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Lysin motif receptor like kinases (LysM-RLKs) are involved in the perception of chitooligosaccharides (COs) and related lipochitooligosaccharides (LCOs) in plants. Expansion and divergence of the gene family during evolution have led to various roles in symbiosis and defence. By studying proteins of the LYR-IA subclass of LysM-RLKs of the Poaceae, we show here that they are high affinity LCO binding proteins with a lower affinity for COs, consistent with a role in LCO perception to establish arbuscular mycorrhiza (AM). In Papilionoid legumes whole genome duplication has resulted in two LYR-IA paralogs, MtLYR1 and MtNFP in Medicago truncatula, with MtNFP playing an essential role in the root nodule symbiosis with nitrogen-fixing rhizobia. We show that MtLYR1 has retained the ancestral LCO binding characteristic and is dispensable for AM. Domain swapping between the three Lysin motifs (LysMs) of MtNFP and MtLYR1 and mutagenesis in MtLYR1 suggest that the MtLYR1 LCO binding site is on the second LysM, and that divergence in MtNFP led to better nodulation, but surprisingly with decreased LCO binding. These results suggest that divergence of the LCO binding site has been important for the evolution of a role of MtNFP in nodulation with rhizobia.

Lipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling the nodulation of legumes by rhizobia. More recently, LCOs were also found in symbiotic fungi and, more surprisingly, very widely in the kingdom Fungi, including in saprophytic and pathogenic fungi. The LCO-V(C18:1, fucosylated/methyl fucosylated), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants such as Medicago truncatula can discriminate between Nod-LCOs and Fung-LCOs. To address this question, we performed a genome-wide association study on 173 natural accessions of M. truncatula, using a root branching phenotype and a newly developed local score approach. Both Nod-LCOs and Fung-LCOs stimulated root branching in most accessions, but the root responses to these two types of LCO molecules were not correlated. In addition, the heritability of the root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, of which only one was common. This suggests that Nod-LCOs and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development., Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by certain bacteria and fungi that establish symbiotic relationships with plants. Here, the authors show that LCOs are produced also by many other, non-symbiotic fungi, and regulate fungal growth and development.

SummaryLipo-chitooligosaccharides (LCOs) were originally found as symbiotic signals called Nod Factors (Nod-LCOs) controlling nodulation of legumes by rhizobia. More recently LCOs were also found in symbiotic fungi and, more surprisingly, very widely in the kingdom fungi including in saprophytic and pathogenic fungi. The LCO-V(C18:1, Fuc/MeFuc), hereafter called Fung-LCOs, are the LCO structures most commonly found in fungi. This raises the question of how legume plants, such asMedicago truncatula, can perceive and discriminate between Nod-LCOs and these Fung-LCOs.To address this question, we performed a Genome Wide Association Study on 173 natural accessions ofMedicago truncatula, using a root branching phenotype and a newly developed local score approach.Both Nod- and Fung-LCOs stimulated root branching in most accessions but there was very little correlation in the ability to respond to these types of LCO molecules. Moreover, heritability of root response was higher for Nod-LCOs than for Fung-LCOs. We identified 123 loci for Nod-LCO and 71 for Fung-LCO responses, but only one was common.This suggests that Nod- and Fung-LCOs both control root branching but use different molecular mechanisms. The tighter genetic constraint of the root response to Fung-LCOs possibly reflects the ancestral origin of the biological activity of these molecules.

International audience; The formation of nitrogen-fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen-fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin-motif receptor-like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild-type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild-type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan-defective strains was achieved by in trans rescue with a Nod factor-deficient S. meliloti mutant. While the Nod factor-deficient strain was always more abundant inside nodules, the succinoglycan-deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.

International audience; A complex network of pathways coordinates nodulation and epidermal root hair infection in the symbiotic interaction between rhizobia and legume plants. Whereas nodule formation was known to be autoregulated, it was so far unclear whether a similar control is exerted on the infection process. We assessed the capacity of Medicago plants nodulated by Sinorhizobium meliloti to modulate root susceptibility to secondary bacterial infection or to purified Nod factors in split-root and volatile assays using bacterial and plant mutant combinations. Ethylene implication in this process emerged from gas production measurements, use of a chemical inhibitor of ethylene biosynthesis and of a Medicago mutant affected in ethylene signal transduction. We identified a feedback mechanism that we named AOI (for Autoregulation Of Infection) by which endosymbiotic bacteria control secondary infection thread formation by their rhizospheric peers. AOI involves activation of a cyclic adenosine 30,50-monophosphate (cAMP) cascade in endosymbiotic bacteria, which decreases both root infectiveness and root susceptibility to bacterial Nod factors. These latter two effects are mediated by ethylene. AOI is a novel component of the complex regulatory network controlling the interaction between Sinorhizobium meliloti and its host plants that emphasizes the implication of endosymbiotic bacteria in fine-tuning the interaction.

International audience; Mycorrhizal fungi form mutualistic associations with the roots of most land plants and provide them with mineral nutrients from the soil in exchange for fixed carbon derived from photosynthesis. The common symbiosis pathway (CSP) is a conserved molecular signaling pathway in all plants capable of associating with arbuscular mycorrhizal fungi. It is required not only for arbuscular mycorrhizal symbiosis but also for rhizobia-legume and actinorhizal symbioses. Given its role in such diverse symbiotic associations, we hypothesized that the CSP also plays a role in ectomycorrhizal associations. We showed that the ectomycorrhizal fungus Laccaria bicolor produces an array of lipochitooligosaccharides (LCOs) that can trigger both root hair branching in legumes and, most importantly, calcium spiking in the host plant Populus in a CASTOR/POLLUX-dependent manner. Nonsulfated LCOs enhanced lateral root development in Populus in a calcium/calmodulin-dependent protein kinase (CCaMK)-dependent manner, and sulfated LCOs enhanced the colonization of Populus by L. bicolor. Compared with the wildtype Populus, the colonization of CASTOR/POLLUX and CCaMK RNA interference lines by L. bicolor was reduced. Our work demonstrates that similar to other root symbioses, L. bicolor uses the CSP for the full establishment of its mutualistic association with Populus.

International audience; . Lipo-chitooligosaccharides (LCOs) are microbial symbiotic signals that also influence root growth. In Medicago truncatula, LCOs stimulate lateral root formation (LRF) synergistically with auxin. However, the molecular mechanisms of this phenomenon and whether it is restricted to legume plants are not known. . We have addressed the capacity of the model monocot Brachypodium distachyon (Brachypodium) to respond to LCOs and auxin for LRF. For this, we used a combination of root phenotyping assays, live-imaging and auxin quantification, and analysed the regulation of auxin homeostasis genes. We show that LCOs and a low dose of the auxin precursor indole-3-butyric acid (IBA) stimulated LRF in Brachypodium, while a combination of LCOs and IBA led to different regulations. Both LCO and IBA treatments locally increased endogenous indole-3-acetic acid (IAA) content, whereas the combination of LCO and IBA locally increased the endogenous concentration of a conjugated form of IAA (IAA-Ala). LCOs, IBA and the combination differentially controlled expression of auxin homeostasis genes. . These results demonstrate that LCOs are active on Brachypodium roots and stimulate LRF probably through regulation of auxin homeostasis. The interaction between LCO and auxin treatments observed in Brachypodium on root architecture opens interesting avenues regarding their possible combined effects during the arbuscular mycorrhizal symbiosis.

Almost all legume plants have the capacity to form two types of root organs: lateral roots and nodules (that will host rhizobia that fix nitrogen). Transcriptomic analyses are useful to understand both the similarities and differences between nodule and LR formation and to compare the LR developmental programs used by Arabidopsis and model legumes such as Medicago truncatula. However, in M. truncatula as in Arabidopsis, root cells "committed" to LR formation programs are scattered along the primary root and localized in the inner most layers of the root. To gain access to these cells, a lateral root-inducible system (LRIS) was first developed in Arabidopsis. This LRIS was recently shown to be effective in maize as well. Here we present a LRIS protocol adapted to the model legume Medicago truncatula. Using the same auxin transporter inhibitor and permeant auxin molecules used for Arabidopsis and maize but with slight modifications in their concentrations, we obtained very efficient enrichment and synchronization in LR development stages in M. truncatula.

Almost all legume plants have the capacity to form two types of root organs: lateral roots and nodules (that will host rhizobia that fix nitrogen). Transcriptomic analyses are useful to understand both the similarities and differences between nodule and LR formation and to compare the LR developmental programs used by Arabidopsis and model legumes such as Medicago truncatula. However, in M. truncatula as in Arabidopsis, root cells "committed" to LR formation programs are scattered along the primary root and localized in the inner most layers of the root. To gain access to these cells, a lateral root-inducible system (LRIS) was first developed in Arabidopsis. This LRIS was recently shown to be effective in maize as well. Here we present a LRIS protocol adapted to the model legume Medicago truncatula. Using the same auxin transporter inhibitor and permeant auxin molecules used for Arabidopsis and maize but with slight modifications in their concentrations, we obtained very efficient enrichment and synchronization in LR development stages in M. truncatula.

International audience; Arbuscular mycorrhiza (AM) is a root endosymbiosis between plants and glomeromycete fungi. It is the most widespread terrestrial plant symbiosis, improving plant uptake of water and mineral nutrients. Yet, despite its crucial role in land ecosystems, molecular mechanisms leading to its formation are just beginning to be unravelled. Recent evidence suggests that AM fungi produce diffusible symbiotic signals. Here we show that Glomus intraradices secretes symbiotic signals that are a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs), which stimulate formation of AM in plant species of diverse families (Fabaceae, Asteraceae and Umbelliferae). In the legume Medicago truncatula these signals stimulate root growth and branching by the symbiotic DMI signalling pathway. These findings provide a better understanding of the evolution of signalling mechanisms involved in plant root endosymbioses and will greatly facilitate their molecular dissection. They also open the way to using these natural and very active molecules in agriculture.

Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.

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.

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 geneMtENOD12 (fromMedicago 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.

The formation of root nodules and arbuscular mycorrhizal (AM) roots is controlled by a common signaling pathway including the calcium/calmodulin-dependent kinase Doesn’t Make Infection3 (DMI3). While nodule initiation by lipochitooligosaccharide (LCO) Nod factors is well characterized, diffusible AM fungal signals were only recently identified as sulfated and nonsulfated LCOs. Irrespective of different outcomes, the perception of symbiotic LCOs in Medicago truncatula is mediated by the LysM receptor kinase M. truncatula Nod factor perception (MtNFP). To shed light on transcriptional responses toward symbiotic LCOs and their dependence on MtNFP and Ca2+ signaling, we performed genome-wide expression studies of wild-type, Nod-factor-perception mutant1, and dmi3 mutant roots challenged with Myc- and Nod-LCOs. We show that Myc-LCOs lead to transient, quick responses in the wild type, whereas Nod-LCOs require prolonged incubation for maximal expression activation. While Nod-LCOs are most efficient for an induction of persistent transcriptional changes, sulfated Myc-LCOs are less active, and nonsulfated Myc-LCOs display the lowest capacity to activate and sustain expression. Although all symbiotic LCOs up-regulated a common set of genes, discrete subsets were induced by individual LCOs, suggesting common and specific functions for these in presymbiotic signaling. Surprisingly, even sulfated fungal Myc-LCOs and Sinorhizobium meliloti Nod-LCOs, having very similar structures, each elicited discrete subsets of genes, while a mixture of both Myc-LCOs activated responses deviating from those induced by single treatments. Focusing on the precontact phase, we identified signaling-related and transcription factor genes specifically up-regulated by Myc-LCOs. Comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNFP and largely requires MtDMI3.

Bradyrhizobium sp. strain ORS285 is a photosynthetic bacterium that forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. The symbiotic interaction of Bradyrhizobium sp. strain ORS285 with certain Aeschynomene spp. depends on the presence of nodulation (nod) genes whereas the interaction with other species is nod gene independent. To study the nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and Aeschynomene spp., we used a nodB-lacZ reporter strain to monitor the nod gene expression with various flavonoids. The flavanones liquiritigenin and naringenin were found to be the strongest inducers of nod gene expression. Chemical analysis of the culture supernatant of cells grown in the presence of naringenin showed that the major Nod factor produced by Bradyrhizobium sp. strain ORS285 is a modified chitin pentasaccharide molecule with a terminal N-C18:1-glucosamine and with a 2-O-methyl fucose linked to C-6 of the reducing glucosamine. In this respect, the Bradyrhizobium sp. strain ORS285 Nod factor is the same as the major Nod factor produced by the nonphotosynthetic Bradyrhizobium japonicum USDA110 that nodulates the roots of soybean. This suggests a classic nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and certain Aeschynomene spp. This is supported by the fact that B. japonicum USDA110 is able to form N2-fixing nodules on both the roots and stems of Aeschynomene afraspera.

International audience; Legumes establish mutualistic associations with mycorrhizal fungi and with nitrogen-fixing rhizobial bacteria. These interactions occur following plant recognition of Nod factor from rhizobial bacteria and Myc factor from mycorrhizal fungi [1-3]. A common symbiosis signaling pathway is involved in the recognition of both Nod factor and Myc factor and is required for the establishment of these two symbioses [4-6]. The outcomes of these associations differ, and therefore, despite the commonality in signaling, there must be mechanisms that allow specificity. In Nod factor signaling, a complex of GRAS-domain transcription factors controls gene expression downstream of the symbiosis signaling pathway. Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. Plants mutated in RAM1 are unable to be colonized by mycorrhizal fungi, with a defect in hyphopodia formation on the surface of the root. RAM1 is specifically required for Myc factor signaling and appears to have no role in Nod factor signaling. RAM1 regulates the expression of RAM2, a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. We conclude that mycorrhizal signaling downstream of the symbiosis-signaling pathway has parallels with nodulation-specific signaling and functions to promote mycorrhizal colonization by regulating cutin biosynthesis.

To analyse the regulation of the nodulation (nod) genes of Rhizobium meliloti RCR2011 we have isolated lacZ gene fusions to a number of common, host-range and regulatory nod genes, using the mini-Mu-lac bacteriophage transposon MudII1734. Common (nodA, nodC, nod region IIa) and host-range (nodE, nodG, nodH) genes were found to be regulated similarly. They were activated (i) by the regulatory nodD1 gene in the presence of flavones such as chrysoeriol, luteolin and 7,3',4'-trihydroxyflavone, (ii) by nodD2 in the presence of alfalfa root exudate but not with the NodD1-activating flavones, and (iii) by the regulatory genes syrM-nodD3 even in the absence of plant inducers. Thus common and host-range nod genes belong to the same regulon. In contrast to the nodD1 gene, the regulatory nodD3 gene was not expressed constitutively and exhibited a complex regulation. It required syrM for expression, was activated by nodD1 in the presence of luteolin and was positively autoregulated.

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.

Sinorhizobium meliloti nodulation factors (NFs) elicit a number of symbiotic responses in alfalfa (Medicago sativa) roots. Using a semiquantitative nodulation assay, we have shown that chemically synthesized NFs trigger nodule formation in the same range of concentrations (down to 10−10m) as natural NFs. The absence of O-sulfate orO-acetate substitutions resulted in a decrease in morphogenic activity of more than 100-fold and approximately 10-fold, respectively. To address the question of the influence of the structure of the N-acyl chain, we synthesized a series of sulfated tetrameric lipo-chitooligosaccharides (LCOs) having fatty acids of different lengths and with unsaturations either conjugated to the carbonyl group (2E) or located in the middle of the chain (9Z). A nonacylated, sulfated chitin tetramer was unable to elicit nodule formation. Acylation with short (C8) chains rendered the LCO active at 10−7m. The optimal chain length was C16, with the C16-LCO being more than 10-fold more active than the C12- and C18-LCOs. Unsaturations were important, and the diunsaturated 2E,9Z LCO was more active than the monounsaturated LCOs. We discuss different hypotheses for the role of the acyl chain in NF perception.

The family Leguminosae is one of the largest families of flowering plants, including more than 18,000 species (Young, Johnston, 1989). Their symbiotic bacterial partners are very diverse and do not form a discrete clade. Although they are collectively referred to as rhizobia, they are now classified in distinct Rhizobium, Bradyrhizobium, Azorhizobium and Sinorhizobium (Young, Haukka, 1996). Some rhizobia are indeed more closely related to nonsymbiotic bacteria than they are to other rhizobia. However, all rhizobial species possess common nodulation (nod) genes, the nodABC genes, and consequently produce nodulation signals, the Nod factors, that belong to the same chemical family: they are chitin oligomers of 4 or 5 glucosamine residues that are mono-N-acylated at the terminal non-reducing end. The common chitin oligomer core can be substituted at both ends of the molecule, and each rhizobial species or biovar produces a set of major Nod factors with characteristic substitutions (Denarie et al., 1996). These substitutions are under the control of specific nod genes. In the case of Sinorhizobium meliloti, it has been shown that the three substitutions are essential for effective infection of the host plant, alfalfa. The structure of major Nod factors has now been determined for most rhizobial species and their comparison indicates that Nod factor structure is related to rhizobial host range (Denarie et al., 1996).

Symbiotic bacteria of the genus Rhizobium synthesize lipo-chitooligosaccharides, called Nod factors (NFs), which act as morphogenic signal molecules on legume hosts. The common nodABC genes, present in all Rhizobium species, are required for the synthesis of the core structure of NFs. NodC is an N -acetylglucosaminyltransferase, and NodB is a chitooligosaccharide deacetylase; NodA is involved in N-acylation of the aminosugar backbone. Specific nod genes are involved in diverse NF substitutions that confer plant specificity. We transferred to R. tropici , a broad host-range tropical symbiont, the ability to nodulate alfalfa, by introducing nod genes of R. meliloti . In addition to the specific nodL and nodFE genes, the common nodABC genes of R. meliloti were required for infection and nodulation of alfalfa. Purified NFs of the R. tropici hybrid strain, which contained chitin tetramers and were partly N-acylated with unsaturated C16 fatty acids, were able to elicit nodule formation on alfalfa. Inactivation of the R. meliloti nodABC genes suppressed the ability of the NFs to nodulate alfalfa. Studies of NFs from nodA , nodB , nodC , and nodI mutants indicate that ( i ) NodA of R. meliloti , in contrast to NodA of R. tropici , is able to transfer unsaturated C16 fatty acids onto the chitin backbone and ( ii ) NodC of R. meliloti specifies the synthesis of chitin tetramers. These results show that allelic variation of the common nodABC genes is a genetic mechanism that plays an important role in signaling variation and in the control of host range.

The symbiotic association between Rhizobium and legumes is specific. For example R. meliloti nodulates Medicago, Melilotus and Trigonella species, R. tropici nodulates Phaseolus and Leucaena, and R. leguminosarum bv viciae ’s hosts belong to the Vicia and Pisum genera (van Rhijn, Vanderleyden, 1995). The bacterial genes controlling nodulation and host specificity (nod, nol and noe genes) have been classified into 3 groups: (i) The regulatory nodD genes activate the expression of the other nodulation genes in response to compounds secreted by the host plants. (ii) The common nodABC genes are found in all rhizobial species studied so far and are considered as functionally conserved between these species, (iii) The species-specific nod genes, such as nodH, nodL or nodFE in R. meliloti, determine the host range. Mutations in the latter genes generally result in an alteration of the host spectrum. In addition, these genes are required for transferring the host range between different rhizobial species

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

Rhizobia elicit the formation of nitrogen-fixing nodules on specific legume hosts. Rhizobium meliloti nodulation (nod) genes control a signal exchange between the two symbiotic partners during infection and the early steps of nodulation. The regulatory nodD1, nodD2 and nodD3 genes are involved in the specific perception of different plant and environmental signals and activate the transcription of the nod operons. Once activated, the structural nod genes specify the synthesis of diffusible lipo-oligosaccharides, the Nod factors, which signal back to the plant. R. meliloti Nod factors are sulfated chito-oligosaccharides which are mono-N-acylated by unsaturated C16 fatty acids or by a series of C18 to C26 (omega-1)-hydroxylated fatty acids. In this paper we show that the regulatory nodD3 gene and another symbiotic regulatory gene, syrM, which mediate bacterial responses to plant signals that differ from those involving nodD1 and nodD2, determine the synthesis of Nod factors with different acyl moieties. nodD3 and syrM are required for the synthesis of Nod factors N-acylated by the (omega-1)-hydroxylated fatty acids. This regulatory mechanism makes possible the qualitative adaptation of bacterial Nod signal production to plant signals in the course of the symbiotic process.

Rhizobium meliloti produces lipochitooligosaccharide nodulation NodRm factors that are required for nodulation of legume hosts. NodRm factors are O-acetylated and N-acylated by specific C16-unsaturated fatty acids. nodL mutants produce non-O-acetylated factors, and nodFE mutants produce factors with modified acyl substituents. Both mutants exhibited a significantly reduced capacity to elicit infection thread (IT) formation in alfalfa. However, once initiated, ITs developed and allowed the formation of nitrogen-fixing nodules. In contrast, double nodF/nodL mutants were unable to penetrate into legume hosts and to form ITs. Nevertheless, these mutants induced widespread cell wall tip growth in trichoblasts and other epidermal cells and were also able to elicit cortical cell activation at a distance. NodRm factor structural requirements are thus clearly more stringent for bacterial entry than for the elicitation of developmental plant responses.

Nodulation (nod) genes of Rhizobium, which control infection, nodulation and host specificity, determine the production of extracellular lipo-oligosaccharidic Nod factors. The Nod factors of various rhizobia, including the narrow host-range temperate R. meliloti and the broad host-range tropical Rhizobium sp. NGR234 belong to the same chemical family: they are mono-N-acylated and substituted chitin oligomers. The common nodC gene codes for a protein which has homology with chitin synthases and may be involved in the synthesis of the chitin backbone. The species-specific nod genes determine the decoration of these core molecules by encoding substitutions with specific groups at particular sites. For example in R. meliloti (i) the nodH and nodPQ genes are involved in the O-sulfation of the lipooligosaccharides on the carbon 6 of the reducing glucosamine; (ii) the nodL gene is required for the O-acetylation of the carbon 6 of the non-reducing terminal glucosamine; (iii) the nodFE genes specify the synthesis of the specific C16:2 N-acyl chain. Rhizobium sp. NGR234 secretes a large family of Nod factors carrying a variety of substituents. The non-reducing end is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups. The reducing glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated, or sulfated, or non-substituted. The O-acetylation and the O-sulfation are mutually exclusive: thus this broad host range strain secretes a variety of charged and uncharged Nod factors. We also present data which strongly suggest that the host range nod genes mediate host specificity by determining the type of substitutions decorating the lipo-oligosaccharidic Nod factors.

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non-legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents. The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups. The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted. All three internal residues are N-acetylated. At pico- to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals.

We have addressed the questions of how nodulation (nod) genes of the symbiotic bacterium R. meliloti operate to determine host specificity, plant (alfalfa) infection and nodulation. Using a root hair deformation assay we have shown that the common nodABC genes and the hostrange nodH and nodQ genes are all involved in the production of an extracellular signal. The structure of the major alfalfa-specific signal, NodRm-1, has been determined by mass spectrometry, NMR spectroscopy, radioactive labelling and chemical modifications. This signal is a sulphated and acylated glucosamine tetrasaccharide. NodRm-1 elicits root hair deformation, cortical cell divisions and nodule formation, on aseptically-grown alfalfa seedlings, at nanomolar concentrations. Hostrange nodH or nodQ mutants are able to infect a non-homologous host, common vetch and were found to excrete another signal, NodRm-2, which differs from NodRm-1 by the absence of the sulphate group. Purified NodRm-2 elicits hair deformation on vetch but not on alfalfa. The function of nodH and nodQ is to make the signal alfalfa-specific by transfer of a sulphate group. We propose that the nod genes of R.meliloti determine host specificity, root hair curling and nodule formation via the production of NodRm-1.

The infection of leguminous plants by Rhizobiwn and the subsequent codifferentiation of both organisms leads ultimately to the formation of the unique nitrogen-fixing plant organ known as the root nodule. Within the nodule the Rhizobium are furnished with photosynthate-derived energy and, in exchange, the endosymbiotic bacterium reduces atmospheric nitrogen to a form which can be assimilated by the host plant. This remarkable symbiosis has been the subject of extensive research for many years, but it is only recently that it has become possible to study, at the molecular level, the role of diffusible factors in plant-bacterial signalling and recognition, with the exciting prospect that such molecules might also be responsible for triggering plant morphogenesis.

The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation (nod) genes determine the production of acylated and sulfated glucosamine oligosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH+ or nodH- strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.

The infection of leguminous plants by Rhizobium and the subsequent co-differentiation of both organisms leads ultimately to the formation of the unique nitrogen-fixing plant organ known as the root nodule (16). Within the nodule, the Rhizobium are furnished with photosynthate-derived energy and, in exchange, the endosymbiotic bacterium reduces atmospheric nitrogen to a form which can be assimilated by the host plant. This remarkable symbiosis has been the subject of extensive research for many years, but it is only recently that it has become possible to study, at the molecular level, the role of diffusible factors in plant-bacterial signalling and recognition, with the exciting prospect that such molecules might also be responsible for triggering plant morphogenesis.

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.

The Rhizobium meliloti nod region IIb is involved in host-range determination: (i) the presence of region IIb is necessary for transfer of alfalfa root hair curling ability to Rhizobium leguminosarum biovar trifolii; (ii) a mutation in region IIb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region IIb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP-binding domain is conserved.

The Rhizobium meliloti nodH gene is involved in determining host range specificity. By comparison with the wild-type strain, NodH mutants exhibit a change in host specificity. That is, although NodH mutants lose the ability to elicit root hair curling (Hac-), infection threads (Inf-), and nodule meristem formation (Nod-) on the homologous host alfalfa, they gain the ability to be Hac+ Inf+ Nod+ on a nonhomologous host such as common vetch. Using root hair deformation (Had) bioassays on alfalfa and vetch, we have demonstrated that sterile supernatant solutions of R. meliloti cultures, in which the nod genes had been induced by the plant flavone luteolin, contained symbiotic extracellular signals. The wild-type strain produced at least one Had signal active on alfalfa (HadA). The NodH- mutants did not produce this signal but produced at least one factor active on vetch (HadV). Mutants altered in the common nodABC genes produced neither of the Had factors. This result suggests that the nodABC operon determines the production of a common symbiotic factor which is modified by the NodH product into an alfalfa-specific signal. An absolute correlation was observed between the specificity of the symbiotic behavior of rhizobial cells and the Had specificity of their sterile filtrates. This indicates that the R. meliloti nodH gene determines host range by helping to mediate the production of a specific extracellular signal.

Rhizobia are soil bacteria which can infect specifically the roots of leguminous hosts and elicit the formation of root nodules in which they reduce dinitrogen into ammonia. The degree of specificity of this relationship is extremely varied. Some tropical strains can nodulate and fix nitrogen on a large number of genera belonging to various tribes of Leguminosae and even on the non-legume genus Parasponia. On the other hand Rhizobium meliloti strains are reported to have a very limited host range : each strain can nodulate effectively only some species of Medicago, Melilotus and Trigonella (see the review of Long 1984). R. meliloti is thus a suitable organism with which to investigate the genetic and molecular basis of a highly specific plant-bacteria interaction.

Rhizobia are symbiotic bacteria that synthesize lipochitooligosaccharide Nod factors (NFs), which act as signal molecules in the nodulation of specific legume hosts. Based on the structure of their N-acyl chain, NFs can be classified into two categories: (i) those that are acylated with fatty acids from the general lipid metabolism; and (ii) those (= alphaU-NFs) that are acylated by specific alpha,beta-unsaturated fatty acids (containing carbonyl-conjugated unsaturation(s)). Previous work has described how rhizobia that nodulate legumes of the Trifolieae and Vicieae tribes produce alphaU-NFs. Here, we have studied the structure of NFs from two rhizobial species that nodulate important genera of the Galegeae tribe, related to Trifolieae and Vicieae. Three strains of Mesorhizobium huakuii, symbionts of Astragalus sinicus, produced as major NFs, pentameric lipochitooligosaccharides O-sulphated and partially N-glycolylated at the reducing end and N-acylated, at the non-reducing end, by a C18:4 fatty acid. Two strains of Rhizobium galegae, symbionts of Galega sp., produced as major NFs, tetrameric O-carbamoylated NFs that could be O-acetylated on the glucosamine residue next to the non-reducing terminal glucosamine and were N-acylated by C18 and C20 alpha,beta-unsaturated fatty acids. These results suggest that legumes nodulated by rhizobia synthesizing alphaU-NFs constitute a phylogenetic cluster in the Galegoid phylum.