Nodulin Genes Research Articles

Cloning and expression analysis of a MAPKKK gene and a novel nodulin gene of Lotus japonicus.

We isolated a cDNA encoding mitogen-activated protein kinase kinase kinase alpha, designated LjM3Kalpha, from Lotus japonicus, a model legume. The gene was expressed constitutively in roots, root nodules, and shoots. We also identified a novel nodulin gene, LjNUF, that shows specific expression in nodules. LjNUF resembles the C-terminal half of a hypothetical protein (pir//D85436), the N-terminal half of which is similar to a portion of mitogen-activated protein kinase kinase kinase gamma. Although LjNUF was predicted to be a secreted protein, its function remains to be clarified.

Promoter of soybean early nodulin gene enod2B is induced by rhizobial Nod factors in transgenic rice

Promoter of soybean early nodulin gene enod2B is induced by rhizobial Nod factors in transgenic rice

Phytohormonal responses in enod40-overexpressing plants of Medicago truncatula and rice.

Phytohormones are well-known regulators of the symbiotic Rhizobium-legume association in the plant host. The enod40 nodulin gene is associated with the earliest phases of the nodule organogenesis programme in the legume host and modifying its expression resulted in perturbations of nodule development in Medicago truncatula. Therefore in our pursuit to mimic the initial signal transduction steps of legume nodulation in the alien physiological set-up of a rice plant, we have expressed the Mtenod40 gene in rice. Molecular data confirm the stable integration, inheritance and transcription of the foreign gene in this non-legume. We have compared the phytohormonal responses of Mtenod40-overexpressing and control plants in a homologous legume background (M. truncatula) and in the non-legume rice. An enod40-mediated root growth response, induced by inhibition of ethylene biosynthesis, was observed in both plants. On the other hand, a significant differential effect of cytokinins was observed only in rice plants. This suggests that ethylene inhibits enod40 action both in legumes and non-legumes and reinforces that some of the early signal transduction steps of the nodule developmental programme may function in rice.

Agrobacterium rhizogenes-mediated transformation of Sesbania rostrata

In order to study the function of nodulin genes and the activity of their promoters, the Agrobacterium rhizogenes transformation strategy was adopted for Sesbania rostrata. Two protocols were selected to generate S. rostrata transgenic roots that can be nodulated efficiently after application of Azorhizobium caulinodans, ORS571. Cotransformation frequencies of 22 and 72% were obtained with the first and second protocol, respectively. The transgenic root nodules showed no apparent differences when compared with the wild-type root nodules. The screening procedure for cotransformation of binary T-DNA in transgenic roots and root nodules was optimized by using an enhanced green-fluorescent protein construct. The A. rhizogenes transformation system was used to analyze the 35S promoter-driven expression of reporter genes in developing root nodules.

Cg12 expression is specifically linked to infection of root hairs and cortical cells during Casuarina glauca and Allocasuarina verticillata actinorhizal nodule development.

cg12 is an early actinorhizal nodulin gene from Casuarina glauca encoding a subtilisin-like serine protease. Using transgenic Casuarinaceae plants carrying cg12-gus and cg12-gfp fusions, we have studied the expression pattern conferred by the cg12 promoter region after inoculation with Frankia. cg12 was found to be expressed in root hairs and in root and nodule cortical cells containing Frankia infection threads. cg12 expression was also monitored after inoculation with ineffective Frankia strains, during mycorrhizae formation, and after diverse hormonal treatments. None of these treatments was able to induce its expression, therefore suggesting that cg12 expression is linked to plant cell infection by Frankia strains. Possible roles of cg12 in actinorhizal symbiosis are discussed.

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.

Expression of the apyrase-like APY1 genes in roots of Medicago truncatula is induced rapidly and transiently by stress and not by Sinorhizobium meliloti or Nod factors.

The model legume Medicago truncatula contains at least six apyrase-like genes, five of which (MtAPY1;1, MtAPY1;2, MtAPY1;3, MtAPY1;4, and MtAPY1;5) are members of a legume-specific family, whereas a single gene (MtAPY2) has closer homologs in Arabidopsis. Phylogenetic analysis has revealed that the proteins encoded by these two plant gene families are more similar to yeast (Saccharomyces cerevisiae) GDA1 and to two proteins encoded by newly described mammalian genes (ENP5 and 6) than they are to mammalian CD39- and CD39-like proteins. Northern analyses and analyses of the frequencies of expressed sequence tags (ESTs) in different cDNA libraries suggest that in roots, leaves, and flowers, the more highly expressed genes are MtAPY1;3/MtAPY2, MtAPY1;3/MtAPY1;5 and MtAPY1;2/MtAPY1;3 respectively. In roots, at least four of the MtAPY1 genes are induced transiently within 3 to 6 h by a stress response that seems to be ethylene independent because it occurs after treatment with an ethylene synthesis inhibitor and also in the skl ethylene-insensitive mutant. This response also occurs in roots of the following symbiotic mutants: dmi1, dmi2, dmi3, nsp, hcl, pdl, lin, and skl. No evidence was obtained for a rapid, transient, and specific induction of the MtAPY genes in roots in response to rhizobia or rhizobial lipochitooligosaccharidic Nod factors. Thus, our data suggest that the apyrase-like genes, which in several legumes have been implicated to play a role in the legume-rhizobia symbiosis (with some members being described as early nodulin genes), are not regulated symbiotically by rhizobia in M. truncatula.

A white clover nodulin gene, dd23b , encoding a cysteine cluster protein, is expressed in roots during the very early stages of interaction with Rhizobium leguminosarum biovar trifolii and after treatment with chitolipooligosaccharide Nod factors

An early nodulin cDNA, dd23b, was isolated from white clover root tissue by differential display RT-PCR. Its full-length sequence of 340 nucleotides encodes a predicted 72-amino-acid protein of molecular mass 8.3 kDa, with a polypeptide region containing cysteine pairs spaced in the manner of a cysteine cluster protein. This feature, which is shared by some other late and early nodulins from pea and broad bean, suggests a role in metal ion binding and membrane transport. Temporal and spatial expression patterns were determined during infection and nodulation by the homologous microsymbiont. No expression was found in unchallenged root tissue over a 7-day sampling period. Expression was first detectable in roots by RT-PCR 6 h post-inoculation with Rhizobium leguminosarum biovar trifolii, placing dd23b among the earliest nodulins to be detected to date. In root nodules, expression occurred primarily in the central symbiotic zone, but also in some host cells within the infection zone. Addition of purified wild-type chitolipooligosaccharide Nod factor to axenic white clover roots induced dd23b expression, providing further evidence for the role of this gene in the early plant response to infection by rhizobia.

Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi.

• An in vitro targeted inoculation technique has been developed for studying the earliest stages of arbuscular endomycorrhizal (AM) infection of Medicago truncatula roots, and in particular the spatio-temporal expression of the early nodulin gene MtENOD11. • Agrobacterium rhizogenes transformed root explants were derived either from Myc + M. truncatula or from the infection-defective Myc - mutant TR26 ( dmi2-2 ), both expressing the pMtENOD11-gusA fusion. The normal positive geotropism of these roots, coupled with the negative geotropism of Gigaspora germ tubes allowed oriented growth of the two symbiotic partners, facilitating the identification of initial fungal/root contacts. • Early infection events at the stage of appressoria and/or internal hyphae could be observed for over 50% of the inoculated explants, revealing that MtENOD11 is expressed transiently in both epidermal and cortical cells at sites of hyphal penetration in Myc + roots, but not in epidermal cells in contact with appressoria in Myc - roots. • We propose that a direct link exists between MtENOD11 gene expression and cellular events required for fungal penetration, thereby extending analogies between rhizobial and AM host root infection processes.

The endosymbiosis-induced genes ENOD40 and CCS52a are involved in endoparasitic-nematode interactions in Medicago truncatula.

Plants associate with a wide range of mutualistic and parasitic biotrophic organisms. Here, we investigated whether beneficial plant symbionts and biotrophic pathogens induce distinct or overlapping regulatory pathways in Medicago truncatula. The symbiosis between Sinorhizobium meliloti and this plant results in the formation of nitrogen-fixing root nodules requiring the activation of specific genes in the host plant. We studied expression patterns of nodule-expressed genes after infection with the root-knot nematode Meloidogyne incognita. Two regulators induced during nodule organogenesis, the early nodulin gene ENOD40 involved in primordium formation and the cell cycle gene CCS52a required for cell differentiation and endoreduplication, are expressed in galls of the host plant. Expression analysis of promoter-uidA fusions indicates an accumulation of CCS52a transcripts in giant cells undergoing endoreduplication, while ENOD40 expression is localized in surrounding cell layers. Transgenic plants overexpressing ENOD40 show a significantly higher number of galls. In addition, out of the 192 nodule-expressed genes tested, 38 genes were upregulated in nodules at least threefold compared with control roots, but only two genes, nodulin 26 and cyclin D3, were found to be induced in galls. Taken together, these results suggest that certain events, such as endoreduplication, cell-to-cell communication with vascular tissues, or water transport, might be common between giant cell formation and nodule development.

Effects of a Nod-factor-overproducing strain of Sinorhizobium meliloti on the expression of the ENOD40 gene in Melilotus alba

We have initiated studies on the molecular biology and genetics of white sweetclover (Melilotus alba Desr.) and its responses to inoculation with the nitrogen-fixing symbiont Sinorhizobium meliloti. Early nodulin genes such as ENOD40 serve as markers for the transition from root to nodule development even before visible stages of nodule formation are evident. Using Northern blot analysis, we found that the ENOD40 gene was expressed within 6 h after inoculation with two different strains of S. meliloti, one of which overproduces symbiotic Nod factors. Inoculation with this strain resulted in an additional increase in ENOD40 gene expression over a typical wild-type S. meliloti strain. Moreover, the increase in mRNA brought about by the Nod-factor-overproducing strain 24 h after inoculation was correlated with lateral root formation by using whole-mount in situ hybridization to localize ENOD40 transcripts in lateral root meristems and by counting lateral root initiation sites. Cortical cell divisions were not detected. We also found that nodulation occurred more rapidly on white sweetclover in response to the Nod-factor-overproducing strain, but ultimately there was no difference in nodulation efficiency in terms of nodule number or the number of roots nodulated by the two strains. Also, the two strains could effectively co-colonize the host when inoculated together, although a few host cells were occupied by both strains.Key words: ENOD40, Nod factor, Melilotus, Sinorhizobium, symbiosis.

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

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

Nod factor structures, responses, and perception during initiation of nodule development.

The onset of nodule development, the result of rhizobia-legume symbioses, is determined by the exchange of chemical compounds between microsymbiont and leguminous host plant. Lipo-chitooligosaccharidic nodulation (Nod) factors, secreted by rhizobia, belong to these signal molecules. Nod factors consist of an acylated chitin oligomeric backbone with various substitutions at the (non)reducing-terminal and/or nonterminal residues. They induce the formation and deformation of root hairs, intra- and extracellular alkalinization, membrane potential depolarization, changes in ion fluxes, early nodulin gene expression, and formation of nodule primordia. Nod factors play a key role during nodule initiation and act at nano- to picomolar concentrations. A correct chemical structure is required for induction of a particular plant response, suggesting that Nod factor-receptor interaction(s) precede(s) a Nod factor-induced signal transduction cascade. Current data on Nod factor structures and Nod factor-induced responses are highlighted as well as recent advances in the characterization of proteins, possibly involved in recognition of Nod factors by the host plant.

Novel aspects of symbiotic nitrogen fixation uncovered by transcript profiling with cDNA arrays.

An array of 2,304 cDNA clones derived from nitrogen-fixing nodules of Lotus japonicus was produced and used to detect differences in relative gene transcript abundance between nodules and uninfected roots. Transcripts of 83 different genes were found to be more abundant in nodules than in roots. More than 50 of these have never before been identified as nodule-induced in any species. Expression of 36 genes was detected in nodules but not in roots. Several known nodulin genes were included among the nodule-induced genes. Also included were genes involved in sucrose breakdown and glycolysis, CO2 recycling, and amino acid synthesis, processes that are known to be accelerated in nodules compared with roots. Genes involved in membrane transport, hormone metabolism, cell wall and protein synthesis, and signal transduction and regulation of transcription were also induced in nodules. Genes that may subvert normal plant defense responses, including two encoding enzymes involved in detoxification of active oxygen species and one that may prohibit phytoalexin synthesis, were also identified. The data represent a rich source of information for hypothesis building and future exploration of symbiotic nitrogen fixation.

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi

The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod(-) and Myc(-) co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod(-) mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod(-) and Myc(-). In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.

Expression of genes encoding late nodulins characterized by a putative signal peptide and conserved cysteine residues is reduced in ineffective pea nodules.

Five nodulin genes, PsN1, PsN6, PsN314, PsN335, and PsN466, with reduced expression in ineffective nodules on the pea (Pisum sativum) mutant E135 (sym13) were characterized. They encode small polypeptides containing a putative signal peptide and conserved cysteine residues and show homology to the nodulins PsENOD3/14 and PsNOD6. For each gene, multiple bands were detected by genomic Southern analysis. Northern analysis showed that all five genes were expressed exclusively in nodules and that their temporal expression patterns were similar to that of the leghemoglobin (Lb) gene during nodule development. Their transcripts were localized predominantly from the interzone II-III to the distal part of nitrogen-fixing zone III in effective nodules, resembling the Lb gene. However, transcripts in ineffective E135 nodules were localized in narrower regions than those in the effective nodules. These results indicate that these nodulins are abundant in pea nodules and that their successive expression during nodule development is associated with nitrogen-fixing activity.

Medicago truncatula plants overexpressing the early nodulin gene enod40 exhibit accelerated mycorrhizal colonization and enhanced formation of arbuscules.

The mutualistic symbiosis between flowering plants and arbuscular mycorrhizal fungi is extremely abundant in terrestrial ecosystems. In this symbiosis, obligately biotrophic fungi colonize the root of the host plants, which can benefit from these fungi by enhanced access to mineral nutrients in the soil, especially phosphorus. One of the main goals of research on this symbiosis is to find plant genes that control fungal development in the host plant. In this work, we show that mycorrhizal colonization is regulated by enod40, an early nodulin gene known to be involved in the nodule symbiosis of legumes with nitrogen-fixing bacteria. Medicago truncatula plants overexpressing enod40 exhibited stimulated mycorrhizal colonization in comparison with control plants. Overexpression of enod40 promoted fungal growth in the root cortex and increased the frequency of arbuscule formation. Transgenic lines with suppressed levels of enod40 transcripts, likely via a cosuppression phenomenon induced by the transgene, exhibited reduced mycorrhizal colonization. Hence, enod40 might be a plant regulatory gene involved in the control of the mycorrhizal symbiosis.

Responses of a model legume Lotus japonicus to lipochitin oligosaccharide nodulation factors purified from Mesorhizobium loti JRL501.

Lotus japonicus has been proposed as a model legume for molecular genetic studies of symbiotic plant-microbe interactions leading to the fixation of atmospheric nitrogen. Lipochitin oligosaccharides (LCOs), or Nod factors, were isolated from the culture of Mesorhizobium loti strain JRL501 (MAFF303099), an efficient microsymbiont of L. japonicus B-129 cv. Gifu. High-performance liquid chromatography and mass spectrometric analyses allowed us to identify at least five different structures of LCOs that were produced by JRL501. The major component was NodMl-V(C18:1, Me, Cb, AcFuc), an N-acetyl-glucosamine pentamer in which the nonreducing residue is N-acylated with a C18:1 acyl moiety, N-methylated, and carries a carbamoyl group and the reducing N-acetylglucosamine residue is substituted with 4-O-acetyl-fucose. Additional novel LCO structures bearing fucose instead of acetyl-fucose at the reducing end were identified. Mixtures of these LCOs could elicit abundant root hair deformation on L. japonicus roots at a concentration of 10(-7) to 10(-9) M. Spot inoculation of a few nanograms of LCOs on L. japonicus roots induced the formation of nodule primordia in which the early nodulin genes, ENOD40 and ENOD2, were expressed in a tissue-specific manner. We also observed the formation of a cytoplasmic bridge (preinfection thread) in the swollen outermost cortical cells. This is the first description of cytoplasmic bridge formation by purified LCOs alone in a legume-forming determinate nodules.

Perception and Signal Transduction of Rhizobial NOD Factors

Referee: Dr. Gary Stacey, Director, Center for Legume Research, Department of Microbiology, M409 Walters Life Science Bldg., University of Tennessee, Knoxville, TN 37966-0845Soil bacteria belonging to genera Rhizobium, Bradyrhizobium, Allorhizobium, Azorhizobium, Mesorhizobium, and Sinorhizobium are able to induce nodule formation on the roots of leguminous plants. In the differentiated root nodules bacteria fix as bacteroids atmospheric nitrogen and deliver it to the host plant. The interaction between bacteria and host plant starts with a complex signal exchange. After induction by plant flavonoids, rhizobia synthesize and secrete lipo-chitooligosaccharides (LCOs), known as Nod factors, which induce morphological changes and expression of early nodulin genes in the roots of host plants. Specific recognition of Nod factors by host plants and early stages of signal transduction are discussed.

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.