Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants.

The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during symbiosis with Medicago sativa has been shown to be required for the proper development of nitrogen-fixing nodules. Cloned DNA from the exo region of R. meliloti is shown to stimulate production of the low-molecular-weight form of this exopolysaccharide, and in this report we show that the symbiotic deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be rescued by the addition of this low-molecular-weight material at the time of inoculation. For exoA and exoH mutants, rescue with a preparation containing low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing nodules which appear somewhat later and at a reduced efficiency compared with wild-type-induced nodules; however, microscopic analysis of these nodules reveals similar nodule morphology and the presence of large numbers of bacteroids in each.     

Genetic analyses of Rhizobium meliloti exopolysaccharides

We have recently obtained strong genetic evidence that the acidic Calcofluor-binding exopolysaccharide (EPS I) of Rhizobium meliloti Rm1021 is required for nodule invasion and possibly for later events in nodule development. Thirteen loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. exoH mutants fail to succinylate their EPS I and form empty Fix- nodules. We have identified two unlinked regulatory loci, exoR and exoS, whose products play negative roles in the regulation of expression of the exo genes. We have recently discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for Rhizobium exopolysaccharides in nodulation are discussed.     

Nodulin gene expression in effective alfalfa nodules and in nodules arrested at three different stages of development

Nodulin gene expression was analyzed in effective and ineffective root nodules of alfalfa (Medicago sativa L. cv Iroquois) elicited by three different Rhizobium meliloti mutants: an exoB mutant having defective acidic exopolysaccharide that does not fluoresce on plates containing the fluorescent brightener Calcofluor; fix21, a spontaneous mutant that has defective lipopolysaccharide and is Calcofluor bright; and a Rhizobium mutant resulting from a Tn5 insertion in the nifH gene of the nif operon. The ineffective nodules elicited by these various mutant rhizobia are blocked at different stages of nodule development and have unique phenotypes. A distinctive pattern of nodulin gene expression as determined by in vitro translations of total nodule RNA characterizes each nodule phenotype. Seventeen nodulins are found in effective nodules including five leghemoglobins. Only one nodulin gene is expressed in the bacteria-free nodules elicited by the exoB mutant. Other nodulin genes (leghemoglobin and nine others) are expressed in fix21-induced nodules. The genes for nodule-enhanced glutamine synthetase as well as for all the other nodulins are expressed in nodules induced by the nifH mutant. The expression of genes for the nodulins, including leghemoglobin, is independent of the nitrogen-fixing ability of the nodule and appears to correlate with the differentiation of densely cytoplasmic host cells in the nodule and, to some extent, with bacterial release from infection threads.     

Genetic analysis of a cluster of genes required for synthesis of the calcofluor-binding exopolysaccharide of Rhizobium meliloti.

Rhizobium meliloti produces an acidic, Calcofluor-binding exopolysaccharide which plays a role in nodulation of alfalfa plants by this bacterium. We constructed and mapped 102 transposon insertions in a 48-kilobase (kb) region previously shown to contain several exo genes. Mutations affecting production of the Calcofluor-binding exopolysaccharide were clustered in a 22-kb region and fell into 12 complementation groups. Strains carrying mutations in seven of the complementation groups (exoA, exoB, exoF, exoL, exoM, exoP, and exoQ) produced no Calcofluor-binding exopolysaccharide and induced non-nitrogen-fixing nodules on alfalfa. Mutants in an eighth complementation group, exoH (Leigh et al., Cell 51:579-587, 1987), produce an altered exopolysaccharide and also induce the formation of non-nitrogen-fixing nodules. Mutants in the remaining four complementation groups produced less Calcofluor-binding material than the wild type. Mutants carrying mutations in two of these complementation groups (exoK and exoN) formed apparently normal, nitrogen-fixing nodules, while mutants in the other two groups (exoG and exoJ) formed normal nodules less efficiently than the wild type.     

Root nodule development: origin, function and regulation of nodulin genes

The symbiotic root nodule, an organ formed on leguminous plants, is a product of successful interactions between the host plant and the soil bacteria, Rhizobium spp. Plant hormones play an important role in the genesis of this organ. The hormonal balance appears to be modulated by the signals produced by bacteria. Many host genes induced during nodule organogenesis and the symbiotic state have been identified and characterized from several legumes. These genes encode nodule-specific proteins (nodulins) which perform diverse functions in root nodule development and metabolism. Formation of a subcellular compartment housing the bacteria is essential to sustain the symbiotic state, and several nodulins are involved in maintaining the integrity and function of this compartment. The bacteroid enclosed in the perbacteroid membrane behaves as an 'organelle,'completely dependent on the host for all its requirements for carbon, nitrogen and other essential elements. Thus it seems likely that the nodulins in the peribacteroid membrane perform specific transport functions. While the function of a few other nodulins is known (e.g. nodulin-100, nodulin-35), a group of uncharacterized nodulins exists in soybean root nodules. These nodulins share structural similarities and seem to have been derived from a common ancestor. Induction of nodulin genes occurs prior to and independent of nitrogen fixation, and thus is a prelude to symbiosis. Although some of the early nodulin genes are induced prior to or during infection, induction of late nodulins requires endocytotic release of bacteria.     

Suppression of the ndv mutant phenotype of Rhizobium meliloti by cloned exo genes

The ndvA and ndvB genes of Rhizobium meliloti are involved in the export and synthesis, respectively, of the small cyclic polysaccharide beta(1,2)glucan. We have previously shown that spontaneous symbiotic pseudorevertants of ndv mutants do not produce periplasmic beta(1,2)glucan. Here we show that the pseudorevertants also do not produce extracellular beta(1,2)glucan, but do show alterations in the amount of the major acidic exopolysaccharide produced. This exopolysaccharide is not detectably different from that produced by the wild type or by the ndv mutants. A cosmid which suppresses the symbiotic defect of both ndvA and ndvB mutants was isolated from a gene bank prepared from DNA of an ndvA pseudorevertant. This cosmid contains a number of exo genes, including exoH and exoF. Subcloning and Tn5 mutagenesis were used to show that the widely separated exoH and exoF genes are both involved in suppression of the ndv mutant phenotype and that the 3.5 kb DNA fragment which contains the exoH gene does not carry the mutation responsible for second site suppression.     

Nodulin-26, a peribacteroid membrane nodulin is expressed independently of the development of the peribacteroid compartment.

The peribacteroid membrane (pbm) of root nodules is derived from the plant cell plasma membrane but contains in addition several nodule-specific host proteins (nodulins). Antibodies raised against purified pbm of soybean were used to immunoprecipitate polysomes to isolate an RNA fraction that served as a template for the synthesis of a cDNA probe for screening a nodule-specific cDNA library. Clone p1B1 was found to encode a 26.5 kDa polypeptide (nodulin-26) which is immunoprecipitable specifically with the anti-pbm serum. Nodulin-26 has features of a transmembrane protein and its structure differs from that of nodulin-24 which appears to be a surface protein of pbm. The expression of these two pbm nodulins was examined in nodules induced by Bradyrhizobium japonicum Tn5 mutants that arrest nodule development at different stages of pbm biosynthesis. Nodules that do not show release of bacteria from the infection thread express nodulin-24 at a very low level. In contrast, the expression of nodulin-26 occurs fully in nodules that form infection threads only and is not affected by the release of bacteria from the threads.     

Developmental regulation of nodule-specific genes in alfalfa root nodules

We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins. Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa. One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa. The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel. Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.     

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules.

Mutants of Rhizobium meliloti SU47 with defects in the production of the Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into alfalfa root nodules. In order to define better the polysaccharide phenotypes of these exo mutants, we analyzed the periplasmic oligosaccharide cyclic (1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a substantial portion of the O side chain. The exoB mutant had a spectrum of LPS species which differed from those of both the wild-type parental strain and the exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF, and exoH mutants eliminated defects in these carbohydrates as explanations for the nodule entry defects of these mutants. We also assayed for high- and low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH completely lacked both forms. For the Calcofluor-dim exoD mutant, the distribution of high- and low-molecular-weight forms depended on the growth medium. The haloless exoH mutant produced high-molecular-weight and only a trace of low-molecular-weight succinoglycan; the succinyl modification was missing, as was expected from the results of previous studies. The implications of these observations with regard to nodule entry are discussed.     

Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii USDA 191

Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria. Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were isolated following Tn5-insertion mutagenesis. Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans. The exopolysaccharides produced by these mutants were analysed for polysaccharide composition by column chromatography and thin-layer chromatography. Two mutants designed exo-3 and exo-5 were deficient in both neutral glucan and exopolysaccharide synthesis, but each induced some functional nodules on Glycine max (Peking). The remaining three mutants (exo-1, exo-2 and exo-4) synthesized neutral glucans at levels higher or lower than those in wild-type and exhibited partial exopolysaccharide deficiencies. The data imply that neither exopolysaccharides nor neutral glucans are essential for the induction of determinate nodules by R. fredii.     

Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation

Spontaneous mutants at a new symbiotic locus in Rhizobium meliloti SU47 are resistant to several phages and are conditionally insensitive to a monoclonal antibody to the bacterial surface, apparently because they are deficient in a wild-type exopolysaccharide. On alfalfa, the mutants do not curl root hairs, but penetrate the epidermis directly, forming nodules that contain no visible infection threads or "bacteroids," have a few bacteria in superficial intercellular spaces only and not within the nodule cells, and fail to fix nitrogen (Fix-). Evidently, infection threads are not essential for cell proliferation and nodule formation, which are here induced by a bacterial signal at a distance and uncoupled from the bacterial differentiation that normally goes on as well.     

Nodulin gene expression in effective root nodules of white sweetclover (Melilotus alba Desr.) and in ineffective nodules elicited by mutant strains of Rhizobium meliloti

Fifteen nodulins and several nodule-stimulated gene productswere expressed in effective, nitrogen-fixing root nodules ofwhite sweetclover (Melilotus alba Desr. cv. U389), as determinedby two-dimensional gel electrophoresis of in vitro translationproducts. The number and gel position of eight leghaemoglobin(Lb) products, as well as a product tentatively identified asnodule-stimulated glutamine synthetase (GS), was similar toprevious reports of alfalfa (Medicago sativa L. cv. Iroquois)nodulins. Three mutants of Rhizobium meliloti, including anexoH mutant, a lipopolysaccharide mutant, and a nifH mutant,elicited ineffective sweetclover nodules blocked at empty (bacteria-free),partially infected, or fully infected stages of nodule development,respectively. In these ineffective nodules, the nodulin Nma30and nodule-stimulated NSTma42 were expressed early in development,while a group of four nodulins and two nodule-stimulated productswere intermediate in order of expression. Lb, GS and the latenodulin Nmal2a were expressed later, following infection. TheexoH mutant, Rm7154, appeared to be a leaky mutant, as a smallpercentage of the plants developed nitrogen-fixing nodules about4 weeks after inoculation. The sequential expression of a largenumber of nodulins and nodule-stimulated products, as well asthe availability of sweetclover nodulation mutants indicatesthat sweetclover is a useful diploid system for analysis ofhost genes essential to the Rhizobium/legume symbiosis. Key words: Nitrogen fixation, nodulation mutants, nodulins     

Nodulins and nodulin genes of Glycine max

Summary Nodulins are organ-specific plant proteins induced during symbiotic nitrogen fixation. Nodulins play both metabolic and structural roles within infected and uninfected nodule cells. In soybean, several nodulin genes, coding for abundant nodulins, have been identified and isolated. Structural analysis of some of these genes has revealed their possible mode of regulation and the subcellar location of the protein product. Studies of ineffective symbiosis based on cultivar-strain genotype differences suggested that both partners influence the expression of nodulin genes. Concomitant with nodule organogenesis, the Rhizobium undergoes substantial differentiation leading to the accumulation of nodule-specific bacterial proteins, bacteroidins. The major structural alteration occuring in the infected cell is the formation of a membrane enclosing the bacteroid (peribacteroid membrane). A number of nodulins are specifically targetted to this membrane during endosymbiosis. The induction of nodulins and bacteroidins leads to the formation of an effective nodule. Nodulin genes can be induced in vitro by factors derived from nodules suggesting that trans-activators may be involved in derepression of the host genes necessary for Rhizobium-legume symbiosis.     

Plant genes induced in the Rhizobium-legume symbiosis

Rhizobium, Bradyrhizobium and Azorhizobium can elicit the formation of N₂-fixing nodules on the roots or stems of their leguminous host plants. The nodule formation involves several developmental steps determined by different sets of genes from both partners, the gene expression being temporally and spatially coordinated. The plant proteins that are specifically synthesised during the formation and function of the nodule are called nodulins. The nodulins that are expressed before the onset of N₂ fixation are termed early nodulins. These proteins are probably involved in the infection process as well as in nodule morphogenesis rather than in nodule function. The nodulins expressed just before or during N₂ fixation are termed late nodulins and they participate in the function of the nodule by creating the physiological conditions required for nitrogen fixation, ammonium assimilation and transport. In this review we will describe nodulins, nodulin genes and the relationship between nodulin gene expression and nodule development. The study of nodulin gene expression may provide insight into root-nodule development and the mechanism of communication between bacteria and host plant.J.A. Muñoz and A.J. Palomares are with the Departamento de Microbiologia y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain. P. Ratet is with the Institut des Sciences Végétales, CNRS, Avenue de la Terrasse, F-91198 Gif-sur-Yvette, Fance     

Glycine-rich proteins encoded by a nodule-specific gene family are implicated in different stages of symbiotic nodule development in Medicago spp

Four genes encoding small proteins with significantly high glycine content have been identified from root nodules of Medicago sativa. All of these proteins as well as their Medicago truncatula homologues carried an amino terminal signal peptide and a glycine-rich carboxy terminal domain. All except nodGRP3 lacked the characteristic repeat structure described for cell wall and stress response-related glycine-rich proteins (GRP). Expression of these GRP genes was undetectable in flower, leaf, stem, and hypocotyl cells, whereas expression was highly induced during root nodule development, suggesting that GRP genes act as nodulins. Moreover, none of these nodule-expressed GRP genes were activated by hormones or stress treatments, which are inducers of many other GRPs. In Rhizobium-free spontaneous nodules and in nodules induced by a noninfective mutant strain of Sinorhizobium meliloti, all these genes were repressed, while they were induced in Fix- nodules, unaffected in bacterial infection, but halted in bacteroid differentiation. These results demonstrated that bacterial infection but not bacteroid differentiation is required for the induction of the nodule-specific GRP genes. Differences in kinetics and localization of gene activation as well as in the primary structure of proteins suggest nonredundant roles for these GRPs in nodule organogenesis.     

Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti

Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.     

The exoD gene of Rhizobium meliloti encodes a novel function needed for alfalfa nodule invasion

During the symbiotic interaction between alfalfa and the nitrogen-fixing bacterium Rhizobium meliloti, the bacterium induces the formation of nodules on the plant roots and then invades these nodules. Among the bacterial genes required for nodule invasion are the exo genes, involved in production of an extracellular polysaccharide, and the ndv genes, needed for production of a periplasmic cyclic glucan. Mutations in the exoD gene result in altered exopolysaccharide production and in a nodule invasion defect. In this work we show that the stage of symbiotic arrest of exoD mutants is similar to that of other exo and ndv mutants. However, the effects of exoD mutations on exopolysaccharide production and growth on various media are different from the effects of other exo and ndv mutations. Finally, exoD mutations behave differently from other exo mutations in their ability to be suppressed or complemented extracellularly. The results suggest that exoD represents a new class of Rhizobium genes required for nodule invasion, distinct from the other exo genes and the ndv genes. We discuss models for the function of exoD.     

Ultrastructural studies on nodules induced by pyrimidine auxotrophs of Sinorhizobium meliloti

Twenty three pyrimidine auxotrophs of Sinorhizobium meliloti Rmd201 were generated by random mutagenesis with transposon Tn5. On the basis of biochemical characters these auxotrophic mutants were classified into car, pyrC and pyrE/pyrF categories. All auxotrophs induced white nodules which were ineffective in nitrogen fixation. Light and electron microscopic studies revealed that the nodules induced by pyrC mutants were more developed than the nodules of car mutants. Similarly the nodules induced by pyrE/pyrF mutants had more advanced structural features than the nodules of pyrC mutants. The nodule development in case of pyrE/pyrF mutants was not to the extent observed in the parental strain. These results indicated that some of the intermediates and/or enzymes of pyrimidine biosynthetic pathway of S. meliloti play a key role in bacteroidal transformation and nodule development.