Nod Gene Induction Research Articles

Production ofSinorhizobium meliloti nodGene Activator and Repressor Flavonoids fromMedicago sativaRoots

During symbiosis between leguminous plants and rhizobia, flavonoids exuded by the plants act as chemoattractants and nodulation (nod) gene regulators in the other partner. To better understand the role of these compounds during the early steps of the alfalfa-Sinorhizobium meliloti symbiosis and the regulation of their production we have isolated nod gene inducers from alfalfa roots. All the compounds that we identified in this study as nod gene inducers in the root are flavonoids, indicating that other compounds with nod gene activator capacity may have little contribution, if any, to nod gene activation. Most of the intermediates of the flavonoid pathway were found in Medicago sativa roots and nodules, but only end products of the flavonoid pathway were identified in the root exudate. We have also studied flavonoid production in different parts of the root and found that it is developmentally regulated during root growth. Finally, we have shown that coumestrol and medicarpin, present in the exudates and previously described as phytoalexins, possess nod gene repressing activity, indicating that the in vivo nod gene inducing activity of the root exudate results from positive as well as negative controls of nod gene expression by the flavonoids.

The Rhizobium sp. BR816 nodD3 gene is regulated by a transcriptional regulator of the AraC/XylS family.

Rhizobium sp. BR816 contains four nodD alleles of which nodD3 is the most important transcriptional regulator for nodulation of Phaseolus vulgaris. Upstream of nodD3 an open reading frame, orf816, was identified. The deduced ORF816 protein shows homology with transcriptional regulators of the AraC/XylS family. The DNA binding domain and the consensus motif, characteristic of the C-terminal region of the members of this family of transcriptional regulators, are present in the deduced ORF816 protein. Activation of nodA gene expression and nodulation of P. vulgaris by Rhizobium sp. NGR234nodD1::omega (Nod-) complemented with the Rhizobium sp. BR816 nodD3 gene were significantly increased in the presence of orf816. This increased nodulation and nod gene induction are mediated through positive regulation of nodD3 expression by ORF816. Expression of orf816 itself is partially RpoN dependent. The role of this transcriptional regulator in the complex cascade regulation of the Rhizobium sp. nodD3 gene is discussed.

Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells.

Reporter strains of soil-borne bacteria were used to test the hypothesis that chemicals released by root border cells can influence the expression of bacterial genes required for the establishment of plant-microbe associations. Promoters from genes known to be activated by plant factors included virE, required for Agrobacterium tumefaciens pathogenesis, and common nod genes from Rhizobium leguminosarum bv viciae and Rhizobium meliloti, required for nodulation of pea (Pisum sativum) and alfalfa (Medicago sativum), respectively. Also included was phzB, an autoinducible gene encoding the biosynthesis of antibiotics by Pseudomonas aureofaciens. The virE and nod genes were activated to different degrees, depending on the source of border cells, whereas phzB activity remained unaffected. The homologous interaction between R. leguminosarum bv viciae and its host, pea, was examined in detail. Nod gene induction by border cells was dosage dependent and responsive to environmental signals. The highest levels of gene induction by pea (but not alfalfa) border cells occurred at low temperatures, when little or no bacterial growth was detected. Detached border cells cultured in distilled water exhibited increased nod gene induction (ini) in response to signals from R. leguminosarum bv viciae.

Identification and characterization of flavonoids in the root exudate of Robinia pseudoacacia

Eight compounds exuded from young roots of black locust (Robinia pseudoacacia) were separated by two-dimensional HPTLC, by HPLC and GC, and were identified by spectroscopic methods (ultraviolet/visible spectroscopy and mass spectrometry) as 4′,7-dihydroxyflavone, apigenin, naringenin, chrysoeriol and isoliquiritigenin. Structural assignments were confirmed by comparison with authentic standards. The capacity to induce β-galactosidase activity in Rhizobium sp. NGR234 containing a nod box::lacZ fusion on plasmid pA27 identified these flavonoids and the chalcone as nod gene inducers. This indicates the important role of these compounds in nodulation of this legume tree.

Inhibition of the expression of Bradyrhizobium japonicum nod genes at low temperatures

The effect of temperature on the induction of nod gene expression in Bradyrhizobium japonicum by the isoflavone genistein was investigated. Experiments were conducted to examine the expression of a nodY-lacZ fusion of B. japonicum USDA110 under suboptimal incubation temperatures. The results of these studies indicated that higher genistein concentrations were required for maximum amounts of nodY-lacZ induction at 10 and 15°C than at 25°C. In addition, the time required to achieve maximal amounts of expression was significantly longer at the suboptimal temperatures. The effects of temperature on nod gene expression correlate with previously described inhibition of soybean nodulation that occurs at low root zone temperatures. The findings reported here indicate that this nodulation inhibition is in large part due to temperature effects on nod gene induction.

Transcriptional activity of the symbiotic plasmid of Rhizobium etli is affected by different environmental conditions

Global patterns of transcriptional activity of the symbiotic plasmid (pSym) of Rhizobium etli were studied under a variety of environmental conditions, including some relevant to the symbiotic process. 32P-labelled single-stranded complementary DNAs synthesized from total RNA were used as hybridization probes against an ordered collection of cosmid clones that covered the whole pSym. Our results showed that, under aerobic conditions, discrete regions of the pSym are differentially transcribed depending on the carbon and nitrogen sources employed. In general, poor carbon or nitrogen sources allowed greater expression than rich ones. Time-course experiments with the nod gene inducer genistein led us to the identification of new regions responsive to this flavonoid. Widespread transcription was observed during microaerobiosis, but not under aerobic conditions, indicating that oxygen concentration is a major effector of transcriptional activity in the pSym. This response was reduced, but not suppressed, in a nifA mutant, indicating the location of regions whose transcription may depend on other oxygen-sensitive regulators. During symbiosis, almost the entire pSym was actively transcribed and the transcription pattern was similar to that observed during microaerobiosis. The experimental approach described allowed the identification and localization of specific regions in the pSym whose expression depends on defined environmental stimuli.

The role of the nodI and nodJ genes in the transport of Nod metabolites in Rhizobium etli

A kinetic analysis of secretion of lipo-chitin oligosaccharides (LCO) produced by Rhizobium etli (Re) wild-type (wt) strain and derivatives carrying disrupted nodI or nodJ genes was performed. LCO were detected in the growth media of the wt strain as early as 1 h after nod gene induction. In contrast, strains carrying nodI or nodJ mutations secreted less LCO, and accumulated LCO metabolites intracellularly after 4 h of induction. These Re mutants presented a delayed nodulation phenotype and a reduction in the maximum number of nodules formed in Phaseolus vulgaris roots.

Assimilation of nod gene inducer 14C-naringenin and the incorporation of labelled carbon atoms into the acyl side chain of a host-specific Nod factor produced by Rhizobium leguminosarum bv. viciae

The fate of 14C-naringenin during its specific activation of nod genes in Rhizobium leguminosarum bv. viciae was examined. After incubation with either strain RBL5560 or its pSym-cured derivative in a medium supplemented with 14 C-naringenin at nod gene-inducing concentrations of 2 nM (ca. 12.5 kBq) plus cold acetate (0.5 μM), a radiocarbon inventory for the cells and supernatant extracts was obtained. The level of 14 C-label incorporation was also determined in the fractionated cellular components. Using 14 C-acetate at 0.5 μM (1036 kBq) and cold naringenin (2 nM) in incubations with strain RBL5560 as a separate treatment, the Nod metabolites were detected by thin layer and high performance liquid chromatographic methods and the data provided the basis for identification of the Nod factors from the supernatant obtained from 14 C-naringenin treatments. Subsequent radio-biochemical and chemical analyses revealed that RBL5560 cells assimilated 14 C-naringenin during the activation of nod genes. Our analysis also showed that labelled carbon atoms from the 14 C-naringenin were incorporated into the acyl moiety of a lipo-oligosaccharide Nod factor, NodRlv IV, present in the culture supernatants of RBL5560. The pSym-cured derivative failed to synthesize any Nod metabolites in a 14 C-naringenin supplemented medium. The tracing of flavonoid-derived carbon atoms to the acyl chain of a host-specific Nod factor, a moiety that defines host specificity for this Rhizobium, adds a new dimension to the signalling function of flavonoids in legume-Rhizobium interactions.

Promotion of nod Gene Inducers and Nodulation in Common Bean (Phaseolus vulgaris) Roots Inoculated with Azospirillum brasilense Cd

Inoculation of Phaseolus vulgaris with Azospirillum brasilense Cd promoted root hair formation in seedling roots and significantly increased total and upper nodule numbers at different concentrations of Rhizobium inoculum. In experiments carried out in a hydroponic system, A. brasilense caused an increase in the secretion of nod gene-inducing flavonoids, as was observed by nod gene induction assays of root exudates fractionated by high-performance liquid chromatography. Possible mechanisms involved in the influence of A. brasilense on this symbiotic system are discussed.

Rhizobium Inoculation Induces Condition-Dependent Changes in the Flavonoid Composition of Root Exudates From Trifolium subterraneum

Rapid induction of chalcone synthase (predominantly CHSS) gene expression occurs within 6 h following the inoculation of Rhizobium leguminosarum bv. trifolii strain ANU843 on Trifolium subterraneum or wounding of plants (C. G. R. Lawson, M. A. Djordjevic, J. J. Weinman and B. G. Rolfe. 1994. Molecular Plant-Microbe Interactions 7, 498-507). Experiments were conducted under the same conditions to examine the time of onset of synthesis and excretion of flavonoids that might result from this early CHS expression. Flavonoids in root tissues and root exudates were examined by HPLC analysis and the ability of fractionated and unfractionated material to induce nodulation gene expression in Rhizobium measured. There were no detectable changes in nod-gene-inducing activity of individual HPLC fractions of root exudates of 1 day dark-grown roots after Rhizobium inoculation. In contrast, after 3 days exposure to Rhizobium, analysis of specific HPLC fractions showed the presence of an additional nod-gene-inducing compound which the data indicate was 4′,7-dihydroxyflavone. A different and additional nod gene inducer was found in inoculated 5 day samples of root exudate of light-grown plants indicating that light exposure changes the HPLC profiles as well as the nod-gene-inducing compound(s). Exudates collected from wounded plants were considerably different from those from Rhizobium-inoculated and uninoculated plants and contained no detectable nod gene inducers. The late detection (at day 3) of Rhizobium-induced flavonoid excretion may occur too late to be directly correlated with the observed expression of CHS 6 h after inoculation. In addition, the data suggest that although the CHS5 promotor responds to both wounding and Rhizobium inoculation, the biochemical consequences of CHS5 induction resulting from these treatments are different.

Metabolic engineering: prospects for crop improvement through the genetic manipulation of phenylpropanoid biosynthesis and defense responses — a review

In leguminous plants such as the forage legume alfalfa, products of the phenylpropanoid pathway of secondary metabolism are involved in interactions with beneficial microorganisms (flavonoid inducers of the Rhizobium symbiosis), and in defense against pathogens (isoflavonoid phytoalexins). In addition, the phenylpropane polymer lignin is a major structural component of secondary vascular tissue and fibers in higher plants. The recent isolation of genes encoding key enzymes of the various phenylpropanoid branch pathways opens up the possibility of engineering important crop plants such as alfalfa for: (a) improved forage digestibility, by modification of lignin composition and/or content; (b) increased or broader-spectrum disease resistance, by introducing novel phytoalexins or structural variants of the naturally occurring phytoalexins, or by modifying expression of transcriptional regulators of phytoalexin pathways; and (c) enhanced nodulation efficiency, by engineering over-production of flavonoid nod gene inducers. The basic biochemistry and molecular biology underlying these strategies is briefly reviewed, and recent progress with transgenic plants summarized. The potential importance of metabolic compartmentation for attempts to engineer phenylpropanoid biosynthetic pathways is also discussed. Over-expression of an alfalfa glucanase-encoding gene confers significant protection against Phytophthora in alfalfa, possibly via indirect effects on phenylpropanoid metabolism.

Inoculation withAzospirillumIncreased Exudation of Rhizobial nod-Gene Inducers by Alfalfa Roots

Inoculation with<i>Azospirillum</i>Increased Exudation of Rhizobial nod-Gene Inducers by Alfalfa Roots

The Rhizobium meliloti region located downstream of the nod box n6 is involved in the specific nodulation of Medicago lupulina

Six nod box regulatory sequences are present in the Rhizobium meliloti genome. We have analysed the DNA region located downstream of nod box n6, and identified three open reading frames, designated nolQa, nolQb and nolS. LacZ fusions in these ORFs are not induced by classical nod gene inducers, which indicates that their expression either is not under the control of the nod box, or involves another regulatory mechanism acting in conjunction with the NodD/nod box regulatory circuit. Mutations in this n6 locus result in a delay in nodule formation on a particular host, Medicago lupulina. As this region is not strictly conserved among different R. meliloti strains, nolQa, nolQb and nolS may constitute auxiliary nodulation genes, for which the selection pressure is limited to particular host plants.

The Rhizobium meliloti region located downstream of the nod box n6 is involved in the specific nodulation of Medicago lupulina.

Six nod box regulatory sequences are present in the Rhizobium meliloti genome. We have analysed the DNA region located downstream of nod box n6, and identified three open reading frames, designated nolQa, nolQb and nolS. LacZ fusions in these ORFs are not induced by classical nod gene inducers, which indicates that their expression either is not under the control of the nod box, or involves another regulatory mechanism acting in conjunction with the NodD/nod box regulatory circuit. Mutations in this n6 locus result in a delay in nodule formation on a particular host, Medicago lupulina. As this region is not strictly conserved among different R. meliloti strains, nolQa, nolQb and nolS may constitute auxiliary nodulation genes, for which the selection pressure is limited to particular host plants.

Isolation, chemical structures and biological activity of the lipo-chitin oligosaccharide nodulation signals from Rhizobium etli

Rhizobium etli is a microsymbiont of plants of the genus Phaseolus. Using mass spectrometry we have identified the lipo-chitin oligosaccharides (LCOs) that are produced by R. etli strain CE3. They are N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with cis-vaccenic acid (C18:1) or stearic acid (C18:0) and carries a carbamoyl group at C4. The reducing residue is substituted at the C6 position with O-acetylfucose. Analysis of their biological activity on the host plant Phaseolus vulgaris shows that these LCOs can elicit the formation of nodule primordia which develop to the stage where vascular bundles are formed. The formation of complete nodule structures, including an organized vascular tissue, is never observed. Considering the very close resemblance of the R. etli LCO structures to those of R. loti (I. M. López-Lara, J. D. J. van den Berg, J. E. Thomas Oates, J. Glushka, B. J. J. Lugtenberg, H. P. Spaink, Mol Microbiol 15: 627-638, 1995) we tested the ability of R. etli strains to nodulate various Lotus species and of R. loti to nodulate P. vulgaris. The results show that R. etli is indeed able to nodulate Lotus plants. However, several Lotus species are only nodulated when an additional flavonoid independent transcription activator (FITA) nodD gene is provided. Phaseolus plants can also be nodulated by R. loti bacteria, but only when the bacteria contain a FITA nodD gene. Apparently, the type of nod gene inducers secreted by the plants is the major basis for the separation of Phaseolus and Lotus into different cross inoculation groups.

Root exuded nod-gene inducing signals limit the nodulation capacity of different alfalfa varieties with Rhizobium meliloti

Different nodulation capacities were found among nine different varieties of alfalfa, cultivated in the Central region of Mexico, by Rhizobium meliloti 2011. A correlation between nodulation capacity and foliar dry weight was observed, which points to a genotype dependance on these parameters. A correlation between the nodulation capacity and the R. meliloti nod-gene inducing activity of the root exudates from the different varieties, as measured by β-galactosidase induction in a test system consisting of a R. meliloti nodC-lacZ strain incubated with each root exudate, was established. When the root exudate from the best nodulating variety was added to the four poorest nodulating varieties, an increase in nodule formation was observed. We conclude that root exuded nod-gene inducing signals are a symbiotically-limiting component in natural populations of the poorest nodulating varieties of alfalfa.

Alfalfa Root Flavonoid Production Is Nitrogen Regulated.

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.

NoIR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis

The synthesis of Rhizobium meliloti Nod signal molecules, encoded by the nod gene products, is finely regulated. A negative control of plasmid-borne nod gene expression is provided by the NolR repressor encoded by the chromosomal nolR gene. NolR was previously shown to downregulate the expression of the activator nodD1 gene and the common nodABC operon by binding to an overlapping region of the two promoters adjacent to the n1 nod-box (Kondorosi et al., 1989). We demonstrate here that NolR also controls the expression of two additional genes, nodD2 and nodM, but does not directly regulate the expression of the host-specific nod genes located downstream of the n2, n3 and n5 nod-boxes. Thus, the nod genes are differentially regulated by NolR and only those providing common nodulation functions, by determining the synthesis of the core Nod factor structure, are subjected to this negative regulation. Furthermore, NolR has a strong negative effect on the production of Nod metabolites, the level of which may serve as a fine-tuning mechanism for optimal nodulation, specific to host-plant genotypes. In addition, it elicits preferential synthesis of Nod factors carrying unsaturated C16 fatty acids. Expression of nolR was high both in the free-living bacterium and in the bacteroid and it was downregulated by its own product and by the nod gene inducer luteolin.

Rhizobium leguminosarum nodulation gene (nod) expression is lowered by an allele-specific mutation in the dicarboxylate transport gene dctB.

To identify host genes that might influence nod (nodulation) gene expression in Rhizobium leguminosarum, a nodC-phoA reporter plasmid (carrying nodD) was introduced into a chemically mutagenized population of a R. leguminosarum strain lacking a symbiotic plasmid. The transconjugants were screened for expression of alkaline phosphatase (PhoA) on plates containing hesperetin, an inducer of nod genes, and a mutant with reduced expression was identified. When the nodC-phoA plasmid was cured from the mutant and the symbiotic plasmid pRL1Jl introduced, the mutant formed nodules, but symbiotic nitrogen fixation was less than 20% of normal. When the nodC-phoA allele was introduced on pRL1Jl a low level of nod gene induction was found. The reduced nodC expression appeared to be caused by a decrease in expression of the regulatory gene nodD, since expression of a nodD-lacZ fusion was also lower in the mutant than in the control. These mutant phenotypes and the low nitrogen fixation were complemented with a plasmid (plJ1848) from a R. leguminosarum cosmid library. DNA hybridization confirmed that plJ1848 was not from the symbiotic plasmid and showed that a DNA insertion was present in the mutant. The complementing region of plJ1848 was defined by transposon mutagenesis; DNA sequencing revealed that it carried the dicarboxylic acid transport (dct) genes. However, the mutant grew well with succinate as sole C-source. Genetic analysis revealed that the mutant appeared to contain IS50 in the regulatory gene dctB and that this mutation caused the reduction in nod gene expression. The effect was allele-specific since other mutations in dctB did not influence nod gene expression. Surprisingly, the mutant had a constitutive high level of succinate transport, indicating that the mutation caused unregulated expression of dctA the structural gene for dicarboxylic acid transport. This in some way appears to have lowered the expression of nodD, indicating that the nodD promoter may be influenced by the metabolic status of the cells or by expression of dctD in the absence of dctB.

Ultrastructural localization of a diprenylated isoflavone inRhizobium lupini-Lupinus albussymbiotic association

Polyclonal IgGs raised against the diprenylated isoflavone, 2'-hydroxylupalbigenin were used in an immunocytochemical study of the root and nodule tissues of white lupin (lupinus albus L.). In the roots, the antigen was detected in the secondary walls of both xylem vessels and pericycle cells. Examination of nodules revealed the presence of the antigen in the inner cortex, associated with a neoparietal material which is deposited as globules on the cell wall and occluding the intercellular spaces. The discrete location of the diprenylated isoflavone in specialized cytoplasmic organelles suggests that its compartmentation within the wall is mediated by membrane vesicles. In the infected cells, more than half of the bacteroids exhibited a specific labelling of their inner core associated with a central fibrillar system, and sometimes surrounding an electron-dense nucleoid region. These results suggest that 2'-hydroxylupalbigenin, and possibly other prenylated isoflavones, may play an important role as a biochemical factor in early symbiotic events other than nod gene induction or inhibition. Its putative biological functions in the post-infectional phases of symbiosis are discussed