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.
The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule ...formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium-host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod- and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.
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.
Legumes can enter into symbiotic relationships with both nitrogen-fixing bacteria (rhizobia) and mycorrhizal fungi. Nodulation by rhizobia results from a signal transduction pathway induced in legume ...roots by rhizobial Nod factors. DMI3, a Medicago truncatula gene that acts immediately downstream of calcium spiking in this signaling pathway and is required for both nodulation and mycorrhizal infection, has high sequence similarity to genes encoding calcium and calmodulin-dependent protein kinases (CCaMKs). This indicates that calcium spiking is likely an essential component of the signaling cascade leading to nodule development and mycorrhizal infection, and sheds light on the biological role of plant CCaMKs.
Legumes form two different types of intracellular root symbioses, with fungi and bacteria, resulting in arbuscular mycorrhiza and nitrogen-fixing nodules, respectively. Rhizobial signalling ...molecules, called Nod factors, play a key role in establishing the rhizobium-legume association and genes have been identified in Medicago truncatula that control a Nod factor signalling pathway leading to nodulation. Three of these genes, the so-called DMI1, DMI2 and DMI3 genes, are also required for formation of mycorrhiza, indicating that the symbiotic pathways activated by both the bacterial and the fungal symbionts share common steps. To analyse possible cross-talk between these pathways we have studied the effect of treatment with Nod factors on mycorrhization in M. truncatula. We show that Nod factors increase mycorrhizal colonization and stimulate lateral root formation. The stimulation of lateral root formation by Nod factors requires both the same structural features of Nod factors and the same plant genes (NFP, DMI1, DMI2, DMI3 and NSP1) that are required for other Nod factor-induced symbiotic responses such as early nodulin gene induction and cortical cell division. A diffusible factor from arbuscular mycorrhizal fungi was also found to stimulate lateral root formation, while three root pathogens did not have the same effect. Lateral root formation induced by fungal signal(s) was found to require the DMI1 and DMI2 genes, but not DMI3. The idea that this diffusible fungal factor might correspond to a previously hypothesized mycorrhizal signal, the 'Myc factor', is discussed.
Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts, in a specific manner, the formation of nodules in which they fix nitrogen. ...Rhizobial nod genes, which determine host specificity, infection, and nodulation, are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins, which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in turn in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule-meristem formation.
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 signalling pathway between nodulation and mycorrhization.
Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia. Several of the plant genes required for transduction of rhizobial signals, the Nod ...factors, are also necessary for mycorrhizal symbiosis. Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula. The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea. The protein is highly conserved in angiosperms and ancestral to land plants. We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.
Using dual cultures of arbuscular mycorrhizal (AM) fungi and Medicago truncatula separated by a physical barrier, we demonstrate that hyphae from germinating spores produce a diffusible factor that ...is perceived by roots in the absence of direct physical contact. This AM factor elicits expression of the Nod factor-inducible gene MtENOD11, visualized using a pMtENOD11-gusA reporter. Transgene induction occurs primarily in the root cortex, with expression stretching from the zone of root hair emergence to the region of mature root hairs. All AM fungi tested (Gigaspora rosea, Gigaspora gigantea, Gigaspora margarita, and Glomus intraradices) elicit a similar response, whereas pathogenic fungi such as Phythophthora medicaginis, Phoma medicaginis var pinodella and Fusarium solani f.sp. phaseoli do not, suggesting that the observed root response is specific to AM fungi. Finally, pMtENOD11-gusA induction in response to the diffusible AM fungal factor is also observed with all three M. truncatula $\text{Nod}^{-}/\text{Myc}^{-}$ mutants (dmi1, dmi2, and dmi3), whereas the same mutants are blocked in their response to Nod factor. This positive response of the $\text{Nod}^{-}/\text{Myc}^{-}$ mutants to the diffusible AM fungal factor and the different cellular localization of pMtENOD11-gusA expression in response to Nod factor versus AM factor suggest that signal transduction occurs via different pathways and that expression of MtENOD11 is differently regulated by the two diffusible factors.
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.
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