques to Nod factorsfrom the broad-host-range species Rhizobium fredii USDA257 andRhizobium spp. NGR234, and the more narrow-host-range Bradyrhizobiumjaponicum USDA110, and have identified a group of novel, relativelyhydrophilic Nod factors from the NGR234 species that may haveimplications for Nod factor biosynthesis. lipo-oligosaccharide Nod factor rhibozobia singals TLC     

Rhizobial Nod factors are required for cortical cell division in the nodule morphogenetic programme of the Aeschynomeneae legume Arachis

Nod factors are among the best-studied molecules implicated in the signal exchange that leads to legume-rhizobia symbiosis. The role of these molecules in symbiosis development has been primarily studied in legumes invaded through infection threads. In these plants, Nod factors generate several responses required for nodulation, including the induction of cortical cell division to form the nodule primordium. Arachis hypogaea L. (peanut) exhibits a specific mode of rhizobial infection and nodule morphogenetic programme in which infection threads are never formed. The role of Nod factors in this particular mechanism is unknown. In this work, a peanut symbiont mutant strain unable to produce Nod factors was obtained and characterised. The strain Bradyrhizobium (Arachis) sp. SEMIA 6144 V2 is altered in the nodC gene, which encodes an N-acetylglucosaminyl transferase involved in the first step of the Nod factor biosynthetic pathway. Further research revealed that, although its ability to colonise peanut roots was unaffected, it is not capable of inducing the division of cortical cells. The results obtained indicate that rhizobial Nod factors are essential for the induction of cortical cell division that leads to nodule primordium formation.     

Nod Bj-V (C18:1, MeFuc) production by Bradyrhizobium japonicum (USDA110, 532C) at suboptimal growth temperatures

Nod factors (Lipo-chitooligosaccharides, or LCOs) act as bacteria-to-plant signal molecules that modulate early events of the Bradyrhizobium-soybean symbiosis. It is known that low root zone temperature inhibits the early stages of this symbiosis; however, the effect of low soil temperature on bacteria-to-plant signaling is largely uninvestigated. We evaluated the effect of low growth temperatures on the production kinetics of Nod factor (LCO) by B. japonicum. Two strains of B. japonicum, 532C and USDA110, were tested for ability to synthesize Nod Bj-V (C(18:1), MeFuc) at three growth temperatures (15, 17 and 28 degrees C). The greatest amounts of the major Nod factor, Nod Bj-V (C(18:1), MeFuc), were produced at 28 degrees C for both strains. At 17 and 15 degrees C, the Nod factor production efficiency, per cell, of B. japonicum 532C and USDA110 was markedly decreased with the lowest Nod factor concentration per cell occurring at 15 degrees C. Strain 532C was more efficient at Nod factor production per cell than strain USDA 110 at all growth temperatures. The biological activity of the extracted Nod factor was unaffected by culture temperature. This study constitutes the first demonstration of reduced Nod factor production efficiency (per cell production) under reduced temperatures, suggesting another way that lower temperatures inhibit establishment of the soybean N(2) fixing symbiosis.     

Nitrogen control of bacterial signal production in Rhizobium meliloti-alfalfa symbiosis

Under nitrogen-depleted conditions nitrogen-fixing soil bacteria of the family Rhizobiaceae are able to induce symbiotic nodules on the roots of leguminous plants where bacteroids convert atmospheric nitrogen to ammonia. The presence of exogenous nitrogen source inhibits the development and the functioning of bacterium-plant symbiosis. Earlier experiments demonstrated that nitrate inhibited all stages of symbiotic interaction, affecting primarily the host functions. The investigation of the possible involvement of the microsymbiont in nitrogen regulation showed that two signalling steps were controlled by ammonium. The synthesis of the first bacterial signal, the Nod factor was repressed by ammonium. The nitrogen signal is conveyed to nodulation (nod) genes by the general nitrogen regulatory (ntr) system and by the nodD3-syrM self-amplifying system. The fine control also involves a negative regulatory factor, ntrR. When ntrR is mutated, more efficient nodule formation and nitrogen fixation is observed in symbiosis with alfalfa even in the presence of ammonium. The biosynthesis of the second bacterial signal succinoglycan is also controlled by ammonium. SyrM, a common regulatory factor for nod and exo gene expression, may contribute to the adjustment of the amount of succinoglycan and the ratio of its biologically active form.     

Contribution of NFP LysM domains to the recognition of Nod factors during the Medicago truncatula/Sinorhizobium meliloti symbiosis

The root nodule nitrogen fixing symbiosis between legume plants and soil bacteria called rhizobia is of great agronomical and ecological interest since it provides the plant with fixed atmospheric nitrogen. The establishment of this symbiosis is mediated by the recognition by the host plant of lipo-chitooligosaccharides called Nod Factors (NFs), produced by the rhizobia. This recognition is highly specific, as precise NF structures are required depending on the host plant. Here, we study the importance of different LysM domains of a LysM-Receptor Like Kinase (LysM-RLK) from Medicago truncatula called Nod factor perception (NFP) in the recognition of different substitutions of NFs produced by its symbiont Sinorhizobium meliloti. These substitutions are a sulphate group at the reducing end, which is essential for host specificity, and a specific acyl chain at the non-reducing end, that is critical for the infection process. The NFP extracellular domain (ECD) contains 3 LysM domains that are predicted to bind NFs. By swapping the whole ECD or individual LysM domains of NFP for those of its orthologous gene from pea, SYM10 (a legume plant that interacts with another strain of rhizobium producing NFs with different substitutions), we showed that NFP is not directly responsible for specific recognition of the sulphate substitution of S. meliloti NFs, but probably interacts with the acyl substitution. Moreover, we have demonstrated the importance of the NFP LysM2 domain for rhizobial infection and we have pinpointed the importance of a single leucine residue of LysM2 in that step of the symbiosis. Together, our data put into new perspective the recognition of NFs in the different steps of symbiosis in M. truncatula, emphasising the probable existence of a missing component for early NF recognition and reinforcing the important role of NFP for NF recognition during rhizobial infection.     

Analysis of Nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus

Nodulation (Nod)-factor signaling molecules are essential for rhizobia to initiate the nitrogen-fixing symbiotic interaction with legumes. Using a dual dye ratiometric calcium imaging technique, we have shown that 10 nM Nod factor added to roots of Lotus japonicus seedlings induces an intracellular calcium increase (calcium flux) that precedes oscillations in intracellular calcium (calcium spiking). The calcium flux was not observed with 1 or 0.1 nM Nod factor, which did induce calcium spiking. The calcium flux was variable in timing of initiation and duration and was observed in approximately half of the root hairs examined. Representatives from 11 complementation groups of symbiotically defective mutants were analyzed for the calcium flux. Mutants from four groups (sym6, ccamk, sym35, and nin) which retained calcium spiking all showed a normal calcium flux. Two classes of mutants (nfr1 and nfr5) lacked both calcium influx and calcium spiking, whereas five classes of mutants (symRK, castor, pollux, nup133, and sym24) defective for calcium spiking retained a calcium flux. There was no correlation between calcium spiking and induction of root hair deformation by Nod factor. We propose that increased bacterial numbers within infection foci in root hairs leads to accumulation of Nod factor to sufficient levels to activate the calcium flux, and this may drive infection thread growth.