Nod factor structures,responses, and perception during initiation of nodule development

The onset of nodule development, the result of rhizobia-legume symbioses, is determined by the exchange of chemical compounds between microsymbiont and leguminous host plant. Lipo-chitooligosaccharidic nodulation (Nod) factors, secreted by rhizobia, belong to these signal molecules. Nod factors consist of an acylated chitin oligomeric backbone with various substitutions at the (non)reducing-terminal and/or nonterminal residues. They induce the formation and deformation of root hairs, intra- and extracellular alkalinization, membrane potential depolarization, changes in ion fluxes, early nodulin gene expression, and formation of nodule primordia. Nod factors play a key role during nodule initiation and act at nano- to picomolar concentrations. A correct chemical structure is required for induction of a particular plant response, suggesting that Nod factor-receptor interaction(s) precede(s) a Nod factor-induced signal transduction cascade. Current data on Nod factor structures and Nod factor-induced responses are highlighted as well as recent advances in the characterization of proteins, possibly involved in recognition of Nod factors by the host plant.     

Root Hair Deformation Activity of Nodulation Factors and Their Fate on Vicia sativa

We used a semiquantitative root hair deformation assay for Vicia sativa (vetch) to study the activity of Rhizobium leguminosarum bv viciae nodulation (Nod) factors. Five to 10 min of Nod factor-root interaction appears to be sufficient to induce root hair deformation. The first deformation is visible within 1 h, and after 3 h about 80% of the root hairs in a small susceptible zone of the root are deformed. This zone encompasses root hairs that have almost reached their maximal size. The Nod factor accumulates preferentially to epidermal cells of the young part of the root, but is not restricted to the susceptible zone. In the interaction with roots, the glucosamine backbone of Nod factors is shortened, presumably by chitinases. NodRlv-IV(C18:4,Ac) is more stable than NodRlv-V(C18:4,Ac). No correlation was found between Nod factor degradation and susceptibility. Degradation occurs both in the susceptible zone and in the mature zone. Moreover, degradation is not affected by NH4NO3 and is similar in vetch and in the nonhost alfalfa (Medicago sativa).     

Rhizobium sp. strain NGR234 NodZ protein is a fucosyltransferase.

Rhizobium sp. strain NGR234 produces a large family of lipochitooligosaccharide Nod factors carrying specific substituents. Among them are 3-O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl substitutions. Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure. Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors. In vitro assays using GDP-L-fucose as the fucose donor show that fucosyltransferase activity is associated with the nodZ gene product (NodZ). NodZ is located in the soluble protein fraction of NGR234 cells. Together with extra copies of the nodD1 gene, the nodZ gene and its associated nod box were introduced into ANU265, which is NGR234 cured of the symbiotic plasmid. Crude extracts of this transconjugant possess fucosyltransferase activity. Fusion of a His6 tag to the NodZ protein expressed in Escherichia coli yielded a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin. NodZ is inactive on monomeric N-acetyl-D-glucosamine and on desulfated Rhizobium meliloti Nod factors. Kinetic analyses showed that the NodZ protein is more active on oligomers of chitin than on nonfucosylated NodNGR factors. Pentameric chitin is the preferred substrate. These data suggest that fucosylation occurs before acylation of the Nod factors.     

Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling

Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.     

The role of nod factor substituents in actin cytoskeleton rearrangements in Phaseolus vulgaris

In order to define the symbiotic role of some of the chemical substituents in the Rhizobium etli Nod factors (NFs), we purified Nod metabolites secreted by the SM25 strain, which carries most of the nodulation genes, and SM17 with an insertion in nodS. These NFs were analyzed for their capabilities to induce root hair curling and cytoskeletal rearrangements. The NFs secreted by strain SM17 lack the carbamoyl and methyl substituents on the nonreducing terminal residue and an acetyl moiety on the fucosyl residue on the reducing-terminal residue as determined by mass spectrometry. We have reported previously that the root hair cell actin cytoskeleton from bean responds with a rapid fragmentation of the actin bundles within 5 min of NF exposure, and also is accompanied by increases in the apical influxes and intracellular calcium levels. In this article, we report that methyl-bearing NFs are more active in inducing root hair curling and actin cytoskeleton rearrangements than nonmethylated NFs. However, the carbamoyl residue on the nonreducing terminal residue and the acetyl group at the fucosyl residue on the reducing terminal residue do not seem to have any effect on root hair curling induction or in actin cytoskeleton rearrangement.     

Calcium oscillations and Nod signal transduction in Rhizobium-legume symbiosis

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting a number of key developmental responses in the roots of legume hosts. One of the earliest responses of root hairs to Nod factors is the induction of sharp oscillations of cytoplasmic calcium ion concentration ("calcium spiking"). This response was first characterised in Medicago sativa and Nod factors were found to be unable to induce calcium spiking in a nodulation-defective mutant of M. sativa. The fact that this mutant lacked any morphological response to Nod factors raised the question of whether calcium spiking could be part of a Nod factor-induced signal transduction pathway leading to nodulation. More recently, calcium spiking has been described in a model legume, Medicago truncatula, and in pea. When nodulation-defective mutants were tested for the induction of calcium spiking in response to Nod factors, three loci of pea and two of M. truncatula were found to be necessary for Nod factor-induced calcium spiking. These loci are also known to be necessary for Nod factor-induction of symbiotic responses such as root hair deformation, nodulin gene expression and cortical cell division. These results therefore constitute strong genetic evidence for the role of calcium spiking in Nod factor transduction. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.     

Activity of Sinorhizobium meliloti NodAB and NodH enzymes on thiochitooligosaccharides

Rhizobium bacteria synthesize signal molecules called Nod factors that elicit responses in the legume root during nodulation. Nod factors, modified N-acylated beta-(1,4)-N-acetylglucosamine, are synthesized by the nodulation (nod) gene products. We tested the ability of three Sinorhizobium meliloti nod gene products to modify Nod factor analogs with thio linkages instead of O-glycosidic bonds in the oligosaccharide backbone.     

Gibberellins are involved in nodulation of Sesbania rostrata

Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.     

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.     

Acetylation of a fucosyl residue at the reducing end of Mesorhizobium loti nod factors is not essential for nodulation of Lotus japonicus

NodMl-V(C(18:1), Me, Cb, AcFuc) is a major component of lipo-chitin oligosaccharides (LCOs), or Nod factors, produced by Mesorhizobium loti. The presence of a 4-O-acetylated fucosyl residue (AcFuc) at the reducing end has been thought to be essential for symbiotic interactions with the compatible host plant, Lotus japonicus. We generated an M. loti mutant in which the nolL gene is disrupted; nolL has been shown to encode acetyltransferase that is responsible for acetylation of the fucosyl residue. The nolL disruptant Ml107 produced LCOs that lacked acetylation of fucosyl residues as expected, but exhibited nodulation performance on L. japonicus as efficiently as the wild-type M. loti strain MAFF303099. We show that LCOs without acetylation of a fucosyl residue purified from Ml107 are also able to induce abundant root hair deformation and nodule primordium formation. These results indicate that NolL-dependent acetylation of a fucosyl residue at the reducing end of M. loti LCOs is not essential for nodulation of L. japonicus.     

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.     

Microbial community analysis of field-grown soybeans with different nodulation phenotypes

Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.     

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.     

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.     

Charcoal and shrubs modify soil processes in ponderosa pine forests of western Montana

In split-root systems of alfalfa (Medicago sativa L.), already existing nodules or arbuscular mycorrhizal roots suppress further establishment of symbiosis in other root parts, a phenomenon named autoregulation. Roots treated with rhizobial nodulation signals (Nod factors) induce a similar systemic suppression of symbiosis.In order to test the hypothesis that flavonoids play a role in this systemic suppression, split-root systems of alfalfa plants were inoculated on one side of the split-root system with Sinorhizobium meliloti or Glomus mosseae or were treated with Nod factor. HPLC-analysis of alfalfa root extracts from both sides of the split-root system revealed a persistent local and systemic accumulation pattern of some flavonoids associated with the different treatments. The two flavonoids, formononetin and ononin, could be identified to be similarily altered after rhizobial or mycorrhizal inoculation or when treated with Nod factor.Exogenous application of formononetin and ononin partially restored nodulation and mycorrhization pointing towards the involvement of these two secondary compounds in the autoregulation of both symbioses.     

Autoregulation of root nodule formation: signals of both symbiotic partners studied in a split-root system of Vicia sativa subsp. nigra

Inhibition of root nodule formation on leguminous plants by already induced or existing root nodules is called autoregulation of root nodule formation (AUT). Optimal conditions for AUT were determined using a split-root technique newly developed for Vicia sativa subsp. nigra. Infection of a root A with nodulating Rhizobium leguminosarum bv. viciae bacteria systemically inhibited nodulation of a spatially separated root B inoculated 2 days later with the same bacteria. This treatment gives complete AUT (total absence of nodules on root B). Only partial AUT of root B was obtained by incubation of root A with mitogenic nodulation (Nod) factors or with a noninfective strain producing normal mitogenic Nod factors. Nonmitogenic Nod factors did not evoke AUT. We identified two systemic plant signals induced by Rhizobium bacteria. Signal 1 (at weak buffering) was correlated with sink formation in root A and induced acidification of B-root medium. This signal is induced by treatment of root A with (i) nodulating rhizobia, (ii) mitogenic Nod factors, (iii) nonmitogenic Nod factors, or (iv) the cytokinin zeatin. Signal 2 (at strong buffering) could only be evoked by treatment with nodulating rhizobia or with mitogenic Nod factors. Most probably, this signal represents the specific AUT signal. Induction of complete AUT appears to require actively dividing nodule cells in nodule primordia, nodule meristems, or both of root A.     

Abscisic acid coordinates nod factor and cytokinin signaling during the regulation of nodulation in Medicago truncatula

Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here, we characterize the nature of abscisic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection, and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signaling and affecting the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signaling through the use of a dominant-negative allele of ABSCISIC ACID INSENSITIVE1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, SENSITIVITY TO ABA, that dictates the sensitivity of the plant to ABA and, as such, impacts the regulation of nodulation. We show that ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin induction of the nodule primordium in the root cortex. Therefore, ABA is capable of coordinately regulating the diverse developmental pathways associated with nodule formation and can intimately dictate the nature of the plants' response to the symbiotic bacteria.     

Four genes of Medicago truncatula controlling components of a nod factor transduction pathway

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting several key developmental responses in the roots of legume hosts. Using nodulation-defective mutants of Medicago truncatula, we have started to dissect the genetic control of Nod factor transduction. Mutants in four genes (DMI1, DMI2, DMI3, and NSP) were pleiotropically affected in Nod factor responses, indicating that these genes are required for a Nod factor-activated signal transduction pathway that leads to symbiotic responses such as root hair deformations, expressions of nodulin genes, and cortical cell divisions. Mutant analysis also provides evidence that Nod factors have a dual effect on the growth of root hair: inhibition of endogenous (plant) tip growth, and elicitation of a novel tip growth dependent on (bacterial) Nod factors. dmi1, dmi2, and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi, indicating that there are at least three common steps to nodulation and endomycorrhization in M. truncatula and providing further evidence for a common signaling pathway between nodulation and mycorrhization.     

Lipochitooligosaccharides and legume Rhizobium symbiosis--a new concept

Legume-Rhizobium symbiosis is a multistep process characterized by the formation of root nodules on the host plant. A number of genes from both symbiotic partners share information during the interaction process. Nodulation genes (nod, nol and noe) have been classified as common nodulation genes and host specific (hsn) nodulation genes. Though common nodulation genes are enough to form root nodules, host specific nodulation genes are needed for specific interaction leading to formation of functional nodules. Core lipochitooligosaccharides (LCOs), the products of common nodulation genes are modified by the action of host specific nodulation genes. LCOs seem to be present in legumes as well as nonlegume and are known to act as a morphogen by acting as auxin-transport inhibitor. The understanding of Nod factor may contribute to reveal complex biological functions such as developmental regulation, signal transduction and plant morphogenesis.