Early Nodulin Gene Expression Research Articles

Reactive Oxygen Species and Nitric Oxide Control Early Steps of the Legume - Rhizobium Symbiotic Interaction.

The symbiotic interaction between legumes and nitrogen-fixing rhizobium bacteria leads to the formation of a new organ, the nodule. Early steps of the interaction are characterized by the production of bacterial Nod factors, the reorientation of root-hair tip growth, the formation of an infection thread (IT) in the root hair, and the induction of cell division in inner cortical cells of the root, leading to a nodule primordium formation. Reactive oxygen species (ROS) and nitric oxide (NO) have been detected in early steps of the interaction. ROS/NO are determinant signals to arbitrate the specificity of this mutualistic association and modifications in their content impair the development of the symbiotic association. The decrease of ROS level prevents root hair curling and ITs formation, and that of NO conducts to delayed nodule formation. In root hairs, NADPH oxidases were shown to produce ROS which could be involved in the hair tip growth process. The use of enzyme inhibitors suggests that nitrate reductase and NO synthase-like enzymes are the main route for NO production during the early steps of the interaction. Transcriptomic analyses point to the involvement of ROS and NO in the success of the infection process, the induction of early nodulin gene expression, and the repression of plant defense, thereby favoring the establishment of the symbiosis. The occurrence of an interplay between ROS and NO was further supported by the finding of both S-sulfenylated and S-nitrosylated proteins during early symbiotic interaction, linking ROS/NO production to a redox-based regulation of the symbiotic process.

Early responses to Nod factors and mycorrhizal colonization in a non-nodulating Phaseolus vulgaris mutant

Legumes can acquire nitrogen through a symbiotic interaction with rhizobial bacteria. The initiation of this process is determined by a molecular dialogue between the two partners. Legume roots exude flavonoids that induce the expression of the bacterial nodulation genes, which encode proteins involved in the synthesis and secretion of signals called Nod factors (NFs). NFs signal back to the plant root and trigger several responses, leading to bacterial invasion and nodule formation. Here, we describe the molecular and cellular characterization of a Phaseolus vulgaris non-nodulating mutant (NN-mutant). Root hair cells of the NN-mutant plant respond with swelling and branching when inoculated with Rhizobium etli, albeit without curling induction. Furthermore, neither initiation of cell division in the outer cortex, nor entrapment of bacteria nor infection thread formation was observed. Both the bean wild-type and the NN-mutant responded with elevated intracellular calcium changes in the root hairs. Although the NN-mutant is deficient in early nodulin gene expression when inoculated with R. etli, it can be effectively colonized by arbuscular mycorrhizal fungi (Glomus intraradices). Our data indicate that the P. vulgaris NN-mutant is not blocked at the NFs early perception stage, but at later downstream stages between Ca(2+) signaling and early nodulin induction. This supports the idea that both microsymbionts are perceived and trigger different downstream pathways in the host plant.

Promoter of soybean early nodulin geneenod2B is induced by rhizobial Nod factors in transgenic rice

Nod factors, which are signaling molecules produced byRhizobia, are the principal determinants of host specificity inRhizobium-legume symbiosis. Nod factors can elicit a number of characteristic developmental responses in the roots of legumes, such as depolarization of the membrane potential in epidermal cells, specific expression of early nodulin genes and changes in the flux of calcium in root hairs, deformation of root hairs, cell division in the root cortex and formation of the nodule primordium. Whether the rice plant can respond to signaling molecules (i.e. Nod factors) is an important question, as it could establish the potential for symbiotic nitrogen fixation in rice. The promoter of the soybean (Glycine max) early nodulin geneGmenod2B fused to the β-glucuronidase (GUS) reporter gene was used as a molecular marker to explore whether Nod factors can be recognized by rice cells as signaling molecules. Transgenic rice plants harboring the chimeric geneGmenod2BP-GUS were obtained via anAgrobacterium tumefaciens-mediated system. NodNGR factors produced by a broad-host-rangeRhizobium strain NGR234(pA28) were used as probes to investigate the activity of theGmenod2B promoter in rice. Our results showed that the early nodulin geneGmenod2B promoter was induced by NodNGR factors in transgenic rice, and that it was specifically expressed in rice plant roots. Moreover, GUS gene expression driven by theGmenod2B promoter in transgenic rice was regulated by nitrogen status. These findings indicated that rice possessed the ability to respond to Nod factor signals, and that this signal transduction system resulted in activation of theGmenod2B promoter. Thus, we predict that the Nod-factor inducible nodulin expression system, which is similar toRhizobium-legume symbiosis, may also exist in rice.

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.

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.

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.

Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved.

Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses-nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

Nod factor-induced expression of leghemoglobin to study the mechanism of NH4NO3 inhibition on root hair deformation.

Nod factors secreted by Rhizobium leguminosarum by, viciae induce root hair deformation, the formation of nodule primordia, and the expression of early nodulin genes in Vicia sativa (vetch). Root hair deformation is induced within 3 h in a small, susceptible zone (+/-2 mm) of the root. NH4NO3, known to be a potent blocker of nodule formation, inhibits root hair deformation, initial cortical cell divisions, and infection thread formation. To test whether NH4NO3 affects the formation of a component of the Nod factor perception-transduction system, we studied Nod factor-induced gene expression. The differential display technique was used to search for marker genes, which are induced within 1 to 3 h after Nod factor application. Surprisingly, one of the isolated cDNA clones was identified as a leghemoglobin gene (VsLb1), which is induced in vetch roots within 1 h after Nod factor application. By using the drug brefeldin A, it was then shown that VsLb1 activation does not require root hair deformation. The pVsLb1 clone was used as a marker to show that in vetch plants grown in the presence of NH4NO3, Nod factor perception and transduction leading to gene expression are unaffected.

Early nodulin gene expression during Nod factor‐induced processes in Vicia sativa

Rhizobium leguminosarum bv. viciae-secreted Nod factors are able to induce root hair deformation, the formation of nodule primordia and the expression of early nodulin genes in Vicia sativa (vetch). To obtain more insight into the mode of action of Nod factors the expression of early nodulin genes was followed during Nod factor-induced root hair deformation and nodule primordium formation. The results of these studies suggested that the expression of VsENOD5 and VsENOD12 is not required for root hair deformation. In the Nod factor-induced primordia both VsENOD12 and VsENOD40 are expressed in a spatially controlled manner similar to that found in Rhizobium-induced nodule primordia. In contrast, VsENOD5 expression has never been observed in Nod factor-induced primordia, showing that the induction of VsENOD5 and VsENOD12 expression are not coupled. VsENOD5 expression is induced in the root epidermis by Nod factors and in Rhizobium-induced nodule primordia only in cells infected by the bacteria, suggesting that the Nod factor does not reach the inner cortical cells.

Lipo‐oligosaccharides of Rhizobium induce infection‐related early nodulin gene expression in pea root hairs

This paper shows that lipo-oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection-related early nodulin genes (PsENOD5 and PsENOD12) in pea root hairs. R. leguminosarum bv. viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1). Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12, but the kinetics of induction are different. The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed. In spite of the host-specific nature of the infection process, heterologous Nod factors of R. meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.

The Relationship between Nodulin Gene Expression and theRhizobium nodGenes inVicia sativaRoot Nodule Development

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.

Rhizobium nod genes are involved in the induction of two early nodulin genes in Vicia sativa root nodules

Nodulin gene expresison was studied in Vicia sativa (common vetch) root nodules induced by several Rhizobium and Agrobacterium strains. An Agrobacterium transconjugant containing a R. leguminosarum symplasmid instead of its Ti-plasmid, that was previously shown to form "empty" nodules on pea, induced nodules on Vicia roots in which nodule cells were infected with bacteria. In the Vicia nodules induced by this transconjugant, two so-called early nodulin genes were found to be expressed, whereas in the nodules formed on pea the expression of only one early nodulin gene was detected. In both cases the majority of the nodulin genes was not expressed.Apparently, an intracellular location of the bacteria is not sufficient for the induction of the majority of the nodulin genes. All nodulin genes were expressed in nodules induced by cured Rhizobium strains containing cosmid clones that have a 10 kb nod region of the sym-plasmid in common. Since in tumours no nodulin gene expression was found at all, the Agrobacterium chromosome does not contribute to the induction of nodulin genes. Therefore it is concluded that the signal for the induction of the expression of the two Vicia early nodulin genes is encoded by the nod-region, and the signal involved in the induction of all other nodulin genes has to be located outside the sym-plasmid, on the Rhizobium chromosome. The apparent difference in early nodulin gene expression between pea and Vicia is discussed in the light of the usefulness of Agrobacterium transconjugants in the study of nodulin gene expression.