N-Acetylglutamic acid: an extracellular nod signal of Rhizobium trifolii ANU843 that induces root hair branching and nodule-like primordia in white clover roots

An extracellular metabolite purified from Rhizobium trifolii ANU843 was established as N-acetylglutamic acid (GluNAc) by 1H NMR and Fourier transform IR spectroscopy, gas chromatography/mass spectrometry of its methylated product, and organic synthesis. TLC analyses indicated that extracellular accumulation of GluNAc by R. trifolii ANU843 grown in defined BIII culture medium was dependent on induction of its bacterial nodulation (nod) genes and the positive regulatory gene nodD on its symbiotic plasmid. 1H NMR analyses showed less GluNAc in fractionated culture supernatants of nodL and nodM mutant derivatives of R. trifolii ANU843. GluNAc induced three morphological responses on axenic roots of white clover seedlings: (i) root hair branching; (ii) tip swelling followed by resumed elongation of root hairs; and (iii) a slight increase in foci of cortical cell divisions, which developed into nodule-like primordia. These biological activities of extracellular GluNAc from R. trifolii ANU843 were confirmed with authentic standards of GluNAc. These results indicate that extracellular accumulation of N-acetylglutamic acid is linked to flavone-dependent metabolism involving nodD, nodL, and nodM in R. trifolii ANU843. This constitutes the first report on the structure of a nod-dependent extracellular signal from R. trifolii that can affect root hair and nodule development in white clover and whose biological activity on this host has been confirmed with authentic standards.     

Structural and functional analysis of two different nodD genes in Bradyrhizobium japonicum USDA110.

Bradyrhizobium japonicum has two closely linked homologs of the nodulation regulatory gene, nodD; these homologs are located upstream of and in divergent orientation to the nodYABCSUIJ gene cluster. We report here the nucleotide sequence and mutational analyses of both nodD copies. The predicted NodD1 and NodD2 proteins shared 62% identical amino acid residues at corresponding positions and exhibited different degrees of homology with NodD proteins of other Bradyrhizobium, Azorhizobium, and Rhizobium strains. Induction of the nodYABCSUIJ operon, as measured by expression of a translational nodC'-'lacZ fusion, required the nodD1 gene, but not nodD2. A B. japonicum mutant deleted for both nodD copies (strain delta 1267) still showed residual nodulation activity; however, nodulation of soybean was significantly delayed, and nodulation of mung bean and siratro resulted in strongly reduced nodule numbers. Fully efficient nodulation of mung bean and siratro by strain delta 1267 was restored by genetic complementation with the nodD1 gene, but not with nodD2. We conclude from these data that nodD1 is the critical gene that contributes to maximal nodulation efficiency, whereas the nodD2 gene does not play any obvious role in nodulation of the host plants tested.     

Studies on the Characterization of Root Morphology of White Clover Infected by Transconjugant of Rhizobium leguminosarum

White clover plants were inoculated with transconjugant strain' 290 which was obtained from introduction of host specific nodulation genes of wild-type Rhizobium trifolii strain ANU 843 to Rhizobium leguminosarum strain 300. The characterization of root morphology of white clover induced by the transconjugant was observed and compared to the plants induced by the parent strains. White clover started tO form a typical root hair curling inoculated with transconjugant strain 290 24h after inoculation, at 48h a part of cell wall of root hair was degradated, infection thread was observed in the infected root hair cell, cortical cell divisions occurred extensively. All these characterizations were similar to that infected by strain ANU 843. Plant inoculation test indicated that no nodule was formed when inoculated by R. leguminosarum strain 300, while plants nodulated when inoculated with transconjugant strain 290 as well as R. trifolii ANU 843. This suggests that introduction of host specific nodulation genes of R. trifolii results in conferring the nodulation ability of R. leguminosarum on white clover.     

Rhizobium meliloti nodD genes mediate host-specific activation of nodABC.

To differentiate among the roles of the three nodD genes of Rhizobium meliloti 1021, we studied the activation of a nodC-lacZ fusion by each of the three nodD genes in response to root exudates from several R. meliloti host plants and in response to the flavone luteolin. We found (i) that the nodD1 and nodD2 products (NodD1 and NodD2) responded differently to root exudates from a variety of hosts, (ii) that NodD1 but not NodD2 responded to luteolin, (iii) that NodD2 functioned synergistically with NodD1 or NodD3, (iv) that NodD2 interfered with NodD1-mediated activation of nodC-lacZ in response to luteolin, and (v) that a region adjacent to and upstream of nodD2 was required for NodD2-mediated activation of nodC-lacZ. We also studied the ability of each of the three R. meliloti nodD genes to complement nodD mutations in R. trifolii and Rhizobium sp. strain NGR234. We found (i) that nodD1 complemented an R. trifolii nodD mutation but not a Rhizobium sp. strain NGR234 nodD1 mutation and (ii) that R. meliloti nodD2 or nodD3 plus R. meliloti syrM complemented the nodD mutations in both R. trifolii and Rhizobium sp. strain NGR234. Finally, we determined the nucleotide sequence of the R. meliloti nodD2 gene and found that R. meliloti NodD1 and NodD2 are highly homologous except in the C-terminal region. Our results support the hypothesis that R. meliloti utilizes the three copies of nodD to optimize the interaction with each of its legume hosts.     

At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.     

Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes

Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.     

Bradyrhizobium (Arachis) sp. strain NC92 contains two nodD genes involved in the repression of nodA and a nolA gene required for the efficient nodulation of host plants.

The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp. strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment. The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD1, nodD2, and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Nodulation assays of nodD1, nodD2, or nolA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers. The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF. nodD1 mutants displayed a 2-day nodulation delay, whereas nodD2 strains were indistinguishable from the wild type. Translational nodA-lacZ, nodD1-lacZ, nodD2-lacZ, and nolA-lacZ fusions were created. Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein. In a nodD1 or nodD2 background, basal expression of the nodA-lacZ fusion increased two- to threefold. The level of expression of the nodD2-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD1, nodD2, and nodD1 nodD2 backgrounds independently of the addition of the inducer genistein. nolA was required for increased expression of the nodD2-lacZ fusion. These data suggest that a common factor is involved in the regulation of nodD2 and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp. strain NC92 in which negative regulation is mediated by the products of the nodD1 and nodD2 genes.     

Regulation of Syrm and Nodd3 in Rhizobium Meliloti

The early steps of symbiotic nodule formation by Rhizobium on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Three different NodD proteins are encoded by Sym plasmid genes in Rhizobium meliloti, the alfalfa symbiont. NodD1 and NodD2 activate nod operons when Rhizobium is exposed to host plant inducers. The third, NodD3, is an inducer-independent activator of nod operons. We previously observed that nodD3 carried on a multicopy plasmid required another closely linked gene, syrM, for constitutive nod operon expression. Here, we show that syrM activates expression of the nodD3 gene, and that nodD3 activates expression of syrM. The two genes constitute a self-amplifying positive regulatory circuit in both cultured Rhizobium and cells within the symbiotic nodule. We find little effect of plant inducers on the circuit or on expression of nodD3 carried on pSyma. This regulatory circuit may be important for regulation of nod genes within the developing nodule.     

Multiple copies of nodD in Rhizobium tropici CIAT899 and BR816.

Rhizobium tropici strains are able to nodulate a wide range of host plants: Phaseolus vulgaris, Leucaena spp., and Macroptilium atropurpureum. We studied the nodD regulatory gene for nodulation of two R. tropici strains: CIAT899, the reference R. tropici type IIb strain, and BR816, a heat-tolerant strain isolated from Leucaena leucocephala. A survey revealed several nodD-hybridizing DNA regions in both strains: five distinct regions in CIAT899 and four distinct regions in BR816. Induction experiments of a nodABC-uidA fusion in combination with different nodD-hybridizing fragments in the presence of root exudates of the different hosts indicate that one particular nodD copy contributes to nodulation gene induction far more than any other nodD copy present. The nucleotide sequences of both nodD genes are reported here and show significant homology to those of the nodD genes of other rhizobia and a Bradyrhizobium strain. A dendrogram based on the protein sequences of 15 different NodD proteins shows that the R. tropici NodD proteins are linked most closely to each other and then to the NodD of Rhizobium phaseoli 8002.     

Flavone-enhanced accumulation and symbiosis-related biological activity of a diglycosyl diacylglycerol membrane glycolipid from Rhizobium leguminosarum biovar trifolii.

Rhizobium leguminosarum bv. trifolii is the bacterial symbiont which induces nitrogen-fixing root nodules on the leguminous host, white clover (Trifolium repens L.). In this plant-microbe interaction, the host plant excretes a flavone, 4',7-dihydroxyflavone (DHF), which activates expression of modulation genes, enabling the bacterial symbiont to elicit various symbiosis-related morphological changes in its roots. We have investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild-type R. leguminosarum bv. trifolii ANU843 when grown with DHF and the biological activities of this glycolipid bacterial factor on host and nonhost legumes. In vivo labeling studies indicated that wild-type ANU843 cells accumulate BF-7 in response to DHF, and this flavone-enhanced alteration in membrane glycolipid composition was suppressed in isogenic nodA::Tn5 and nodD::Tn5 mutant derivatives. Seedling bioassays performed under microbiologically controlled conditions indicated that subnanomolar concentrations of purified BF-7 elicit various symbiosis-related morphological responses on white clover roots, including thick short roots, root hair deformation, and foci of cortical cell divisions. Roots of the nonhost legumes alfalfa and vetch were much less responsive to BF-7 at these low concentrations. A structurally distinct diglycosyl diacylglycerol did not induce these responses on white clover, indicating structural constraints in the biological activity of BF-7 on this legume host. In bioassays using aminoethoxyvinylglycine to suppress plant production of ethylene, BF-7 elicited a meristematic rather than collaroid type of mitogenic response in the root cortex of white clover. These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 and a potent ability of this diglycosyl diacylglycerol glycolipid to perform as a bacterial factor enabling R. leguminosarum bv. trifolii to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.     

Role of the nodD and syrM genes in the activation of the regulatory gene nodD3, and of the common and host-specific nod genes of Rhizobium meliloti

To analyse the regulation of the nodulation (nod) genes of Rhizobium meliloti RCR2011 we have isolated lacZ gene fusions to a number of common, host-range and regulatory nod genes, using the mini-Mu-lac bacteriophage transposon MudII1734. Common (nodA, nodC, nod region IIa) and host-range (nodE, nodG, nodH) genes were found to be regulated similarly. They were activated (i) by the regulatory nodD1 gene in the presence of flavones such as chrysoeriol, luteolin and 7,3',4'-trihydroxyflavone, (ii) by nodD2 in the presence of alfalfa root exudate but not with the NodD1-activating flavones, and (iii) by the regulatory genes syrM-nodD3 even in the absence of plant inducers. Thus common and host-range nod genes belong to the same regulon. In contrast to the nodD1 gene, the regulatory nodD3 gene was not expressed constitutively and exhibited a complex regulation. It required syrM for expression, was activated by nodD1 in the presence of luteolin and was positively autoregulated.     

Induction of nitrogen-fixing nodules on clover requires only 32 kilobase pairs of DNA from the Rhizobium trifolii symbiosis plasmid.

Overlapping subclones from the Rhizobium trifolii symbiosis plasmid pRt843a were generated by using in vivo and in vitro methods. Subclones were assayed for symbiotic phenotype by introducing them into a derivative of R. trifolii ANU843 cured of its symbiosis plasmid and testing the transconjugant strains for the ability to induce nitrogen-fixing nodules on clover. One subclone spanning 32 kilobase pairs (kb) of DNA from pRt843a was found to restore nitrogen fixation ability. This subclone included all known nodulation genes of R. trifolii ANU843 and the nitrogenase structural genes nifHDK. In addition, regions homologous to fixABC, nifA, nifB, nifE, and nifN genes of other nitrogen-fixing bacteria were identified in this 32-kb subclone by DNA-DNA hybridization. Transposon mutagenesis of this subclone confirmed that regions containing these nif and fix genes were required for induction of nitrogen-fixing nodules on clover. In addition, a region located 5 kb downstream of the nifK gene was found to be required for induction of nitrogen-fixing nodules. No homology to known nif and fix genes could be detected in this latter region.     

A cultivar-specific interaction between Rhizobium leguminosarum bv. trifolii and subterranean clover is controlled by nodM, other bacterial cultivar specificity genes, and a single recessive host gene.

Insertion mutagenesis identified two negatively acting gene loci which restrict the ability of Rhizobium leguminosarum bv. trifolii TA1 to infect the homologous host Trifolium subterraneum cv. Woogenellup. One locus was confirmed by DNA sequence analysis as the nodM gene, while the other locus, designated csn-1 (cultivar-specific nodulation), is not located on the symbiosis plasmid. The presence of these cultivar specificity loci could be suppressed by the introduction of the nodT gene from ANU843, a related R. leguminosarum bv. trifolii strain. Other nod genes, present in R. leguminosarum bv. viciae (including nodX) and R. meliloti, were capable of complementing R. leguminosarum bv. trifolii TA1 for nodulation on cultivar Woogenellup. Nodulation studies conducted with F2 seedlings from a cross between cultivar Geraldton and cultivar Woogenellup indicated that a single recessive gene, designated rwt1, is responsible for the Nod- association between strain TA1 and cultivar Woogenellup. Parallels can be drawn between this association and gene-for-gene systems common in interactions between plants and biotrophic pathogens.