Meliloti Nod Factors Research Articles

The Medicago truncatula hydrolase MtCHIT5b degrades Nod factors of Sinorhizobium meliloti and cooperates with MtNFH1 to regulate the nodule symbiosis.

Nod factors secreted by nitrogen-fixing rhizobia are lipo-chitooligosaccharidic signals required for establishment of the nodule symbiosis with legumes. In Medicago truncatula, the Nod factor hydrolase 1 (MtNFH1) was found to cleave Nod factors of Sinorhizobium meliloti. Here, we report that the class V chitinase MtCHIT5b of M. truncatula expressed in Escherichia coli can release lipodisaccharides from Nod factors. Analysis of M. truncatula mutant plants indicated that MtCHIT5b, together with MtNFH1, degrades S. meliloti Nod factors in the rhizosphere. MtCHIT5b expression was induced by treatment of roots with purified Nod factors or inoculation with rhizobia. MtCHIT5b with a fluorescent tag was detected in the infection pocket of root hairs. Nodulation of a MtCHIT5b knockout mutant was not significantly altered whereas overexpression of MtCHIT5b resulted in fewer nodules. Reduced nodulation was observed when MtCHIT5b and MtNFH1 were simultaneously silenced in RNA interference experiments. Overall, this study shows that nodule formation of M. truncatula is regulated by a second Nod factor cleaving hydrolase in addition to MtNFH1.

Detection of fungal and bacterial carbohydrates: Do the similar structures of chitin and peptidoglycan play a role in immune dysfunction?

Carbohydrate recognition is fundamental to a wide variety of interkingdom interactions. For example, bacterial peptidoglycan, an N-acetyl-D-glucosamine (GlcNAc)–N-acetylmuramic acid (MurNAc) polymer [1], and fungal chitin, a GlcNAc polymer [2, 3], are both immunostimulatory to vertebrates. In addition, bacterially produced Nodulation (Nod) factors, which consist of a modified GlcNAc, play a key role in symbiotic interactions with plants [4]. The structural similarity of these GlcNAc-containing molecules (Fig 1) suggests possible overlap in their physiological action, although many of the mechanisms underlying the detection of these molecules in different organisms appear unrelated. However, a single phylogenetically conserved domain, called Lysin (LysM), is found in specific receptors in signaling pathways responsive to one or more of these three related carbohydrates in bacteria, plants, and fungi and possibly mammalian systems. Thus, promiscuous activation could occur when a structurally similar but physiologically inappropriate ligand binds and thereby aberrantly activates an incorrect LysM domain-containing receptor. Here, I will discuss this possibility and its implications for immune pathologies such as asthma in which chitin is relevant. Open in a separate window Fig 1 Bacterial and fungal carbohydrates. A. Monomer of L-Lys containing peptidoglycan from Staphlycoccus aureus. B. Fungal chitin. C. Sinorhizobium meliloti Nod factor NodSmIV. Lys, Lysin; Nod, Nodulation.

Legume nodulation

Legume nodulation

Short‐chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone

The primary objective of this study was to identify the molecular signals present in arbuscular mycorrhizal (AM) germinated spore exudates (GSEs) responsible for activating nuclear Ca(2+) spiking in the Medicago truncatula root epidermis. Medicago truncatula root organ cultures (ROCs) expressing a nuclear-localized cameleon reporter were used as a bioassay to detect AM-associated Ca(2+) spiking responses and LC-MS to characterize targeted molecules in GSEs. This approach has revealed that short-chain chitin oligomers (COs) can mimic AM GSE-elicited Ca(2+) spiking, with maximum activity observed for CO4 and CO5. This spiking response is dependent on genes of the common SYM signalling pathway (DMI1/DMI2) but not on NFP, the putative Sinorhizobium meliloti Nod factor receptor. A major increase in the CO4/5 concentration in fungal exudates is observed when Rhizophagus irregularis spores are germinated in the presence of the synthetic strigolactone analogue GR24. By comparison with COs, both sulphated and nonsulphated Myc lipochito-oligosaccharides (LCOs) are less efficient elicitors of Ca(2+) spiking in M. truncatula ROCs. We propose that short-chain COs secreted by AM fungi are part of a molecular exchange with the host plant and that their perception in the epidermis leads to the activation of a SYM-dependent signalling pathway involved in the initial stages of fungal root colonization.

Contribution of NFP LysM Domains to the Recognition of Nod Factors during the Medicago truncatula/Sinorhizobium meliloti Symbiosis

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

TheMedicago truncatulaE3 Ubiquitin Ligase PUB1 Interacts with the LYK3 Symbiotic Receptor and Negatively Regulates Infection and Nodulation

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

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.

MtENOD11 Gene Activation During Rhizobial Infection and Mycorrhizal Arbuscule Development Requires a Common AT-Rich-Containing Regulatory Sequence

The MtENOD11 gene from the model legume Medicago truncatula is transcriptionally activated both in response to Sinorhizobium meliloti Nod factors and throughout infection of root tissues by the nitrogen-fixing microsymbiont. To identify the regulatory sequences involved in symbiosis-related MtENOD11 expression, a series of promoter deletions driving the beta-glucuronidase reporter gene were analyzed in transgenic M. truncatula roots. These studies have revealed that distinct regulatory regions are involved in infection-related MtENOD11 expression compared with preinfection (Nod factor-mediated) expression. In particular, the 257-bp promoter sequence immediately upstream from the start codon is sufficient for infection-related expression, but is unable to drive gene transcription in response to the Nod factor elicitor. This truncated promoter is also sufficient to confer MtENOD11 expression during both the arbuscular mycorrhizal (AM) association and the parasitic interaction with root-knot nematodes. Site-directed mutagenesis further showed that a previously identified nodule-specific AT-rich motif is required for high-level MtENOD11 expression during S. meliloti infection as well as during AM colonization. However, mutation of this motif does not affect gene expression associated with nematode-feeding sites. Taken together, these results suggest a close link between regulatory mechanisms controlling transcriptional early nodulin gene activation during both rhizobial and AM root endosymbioses.

Sinorhizobium meliloti sulfotransferase that modifies lipopolysaccharide.

Sinorhizobium meliloti is a gram-negative soil bacterium found either in free-living form or as a nitrogen-fixing endosymbiont of a plant structure called the nodule. Symbiosis between S. meliloti and its plant host alfalfa is dependent on bacterial transcription of nod genes, which encode the enzymes responsible for synthesis of Nod factor. S. meliloti Nod factor is a lipochitooligosaccharide that undergoes a sulfate modification essential for its biological activity. Sulfate also modifies the carbohydrate substituents of the bacterial cell surface, including lipopolysaccharide (LPS) and capsular polysaccharide (K-antigen) (R. A. Cedergren, J. Lee, K. L. Ross, and R. I. Hollingsworth, Biochemistry 34:4467-4477, 1995). We utilized the genomic sequence of S. meliloti to identify an open reading frame, SMc04267 (which we now propose to name lpsS), which encodes an LPS sulfotransferase activity. We expressed LpsS in Escherichia coli and demonstrated that the purified protein functions as an LPS sulfotransferase. Mutants lacking LpsS displayed an 89% reduction in LPS sulfotransferase activity in vitro. However, lpsS mutants retain approximately wild-type levels of sulfated LPS when assayed in vivo, indicating the presence of an additional LPS sulfotransferase activity(ies) in S. meliloti that can compensate for the loss of LpsS. The lpsS mutant did show reduced LPS sulfation, compared to that of the wild type, under conditions that promote nod gene expression, and it elicited a greater number of nodules than did the wild type during symbiosis with alfalfa. These results suggest that sulfation of cell surface polysaccharides and Nod factor may compete for a limiting pool of intracellular sulfate and that LpsS is required for optimal LPS sulfation under these conditions.

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae.

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.

The HCL gene of Medicago truncatula controls Rhizobium-induced root hair curling.

The symbiotic infection of the model legume Medicago truncatula by Sinorhizobium meliloti involves marked root hair curling, a stage where entrapment of the microsymbiont occurs in a chamber from which infection thread formation is initiated within the root hair. We have genetically dissected these early symbiotic interactions using both plant and rhizobial mutants and have identified a M. truncatula gene, HCL, which controls root hair curling. S. meliloti Nod factors, which are required for the infection process, induced wild-type epidermal nodulin gene expression and root hair deformation in hcl mutants, while Nod factor induction of cortical cell division foci was reduced compared to wild-type plants. Studies of the position of nuclei and of the microtubule cytoskeleton network of hcl mutants revealed that root hair, as well as cortical cells, were activated in response to S. meliloti. However, the asymmetric microtubule network that is typical of curled root hairs, did not form in the mutants, and activated cortical cells did not become polarised and did not exhibit the microtubular cytoplasmic bridges characteristic of the pre-infection threads induced by rhizobia in M. truncatula. These data suggest that hcl mutations alter the formation of signalling centres that normally provide positional information for the reorganisation of the microtubular cytoskeleton in epidermal and cortical cells.

Nod factors and chitooligomers elicit an increase in cytosolic calcium in aequorin-expressing soybean cells.

Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants. NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end. Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity. Transgenic suspension-cultured soybean (Glycine max [L.] Merr.) cells expressing aequorin have been used to record cytosolic [Ca(2+)] changes upon treatment with purified NFs and chitin fragments. Both compounds elicited an increase of cytosolic [Ca(2+)] at nanomolar concentrations. The shape and amplitude of cytosolic [Ca(2+)] changes was similar to the response elicited by un-derivatized chitin oligomers. Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa. Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs. The capacity of NFs to elicit the calcium response depended on their structure. The presence of reducing end substituents in methylfucosylated NFs from Rhizobium sp. NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes. The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity. Pentameric S. meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive. Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers.

Alfalfa and tobacco cells react differently to chitin oligosaccharides and sinorhizobium meliloti nodulation factors

Alfalfa (Medicago sativa L.) suspension cultures respond to yeast elicitors with a strong alkalinization of the culture medium, a transient synthesis of activated oxygen species, and typical late defence reactions such as phytoalexin accumulation and increased peroxidase activity. The alkalinization reaction as well as the oxidative burst were also observed when tobacco (Nicotiana tabacum L. ) cell-suspension cultures were treated with yeast elicitors. Depending on the degree of polymerization, N-acetyl chitin oligomers induced the alkalinization response in both plant cell-suspension cultures, while only tobacco cell cultures developed an oxidative burst. Suspension-cultured tobacco cells responded to Sinorhizobium meliloti nodulation factors with a maximal alkalinization of 0.25 pH units and a remarkable oxidative burst. In contrast, addition of Sinorhizobium meliloti nodulation factors to suspension-cultured alfalfa cells induced a slight acidification of the culture medium, instead of an alkalinization, but no oxidative burst.

MtENOD20, a Nod Factor-Inducible Molecular Marker for Root Cortical Cell Activation

The spatio-temporal expression pattern of the Medicago truncatula ENOD20 gene during early stages of nodulation has been analyzed with transgenic alfalfa (M. varia) expressing a pMtENOD20-GUS chimeric fusion. Our results show that transcriptional activation of this gene occurs initially in dividing inner cortical cells corresponding to sites of nodule primordium formation and subsequently in root hairs containing infection threads. Use of Sinorhizobium meliloti nod gene mutants that uncouple nodule organogenesis from infection has confirmed that early MtENOD20 transcription is tightly linked to cortical cell activation (CCA). Furthermore, these experiments have revealed that an S. meliloti nodH mutant, defective in Nod factor sulfation and epidermal cell activation, is nevertheless able to elicit low-level CCA. Purified S. meliloti Nod factors trigger MtENOD20 transcription very rapidly (within 12 to 24 h) in the root cortex, and studies with transgenic alfalfa show that Nod factors are able to elicit gene expression coupled to CCA at concentrations as low as 10-11 M. Finally, we have made use of a range of synthetic lipo-chitooligosaccharides to show that fatty acid chain length is an important structural parameter in the capacity of Nod factors to elicit CCA. Taken together, our results suggest that pMtENOD20-GUS transgenic lines should prove valuable tools in future studies of Rhizobium and Nod factor-elicited CCA.

Purification and characterization of a novel chitinase-lysozyme, of another chitinase, both hydrolysing Rhizobium meliloti Nod factors, and of a pathogenesis-related protein from Medicago sativa roots.

The symbiosis between Rhizobium meliloti and Medicago sativa (Leguminosae) involves the interaction of lipochito-oligosaccharides (Nod factors) excreted by bacteria with specific proteins of the host plant. The cleavage of Nod factors can be used as an enzymic assay to identify novel hydrolytic enzymes. Here a soluble extract of 3-day-old roots was fractionated by anion exchange, affinity chromatography, gel filtration and native electrophoresis. Two acidic chitinases (pI 4.6-5.4), CHIT24 and CHIT36, designated in accordance with their molecular mass in kDa, were separated. CHIT24 cleaves all tested Nod factors to produce lipotrisaccharides with the preference NodRm-V(S)>NodRm-IV >NodRm-IV(S)>=NodRm-IV(Ac,S); it also hydrolyses colloidal 3H-chitin and has lysozyme activity. The kinetics of Nod factor degradation by CHIT24 depends on substrate structural parameters, namely the length of the oligosaccharide chain and sulphation (S) at the reducing end, but not much on acetylation (Ac) at the non-reducing end. The 25-residue N-terminal sequence of CHIT24 has no similarity with known chitinases or lysozymes, indicating that it is a novel type of hydrolase. CHIT36 also hydrolyses NodRm-V(S) into NodRm-III, but it is inactive towards NodRm-IV(S) and NodRm-IV(Ac,S) formed by R. meliloti. Finally, a 17 kDa protein, P17, was co-purified with CHIT24. It neither degrades Nod factors nor exhibits lysozyme activity and shows complete identity, at the 15-residue N-terminal sequence, with a class 10 pathogenesis-related protein, PR-10.

The role of ion fluxes in Nod factor signalling in Medicago sativa

SummaryUsing ion‐selective microelectrodes, the basis of Nod factor‐induced changes in the plasma membrane potential was analysed by measuring the extracellular free concentrations of Ca2+, K+, H+ and Cl– in the root hair zone of alfalfa. After addition of the Rhizobium meliloti Nod factor NodRm‐IV(C16:2,S) at a concentration of 0.1 μM, a decrease in [Ca2+] was observed first, which was followed after a few seconds by an increase of [Cl–], by an alkalinization, and then by a delayed increase of [K+], all of which were transient changes. Simultaneously with the appearance of Cl– ions in the root hair zone, a decrease in cytosolic [Cl–] was measured. It was concluded that the depolarization was caused by temporary short‐circuiting of the proton pump through the rapid release of Cl– ions along their steep electrochemical gradient. Since under resting conditions the driving force for K+ ions was inwardly directed, their release was delayed until their driving force was inverted. This indicates that K+ serves as a charge balance that eventually stops depolarization and initiates repolarization. Since the decrease in [Ca2+] was observed seconds before the increase in [Cl–] and the depolarization, it is argued that Ca2+ entering into the cell does not cause the depolarization directly, but might initiate it by triggering the activation of an anion channel that then releases the chloride ions. The observations that the Ca2+ ionophore A23187 mimicks the Nod factor response, and that the Ca2+ channel antagonist nifedipine inhibits this response, support the idea that Ca2+ plays a primary role in the transduction of the Nod signal in alfalfa.

A persistent meristem is formed in nodular structures elicited by Nod factor or by a Rhizobium meliloti exopolysaccharide mutant in alfalfa plants which nodulate spontaneously

Certain alfalfa ( Medicago sativa L.) plants are able to nodulate spontaneously (Nar character) and form well-developed, elongated nodules in the absence of Rhizobium. We selected and selfed Nar + and Nar − plants, obtaining S 1 populations with either a high or a very low frequency of Nar + plants, respectively. To assess the influence of the Nar character on nodule development, we tested whether Nar + and Nar − progenies treated by Rhizobium meliloti Nod factor or inoculated with a R. meliloti exopolysaccharide mutant differed in terms of nodule meristematic activity. Following addition of NodRmIV(Ac,S,C 16:2), only small nodular outgrowths were formed on Nar − progenies, whereas an increased number of elongated nodules was observed on Nar + progenies. Nodules induced by a R. meliloti exo − mutant on Nar − progenies lacked a persistent meristem and arrested in development soon after emergence, whereas on Nar + progenies they possessed a persistent meristem and elongated. From both experiments, we conclude that in Nar + plants, the capacity to induce the persistent meristem observed during spontaneous nodulation is conserved in nodules elicited by Nod factor or exo − mutant.

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.

Distinct response of Medicago suspension cultures and roots to Nod factors and chitin oligomers in the elicitation of defense‐related responses

The induction of plant defense‐related responses by chitin oligomers and the Rhizobium meliloti lipo‐chito‐oligosaccharide nodulation signals (Nod factors) in Medicago cell cultures and roots was investigated by following the expression of genes encoding enzymes of the isoflavonoid biosynthetic pathway, such as chalcone synthase, chalcone reductase, isoflavone reductase, as well as genes encoding a pathogenesis‐related protein and a peroxidase. In suspension‐cultured cells, all genes except the peroxidase gene were induced by both the R. meliloti Nod factor NodRm‐IV(C16:2,S) and chitin oligomers with a minimum of three sugar residues. However, activation of these genes was not elicited by the symbiotically inactive, desulfated NodRm‐IV(C16:2). Moreover, the cells were more sensitive to the chitin oligosaccharides than to the Nod factor. Analysis of flavonoids in Medicago microcallus cultures revealed differences between cells treated with N‐acetyl‐chitotetraose and those treated with Nod factor and demonstrated increased production of the phytoalexin medicarpin in the presence of Nod factor. In Medicago roots, none of the tested genes was activated by the N‐acetylchitotetraose, whereas the Nod factor at micro‐molar concentration enhanced transient expression of the isoflavonoid biosynthetic genes. The differential responses to Nod factors and chitin oligomers suggest that Medicago cells possess distinct perception systems for these related molecules.

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 (omega-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.