Nodulation outer proteins: double-edged swords of symbiotic rhizobia.

Rhizobia are nitrogen-fixing bacteria that establish a nodule symbiosis with legumes. Nodule formation depends on signals and surface determinants produced by both symbiotic partners. Among them, rhizobial Nops (nodulation outer proteins) play a crucial symbiotic role in many strain-host combinations. Nops are defined as proteins secreted via a rhizobial T3SS (type III secretion system). Functional T3SSs have been characterized in many rhizobial strains. Nops have been identified using various genetic, biochemical, proteomic, genomic and experimental approaches. Certain Nops represent extracellular components of the T3SS, which are visible in electron micrographs as bacterial surface appendages called T3 (type III) pili. Other Nops are T3 effector proteins that can be translocated into plant cells. Rhizobial T3 effectors manipulate cellular processes in host cells to suppress plant defence responses against rhizobia and to promote symbiosis-related processes. Accordingly, mutant strains deficient in synthesis or secretion of T3 effectors show reduced symbiotic properties on certain host plants. On the other hand, direct or indirect recognition of T3 effectors by plant cells expressing specific R (resistance) proteins can result in effector triggered defence responses that negatively affect rhizobial infection. Hence Nops are double-edged swords that may promote establishment of symbiosis with one legume (symbiotic factors) and impair symbiotic processes when bacteria are inoculated on another legume species (asymbiotic factors). In the present review, we provide an overview of our current understanding of Nops. We summarize their symbiotic effects, their biochemical properties and their possible modes of action. Finally, we discuss future perspectives in the field of T3 effector research.

Lotus japonicus symRK-14 uncouples the cortical and epidermal symbiotic program.

SYMRK is a leucine-rich-repeat (LRR)-receptor kinase that mediates intracellular symbioses of legumes with rhizobia and arbuscular mycorrhizal fungi. It participates in signalling events that lead to epidermal calcium spiking, an early cellular response that is typically considered as central for intracellular accommodation and nodule organogenesis. Here, we describe the Lotus japonicus symRK-14 mutation that alters a conserved GDPC amino-acid sequence in the SYMRK extracellular domain. Normal infection of the epidermis by fungal or bacterial symbionts was aborted in symRK-14. Likewise, epidermal responses of symRK-14 to bacterial signalling, including calcium spiking, NIN gene expression and infection thread formation, were significantly reduced. In contrast, no major negative effects on the formation of nodule primordia and cortical infection were detected. Cumulatively, our data show that the symRK-14 mutation uncouples the epidermal and cortical symbiotic program, while indicating that the SYMRK extracellular domain participates in transduction of non-equivalent signalling events. The GDPC sequence was found to be highly conserved in LRR-receptor kinases in legumes and non-legumes, including the evolutionarily distant bryophytes. Conservation of the GDPC sequence in nearly one-fourth of LRR-receptor-like kinases in the genome of Arabidopsis thaliana suggests, however, that this sequence might also play an important non-symbiotic function in this plant.

Nodulation of Sesbania species by Rhizobium (Agrobacterium) strain IRBG74 and other rhizobia.

Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a bacterial strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridization with R. radiobacter, R. rubi, R. vitis and R. huautlense gave only 44%, 5%, 8% and 8% similarity respectively, suggesting that IRBG74 is potentially a new species. Additionally, it contained no vir genes and lacked tumour-forming ability, but harboured a sym-plasmid containing nifH and nodA genes similar to those in other Sesbania symbionts. Indeed, IRBG74 effectively nodulated S. cannabina and seven other Sesbania spp. that nodulate with Ensifer (Sinorhizobium)/Rhizobium strains with similar nodA genes to IRBG74, but not species that nodulate with Azorhizobium or Mesorhizobium. Light and electron microscopy revealed that IRBG74 infected Sesbania spp. via lateral root junctions under flooded conditions, but via root hairs under non-flooded conditions. Thus, IRBG74 is the first confirmed legume-nodulating symbiont from the Rhizobium (Agrobacterium) clade. Cross-inoculation studies with various Sesbania symbionts showed that S. cannabina could form fully effective symbioses with strains in the genera Rhizobium and Ensifer, only ineffective ones with Azorhizobium strains, and either partially effective (Mesorhizobium huakii) or ineffective (Mesorhizobium plurifarium) symbioses with Mesorhizobium. These data are discussed in terms of the molecular phylogeny of Sesbania and its symbionts.

Identification of Rhizobium plasmid sequences involved in recognition of Psophocarpus, Vigna, and other legumes.

Symbiotic DNA sequences involved in nodulation by Rhizobium must include genes responsible for recognizing homologous hosts. We sought these genes by mobilizing the symbiotic plasmid of a broad host-range Rhizobium MPIK3030 (= NGR234) that can nodulate Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, etc., into two Nod- Rhizobium mutants as well as into Agrobacterium tumefaciens. Subsequently, cosmid clones of pMPIK3030a were mobilized into Nod+ Rhizobium that cannot nodulate the chosen hosts. Nodule development was monitored by examining the ultrastructure of nodules formed by the transconjugants. pMPIK3030a could complement Nod- and Nif- deletions in R. leguminosarum and R. meliloti as well as enable A. tumefaciens to nodulate. Three non-overlapping sets of cosmids were found that conferred upon a slow-growing Rhizobium species, as well as on R. loti and R. meliloti, the ability to nodulate Psophocarpus and Vigna, thus pointing to the existence of three sets of host-specificity genes. Recipients harboring these hsn regions had truly broadened host-range since they could nodulate both their original hosts as well as MPIK3030 hosts.

Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development.

To identify bacterial genes involved in symbiotic nodule development, ineffective nodules of alfalfa (Medicago sativa) induced by 64 different Fix-mutants of Rhizobium meliloti were characterized by assaying for symbiotic gene expression and by morphological studies. The expression of leghemoglobin and nodulin-25 genes from alfalfa and of the nifHD genes from R. meliloti were monitored by hybridizing the appropriate DNA probes to RNA samples prepared from nodules. The mutants were accordingly divided into three groups. In group I none of the genes were expressed, in group II only the plant genes were expressed and in group III all three genes were transcribed. Light and electron microscopical analysis of nodules revealed that nodule development was halted at different stages in nodules induced by different group I mutants. In most cases nodules were empty lacking infection threads and bacteroids or nodules contained infection threads and a few released bacteroids. In nodules induced by a third mutant class bacteria were released into the host cells, however the formation of the peribacteroid membrane was not normal. On this basis we suggest that peribacteroid membrane formation precedes leghemoglobin and nodulin-25 induction, moreover, after induction of nodulation by the nod genes at least two communication steps between the bacteria and the host plants are necessary for the development of the mature nodule. By complementing each mutant of group I with a genomic R. meliloti library made in pLAFRl, four new fix loci were identified, indicating that several bacterial genes are involved in late nodule development.

Scanning electron microscopic observations on micro-organisms in the root nodules of Tribulus terrestris L. (Zygophyllaceae).

Scanning electron microscopic observations were made on the micro-organisms of root nodules of Tribulus terrestris L. The results showed that nodules of T. terrestris contained dual infection consisting of Rhizobium sp. and Newmania karachiensis. Based on these observations, T. terrestris should be grouped with nonlegume Parasponia-type bacterial nodules.

Rhizobium strains differ considerably in outer membrane permeability and polymyxin B resistance.

Six rhizobium (Rhizobium leguminosarum bv. Trifolii TA1, Sinorhizobium meliloti 1021, Mesorhizobium huakuii IFO 15243(T), Ochrobactrum lupini LUP 21(T), Bradyrhizobium japonicum USDA110 and B. elkanii USDA 76) and two Escherichia coli strains (E. coli ATCC 25922 and E. coli HB 101) were compared in respect to polymyxin B and EDTA resistance, as well as bacterial outer membrane (OM) permeability to a fluorescent hydrophobic agent (N-phenyl-1-naphthylamine - NPN). TEM (Transmission Electron Microscopy) and a microbial test demonstrated that all the rhizobia were much more resistant to polymyxin B in comparison with E. coli strains. EDTA and polymyxin B enhance permeability of B. japonicum and O. lupini OM. Other rhizobia incorporated NPN independently of the presence of membrane-deteriorating agents; however, the level of fluorescence (measured as NPN absorption) was strain dependent.

Purification and biochemical properties of complex flagella isolated from Rhizobium lupini H13-3.

1. The complex flagella of Rhizobium lupini H13-3 differ from plain bacterial flagella in the fine structure of their filaments dominated by conspicuous helical bands, in their fragility and their resistance against heat decomposition. To elucidate the basis of these differences, the composition of complex filaments and their subunits was analysed. 2. Isolated complex flagella containing the filament and hook protions were purified by differential centrifugation. Hooks were separated by ultracentrifugation after acid degradation of filaments at pH 2. The complex filaments consist of 43 000 dalton monomers (cx-flagellin), the hooks are composed of 41 000 dalton subunits. 3. Amino acid analysis of cx-flagellin indicated the presence of approx. 417 amino acid residues. These comprise 47% hydrophobic residues and 21% Asp and Glu (or amides), but no Cys, His, Pro and Trp. No carbohydrate, phosphate or lipid moieties have been detected. Fingerprint analysis after tryptic digestion yields approx. 36 peptides, about half of them clustered in the neutral region. A comparison with the composition of varous known flagellins from plain flagella indicates a 7% higher content of hydrophobic amino acid residues in complex filaments; this is largely compensated for by the higher content of Glu and Asp (presumably as Gln and Asn) in plain filaments. 4. Immunodiffusion and immunoelectrophoresis of cx-flagellin yield single precipitin bands indicating homogeneity. In contrast, isoelectric focusing lead to three close-running bands around pH4.7. When isolated, the two major bands again produced an "isoelectric spectrum" suggesting that it reflects an allomorphism of cx-flagellin. 5. Self-assembly experiments with cx-flagellin lead to coiled fibres including helical regions, but not to intact filaments. The products resemble heat-denatured complex filaments and may represent intermediates between monomers and complete polymers.

NopA is associated with cell surface appendages produced by the type III secretion system of Rhizobium sp. strain NGR234.

Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, utilizes a variety of signaling molecules to establish nitrogen-fixing symbioses. Among these are nodulation outer proteins (Nops) that transit through a type III secretion system (TTSS). Abolition of Nop secretion affects nodulation of certain legumes. Under free-living conditions, the secretion of Nops can be induced by the addition of flavonoids. Here, we show that an in-frame deletion of nopA abolishes secretion of all other Nops and has the same impact on nodule formation as mutations that lead to a nonfunctional TTSS. This secretion-minus phenotype of the nopA mutant, as well as bioinformatics analysis of NopA itself, suggests that NopA could be an external component of the TTSS. Electron microscopy showed that NGR234 synthesizes fibrillar structures on the cell surface in a flavonoid-inducible and NopA-dependent manner. Purification of the macromolecular surface appendages revealed that NopA is a major component of these structures.

Role of programmed cell death in development.

Programmed cell death (PCD) is an integral part of both animal and plant development. In animals, model systems such as Caenorhabditis elegans, Drosophila melanogaster, and mice have shown a general cell death profile of induction, caspase mediation, cell death, and phagocytosis. Tremendous strides have been made in cell death research in animals in the past decade. The ordering of the C. elegans genes Ced-3, 4 and 9, identification of caspase-activated DNase that degrades nuclear DNA during PCD, identification of signal transduction modules involving caspases as well as the caspase-independent pathway, and the involvement of mitochondria are some of the findings of immense value in understanding animal PCDs. Similarly, the caspase inactivation mechanisms of infecting viruses to stall host cell death give a new dimension to the viral infection process. However, plant cell death profiles provide an entirely different scenario. The presence of a cell wall that cannot be phagocytosed, absence of the hallmarks of animal PCDs such as DNA laddering, formation of apoptotic bodies, a cell-death-specific nuclease, a biochemical machinery of killer enzymes such as caspases all point to novel ways of cell elimination. Large gaps in our understanding of plant cell death have prompted speculative inferences and comparisons with animal cell death mechanisms. This paper deals with both animals and plants for a holistic view on cell death in eukaryotes.

Pairwise perturbation of flagellin subunits. The structural basis for the differences between plain and complex bacterial flagellar filaments.

Although plain and complex bacterial flagellar filaments differ in their physical properties and helical symmetry, they both appear to derive from a common underlying structure. Analysis of electron micrographs of complex filaments of Rhizobium lupini revealed that the unit cell has twice the length of that of plain filaments, with a corresponding reduction in helical symmetry whereby the six-start helical family present in plain filaments collapses into a three-start family. Mass per unit length measurements were made by scanning transmission electron microscopy. These, together with the unit cell dimensions and the molecular weight of the flagellin monomer, enabled the number of monomers per unit cell to be estimated. Whereas plain filaments have a single monomer per unit cell, complex filaments have two. These results suggest that complex filament structure differs from plain filament structure by a pairwise perturbation, or interaction, of the flagellin monomers. The additional bonding interactions involved in the perturbation in the complex filament may make it more rigid than the plain filament, which has no such perturbation.

Three-dimensional structure of the complex flagellar filament of Rhizobium lupini and its relation to the structure of the plain filament.

Electron micrographs of negatively stained preparations were used to obtain a three-dimensional reconstruction of the complex flagellar filament of Rhizobium lupini H13-3. The complex filament has an organization similar to that of the more common plain filament, but the subunits are perturbed in a pairwise fashion to generate a very distinctive set of three continuous ridges of density along the outer surface of the filament. In the three-dimensional map, the design of the complex filament is similar to that of the plain filament described in the accompanying paper. The structures consist of 11 segmented rods of density lying at a radius of 65 to 70 A. The exterior surfaces of both kinds of filaments consist of features that protrude from the segmented rods. The interiors of both consist of arms that extend inwards from the rods. In the case of the complex filament, but not of the plain filament, the inner arms interact to generate three tubular features, which, together with the three outer ridges, may account for the more brittle and, by implication, stiffer nature of the complex filament.

Cell wall degradation during infection thread formation by the root nodule bacterium Rhizobium leguminosarum is a two-step process.

In the nitrogen-fixing root-nodule symbiosis of Rhizobium leguminosarum biovar viciae and its host plants pea and vetch, the bacteria enter one root cortical cell after another via a tip-growing structure, the infection thread. Rhizobial Nod (nodulation) factors induce the formation of preinfection thread structures (Van Brussel, A.A.N., R. Bakhuizen, P.C. van Spronsen, H.P. Spaink, T. Tak, B.J.J. Lugtenberg, J.W. Kijne, Science 257, 70-72 (1992)), but formation of infection threads requires the presence of bacterial cells. Passing of an infection thread from cell to cell requires local cell wall degradation. We compared at the ultrastructural level local cell wall changes in the outer root cortex of pea and vetch related to preinfection thread formation and infection thread formation, respectively. Cell wall modifications in the outer periclinal walls of root cortical cells induced by Nod factors appeared to be similar to those induced by rhizobia. These modifications take place opposite cytoplasmic bridges and are probably related to induction of tip growth. However, complete cell wall degradation was never observed in the absence of rhizobia. We propose a two-step cell wall degradation process for infection thread formation. The first step is a local cell wall modification by plant enzymes, induced by rhizobial Nod factors. The second step is complete cell wall degradation in the presence of rhizobia.

Auxotrophic mutant strains of Rhizobium etli reveal new nodule development phenotypes.

We report here the isolation and characterization of amino acid-requiring mutant strains of Rhizobium etli. We observe that the phenotype of most mutations, even when causing a strict auxotrophy, is overcome by cross-feeding from the host plant Phaseolus vulgaris, thereby allowing bacterial production of Nod factors and, consequently, nodule induction. Conversely, light and electron microscopy analysis reveals that the nodules induced by all mutants, including those with normal external morphology, are halted or strongly altered at intermediate or late stages of development. Moreover, some mutants induce nodules that display novel symbiotic phenotypes, such as specific alterations of the invaded cells or the presence of a reduced number of abnormally shaped uninvaded cells. Other mutants induce nodules showing an early and vast necrosis of the central tissue, a phenotype not previously observed in bean nodules, not even in nodules induced by a Fix- mutant. These observations indicate that amino acid auxotrophs represent a powerful tool to study the development of globose determinate-type nodules and emphasize the importance of establishing their histology and cytology before considerations of metabolic exchange are made.

How is leghemoglobin involved in peribacteroid membrane degradation during nodule senescence?

An increase in the rate of succinate and glutamate uptake by isolated symbiosomes from French bean nodules was observed in the presence of iron plus H2O2. The lipid bilayer, and not proteins involved in transport, seems to be the major target of radical attack. Leghemoglobin in the presence of a 6-fold excess of H2O2 (where heme breakdown and iron release occurred) provoked also an increase in peribacteroid membrane permeability. In contrast, this hemoprotein in the presence of a 2-fold excess of H2O2 (where a protein radical was generated) was without effect. We suggest that in vivo the release of heme iron may constitute the major process concerning the involvement of leghemoglobin in the degradation of the peribacteroid membrane during nodule senescence.

A phenomenon of photoenhancement of acridine orange-induced fluorescence.

It is a common experience that, with exposure to exciting radiation of the fluorescence microscope, the acridine orange-induced red fluorescence of the nucleus, produced by Feulgen hydrolysis, fades with a concomitant shift to green. The present investigation reports a phenomenon of photoenhancement observed in the hydrolyzed cytoplasm where pale green fluorescence increases in intensity with exposure to exciting radiation. The phenomenon has been noticed in Rhizobium, Oscillatoria, tomato root tip cells and human buccal epithelial cells. It is tentatively concluded that the gain in fluorescence yield is due to certain conformational changes of the acridine orange-protein complex induced by ultraviolet light flux.

Localization of the protein VirB1 involved in contact formation during conjugation among Agrobacterium cells.

Supramembrane structures of Agrobacterium, which link cells during mating, were for the first time visualized using transmission electron microscopy. The initial cell contact was found to be mediated by long pili. Using colloidal gold-labeled, VirB1-specific antibodies, it was established that VirB1 proteins enter into the composition of short pilus-like structures, which emerge at the poles of acetosyringone (AS)-induced agrobacterial cells. Labeling of non-centrifuged agrobacterial cells on a nitrocellulose membrane using colloidal gold-conjugated antibodies to VirB1 showed that the labeled complex could bind to AS-induced cells, but failed to form red stains during incubation with cells of the Ti plasmidless A. tumefaciens strains LBA288 and UBAPF-2.

The region for exopolysaccharide synthesis in Rhizobium leguminosarum biovar trifolii is located on the non-symbiotic plasmid.

An Exo- mutant of Rhizobium leguminosarum biovar trifolii was isolated which did not produce acidic exopolysaccharide and induced defective, non-fixing nodules on clover plants. The nodules were defective at a late stage of development, they contained infection threads and bacteria were released into the host cells. Cosmid pARF136 capable of complementing the Exo- mutation was isolated from a cosmid bank made from total R. trifolii DNA. Hybridization between DNA of pARF136 and plasmids of R. trifolii strains separated by Eckhardt's technique suggested that the exo locus is located on a 300 kb megaplasmid, and nodDABC and nifKDH genes are located on another 180 kb pSym plasmid. A 5.4 kb BamH1 fragment of the recombinant cosmid pARF136 was able to restore exopolysaccharide synthesis in Exo- mutant of R. trifolii 93 but it did not complement the symbiotic defect.

Effect of cadmium on nodulation and N2-fixation of soybean in contaminated soils.

The effects of cadmium stress on nodulation, N2-fixation capabilities of the root nodule, the change in ultrastructure of the root nodule, soybean growth, and the distribution of cadmium in plants were studied. The results obtained show that the nodulation of soybean roots was greatly inhibited by the addition of Cd, especially at the addition level of 10 and 20 mg kg(-1) soil. The inhibition of plant growth, especially the root growth, increased as the cadmium concentration increased, with deleterious effects observed for the roots. The weight ratio of soybean root/leaf decreased as the Cd concentration increased, which might explain the reason for nodulation decreases. The results also indicate that N2-fixation of root nodule was stimulated to some extent at the low levels of Cd addition, but decreased sharply with further increase of the Cd concentration. High Cd levels were also associated with changes in the ultrastructure of root nodule, in which the effective N2-fixing area was reduced and the N2-fixing cells in the area also reduced. In addition, the results also reveal that the content of Cd in different parts of the plants was as follows: roots >> stems > seeds, indicating that the accumulation of Cd by roots is much larger than that by any other part of the soybean plant, and might cause deleterious effects to root systems.

[An current question in the molecular genetics of bacteria]. / Ob odnom aktual'nom voprose molekuliarnoi genetiki bakterii

The author considers the possibilities and limits of extrapolation of the data by the genetics of one species of bacteria to the other. It is emphasized that even in the related bacterial species a similar localization on chromosomes was inherent only to some of the unitypical genes, by in this case as well not all the genes were grouped in the same way, and differed by their delicate structure. An idea on the significant role of genetic metabolism in the microbial evolution is being developed; particular significance is attributed to plasmides. It is supposed that foreign plasmides, particularly transmissive factors of multiple drug resistance could aid in charting the chromosomes of bacteria in which the routes of transmission of genetic information are still unknown. A conclusion was drawn on the necessity of intensification and widening the investigations on the molecular genetics of bacteria of significance for public health and public economy.