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.     

The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids

Rhizobia synthesize mono- N -acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation ( nod ) genes, including the nodABC genes present in all rhizobial species. The N -acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti , an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (ω-1) hydroxylated fatty acid, whereas in Rhizobium tropici , a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non-polar deletion of nodA , and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N -acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.     

Plant responses to nodulation factors

The focus of research on signalling in Rhizobium-legume interactions has moved from understanding the structure and synthesis of rhizobially made Nod factors, towards an analysis of how they function in plants. Nod-factor-induced changes in ion fluxes across membranes, followed by establishment of an oscillation of intracellular Ca(2+) concentration, point to the involvement of a receptor-mediated signal transduction pathway. Progress towards the identification of components in this pathway is being made by identifying Nod-factor binding proteins, isolating plant mutants that are defective in signalling and analysing plant responses to Nod factors.     

Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti

Medicago truncatula is a model legume plant that interacts symbiotically with Sinorhizobium meliloti, the alfalfa symbiont. This process involves a molecular dialogue between the bacterium and the plant. Legume roots exude flavonoids that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range. In turn, nod genes control the synthesis of lipo-chito-oligosaccharides (LCOs), Nod factors, which are bacteria-to-plant signal molecules mediating recognition and nodule organogenesis. M. truncatula roots or seeds have been treated with Nod factors and hydroponically growing seedlings have been inoculated with a limiting population of S. meliloti. It has been shown that submicromolar concentrations of Nod factors increase the number of nodules per plant on M. truncatula. Compared with roots, this increase is more noticeable when seeds are treated. M. truncatula seeds are receptive to submicromolar concentrations of Nod factors, suggesting the possibility of a high affinity LCO perception system in seeds or embryos as well.     

Genetic and genomic analysis in model legumes bring Nod-factor signaling to center stage

The control of host-specificity in the Rhizobium-legume symbiosis has been a topic of long-standing interest to plant biologists. By the early 1990s, biologists had deciphered the chemical signals that trigger early symbiotic responses. Flavonoids from the plant root trigger bacterial gene expression and the production of lipo-chitooligosaccharide signals (called Nod factors) that are recognized by the plant host. Genetic differences between bacterial strains modify the oligosaccharide backbone, for example by the addition of sulfate, acetate or fucose, and simultaneously alter the host-specificity of the purified Nod factor and the bacterium. Recent studies have begun to reveal the genetic and molecular basis of Nod-factor perception in legumes, a signaling system that also controls plant interactions with mycorrhizal fungi.     

Legume agglutinins that bind to Rhizobium meliloti.

A protein found in seeds and roots of alfalfa (Medicago sativa) was implicated in the specificity of the infection process, based on its binding to the symbiont Rhizobium meliloti. We found an agglutinin with similar properties in seeds and roots of sweet clover (Melilotis alba). The sweet clover differed from alfalfa in nodulation by a mutant strain of R. meliloti, but the agglutinins were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Rhizobium agglutination, and cross-reactivity to antibodies. Similar agglutinins binding R. meliloti were found in seeds of legumes from different cross-inoculation groups, including soybean (Glycine max), cowpea (Vigna unguiculata), pea (Pisum sativum L), and mung bean (Vigna mungo). The agglutinins from these legumes were recognized by antibodies raised against the agglutinins of alfalfa and sweet clover. Seeds of corn (Zea mays) and tomato (Lycopersicon esculentum) contained a protein similar to the legume agglutinin, but it did not react with the antibodies. We conclude that the alfalfa agglutinin is representative of a common legume protein and that there is no evidence for its role in specificity or nodule initiation.     

Inactivation of the nodH gene in Sinorhizobium sp. BR816 enhances symbiosis with Phaseolus vulgaris L

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.     

Inhibition of the catalase activity from Phaseolus vulgaris and Medicago sativa by sodium chloride.

Changes in catalase activity during the development of the Rhizobium-legume symbiosis as well as its response in salinized plants of Phaseolus vulgaris and Medicago sativa, was studied. Besides, it was examined the behavior of the enzyme, isolated from leaves and root nodules, during in vitro incubation with NaCl doses. Nodule catalase activities of both legumes were assayed with several enzyme inhibitors and also purified. Leaf catalase activity of Phaseolus vulgaris and Medicago sativa decreased and increased respectively throughout the ontogeny, but root nodule catalase kept a high and stable value. This last result suggests that both legumes require the maintenance of high nodule catalase in nitrogen-fixing nodules. Under salt stress conditions leaf and nodule catalase activity decreased in both, grain and pasture legumes. Because catalase from leaf of Medicago sativa and nodules of Phaseolus vulgaris were relatively sensitive to NaCl during in vitro experiments, the detoxifying role of this enzyme for H(2)O(2) should be limited in such conditions. Both catalases, from determinate and indeterminate nodules, were affected neither by oxygen nor superoxide radicals but showed a strong (Phaseolus vulgaris) or partial (Medicago sativa) inhibition with dithiothreitol, dithionite and beta-mercaptoethanol. Besides, cyanide was the most potent inhibitor of nodule catalases. Finally, catalases partially purified by immobilized metal ion affinity chromatography migrated at 42 (Phaseolus vulgaris) and 46kDa (Medicago sativa) on SDS-PAGE, whereas native forms on sephacryl S-300 columns exhibited a molecular mass of 59 and 48kDa (Phaseolus vulgaris) and 88 and 53kDa (Medicago sativa).     

Rhizobial lipochitooligosaccharide nodulation factors activate expression of the legume early nodulin gene ENOD12 in rice

Lipochitooligosaccharide nodulation factors (Nod factors) produced by rhizobia are a major host range determinant. These factors play a pivotal role in the molecular signal exchange, infection and induction of symbiotic developmental responses in legumes leading to the formation of a nodule in which rhizobia carry out N₂ fixation. Determining whether rice ( Oryza sativa ) can respond to Nod factors could lead to strategies that would make rice amenable to develop a nitrogen-fixing endosymbiotic association with rhizobia. We introduced into rice the promoter of the infection-related gene MtENOD12 (from Medicago truncatula ) fused to the β-glucuronidase (GUS) reporter gene to serve as a molecular marker to aid in the detection of Nod factor signal perception by rice cells. Treatment of the transgenic rice roots with Nod factors (10^–6–10^–9 m ) under nitrogen-limiting conditions induced MtENOD12 -GUS expression in cortical parenchyma, endodermis and pericycle. In contrast, chitooligosaccharide backbone alone failed to elicit such a response in the root tissues. These findings demonstrate that rice roots perceive Nod factors and that these lipochitooligosaccharides, but not simple chitin oligomers, act as signal molecules in activating MtENOD12 in cortical parenchyma as in legumes. Exogenous application of N -naphthaleneacetic acid mimicked the Nod factor-elicited tissue-specific expression of MtENOD12 in roots while cytokinins inhibited it, thus evidencing that Nod factors, auxin and cytokinins probably act on similar signaling elements responsible for the regulation of MtENOD12 activation in rice. Taken together, these results suggest that at least a portion of the signal transduction machinery important for legume nodulation is likely to exist in rice.      

Phaseolus vulgaris recognizes Azorhizobium caulinodans Nod factors with a variety of chemical substituents.

Phaseolus vulgaris is a promiscuous host plant that can be nodulated by many different rhizobia representing a wide spectrum of Nod factors. In this study, we introduced the Rhizobium tropici CFN299 Nod factor sulfation genes nodHPQ into Azorhizobium caulinodans. The A. caulinodans transconjugants produce Nod factors that are mostly if not all sulfated and often with an arabinosyl residue as the reducing end glycosylation. Using A. caulinodans mutant strains, affected in reducing end decorations, and their respective transconjugants in a bean nodulation assay, we demonstrated that bean nodule induction efficiency, in decreasing order, is modulated by the Nod factor reducing end decorations fucose, arabinose or sulfate, and hydrogen.     

Characterization of a binding site for chemically synthesized lipo-oligosaccharidic NodRm factors in particulate fractions prepared from roots

This paper describes the characteristics of a binding site for the major, lipo-oligosaccharide Nod factor of Rhizobium meliloti in roots of the symbiotic host plant, Medicago truncatula. Chemically synthesized NodRm-IV(Ac, S, C16:2) was labelled by tritiation to a specific activity of 56 Ci mmol^?1 and this ligand was shown to be biologically active in the root hair deformation assay at 10^?11 M. Binding of the ligand to a particulate fraction from roots of M. truncatula was found to be saturable and reversible with an affinity (K_d) of 86 nM and the binding characteristics were consistent with a single class of binding sites. Competition with modified Nod factors showed that the binding was independent of both the O-acetyl and the sulphyl group and did not depend on the unsaturation of the fatty acid. However, both moieties of the lipo-oligosaccharide are required for high-affinity binding since tetra-N-acetyl-chitotetraose and palmitate were found to be poor competitors of ligand binding. A binding site with analogous characteristics was also found in a similarly prepared particulate fraction of tomato roots. This binding site for Nod factors, termed NFBS1, which is present in both a leguminous and a non-leguminous plant, may have a more general role than symbiosis.     

In Rhizobium meliloti, the operon associated with the nod box n5 comprises nodL, noeA and noeB, three host-range genes specifically required for the nodulation of particular Medicago species

In Rhizobium meliloti , the genes required for nodulation of legume hosts are under the control of DNA regulatory sequences called nod boxes. In this paper, we have characterized three host-specific nodulation genes, which form a flavonoid-inducible operon down-stream of the nod box n5. The first gene of this operon is identical to the nodL gene identified by Baev and Kondorosi (1992) in R. meliloti strain AK631. The product of the second gene, NoeA, presents some homology with a methyl transferase. nodL mutants synthesize Nod factors lacking the O -acetate substituent. In contrast, in strains carrying a mutation in either noeA or noeB , no modification in Nod-factor structure or production could be detected. On particular hosts, such as Medicago littoralis , mutants of the n5 operon showed a very weak nodule-forming ability, associated with a drastic decrease in the number of infection threads, while nodulation of Medicago truncatula or Melilotus alba was not affected. Thus, nodL , noeA and noeB are host-specific nodulation genes. By using a gain-of-function approach, we showed that the presence of nodL , and hence of O -acetylated Nod factors, is a major prerequisite for confering the ability to nodulate alfalfa upon the heterologous bacterium Rhizobium tropici .     

Nod factor elicits two separable calcium responses in Medicago truncatula root hair cells

Modulation of intracellular calcium levels plays a key role in the transduction of many biological signals. Here, we characterize early calcium responses of wild-type and mutant Medicago truncatula plants to nodulation factors produced by the bacterial symbiont Sinorhizobium meliloti using a dual-dye ratiometric imaging technique. When presented with 1 nM Nod factor, root hair cells exhibited only the previously described calcium spiking response initiating 10 min after application. Nod factor (10 nM) elicited an immediate increase in calcium levels that was temporally earlier and spatially distinct from calcium spikes occurring later in the same cell. Nod factor analogs that were structurally related, applied at 10 nM, failed to initiate this calcium flux response. Cells induced to spike with low Nod factor concentrations show a calcium flux response when Nod factor is raised from 1 to 10 nM. Plant mutants previously shown to be deficient for the calcium spiking response (dmi1 and dmi2) exhibited an immediate, truncated calcium flux with 10 nM Nod factor, demonstrating a competence to respond to Nod factor but an impaired ability to generate a full biphasic response. These results demonstrate that the legume root hair cell exhibits two independent calcium responses to Nod factor triggered at different agonist concentrations and suggests an early branch point in the Nod factor signal transduction pathway.     

Characterization of Rhizobia from Ineffective Alfalfa Nodules: Ability to Nodulate Bean Plants [Phaseolus vulgaris (L.) Savi.

This study was initiated to characterize Rhizobium isolates obtained from root nodules of ineffectively nodulated, field-grown alfalfa (Medicago sativa L.) plants. The purpose was to determine if these isolates possessed characteristics which would explain either their ineffectiveness in N(2) fixation or their apparent ability to tolerate the moderately acid soil conditions from which they originated. Isolates were characterized by analysis of growth rate, 39 degrees C tolerance, acid production on conventional media, and symbiotic performance. All isolates were ineffective in N(2) fixation on alfalfa, and they contained one or more anomalous characteristics. These included either slow growth rate, lack of 39 degrees C tolerance, or lack of acid production on conventional media. Infectiveness tests on a broad range of legumes revealed that the isolates formed root nodules on M. sativa, Medicago lupulina L., and Phaseolus vulgaris (L.) Savi. (common bean). These results provide evidence that, in some situations, ineffective nodulation of M. sativa in the field may be due to the presence of promiscuous, native Rhizobium species.     

Identical accumulation and immobilization of sulfated and nonsulfated Nod factors in host and nonhost root hair cell walls

Nod factors are signaling molecules secreted by Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host. How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs. The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM. Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM. When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium. Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M. truncatula and V. sativa, respectively. Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M. truncatula and V. sativa. The implications for the mode of Nod factor perception are discussed.     

Capturing cold-stress-related sequence diversity from a wild relative of common bean (Phaseolus angustissimus)

One restriction to the cultivation of common bean, Phaseolus vulgaris L., is its limited tolerance to low temperatures. In the present study, subtraction suppression hybridization was employed to enrich for stress responsive genes in both a chilling-susceptible common bean and a relatively more chilling-tolerant wild bean species, Phaseolus angustissimus. For each species, approximately 11 000 expressed sequence tags were generated. Comparative sequence analysis of the EST collection with the available annotated genome sequences of the model Fabaceae species Medicago truncatula and Glycine max identified protein homologues for approximately 65% and 80% of the Phaseolus sequences, respectively. This difference reflects the closer phylogenetic relationship between the genera Phaseolus and Glycine compared with Medicago. Annotation of the Phaseolus sequences was facilitated through this comparative analysis and indicated that several heat shock proteins, cytochrome P450s, and DNA binding factors were uniquely found among the sequences from the wild species P. angustissimus. The Phaseolus sequences have been made available on a GBrowse implementation using M. truncatula as the reference genome, providing rapid access to the sequence data and associated comparative genome data.     

Lipo-chitooligosaccharide Nodulation Signals from Rhizobium meliloti Induce Their Rapid Degradation by the Host Plant Alfalfa

Extracellular enzymes from alfalfa (Medicago sativa L.) involved in the degradation of nodulation (Nod) factors could be distinguished by their different cleavage specificities and were separated by lectin affinity chromatography. A particular glycoprotein was able to release an acylated lipo-disaccharide from all tested Nod factors having an oligosaccharide chain length of four or five residues. Structural modifications of the basic lipo-chitooligosaccharide did not affect the cleavage site and had only weak influence on the cleavage efficiency of Nod factors tested. The acylated lipo-trisaccharide was resistant to degradation. When alfalfa roots were preincubated with Nod factors at nanomolar concentrations, the activity of the dimer-forming enzyme was stimulated up to 6-fold within a few hours. The inducing activity of Nod factors decreased in the order NodRm-IV(C16:2,Ac,S) > NodRm-IV(C16:2,S) and NodRm-V(C16:2,Ac,S) > NodRm-V(C16:2,S) > NodRm-IV(C16:0,S) > NodRm-IV(C16:2). Pretreatment with NodRm-III(C16:2) as well as unmodified chitooligosaccharides did not stimulate the dimer-forming enzyme. Roots preincubated with Rhizobium meliloti showed similar stimulation of the dimer-forming activity. Mutant strains unable to produce Nod factors did not enhance the hydrolytic activity. These results indicate a rapid feedback inactivation of Nod signals after their perception by the host plant alfalfa.     

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.     

LysM domains of Medicago truncatula NFP protein involved in Nod factor perception. Glycosylation state, molecular modeling and docking of chitooligosaccharides and Nod factors

The establishment of the symbiosis between legume plants and rhizobial bacteria depends on the production of rhizobial lipo-chitooligosaccharidic signals (the Nod factors) that are specifically recognized by roots of the host plant. In Medicago truncatula, specific recognition of Sinorhizobium meliloti and its Nod factors requires the NFP (Nod factor perception) gene, which encodes a putative serine/threonine receptor-like kinase (RLK). The extracellular region of this protein contains three tandem lysin motifs (LysMs), a short peptide domain that is implicated in peptidoglycan or chitin binding in various bacterial or eukaryotic proteins, respectively. We report here the homology modeling of the three LysM domains of M. truncatula NFP based on the structure of a LysM domain of the Escherichia coli membrane-bound lytic murein transglycosidase D (MltD). Expression of NFP in a homologous system (M. truncatula roots) revealed that the protein is highly N-glycosylated, probably with both high-mannose and complex glycans. Surface analysis and docking calculations performed on the models of the three domains were used to predict the most favored binding modes for chitooligosaccharides and Nod factors. A convergent model can be proposed where the sulfated, O-acetylated lipo-chitooligosaccharidic Nod factor of S. meliloti binds in similar orientation to the three LysM domains of M. truncatula NFP. N-glycosylation is not expected to interfere with Nod factor binding in this orientation.