Expression of the Medicago truncatula DM12 gene suggests roles of the symbiotic nodulation receptor kinase in nodules and during early nodule development

The Medicago truncatula DMI2 gene encodes a receptorlike kinase required for establishing root endosymbioses. The DMI2 gene was shown to be expressed much more highly in roots and nodules than in leaves and stems. In roots, its expression was not altered by nitrogen starvation or treatment with lipochitooligosaccharidic Nod factors. Moreover, the DMI2 mRNA abundance in roots of the nfp, dmil, dmi3, nsp1, nsp2, and hcl symbiotic mutants was similar to the wild type, whereas lower levels in some dmi2 mutants could be explained by regulation by the nonsense-mediated decay, RNA surveillance mechanism. Using pDMI2::GUS fusions, the expression of DMI2 in roots appeared to be localized primarily in the cortical and epidermal cells of the younger, lateral roots and was not observed in the root apices. Following inoculation with Sinorhizobium meliloti, the DMI2 gene was induced in the nodule primordia, before penetration by the infection threads. No increased expression was seen in lateral-root primordia. In nodules, expression was observed primarily in a few cell layers of the pre-infection zone. These results are consistent with the DMI2 gene mediating Nod factor perception and transduction leading to rhizobial infection, not only in root epidermal cells but also during nodule development.     

Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling

Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.     

Rhizobium meliloti host range nodH gene determines production of an alfalfa-specific extracellular signal.

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.     

A non-nodulating alfalfa mutant displays neither root hair curling nor early cell division in response to Rhizobium meliloti.

The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.     

The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation

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.     

Four genes of Medicago truncatula controlling components of a nod factor transduction pathway

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.     

Symbiotic characterization of isoleucine+valine and leucine auxotrophs of Sinorhizobium meliloti

Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.     

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.     

Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase

The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11::GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.     

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.     

Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements.

In situ immunolocalization of tubulin revealed that important rearrangements occur during all the early symbiotic steps in the Medicago/R. meliloti symbiotic interaction. Microtubular cytoskeleton (MtC) reorganizations were observed in inner tissues, first in the pericycle and then in the inner cortex where the nodule primordium forms. Subsequently, major MtC changes occurred in outer tissues, associated with root hair activation and curling, the formation of preinfection threads (PITs) and the initiation and the growth of an infection network. From the observed sequence of MtC changes, we propose a model which aims to better define, at the histological level, the timing of the early symbiotic stages. This model suggests the existence of two opposite gradients of cell differentiation controlling respectively the formation of division centers in the inner cortex and plant preparation for infection. It implies that (i) MtC rearrangements occur in pericycle and inner cortex earlier than in the root hair, (ii) the infection process proceeds prior to the formation of the nodule meristem, (iii) the initial primordium prefigures the future zone II of the mature nodule and (iv) the nodule meristem derives from the nodule primordium. Finally, our data also strongly suggest that in alfalfa PIT differentiation, a stage essential for successful infection, requires complementary signaling additional to Nod factors.     

Calcium oscillations and Nod signal transduction in Rhizobium-legume symbiosis

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting a number of key developmental responses in the roots of legume hosts. One of the earliest responses of root hairs to Nod factors is the induction of sharp oscillations of cytoplasmic calcium ion concentration ("calcium spiking"). This response was first characterised in Medicago sativa and Nod factors were found to be unable to induce calcium spiking in a nodulation-defective mutant of M. sativa. The fact that this mutant lacked any morphological response to Nod factors raised the question of whether calcium spiking could be part of a Nod factor-induced signal transduction pathway leading to nodulation. More recently, calcium spiking has been described in a model legume, Medicago truncatula, and in pea. When nodulation-defective mutants were tested for the induction of calcium spiking in response to Nod factors, three loci of pea and two of M. truncatula were found to be necessary for Nod factor-induced calcium spiking. These loci are also known to be necessary for Nod factor-induction of symbiotic responses such as root hair deformation, nodulin gene expression and cortical cell division. These results therefore constitute strong genetic evidence for the role of calcium spiking in Nod factor transduction. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.     

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Identification of symbiotically defective mutants of Lotus japonicus affected in infection thread growth

During the symbiotic interaction between legumes and rhizobia, the host cell plasma membrane and associated plant cell wall invaginate to form a tunnel-like infection thread, a structure in which bacteria divide to reach the plant root cortex. We isolated four Lotus japonicus mutants that make infection pockets in root hairs but form very few infection threads after inoculation with Mesorhizobium loti. The few infection threads that did initiate in the mutants usually did not progress further than the root hair cell. These infection-thread deficient (itd) mutants were unaffected for early symbiotic responses such as calcium spiking, root hair deformation, and curling, as well as for the induction of cortical cell division and the arbuscular mycorrhizal symbiosis. Complementation tests and genetic mapping indicate that itd2 is allelic to Ljsym7, whereas the itdl, itd3, and itd4 mutations identified novel loci. Bacterial release into host cells did occur occasionally in the itdl, itd2, and itd3 mutants suggesting that some infections may succeed after a long period and that infection of nodule cells could occur normally if the few abnormal infection threads that were formed reached the appropriate nodule cells.     

The role of nod factor substituents in actin cytoskeleton rearrangements in Phaseolus vulgaris

In order to define the symbiotic role of some of the chemical substituents in the Rhizobium etli Nod factors (NFs), we purified Nod metabolites secreted by the SM25 strain, which carries most of the nodulation genes, and SM17 with an insertion in nodS. These NFs were analyzed for their capabilities to induce root hair curling and cytoskeletal rearrangements. The NFs secreted by strain SM17 lack the carbamoyl and methyl substituents on the nonreducing terminal residue and an acetyl moiety on the fucosyl residue on the reducing-terminal residue as determined by mass spectrometry. We have reported previously that the root hair cell actin cytoskeleton from bean responds with a rapid fragmentation of the actin bundles within 5 min of NF exposure, and also is accompanied by increases in the apical influxes and intracellular calcium levels. In this article, we report that methyl-bearing NFs are more active in inducing root hair curling and actin cytoskeleton rearrangements than nonmethylated NFs. However, the carbamoyl residue on the nonreducing terminal residue and the acetyl group at the fucosyl residue on the reducing terminal residue do not seem to have any effect on root hair curling induction or in actin cytoskeleton rearrangement.     

Early symbiotic responses induced by Sinorhizobium meliloti iIvC mutants in alfalfa

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

The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti

Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.     

Nod factor elicits two separable calcium responses in Medicago truncatula root hair cells

Modulation of intracellular calcium levels plays a key role in the transduction of many biological signals. Here, we characterize early calcium responses of wild-type and mutant Medicago truncatula plants to nodulation factors produced by the bacterial symbiont Sinorhizobium meliloti using a dual-dye ratiometric imaging technique. When presented with 1 nM Nod factor, root hair cells exhibited only the previously described calcium spiking response initiating 10 min after application. Nod factor (10 nM) elicited an immediate increase in calcium levels that was temporally earlier and spatially distinct from calcium spikes occurring later in the same cell. Nod factor analogs that were structurally related, applied at 10 nM, failed to initiate this calcium flux response. Cells induced to spike with low Nod factor concentrations show a calcium flux response when Nod factor is raised from 1 to 10 nM. Plant mutants previously shown to be deficient for the calcium spiking response (dmi1 and dmi2) exhibited an immediate, truncated calcium flux with 10 nM Nod factor, demonstrating a competence to respond to Nod factor but an impaired ability to generate a full biphasic response. These results demonstrate that the legume root hair cell exhibits two independent calcium responses to Nod factor triggered at different agonist concentrations and suggests an early branch point in the Nod factor signal transduction pathway.