Cloning of hemA from Rhizobium sp. NGR234 and symbiotic phenotype of a gene-directed mutant in diverse legume genera

Summary The hemA gene which encodes -aminolaevulinic acid synthase (ALAS), was cloned and characterized from the broad host-range Rhizobium strain NGR234. A cosmid, identified by hybridization with the cloned gene of R. meliloti and complementation of an R. meliloti hemA mutant, was subcloned to yield a 5.5 kb fragment containing the entire NGR234 gene. A physical-genetic map was made and the interposon was introduced into a single EcoRI site which bisects the gene. The mutated gene was homogenotized into NGR234 to generate a hemA mutant, with a view to evaluating the role of rhizobial bacteroid ALAS activity for a wide variety of legume symbioses. The mutant strain formed an ineffective (Fix^–) symbiosis with all tested host plants. These included tropical legumes that produce either indeterminate (Leucaena) or determinate (Desmodium, Macroptilium, Lablab, Vigna) root nodules.Abbreviations ALA -aminolaevulinic acid - ALAS aminolaevulinic acid synthase - Lb leghaemoglobin - Lb-haem haem moiety of leghaemoglobin     

Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes

The role of the dicarboxylic acid transport (dct) system in the Rhizobium meliloti-Alfalfa symbiosis was investigated. Mutants of R. meliloti CM2 unable to grow on medium containing succinate as the sole carbon source were isolated following chemical and transposon mutagenesis. These mutants were also unable to utilize malate or fumarate as the sole source of carbon. Transport studies with ¹⁴C-labelled succinate showed that the mutants were specifically defective in succinate transport. Revertants of both chemical and transposon mutants were obtained at a frequency of 10^-5–10^-6. The R. meliloti dct mutants were able to nodulate Alfalfa plants but the nodules formed were unable to fix nitrogen. Revertants of the mutants were fully effective on plants. The mutants unable to transport succinate were used to isolate dct genes from a R. meliloti gene bank. Two plasmids containing a common 26.5 Mdal insert were found to complement some of the mutants. The presence of this DNA insert in the complementing mutant strains restored their effectivenss of plants. This DNA fragment encoding succinate transport function(s) was used to produce genetically engineered R. meliloti strains with an increased rate of succinate uptake.Abbreviation dct dicarboxylic acid transport     

Expression of host genes during root nodule development in soybeans

Summary Nine unique nodulin cDNA clones from soybean have been characterized with regard to the size of the RNA and the corresponding protein products. Based on the sequence homology between clones C51 and E27 and the multiple RNA species corresponding to clones D41 and E41, it is suggested that some of the nodulin genes represent members of small gene families. The amino acid sequence deduced from the nucleotide sequence of clones C51 and E27 revealed the presence of a signal peptide and no stop transfer signal, typical of membrane proteins, suggesting that the proteins encoded by these clones are localized in organelles and as such probably involved in ureide biosynthesis (Boland et al. 1982; Schubert and Boland 1984). Based on the timing of appearance of RNA corresponding to the nodulin clones and the pattern of their accumulation, at least three sets of nodulin genes are being represented here. Al1 the nodulin RNAs examined were made in Fix^- nodules formed by strain Ag168 (which does not make Cl component of nitrogenase) at a level comparable to that in Fix⁺ nodules and at a very reduced level in Fix^- nodules formed by strain HS124 (which show very few infected cells). It is concluded that all the nodulin genes examined here are induced independent of nitrogenase activity.     

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN of Rhizobium meliloti involved in symbiotic nitrogen fixation.

Summary TheRhizobium meliloti fdxN gene, which is part of thenifA-nifB fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix^– phenotype of anR. meliloti fdxN: :[Tc] mutant could be rescued by theR. leguminosarum fdxN gene, whereas no complementation was observed withnif-associated genes encoding ferredoxins fromBradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum andRhodobacter capsulatus. In addition to these heterologous genes, severalR. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of theR. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X₂-C-X₂-C-X₃-C,nif-associated ferredoxins, includingR. meliloti FdxN, are characterized by two different cysteine motifs. Six additional amino acids separate the second (Cys₄₂) and the third cysteine (Cys₅₁) in the C-terminal motif (C-X₂-C-X₈-C-X₃-C). By molecular modelling, it was predicted that these amino acid residues form a loop, which does not alter the relative positions of the neighbouring cysteines. Deletion of this loop resulted in anR. meliloti FdxN mutant protein, which exhibited almost 70% wild-type activity, indicating that the predicted loop does not affect Fe-S cluster binding and plays no crucial role in activity of the FdxN protein.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Etude expérimentale de la symbiose entre la luzerne etRhizobium meliloti. Mise au point d'une miniserre

Resumé Des mutants non mobiles (mob^–) et non chimiotactiques (chem^–) deRhizobium meliloti Ve 26 ont été isolés afin de connaître l'importance de ces caractères dans la nodulation de la luzerne en milieu liquide et en milieu solide. En milieu liquide les mutants produisent un nombre de nodules équivalent à ceux des souches parentales; par contre, en milieu solide (perlite, sol) l'étude réalisée dans une miniserre expérimentale adaptée montre que les caractères de mobilité et de chimiotactisme favorisent la nodulation et augmentent notablement le pouvoir compétitif des souches qui les possèdent.

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Experimental study of symbiosis between lucerne and Rhizobium meliloti: a new technique in a mini-greenhouse

Summary Non mobile (mob^–) and non chemotactic (chem^–) mutants ofRhizobium meliloti Ve 26 have been isolated so that the importance of these characters in lucerne nodulation in liquid medium and in solid medium might be known. In liquid medium the mutants developed a number of nodules equal to those obtained from parental strains; on the opposite, in solid medium (perlite, soil) the study in an adapted experimental mini-greenhouse showed that the mobility and chemotactic characters improved the nodulation and greatly increased the competitive capacity of the strains that possessed them.

     

Molecular cloning of thentrA gene of the broad host-rangeRhizobium sp. NGR234, and phenotypes of a site-directed mutant

Summary The clonedntrA (rpoN) gene andntrA mutants ofRhizobium meliloti were used to isolate the homologous gene from the broad-host rangeRhizobium sp. NGR234 by hybridization and interspecies complementation. The NGR234 locus was analyzed by deletion and insertional mutagenesis. A site-directedntrA mutant, NGR234rn1, was made with an interposon, GmI, and its phenotype was examined ex planta and in symbiosis. NGR234rn1 formed Fix⁻ nodules on six genera tested from among its legume hosts, including both indeterminate and determinate nodule-type plants. Formation of nodules onMacroptilium was delayed, and expression of anR. meliloti nodABC-lacZ fusion was reduced by the mutant allele.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

The two megaplasmids of Rhizobium meliloti are involved in the effective nodulation of alfalfa

Summary We have shown by physical and genetic means that there are two megaplasmids in all strains of Rhizobium meliloti that we have studied. Megaplasmids from several strains of R. meliloti were mobilized to Agrobacterium tumefaciens and to other Rhizobium strains using the Tn5-Mob system. We were also able to resolve these two megaplasmids in agarose gels for most strains, and to show that only one of them hybridized to nif and nod genes. Transfer of this plasmid, the pSym, to Agrobacterium, R. leguminosarum, and R. trifolii strains conferred on these recipients the ability to nodulate alfalfa ineffectively. The second megaplasmid did not appear to have a direct role in nodule initiation. However, we were able to complement extracellular polysaccharide (EPS^-) mutants of R. meliloti by transferring this second megaplasmid into them. Furthermore, Tn5-induced EPS^- mutants of R. meliloti 2011, which produced ineffective (Fix^-) nodules of abnormal morphology, were shown by hybridization and complementation to carry mutations in this second megaplasmid. This demonstrates that both megaplasmids of R. meliloti are necessary for the effective nodulation of alfalfa.     

Regulation of Nodulation by Rhizobium meliloti 102F15 on Its Mutant Which Forms an Unusually High Number of Nodules on Alfalfa

A mutant (WL3A150) of Rhizobium meliloti 102F51 that elicits an unusually high number of nodules on its host, alfalfa (Medicago sativa), supports the idea that the host may rely on early bacteroid development in the nodule or on metabolites produced in the infection thread as one of the signals to control further nodulation. This mutant was initially isolated because of its Fix⁻ phenotype. It consistently formed many more nodules than all the other Fix⁻ mutants isolated from strain 102F51 (a total of 11 mutants). Nodules formed by this mutant were small and white and were indistinguishable in appearance from nodules formed by the other Fix⁻ mutants. An ultrastructural study of the nodules, however, showed that this mutant, although forming numerous infection threads, failed to develop into bacteroids. The ability of the mutant to form an unusually high number of nodules coulde be suppressed in a time-dependent manner by the presence of the wild type.     

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.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Isolation of pea nodule bacteroids utilizing silica sol (Percoll) density gradient centrifugation

Isolation of bacteroids from effective (Fix⁺) and ineffective (Fix^–) pea nodules, inoculated withRhizobium leguminosarum K, were performed by a density gradient centrifugation method using silica sol (Percoll). Only one zone (=1.064–1.072; n-zone) was recognized in the Fix⁺ nodule which contained typical Y-shaped bacteroids while two zones (n-zone and =1.125–1.145; n'-zone) were obtained from the Fix^– nodule. The cells in the n'-zone, which are long rods differed morphologically from free-living cells at any growth phase (=1.108–1.125; f-zone and =1.074–1.078; f'-zone), and differed from Y-shaped bacteroids by cell density. The esterase isozyme pattern of bacteroids in the n-and n'-zones also showed clear differences from that of f-and f'-zone of free-living cells.     

Exopolysaccharide-deficient mutants of Astragali rhizobia are symbiotically effective on Astragalus sinicus, an indeterminate nodulating host

Five exopolysaccharide-deficient mutants were isolated after rhizobial strain 107 was subjected to transposon Tn5 mutagenesis. The amount of EPS produced by the mutants was dramatically decreased to between 3% and 6% of wild-type level. All mutants carried a singel copy of Tn5. Two mutants (NA3 and NA10) were complemented by the R. meliloti exoA gene and the functionally equivalent exoD gene of Rhizobium sp. strain NGR234. Two other mutants (NA7 and NA8) were complemented by the R. meliloti exoB gene and the functionally equivalent NGR234 exoC gene. The remaining mutant (NA11) was not complemented by any exo genes of R. meliloti or Rhizobium NGR234. All mutants induced normal nitrogen-fixing nodules on Astragalus sinicus, an indeterminate nodulating host.     

Identification and cloning of nodulation genes from the wide host range Rhizobium strain MPIK3030

Summary A 70 kbp segment of the megaplasmid from a broad host range Rhizobium strain (MPIK3030) was mapped with the aid of cosmid clones made in the vector pJB8. A 7.9 kbp EcoRI fragment from this region, 55 kbp away from the nif gene cluster, was shown to hybridize to the common nod genes from R. meliloti. Using several R. meliloti nod probes it was possible to delimit an 830 bp region as being the center of greatest homology. Sequence data from two sections of this region gave a nucleotide homology of 73.7% to the nodC gene of R. meliloti. Using Tn5 mutagenesis a clone was isolated carrying Tn5 in the highly homologous region. When tested on Macroptilium atropurpureum, this MPIK3030 derivative was shown to have a Nod^– phenotype. When the wild-type allele was reintroduced into the Tn5 mutant, nodulation was restored. Interspecies complementation also showed that both R. meliloti and Rhizobium sp. MPIK3030 nod regions were able to restore nodulation to Tn5-induced nodC mutants from either strain.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Nodulin gene expression during soybean (Glycine max) nodule development

In vitro translation products of total RNA isolated from soybean nodules at successive stages of nodule development were analyzed by two-dimensional gel electrophoresis. In that way the occurrence of over 20 mRNAs specifically transcribed from nodulin genes was detected. The nodulin genes could be divided into two classes according to the time of expression during nodule development. Class A comprises at least 4 nodulin mRNAs which are found when a globular meristem is present in the root cortex. These class A nodulin genes have a transient expression. Class B nodulin genes are expressed when the formation of a nodule structure has been completed. Bradyrhizobium japonicum nod ⁺ fix^-mutants, with large deletions spanning the nif H,DK region, still induced nodules showing normal expression of all nodulin genes, indicating that the nif H,DK region is not involved in the induction of nodulin genes. In nodules induced by Bradyrhizobium japonicum nod ⁺ fix^-mutant HS124 the bacteria are rarely released from the infection thread and the few infected cells appear to be collapsed. All class A and class B nodulin genes are expressed in HS124 nodules with the exception of 5 class B genes.     

Plant genetic suppression of the non-nodulation phenotype of Rhizobium meliloti host-range nodH mutants: gene-for-gene interaction in the alfalfa-Rhizobium symbiosis?

Summary Rhizobium nodulation genes can produce active extracellular signals for legume nodulation. The R. meliloti host-range nodH gene has been postulated to mediate the transfer of a sulfate to a modified lipo-oligosaccharide, which in its sulfated form is a specific nodulation factor for alfalfa (Medicago sativa L.). We found that alfalfa was capable of effective nodulation with signal-defective and non-nodulating nodH mutants (Nnr) defining a novel gene-for-gene interaction that conditions nodulation. Bacteria-free nodules that formed spontaneously at about a 3–5% rate in unselected seed populations of alfalfa cv Vernal in the total absence of Rhizobium (Nar) exhibited all the histological, regulatory and ontogenetic characteristics of alfalfa nodules. Inoculation of such populations with nodH mutants, but not with nodA or nodC mutants, produced a four- to five-fold increase in the percentage of nodulated plants. Some 10–25% of these nodulated plants formed normal pink nitrogen-fixing nodules instead of white empty nodules. About 70% of the S₁ progeny of such Nnr⁺ plants retained the parental phenotype; these plants were also able to form nodules in the absence of Rhizobium. If selected Nar⁺ plants were self-pollinated almost the entire progeny exhibited the parental Nar⁺ phenotype. Segregation analysis of S₁ and S₂ progeny from selected Nar⁺ plants suggests that the Nar character is monogenic dominant and that the nodulation phenotype is controlled by a gene dose effect. The inoculation of different S₁ Nar⁺ progeny with nodH mutant bacteria gave only empty non-fixing nodules. Our results indicate that certain alfalfa genotypes can be selected for suppression of the non-nodulation phenotype of nodH mutants. The fact that the Nnr plant phenotype behaved as a dominant genetic trait and that it directly correlated with the ability of the selected plants to form nodules in the absence of Rhizobium suggests that the interaction of plant and bacterial alleles occurs early during signal transduction through the alteration of a signal reception component of the plant so that it responds to putative signal precursors.Abbreviations Nara Nodulation in the absence of Rhizobium - Nara nodulation with non-nodulating Rhizobium