Nod Factor Signaling Research Articles

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.

Nod factor induction of reactive oxygen species production is correlated with expression of the early nodulin gene rip1 in Medicago truncatula.

Plant genes that are specifically activated by the rhizobial lipochitooligosaccharide signal molecule (Nod factor) in legume hosts are collectively referred to as nodulins. Although nodulin gene expression is both spatially and temporally correlated with symbiosis, the function of these genes and the molecular events underlying their expression remain unknown. Sequence analysis of rip1, an early nodulin gene encoding a putative peroxidase protein, revealed the existence of sequence motifs with homology to reactive oxygen species (ROS) responsive cis elements. Here we report that recognition of compatible Nod factor rapidly stimulates a spatially localized production of reactive oxygen species in legume roots. Sinorhizobium meliloti mutants that produce an altered Nod factor structure and a nonnodulating plant mutant, dmi1-1, that is implicated in Nod factor signal transduction are equally impaired in the ability to elicit ROS production and rip1 expression. Interestingly, both rip1 transcription and ROS production exhibit the same tissue-specific pattern of localization. Moreover, exogenous hydrogen peroxide is sufficient to activate rip1 transcription. Taken together, these results suggest that ROS production is a consequence of specific Nod factor perception and implicate H2O2 produced during this response as a mediator of Nod factor-induced rip1 expression.

Structure-function analysis of nod factor-induced root hair calcium spiking in Rhizobium-legume symbiosis.

In the Rhizobium-legume symbiosis, compatible bacteria and host plants interact through an exchange of signals: Host compounds promote the expression of bacterial biosynthetic nod (nodulation) genes leading to the production of a lipochito-oligosaccharide signal, the Nod factor (NF). The particular array of nod genes carried by a given species of Rhizobium determines the NF structure synthesized and defines the range of legume hosts by which the bacterium is recognized. Purified NF can induce early host responses even in the absence of live Rhizobium One of the earliest known host responses to NF is an oscillatory behavior of cytoplasmic calcium, or calcium spiking, in root hair cells, initially observed in Medicago spp. and subsequently characterized in four other genera (D.W. Ehrhardt, R. Wais, S.R. Long [1996] Cell 85: 673-681; S.A. Walker, V. Viprey, J.A. Downie [2000] Proc Natl Acad Sci USA 97: 13413-13418; D.W. Ehrhardt, J.A. Downie, J. Harris, R.J. Wais, and S.R. Long, unpublished data). We sought to determine whether live Rhizobium trigger a rapid calcium spiking response and whether this response is NF dependent. We show that, in the Sinorhizobium meliloti-Medicago truncatula interaction, bacteria elicit a calcium spiking response that is indistinguishable from the response to purified NF. We determine that calcium spiking is a nod gene-dependent host response. Studies of calcium spiking in M. truncatula and alfalfa (Medicago sativa) also uncovered the possibility of differences in early NF signal transduction. We further demonstrate the sufficiency of the nod genes for inducing calcium spiking by using Escherichia coli BL21 (DE3) engineered to express 11 S. meliloti nod genes.

Pharmacological analysis of nod factor-induced calcium spiking in Medicago truncatula. Evidence for the requirement of type IIA calcium pumps and phosphoinositide signaling.

Bacterial Nod factors trigger a number of cellular responses in root hairs of compatible legume hosts, which include periodic, transient increases in cytosolic calcium levels, termed calcium spiking. We screened 13 pharmaceutical modulators of eukaryotic signal transduction for effects on Nod factor-induced calcium spiking. The purpose of this screening was 2-fold: to implicate enzymes required for Nod factor-induced calcium spiking in Medicago sp., and to identify inhibitors of calcium spiking suitable for correlating calcium spiking to other Nod factor responses to begin to understand the function of calcium spiking in Nod factor signal transduction. 2-Aminoethoxydiphenylborate, caffeine, cyclopiazonic acid (CPA), 2,5-di-(t-butyl)-1,4-hydroquinone, and U-73122 inhibit Nod factor-induced calcium spiking. CPA and U-73122 are inhibitors of plant type IIA calcium pumps and phospholipase C, respectively, and implicate the requirement for these enzymes in Nod factor-induced calcium spiking. CPA and U-73122 inhibit Nod factor-induced calcium spiking robustly at concentrations with no apparent toxicity to root hairs, making CPA and U-73122 suitable for testing whether calcium spiking is causal to subsequent Nod factor responses.

Nodulation by numbers: the role of ethylene in symbiotic nitrogen fixation

Nitrogen fixation in root nodules resulting from the symbiotic interaction of leguminous plants and rhizobial bacteria meets ∼70% of the host's nitrogen requirements. Nodulation is triggered when bacterial Nod factors, produced in response to chemical signals released by the plant roots, initiate signalling pathways in the plant, leading to infection and nodule morphogenesis. Although Nod factors are considered to be the primary morphogens, the host is known to regulate the number of nodules strictly via an unknown feedback mechanism termed nodule autoregulation. Ethylene has been implicated in this control following observations that its production is induced in legumes in response to rhizobial infection and that it inhibits nodule number in some interactions. By determining the ethylene sensitivity of specific events during nodule morphogenesis in wild-type Medicago trunculata and an ethylene-insensitive super-nodulating mutant sickle (skl), Giles Oldroyd and colleagues 1 Oldroyd G.E.D. et al. Ethylene inhibits the Nod factor signal transduction pathway of Medicago trunculata. Plant Cell. 2001; 13: 1835-1849 PubMed Google Scholar now present evidence that ethylene might regulate nodulation by modulating one of the earliest responses in the Nod factor signal transduction pathway – Ca2+ spiking.

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.

Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula.

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.

Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation.

Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [(32)P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.

Nod factor‐induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation

SummaryRhizobium‐secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [32P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.

Localization of a Nod factor-binding protein in legume roots and factors influencing its distribution and expression.

The roots of the legume Dolichos biflorus contain a lectin/nucleotide phosphohydrolase (Db-LNP) that binds to the Nod factor signals produced by rhizobia that nodulate this plant. In this study we show that Db-LNP is differentially distributed along the surface of the root axis in a pattern that correlates with the zone of nodulation of the root. Db-LNP is present on the surface of young and emerging root hairs and redistributes to the tips of the root hairs in response to treatment of the roots with a rhizobial symbiont or with a carbohydrate ligand. This redistribution does not occur in response to a non-symbiotic rhizobial strain or a root pathogen. Db-LNP is also present in the root pericycle where its level decreases upon initiation of nodule formation. Maximum levels of Db-LNP are found in 2-d-old roots, and the expression of this root protein is increased when the plants are grown in the absence of NO(3)(-) and NH(4)(+). These results support the possibility that Db-LNP is involved in the initiation of the Rhizobium legume symbiosis.

Nod factors modulate the concentration of cytosolic free calcium differently in growing and non-growing root hairs of Medicago sativa L.

Using Ca(2+)-selective microelectrodes, the concentration of free calcium ([Ca(2+)]) in the cytosol has been measured in root hair cells of Medicago sativa L. in the presence of nodulation (Nod) factors. Growing root hairs of M. sativa displayed a steep apical [Ca(2+)] gradient, i.e. 604-967 nM in the tip compared with 95-235 nM in the basal region. When tested within the first 5 to 10 µm of the tip, addition of NodRm-IV(C16:2,S) decreased the cytosolic [Ca(2+)], whereas an increase was observed when tested behind the tip. Overall, this led to a partial dissipation of the [Ca(2+)] gradient. The Ca(2+) response was specific: it was equally well observed in the presence of NodRm-IV(Ac,C16:2,S), reduced with NodRm-IV(C16:0,S), but not with chitotetraose, the nonactive glucosamine backbone. In contrast to growing root hairs, non-growing root hairs without a tip-to-base cytosolic [Ca(2+)] gradient responded to NodRm-IV(C16:2,S) with an increase in cytosolic [Ca(2+)] at the tip as well as at the root hair base. We suggest that the response to Nod factors depends on the stage of development of the root hairs, and that changes in cytosolic [Ca(2+)] may play different roles in Nod-factor signaling: changes of cytosolic [Ca(2+)] in the apical part of the root hair may be related to root hair deformation, while the increase in [Ca(2+)] behind the tip may be essential for the amplification of the Nod signal, for its propagation and transduction to trigger downstream events.

Recent advances in the study of Nod factor perception and signal transduction

Rhizobial lipochitooligosaccharidic Nod factors mediate the specific recognition between leguminous plants and their prokaryotic symbionts. This review summarizes recent findings on the way plants could perceive and transduce these bacterial signals. It starts by summarizing knowledge about Nod factor binding sites, before moving to the potential implications in Nod factor signal transduction of G proteins, root-hair plasma membrane depolarisation, cytoplasmic and extracellular alkalinisation and finally variations in cytoplasmic calcium concentration.

Roles for Azorhizobial Nod Factors and Surface Polysaccharides in Intercellular Invasion and Nodule Penetration, Respectively

In the symbiotic interaction between Azorhizobium caulinodans and Sesbania rostrata root and stem-borne nodules are formed. The bacteria enter the host via intercellular spaces at lateral or adventitious root bases and form infection pockets in outer cortical layers. Infection threads guide the bacteria to nodule primordia where plant cells are invaded. To identify bacterial functions that are required for this infection process, two mutants defective in nodulation were studied; one produced no Nod factors (nodA mutant), the other had altered surface polysaccharides (SPS) and induced the formation of pseudo-nodules. Bacteria were visualized with the help of a nodA-uidA reporter fusion that was functional during nodule development and in bacteroids. In contrast to the SPS mutant, nodA mutants were unable to colonize outer cortical regions. In mixed inoculations with both mutants, functional nodules were formed, the central tissue of which was occupied by the nodA mutant. These observations suggest that SPS play a role in deeper invasion and that Nod factors are necessary for entry. Simultaneous application of purified Nod factors and nodA mutant bacteria restored the formation of outer cortical infection pockets leading to the conclusion that intercellular infection is an active process that is dependent on bacterial Nod factor signaling.

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

Summary 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 (10–6–10–9m) 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 activatingMtENOD12 in cortical parenchyma as in legumes. Exogenous application ofN‐naphthaleneacetic acid mimicked the Nod factor‐elicited tissue‐specific expression ofMtENOD12 in roots while cytokinins inhibited it, thus evidencing that Nod factors, auxin and cytokinins probably act on similar signaling elements responsible for the regulation ofMtENOD12 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.

Rhizobium nod factor signaling. Evidence for a g protein-mediated transduction mechanism

Rhizobium nodulation (Nod) factors are lipochitooligosaccharide signals that elicit key symbiotic developmental responses in the host legume root. In this study, we have investigated Nod factor signal transduction in the Medicago root epidermis by using a pharmacological approach in conjunction with transgenic plants expressing the Nod factor-responsive reporter construct pMtENOD12-GUS. Evidence for the participation of heterotrimeric G proteins in Nod factor signaling has come from three complementary observations: (1) the amphiphilic peptides mastoparan and Mas7, known G protein agonists, are able to mimic Nod factor-induced epidermal MtENOD12 expression; (2) growth of plants in nodulation-inhibiting conditions (10 mM NH4NO3) leads to a dramatic reduction in both Nod factor- and mastoparan-elicited gene expression; and (3) bacterial pertussis toxin, a well-characterized G protein antagonist, blocks the activities of both the Nod factor and mastoparan. In addition, we have found that antagonists that interfere with phospholipase C activity (neomycin and U73122) and Ca2+ influx/release (EGTA, La3+, and ruthenium red) block Nod factor/mastoparan activity. Taken together, these results are consistent with a Nod factor signal transduction mechanism involving G protein mediation coupled to the activation of both phosphoinositide and Ca2+ second messenger pathways.

Rhizobium Nod Factor Signaling: Evidence for a G Protein-Mediated Transduction Mechanism

Rhizobium Nod Factor Signaling: Evidence for a G Protein-Mediated Transduction Mechanism

Dictyostelium discoideum Fatty-acyl Amidase II Has Deacylase Activity on Rhizobium Nodulation Factors

Dictyostelium discoideum (Amoebidae) secretes cell-lysing enzymes: esterases, amidases, and glycosylases, many of which degrade soil bacteria to provide a source of nutrients. Two of these enzymes, fatty-acyl amidases FAA I and FAA II, act sequentially on the N-linked long chain acyl groups of lipid A, the lipid anchor of Gram-negative bacterial lipopolysaccharide. FAA I selectively hydrolyzes the 3-hydroxymyristoyl group N-linked to the proximal glucosamine residue of de-O-acylated lipid A. Substrate specificity for FAA II is less selective, but does require prior de-N-acylation of the proximal sugar, i.e. bis-N-acylated lipid A is not a substrate. We have synthesized a 14C-labeled substrate analog for FAA II and used this in a novel assay to monitor its purification. Inhibitory studies indicate that FAA II is not a serine protease, but may have a catalytic mechanism similar to metalloprotein de-N-acetylases such as LpxC. Interestingly, rhizobial Nod factor signal oligosaccharides that induce root nodules on leguminous plants have many of the structural requirements for substrate recognition by FAA II. In vitro evidence indicates that Rhizobium fredii Nod factors are selectively de-N-acylated by purified FAA II, suggesting that the enzyme may reduce the N2-fixing efficiency of Rhizobium-legume symbioses. In contrast, N-methylated Nod factors from transgenic R. fredii carrying the rhizobial nodS gene were resistant to FAA II, suggesting a mechanism by which Nod factors may be protected from enzymatic de-N-acylation. Since FAA II and Nod factors are both secreted, and Nod factors that lack the N-acyl group are unable to induce nodules, dictyostelial FAA II may decrease the efficiency of symbiotic nitrogen fixation in the environment by reducing the available biologically active nodule inducer signal.

Molecular mechanisms of Nod factor diversity.

The rhizobia-legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo-chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non-allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co-evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on 'symbiosis islands', which are symbiotic regions associated with movable elements.

The genetic and biochemical basis for nodulation of legumes by rhizobia.

Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium are collectively termed rhizobia. They share the ability to penetrate legume roots and elicit morphological responses that lead to the appearance of nodules. Bacteria within these symbiotic structures fix atmosphere nitrogen and thus are of immense ecological and agricultural significance. Although modern genetic analysis of rhizobia began less than 20 years ago, dozens of nodulation genes have now been identified, some in multiple species of rhizobia. These genetic advances have led to the discovery of a host surveillance system encoded by nodD and to the identification of Nod factor signals. These derivatives of oligochitin are synthesized by the protein products of nodABC, nodFE, NodPQ, and other nodulation genes; they provoke symbiotic responses on the part of the host and have generated immense interest in recent years. The symbiotic functions of other nodulation genes are nonetheless uncertain, and there remain significant gaps in our knowledge of several large groups of rhizobia with interesting biological properties. This review focuses on the nodulation genes of rhizobia, with particular emphasis on the concept of biological specificity of symbiosis with legume host plants.

Plant genes involved in root-nodule development on legumes

Rhizobium is able to induce the formation of a new organ on roots of leguminous plants, the root nodule, in which the penetrated bacteria fix atmospheric nitrogen. This process is initiated by specific lipo-oligosaccharides, called Nod factors, secreted by the bacterium. Nodule formation proceeds through distinct steps like infection thread formation and activation of mitotic activity in cortical cells. During these steps specific plant genes, nodulin genes, are induced and several of these have been identified and characterized. Nodulin genes are used now as markers to study Nod factor perception and signal transduction.