Induce Root Hair Deformation Research Articles

Biological activity of Nod factors

Chemically, the Nod factors (NFs) are lipochitooligosaccharides, produced mainly by bacteria of the Rhizobium genus. They are the main signaling molecules involved in the initiation of symbiosis between rhizobia and legume plants. Nod factors affect plant tissues at very low concentrations, even as low as 10-12 mol/L. They induce root hair deformation, cortical cell division, and root nodules' formation in the host plant. At the molecular level, the cytoskeleton is reorganized and expression of genes encoding proteins called nodulins is induced in response to Nod factors in the cell. Action of Nod factors is highly specific because it depends on the structure of a particular Nod factor involved, as well as the plant receptor reacting with it.

The plant-growth-promoting actinobacteria of the genus Nocardia induces root nodule formation in Casuarina glauca.

Actinorhizal plants form a symbiotic association with the nitrogen-fixing actinobacteria Frankia. These plants have important economic and ecological benefits including land reclamation, soil stabilization, and reforestation. Recently, many non-Frankia actinobacteria have been isolated from actinorhizal root nodules suggesting that they might contribute to nodulation. Two Nocardia strains, BMG51109 and BMG111209, were isolated from Casuarina glauca nodules, and they induced root nodule-like structures in original host plant promoting seedling growth. The formed root nodule-like structures lacked a nodular root at the apex, were not capable of reducing nitrogen and had their cortical cells occupied with rod-shaped Nocardiae cells. Both Nocardia strains induced root hair deformation on the host plant. BMG111209 strain induced the expression of the ProCgNin:Gus gene, a plant gene involved in the early steps of the infection process and nodulation development. Nocardia strain BMG51109 produced three types of auxins (Indole-3-acetic acid [IAA], Indole-3-Byturic Acid [IBA] and Phenyl Acetic Acid [PAA]), while Nocardia BMG111209 only produced IAA. Analysis of the Nocardia genomes identified several important predicted biosynthetic gene clusters for plant phytohormones, secondary metabolites, and novel natural products. Co-infection studies showed that Nocardia strain BMG51109 plays a role as a "helper bacteria" promoting an earlier onset of nodulation. This study raises many questions on the ecological significance and functionality of Nocardia bacteria in actinorhizal symbioses.

Legume nodulation

Legume nodulation

Paenibacillus polymyxa Rhizobacteria and Their Synthesized Exoglycans in Interaction with Wheat Roots: Colonization and Root Hair Deformation

We examined the ability of several Paenibacillus polymyxa strains to colonize wheat roots and the ability of P. polymyxa exoglycans to induce root hair deformation. For the first time, exopolysaccharides isolated from P. polymyxa were found to produce, with different intensities, various morphological changes in the root hairs of wheat seedlings, which are some of the earliest responses of plants to bacteria in the surrounding milieu. P. polymyxa 1465, giving the highest exopolysaccharide yield and the highest viscosity of aqueous exopolysaccharide solutions, was best able to colonize wheat seedling roots, and its exopolysaccharide proved to be the best in producing root hair deformation. It is suggested that P. polymyxa exoglycans have an active role in the establishment of plant-microbe associations.

Rhizobial and mycorrhizal symbioses in Lotus japonicus require lectin nucleotide phosphohydrolase, which acts upstream of calcium signaling.

Nodulation in legumes requires the recognition of rhizobially made Nod factors. Genetic studies have revealed that the perception of Nod factors involves LysM domain receptor-like kinases, while biochemical approaches have identified LECTIN NUCLEOTIDE PHOSPHOHYDROLASE (LNP) as a Nod factor-binding protein. Here, we show that antisense inhibition of LNP blocks nodulation in Lotus japonicus. This absence of nodulation was due to a defect in Nod factor signaling based on the observations that the early nodulation gene NODULE INCEPTION was not induced and that both Nod factor-induced perinuclear calcium spiking and calcium influx at the root hair tip were blocked. However, Nod factor did induce root hair deformation in the LNP antisense lines. LNP is also required for infection by the mycorrhizal fungus Glomus intraradices, suggesting that LNP plays a role in the common signaling pathway shared by the rhizobial and mycorrhizal symbioses. Taken together, these observations indicate that LNP acts at a novel position in the early stages of symbiosis signaling. We propose that LNP functions at the earliest stage of the common nodulation and mycorrhization symbiosis signaling pathway downstream of the Nod factor receptors; it may act either by influencing signaling via changes in external nucleotides or in conjunction with the LysM receptor-like kinases for recognition of Nod factor.

Sinorhizobium meliloti 1021 Loss-of-Function Deletion Mutation in chvI and Its Phenotypic Characteristics

Bacterial two-component regulatory systems (TCS) are common components of complex regulatory networks and cascades. In Sinorhizobium meliloti, the TCS ExoS/ChvI controls exopolysaccharide succinoglycan production and flagellum biosynthesis. Although this system plays a crucial role in establishing the symbiosis between S. meliloti and its host plant, it is not well characterized. Attempts to generate complete loss-of-function mutations in either exoS or chvI in S. meliloti have been unsuccessful; thus, it was previously suggested that exoS or chvI are essential genes for bacterial cell growth. We constructed a chvI mutant by completely deleting the open reading frame encoding this gene. The mutant strain failed to grow on complex medium, exhibited lower tolerance to acidic condition, produced significantly less poly-3-hydroxybutyrate than the wild type, was hypermotile, and exhibited an altered lipopolysaccharide profile. In addition, this mutant was defective in symbiosis with Medicago truncatula and M. sativa (alfalfa), although it induced root hair deformation as efficiently as the wild type. Together, our results demonstrate that ChvI is intimately involved in regulatory networks involving the cell envelope and metabolism; however, its precise role within the regulatory network remains to be determined.

NPR1 Protein Regulates Pathogenic and Symbiotic Interactions between Rhizobium and Legumes and Non-Legumes

BackgroundLegumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs) produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non-legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA). SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1.Methodology/Principal FindingsWe used Arabidopsis mutants in SA defense pathway to test the role of NPR1 in symbiotic interactions. Inoculation of Sinorhizobium meliloti or purified NF on Medicago truncatula or nim1/npr1 A. thaliana mutants induced root hair deformation and transcription of early and late nodulins. Application of S. meliloti or NF on M. truncatula or A. thaliana roots also induced a strong oxidative burst that lasted much longer than in plants inoculated with pathogenic or mutualistic bacteria. Transient overexpression of NPR1 in M. truncatula suppressed root hair curling, while inhibition of NPR1 expression by RNAi accelerated curling.Conclusions/SignificanceWe show that, while NPR1 has a positive effect on pathogen resistance, it has a negative effect on symbiotic interactions, by inhibiting root hair deformation and nodulin expression. Our results also show that basic plant responses to Rhizobium inoculation are conserved in legumes and non-legumes.

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–RhizobiumSymbiotic Association

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.

Pharmacological evidence for activation of phospholipid and small GTP binding protein signalling cascades by Nod factors

The effects of lipo-chitin oligosaccharide Nod factors (NodNGR[S] from Rhizobium sp. NGR234) on root hair deformation in Vigna unguiculata (L.) Walp. were studied using pharmacological agents to mimic and/or inhibit their action. It was hypothesised that the rearrangement of the cytoskeleton seen during Nod factor induced root hair deformation is modulated by protein kinase C, monomeric G proteins of the Rho superfamily and the location and amount of phosphatidylinositol 3-phosphates (PI3Ps). This hypothesis is supported by the following observations. The protein kinase C activators, 12-deoxyphorbol 13-acetate (DPA) and diacylglycerol kinase inhibitor 1, stimulated root hair deformation to a level similar to that seen with Nod factors or mastoparan, whereas the inhibitor Gö 6976 inhibited root hair deformations induced by NodNGR[S], mastoparan, DPA and diacylglycerol kinase inhibitor 1. The Ras antagonists mevastatin and sulindac sulphide, and the Rho antagonist exoenzyme C3 toxin from Clostridium botulinum all inhibited Nod factor stimulated root hair deformation. Pasteurella multocida toxin activates Rho and stimulated root hair deformation, this stimulation was inhibited by both neomycin and exoenzyme C3 toxin. The PI3 kinase inhibitors, wortmannin and LY-294002 attenuated Nod factor induced root hair deformation. These studies were complemented with actin immunoprecipitations of root hair enriched microsomal membrane preparations from V. unguiculata which pulled down small GTP binding proteins. Root hair deformation is an important early stage in the formation of nitrogen fixing nodules and this study highlights that these processes may depend on signalling cascades involving phospholipids and small GTP binding proteins.

Nod factor-induced root hair curling: continuous polar growth towards the point of nod factor application.

A critical step in establishing a successful nitrogen-fixing symbiosis between rhizobia and legume plants is the entrapment of the bacteria between root hair cell walls, usually in characteristic 180 degrees to 360 degrees curls, shepherd's crooks, which are formed by the host's root hairs. Purified bacterial signal molecules, the nodulation factors (NFs), which are lipochitooligosaccharides, induce root hair deformation in the appropriate host legume and have been proposed to be a key player in eliciting root hair curling. However, for curling to occur, the presence of intact bacteria is thought to be essential. Here, we show that, when spot applied to one side of the growing Medicago truncatula root hair tip, purified NF alone is sufficient to induce reorientation of the root hair growth direction, or a full curl. Using wild-type M. truncatula containing the pMtENOD11::GUS construct, we demonstrate that MtENOD11::GUS is expressed after spot application. The data have been incorporated into a cell biological model, which explains the formation of shepherd's crook curls around NF-secreting rhizobia by continuous tip growth reorientation.

A cell-specific, prenylation-independent mechanism regulates targeting of type II RACs.

The RHO proteins, which regulate numerous signaling cascades, undergo prenylation, facilitating their interaction with membranes and with proteins called RHO.GDP dissociation inhibitors. It has been suggested that prenylation is required for RHO function. Eleven RHO-related proteins were identified in Arabidopsis. Eight of them are putatively prenylated. We show that targeting of the remaining three proteins, AtRAC7, AtRAC8, and AtRAC10, is prenylation independent, requires palmitoylation, and occurs by a cell-specific mechanism. AtRAC8 and AtRAC10 could not be prenylated by either farnesyltransferase or geranylgeranyltransferase I, whereas AtRAC7 could be prenylated by both enzymes in yeast. The association of AtRAC7 with the plasma membrane in plants did not require farnesyltransferase or a functional CaaX box. Recombinant AtRAC8 was palmitoylated in vitro, and inhibition of protein palmitoylation relieved the association of all three proteins with the plasma membrane. Interestingly, AtRAC8 and a constitutively active mutant, Atrac7mV(15), were not associated with the plasma membrane in root hair cells, whose elongation requires the localization of prenylated RHOs in the plasma membrane at the cell tip. Moreover, Atrac7mV(15) did not induce root hair deformation, unlike its prenylated homologs. Thus, AtRAC7, AtRAC8, and AtRAC10 may represent a group of proteins that have evolved to fulfill unique functions.

Nod factors activate both heterotrimeric and monomeric G-proteins in Vigna unguiculata (L.) Walp.

Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]- (from Rhizobiumsp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata(L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [(35)S]GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N- N'- N"- N'"-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPgammaS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(common) antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.

Early symbiotic responses induced by Sinorhizobium meliloti iIvC mutants in alfalfa.

A mutation in the ilvC gene of Sinorhizobium meliloti 1021 determines a symbiotically defective phenotype. ilvC mutants obtained from different S. meliloti wild-type strains are able to induce root hair deformation on alfalfa roots and show variable activation of the common nodulation genes nodABC. All of these mutants are noninfective. The presence of extra copies of nodD3-syrM in an IlvC- background does not promote nod expression but allows the detection of low levels of Nod factor production. The sulphation of the Nod factor metabolites, however, is not affected. Furthermore, IlvC- strains induce a specific pattern of starch accumulation on alfalfa roots as well as of early nodulin expression. Hence, the pleiotropic action of the ilvC gene in S. meliloti may reveal novel complexities involved in the symbiotic interaction.

In vivo fluorescence correlation microscopy (FCM) reveals accumulation and immobilization of Nod factors in root hair cell walls.

Fluorescence correlation microscopy (FCM) is a new single-molecule detection technique based on the confocal principle to quantify molecular diffusion and concentration of fluorescent molecules (particles) with sub-micron resolution. In this study, FCM is applied to examine the diffusional behaviour of fluorescent Nod factor analogues on living Vicia sativa root hairs. Three recently described Nod factors with a fluorescent acyl chain (Goedhart et al. Biochemistry 1999, 38, 10898-10907) were used. Plasmolysis of fluorescently labelled root hairs showed that the Nod factors are predominantly located in the cell wall, as hardly any fluorescence could be detected in the plasma membrane. After Nod factor-induced root hair deformation, the new outgrowth was not labelled, indicating a lack of migration of Nod factors to the newly synthesized cell wall. In agreement, FCM showed a > 1,000-fold reduction of molecular mobility of the fluorescence Nod factors upon binding to the cell wall. In addition, FCM demonstrated that Nod factors, when exogenously applied in aqueous solution at 10 nM, markedly concentrate in the cell wall of root hairs (up to 50-fold). The feasibility of applying FCM for the study of living plant cells as well as the implications of our results for the perception of Nod factors are discussed.

Rapid, Plateau-like Increases in Intracellular Free Calcium Are Associated with Nod-Factor—Induced Root-Hair Deformation

Rhizobia excrete variously substituted lipo-oligosaccha-ride Nod factors into the legume rhizosphere. Homologous legumes respond to these signals through deformation of the root hairs and the development of nodulation foci in the root cortex. Cellular events in root hairs from the susceptible zone of nearly mature root hairs were studied in root segments loaded with the calcium indicators Fura-2 or Fluo-3. Application of 10-9 M Nod factors of the broad-host-range Rhizobium sp. NGR234 to the homologous legume Vigna unguiculata resulted, within seconds, in plateau-like increases in intracellular free calcium ([Ca2+]i) in the root hairs and root epidermal cells. Nod factors of R. meliloti at 10-9 M caused equally rapid increases in [Ca2+]i in the root hairs and epidermal cells of the nonhost V. unguiculata, and also induced root-hair deformation. The chitin tetramer, N-N′-N″-N′″-tetracetylchitotetraose, which represents the backbone of Nod factors, induced neither root-hair deformation nor changes in [Ca2+]i in V. unguiculata. Root hairs and epidermal cells of the nonlegume non-host Arabidopsis thaliana showed neither [Ca2+]i increases nor root-hair deformation in response to both factors.

Ethylene provides positional information on cortical cell division but is not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction.

Nod factors secreted by Rhizobium leguminosarum bv. viciae induce root hair deformation, involving a reinitiation of tip growth, and the formation of nodule primordia in Vicia sativa (vetch). Ethylene is a potent inhibitor of cortical cell division, an effect that can be counteracted by applying silver ions (Ag+) or aminoethoxy-vinylglycine (AVG). In contrast to the inhibitory effect on cortical cell division, ethylene promotes the formation of root hairs (which involves tip growth) in the root epidermis of Arabidopsis. We investigate the possible paradox concerning the action of ethylene, putatively promoting Nod factor induced tip growth whilst, at the same time, inhibiting cortical cell division. We show, by using the ethylene inhibitors AVG and Ag+, that ethylene has no role in the reinitiation of root hair tip growth induced by Nod factors (root hair deformation) in vetch. However, root hair formation is controlled, at least in part, by ethylene. Furthermore, we show that ACC oxidase, which catalizes the last step in ethylene biosynthesis, is expressed in the cell layers opposite the phloem in that part of the root where nodule primordia are induced upon inoculation with Rhizobium. Therefore, we test whether endogenously produced ethylene provides positional information controlling the site where nodule primordia are formed by determining the position of nodules formed on pea roots grown in the presence of AVG or Ag+.

Nod factor-induced expression of leghemoglobin to study the mechanism of NH4NO3 inhibition on root hair deformation.

Nod factors secreted by Rhizobium leguminosarum by, viciae induce root hair deformation, the formation of nodule primordia, and the expression of early nodulin genes in Vicia sativa (vetch). Root hair deformation is induced within 3 h in a small, susceptible zone (+/-2 mm) of the root. NH4NO3, known to be a potent blocker of nodule formation, inhibits root hair deformation, initial cortical cell divisions, and infection thread formation. To test whether NH4NO3 affects the formation of a component of the Nod factor perception-transduction system, we studied Nod factor-induced gene expression. The differential display technique was used to search for marker genes, which are induced within 1 to 3 h after Nod factor application. Surprisingly, one of the isolated cDNA clones was identified as a leghemoglobin gene (VsLb1), which is induced in vetch roots within 1 h after Nod factor application. By using the drug brefeldin A, it was then shown that VsLb1 activation does not require root hair deformation. The pVsLb1 clone was used as a marker to show that in vetch plants grown in the presence of NH4NO3, Nod factor perception and transduction leading to gene expression are unaffected.

Structural Requirements of Synthetic and Natural Product Lipo-Chitin Oligosaccharides for Induction of Nodule Primordia on Glycine soja

Rhizobia synthesize a class of lipo-chitin oligosaccharides that induce root hair deformation and induce the initiation of nodule structures on legume roots. These lipo-chitin oligosaccharides are tetra- and penta-lipo-oligosaccharides of N-acetylglucosamine with an acyl substitution on the nonreducing end and are commonly known as Nod factors. In this study, we demonstrate that synthetic analogs of natural product Nod factors have the same biological activities. To determine structure-activity relationships, a collection of synthetic and natural product lipo-chitin oligosaccharides was assayed on Glycine soja. All biologically active lipo-chitin oligosaccharides induced both root hair deformation and nodule initiations on G. soja. The most active lipo-chitin oligosaccharides deformed root hairs at 10(-15) M and induced nodules at 1 ng of lipo-chitin oligosaccharide per spot inoculation. Plant responses demonstrate an interdependence of backbone length and the presence of substitutions on the reducing end. Lipo-chitin oligosaccharides containing four N-acetylglucosamine residues were active only without a reducing end modification, whereas lipo-chitin oligosaccharides containing five N-acetylglucosamine residues were active only with reducing end modification. The plant thus recognizes lipo-chitin oligosaccharides without reducing end substitutions despite the importance of these modifications for host range.

Early nodulin gene expression during Nod factor‐induced processes in Vicia sativa

Rhizobium leguminosarum bv. viciae-secreted Nod factors are able to induce root hair deformation, the formation of nodule primordia and the expression of early nodulin genes in Vicia sativa (vetch). To obtain more insight into the mode of action of Nod factors the expression of early nodulin genes was followed during Nod factor-induced root hair deformation and nodule primordium formation. The results of these studies suggested that the expression of VsENOD5 and VsENOD12 is not required for root hair deformation. In the Nod factor-induced primordia both VsENOD12 and VsENOD40 are expressed in a spatially controlled manner similar to that found in Rhizobium-induced nodule primordia. In contrast, VsENOD5 expression has never been observed in Nod factor-induced primordia, showing that the induction of VsENOD5 and VsENOD12 expression are not coupled. VsENOD5 expression is induced in the root epidermis by Nod factors and in Rhizobium-induced nodule primordia only in cells infected by the bacteria, suggesting that the Nod factor does not reach the inner cortical cells.

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).