Application of Strigolactones to Plant Roots to Influence Formation of Symbioses.

Strigolactones play a potent role in the rhizosphere as a signal to symbiotic microbes including arbuscular mycorrhizal fungi and rhizobial bacteria. This chapter outlines guidelines for application of strigolactones to pea roots to influence symbiotic relationships, and includes careful consideration of type of strigolactones applied, solvent use, frequency of application and nutrient regime to optimize experimental conditions.

Airborne exposure of Rhizobium leguminosarum strain E20-8 to volatile monoterpenes: Effects on cells challenged by cadmium.

Volatile organic compounds (VOCs) are produced by plants, fungi, bacteria and animals. These compounds are metabolites originated mainly in catabolic reactions and can be involved in biological processes. In this study, the airborne effects of five monoterpenes (α-pinene, limonene, eucalyptol, linalool, and menthol) on the growth and oxidative status of the rhizobial strain Rhizobium leguminosarum E20-8 were studied, testing the hypothesis that these VOCs could influence Rhizobium growth and tolerance to cadmium. The tested monoterpenes were reported to have diverse effects, such as antibacterial activity (linalool, limonene, α-pinene, eucalyptol), modulation of antioxidant response or antioxidant properties (α-pinene and menthol). Our results showed that non-stressed cells of Rhizobium E20-8 have different responses (growth, cell damage and biochemistry) to monoterpenes, with α-pinene and eucalyptol increasing colonies growth. In stressed cells the majority of monoterpenes failed to minimize the detrimental effects of Cd and increased damage, decreased growth and altered cell biochemistry were observed. However, limonene (1 and 100 mM) and eucalyptol (100 nM) were able to increase the growth of Cd-stressed cells. Our study evidences the influence at-a-distance that organisms able to produce monoterpenes may have on the growth and tolerance of bacterial cells challenged by different environmental conditions.

Characterisation of SalRAB a salicylic acid inducible positively regulated efflux system of Rhizobium leguminosarum bv viciae 3841.

Salicylic acid is an important signalling molecule in plant-microbe defence and symbiosis. We analysed the transcriptional responses of the nitrogen fixing plant symbiont, Rhizobium leguminosarum bv viciae 3841 to salicylic acid. Two MFS-type multicomponent efflux systems were induced in response to salicylic acid, rmrAB and the hitherto undescribed system salRAB. Based on sequence similarity salA and salB encode a membrane fusion and inner membrane protein respectively. salAB are positively regulated by the LysR regulator SalR. Disruption of salA significantly increased the sensitivity of the mutant to salicylic acid, while disruption of rmrA did not. A salA/rmrA double mutation did not have increased sensitivity relative to the salA mutant. Pea plants nodulated by salA or rmrA strains did not have altered nodule number or nitrogen fixation rates, consistent with weak expression of salA in the rhizosphere and in nodule bacteria. However, BLAST analysis revealed seventeen putative efflux systems in Rlv3841 and several of these were highly differentially expressed during rhizosphere colonisation, host infection and bacteroid differentiation. This suggests they have an integral role in symbiosis with host plants.

Synergistic interaction of Rhizobium leguminosarum bv. viciae and arbuscular mycorrhizal fungi as a plant growth promoting biofertilizers for faba bean (Vicia faba L.) in alkaline soil.

Egyptian soils are generally characterized by slightly alkaline to alkaline pH values (7.5-8.7) which are mainly due to its dry environment. In arid and semi-arid regions, salts are less concentrated and sodium dominates in carbonate and bicarbonate forms, which enhance the formation of alkaline soils. Alkaline soils have fertility problems due to poor physical properties which adversely affect the growth and the yield of crops. Therefore, this study was devoted to investigating the synergistic interaction of Rhizobium and arbuscular mycorrhizal fungi for improving growth of faba bean grown in alkaline soil. A total of 20 rhizobial isolates and 4 species of arbuscular mycorrhizal fungi (AMF) were isolated. The rhizobial isolates were investigated for their ability to grow under alkaline stress. Out of 20 isolates 3 isolates were selected as tolerant isolates. These 3 rhizobial isolates were identified on the bases of the sequences of the gene encoding 16S rRNA and designated as Rhizobium sp. Egypt 16 (HM622137), Rhizobium sp. Egypt 27 (HM622138) and Rhizobium leguminosarum bv. viciae STDF-Egypt 19 (HM587713). The best alkaline tolerant was R. leguminosarum bv. viciae STDF-Egypt 19 (HM587713). The effect of R. leguminosarum bv. viciae STDF-Egypt 19 and mixture of AMF (Acaulospora laevis, Glomus geosporum, Glomus mosseae and Scutellospora armeniaca) both individually and in combination on nodulation, nitrogen fixation and growth of Vicia faba under alkalinity stress were assessed. A significant increase over control in number and mass of nodules, nitrogenase activity, leghaemoglobin content of nodule, mycorrhizal colonization, dry mass of root and shoot was recorded in dual inoculated plants than plants with individual inoculation. The enhancement of nitrogen fixation of faba bean could be attributed to AMF facilitating the mobilization of certain elements such as P, Fe, K and other minerals that involve in synthesis of nitrogenase and leghaemoglobin. Thus it is clear that the dual inoculation with Rhizobium and AMF biofertilizer is more effective for promoting growth of faba bean grown in alkaline soils than the individual treatment, reflecting the existence of synergistic relationships among the inoculants.

Effects of nano-TiO2 on the agronomically-relevant Rhizobium-legume symbiosis.

The impact of nano-TiO2 on Rhizobium-legume symbiosis was studied using garden peas and the compatible bacterial partner Rhizobium leguminosarum bv. viciae 3841. Exposure to nano-TiO2 did not affect the germination of peas grown aseptically, nor did it impact the gross root structure. However, nano-TiO2 exposure did impact plant development by decreasing the number of secondary lateral roots. Cultured R. leguminosarum bv. viciae 3841 was also impacted by exposure to nano-TiO2, resulting in morphological changes to the bacterial cells. Moreover, the interaction between these two organisms was disrupted by nano-TiO2 exposure, such that root nodule development and the subsequent onset of nitrogen fixation were delayed. Further, the polysaccharide composition of the walls of infected cells of nodules was altered, suggesting that the exposure induced a systemic response in host plants. Therefore, nano-TiO2 contamination in the environment is potentially hazardous to the Rhizobium-legume symbiosis system.

Rhizobium leguminosarum bv. viciae 3841 Adapts to 2,4-Dichlorophenoxyacetic Acid with "Auxin-Like" Morphological Changes, Cell Envelope Remodeling and Upregulation of Central Metabolic Pathways.

There is a growing need to characterize the effects of environmental stressors at the molecular level on model organisms with the ever increasing number and variety of anthropogenic chemical pollutants. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as one of the most widely applied pesticides in the world, is one such example. This herbicide is known to have non-targeted undesirable effects on humans, animals and soil microbes, but specific molecular targets at sublethal levels are unknown. In this study, we have used Rhizobium leguminosarum bv. viciae 3841 (Rlv) as a nitrogen fixing, beneficial model soil organism to characterize the effects of 2,4-D. Using metabolomics and advanced microscopy we determined specific target pathways in the Rlv metabolic network and consequent changes to its phenotype, surface ultrastructure, and physical properties during sublethal 2,4-D exposure. Auxin and 2,4-D, its structural analogue, showed common morphological changes in vitro which were similar to bacteroids isolated from plant nodules, implying that these changes are related to bacteroid differentiation required for nitrogen fixation. Rlv showed remarkable adaptation capabilities in response to the herbicide, with changes to integral pathways of cellular metabolism and the potential to assimilate 2,4-D with consequent changes to its physical and structural properties. This study identifies biomarkers of 2,4-D in Rlv and offers valuable insights into the mode-of-action of 2,4-D in soil bacteria.

Mutation in the pssA gene involved in exopolysaccharide synthesis leads to several physiological and symbiotic defects in Rhizobium leguminosarum bv. trifolii.

The symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. trifolii 24.2 secretes large amounts of acidic exopolysaccharide (EPS), which plays a crucial role in establishment of effective symbiosis with clover. The biosynthesis of this heteropolymer is conducted by a multi-enzymatic complex located in the bacterial inner membrane. PssA protein, responsible for the addition of glucose-1-phosphate to a polyprenyl phosphate carrier, is involved in the first step of EPS synthesis. In this work, we characterize R. leguminosarum bv. trifolii strain Rt270 containing a mini-Tn5 transposon insertion located in the 3'-end of the pssA gene. It has been established that a mutation in this gene causes a pleiotropic effect in rhizobial cells. This is confirmed by the phenotype of the mutant strain Rt270, which exhibits several physiological and symbiotic defects such as a deficiency in EPS synthesis, decreased motility and utilization of some nutrients, decreased sensitivity to several antibiotics, an altered extracellular protein profile, and failed host plant infection. The data of this study indicate that the protein product of the pssA gene is not only involved in EPS synthesis, but also required for proper functioning of Rhizobium leguminosarum bv. trifolii cells.

Functional and expression analysis of the metal-inducible dmeRF system from Rhizobium leguminosarum bv. viciae.

A gene encoding a homolog to the cation diffusion facilitator protein DmeF from Cupriavidus metallidurans has been identified in the genome of Rhizobium leguminosarum UPM791. The R. leguminosarum dmeF gene is located downstream of an open reading frame (designated dmeR) encoding a protein homologous to the nickel- and cobalt-responsive transcriptional regulator RcnR from Escherichia coli. Analysis of gene expression showed that the R. leguminosarum dmeRF genes are organized as a transcriptional unit whose expression is strongly induced by nickel and cobalt ions, likely by alleviating the repressor activity of DmeR on dmeRF transcription. An R. leguminosarum dmeRF mutant strain displayed increased sensitivity to Co(II) and Ni(II), whereas no alterations of its resistance to Cd(II), Cu(II), or Zn(II) were observed. A decrease of symbiotic performance was observed when pea plants inoculated with an R. leguminosarum dmeRF deletion mutant strain were grown in the presence of high concentrations of nickel and cobalt. The same mutant induced significantly lower activity levels of NiFe hydrogenase in microaerobic cultures. These results indicate that the R. leguminosarum DmeRF system is a metal-responsive efflux mechanism acting as a key element for metal homeostasis in R. leguminosarum under free-living and symbiotic conditions. The presence of similar dmeRF gene clusters in other Rhizobiaceae suggests that the dmeRF system is a conserved mechanism for metal tolerance in legume endosymbiotic bacteria.

ABC transport is inactivated by the PTS(Ntr) under potassium limitation in Rhizobium leguminosarum 3841.

PTS(Ntr) is a regulatory phosphotransferase system in many bacteria. Mutation of the PTS(Ntr) enzymes causes pleiotropic growth phenotypes, dry colony morphology and a posttranslational inactivation of ABC transporters in Rhizobium leguminosarum 3841. The PTS(Ntr) proteins EI(Ntr) and 2 copies of EIIA(Ntr) have been described previously. Here we identify the intermediate phosphocarrier protein NPr and show its phosphorylation by EI(Ntr) in vitro. Furthermore we demonstrate that phosphorylation of EI(Ntr) and NPr is required for ABC transport activation and that the N-terminal GAF domain of EI(Ntr) is not required for autophosphorylation. Previous studies have shown that non-phosphorylated EIIA(Ntr) is able to modulate the transcriptional activation of the high affinity potassium transporter KdpABC. In R. leguminosarum 3841 kdpABC expression strictly depends on EIIA(Ntr). Here we demonstrate that under strong potassium limitation ABC transport is inactivated, presumably by non-phosphorylated EIIA(Ntr). This is to our knowledge the first report where PTS(Ntr) dictates an essential cellular function. This is achieved by the inverse regulation of two important ATP dependent transporter classes.

Quantitative analysis of the naringenin-inducible proteome in Rhizobium leguminosarum by isobaric tagging and mass spectrometry.

The rhizobium-legume interaction is a critical cornerstone of crop productivity and environmental sustainability. Its potential improvement relies on elucidation of the complex molecular dialogue between its two partners. In the present study, the proteomic patterns of gnotobiotic cultures of Rhizobium leguminosarum bv. viciae 3841 grown for 6 h in presence or absence of the nod gene-inducing plant flavonoid naringenin (10 µM) were analyzed using the iTRAQ approach. A total of 1334 proteins were identified corresponding to 18.67% of the protein-coding genes annotated in the sequenced genome of bv. viciae 3841. The abundance levels of 47 proteins were increased upon naringenin treatment showing fold change ratios ranging from 1.5 to 25 in two biological replicates. Besides the nod units, naringenin enhanced the expression of a number of other genes, many of which organized in operons, including ß(1-2) glucan production and secretion, succinoglycan export, the RopA outer membrane protein with homology to an oligogalacturonide-specific porin motif, other enzymes for carbohydrate and amino acid metabolism, and proteins involved in the translation machinery. Data were validated at the transcriptional and phenotypic levels by RT-PCR and an assay of secreted sugars in culture supernatants, respectively. The current approach provides not only a high-resolution analysis of the prokaryotic proteome but also unravels the rhizobium molecular dialogue with legumes by detecting the enhanced expression of several symbiosis-associated proteins, whose flavonoid-dependency had not yet been reported.

[Role of allelopathic compositions in the regulation and development of legume-rhizobial symbiosis].

It was discovered that aromatic compounds isolated from root exudates of three legume species (Pisum sativum L., Vicia faba L. var. major Hartz, and Glycine max L. MERR) and identified as N-phenyl-2-naphthyl amine, dibutyl, and dioctyl esters of orthophthalic acid, which are known to work as negative allelopathic substances, are involved in the regulation of legume-rhizobial symbiosis formation after the inoculation of roots with rhizobia under unfavorable conditions for symbiosis.

Synthesis of microbial signaling molecules and their stereochemistry-activity relationships.

Microbial signaling molecules such as autoinducers and microbial hormones play important roles in intercellular communication in microorganisms. Information transfer between the individual cells of a microorganism is one of the most important biological events among them. Researchers often suffer from extremely low levels of microbial signaling molecule contents, which prevent them from understanding chemistry and biology of intercellular communication in microorganisms. Chemical synthesis is a powerful tool to obtain sufficient amounts of sample and to clarify the structure of a molecule. This review focuses on the synthesis and stereochemistry-bioactivity relationships of five microbial signaling molecules, Vibrio cholerae autoinducer-1 (CAI-1), AI-2 precursor (DPD), an acylhomoserine lactone from Rhizobium leguminosarum (small bacteriocin), a diffusible extracellular factor of Xanthomondas campestris pv. campestris, and Phytophthora mating hormone α1.

An acpXL mutant of Rhizobium leguminosarum bv. phaseoli lacks 27-hydroxyoctacosanoic acid in its lipid A and is developmentally delayed during symbiotic infection of the determinate nodulating host plant Phaseolus vulgaris.

Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membrane-derived subcellular compartments. Notably, an unusual very-long-chain fatty acid (VLCFA) is found in the lipid A of R. leguminosarum as well as in the lipid A of the medically relevant pathogens Brucella abortus, Brucella melitensis, Bartonella henselae, and Legionella pneumophila, which are also able to persist within intracellular host-derived membranes. These bacterial symbionts and pathogens each contain a homologous gene region necessary for the synthesis and transfer of the VLCFA to the lipid A. Within this region lies a gene that encodes the specialized acyl carrier protein AcpXL, on which the VLCFA is built. This study describes the biochemical and infection phenotypes of an acpXL mutant which lacks the VLCFA. The mutation was created in R. leguminosarum bv. phaseoli strain 8002, which forms symbiosis with Phaseolus vulgaris, a determinate nodulating legume. Structural analysis using gas chromatography and mass spectrometry revealed that the mutant lipid A lacked the VLCFA. Compared to the parent strain, the mutant was more sensitive to the detergents deoxycholate and dodecyl sulfate and the antimicrobial peptide polymyxin B, suggesting a compromise to membrane stability. In addition, the mutant was more sensitive to higher salt concentrations. Passage through the plant restored salt tolerance. Electron microscopic examination showed that the mutant was developmentally delayed during symbiotic infection of the host plant Phaseolus vulgaris and produced abnormal symbiosome structures.

Development of a real-time PCR assay for detection and quantification of Rhizobium leguminosarum bacteria and discrimination between different biovars in zinc-contaminated soil.

Primers were designed to target 16S rRNA and nodD genes of Rhizobium leguminosarum from DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by both nodD and the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional gene nodD, providing compelling evidence of a toxic effect of Zn on R. leguminosarum populations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH4NO3-extractable and soil solution Zn. R. leguminosarum bv. viciae nodD gene copies were generally less sensitive to Zn than R. leguminosarum bv. trifolii nodD. The latter were generally below detection limits at Zn levels of >250 mg kg(-1). Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.

Characterization of swarming motility in Rhizobium leguminosarum bv. viciae.

We have characterized swarming motility in Rhizobium leguminosarum strains 3841 and VF39SM. Swarming was dependent on growth on energy-rich media, and both agar concentration and incubation temperature were critical parameters for surface migration. A cell density-dependent lag period was observed before swarming motility was initiated. Surface migration began 3-5 days after inoculation and a full swarming phenotype was observed 3 weeks after inoculation. The swarming front was preceded by a clear extracellular matrix, from which we failed to detect surfactants. The edge of the swarming front formed by VF39SM was characterized by hyperflagellated cells arranged in rafts, whereas the cells at the point of inoculation were indistinguishable from vegetative cells. Swarmer cells formed by 3841, in contrast, showed a minor increase in flagellation, with each swarmer cell exhibiting an average of three flagellar filaments, compared with an average of two flagella per vegetative cell. Reflective of their hyperflagellation, the VF39SM swarmer cells demonstrated an increased expression of flagellar genes. VF39SM swarmed better than 3841 under all the conditions tested, and the additional flagellation in VF39SM swarm cells may contribute to this difference. Metabolism of the supplemented carbon source appeared to be necessary for surface migration as strains incapable of utilizing the carbon source failed to swarm. We also observed that swarmer cells have increased resistance to several antibiotics.

[Effect of nitric oxide and other nitrogen compounds on the adhesion and penetration of nodule bacteria into root tissues and on growth of etiolated pea seedlings].

The action of sodium nitroprusside, a nitric oxide donor, and other nitrogen compounds (KNO3, KNO2, and (NH4)2SO4) on adhesion and penetration of nodule bacteria into root tissues of etiolated pea seedlings was studied. Only nitroprusside displayed a clearly negative effect on rhizobium adhesion and penetration and seedling growth. This effect was not observed with other nitrogen compounds even at high (20 mM) concentrations. Hemoglobin attenuated the negative effect of nitroprusside on bacteria and seedlings. The results are discussed in the context of the role of nitric oxide in the life of plants and nodule bacteria.

Synthesis and stereochemistry-activity relationship of small bacteriocin, an autoinducer of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum.

The four stereoisomers of small bacteriocin, an autoinducer of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum, were synthesized via a versatile methodology for 3'-hydroxyacyl homoserine lactones based on the Nagao asymmetric aldol reaction. The synthetic isomers were much less effective at inhibiting the growth of R. leguminosarum RBL5523 than the natural isomer, showing the importance of stereochemistry for activity.

Effects of heavy metal toxicity on growth, symbiosis, seed yield and metal uptake in pea grown in metal amended soil.

Soils contaminated with heavy metals present a major threat to sustainable agriculture. Understanding the effects of these metals on pea productivity will be useful. We studied the effects of cadmium, chromium and copper used both separately and as mixtures, on over all growth of pea plants inoculated with Rhizobium sp. Among the metals, copper was most toxic for pea plants and decreased the seed yield by 15% at 1,338 mg kg(-1) compared to control plants whereas cadmium and chromium in general, increased the measured parameters. The metal accumulation in roots and shoots at 90 d and in grains at 120 d differed among treatments.

Mutagenesis of the carboxy terminal protease CtpA decreases desiccation tolerance in Rhizobium leguminosarum.

To better understand the role of proteases in Rhizobium leguminosarum biovar viciae, a gene with homology to the carboxy-terminal protease (CtpA), which belongs to a novel group of serine proteases, was studied. The ctpA gene was cloned and mutated using allelic exchange and a gusA reporter gene was used to study ctpA expression. Mutational analysis shows that ctpA is critical for the viability of R. leguminosarum when cells are grown on complex semi-solid media but is dispensable when cells are grown in complex liquid media and that this is likely due to an increase in susceptibility to desiccation on semi-solid media. The ctpA mutant also displayed an increased sensitivity to detergents, indicating an alteration in the permeability of the cell envelope. This is the first characterization of a ctpA gene within the Rhizobiaceae and the first report of a ctpA mutant that exhibits an increased sensitivity to desiccation.

Response of rhizobial populations to moderate copper stress applied to an agricultural soil.

The use of pesticides in agricultural soils may affect the soil microbiota. The effect of repeated application of copper sulfate in soil on indigenous populations of rhizobia was assessed in a medium-term field experiment. Copper sulfate was applied over 8 years at two different rates, 12.5 and 50 kg of CuSO4 ha(-1) year(-1), in the field. The concentrations of total copper in soil varied between 14.0 (control plots that did not receive copper sulfate) and 91.0 mg kg(-1) (the most contaminated plots) at the time of sampling, 3 years after the end of the copper treatments. All the other physicochemical parameters were similar among the plots that also shared the same cropping history. The target rhizobia were monospecific populations of Rhizobium leguminosarum bv. viciae nodulating Vicia sativa and communities of rhizobial species nodulating Phaseolus vulgaris. The size of the vetch rhizobial populations was significantly reduced in the soils with the higher Cu content, whereas the size of the Phaseolus rhizobial populations was not significantly affected. However, the number of nodules formed on both vetches and common beans were reduced for the plants grown in the most contaminated soils, suggesting an additional toxic effect of copper on plant physiology. The diversity (Simpson's indices) of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer (IGS), was not influenced by copper application. Also, the genetic structure of the R. leguminosarum bv. viciae populations was not modified by copper treatments. By contrast, a shift was observed in the composition of the Phaseolus-nodulating communities in relation to soil copper content. The communities were composed of three 16S rDNA haplotypes: one corresponding to the R. leguminosarum (biovar phaseoli) species, the two others forming a new lineage of Phaseolus rhizobia based on 16S rDNA sequence analysis. The reduced frequency of the R. leguminosarum species in the Phaseolus-nodulating communities from the copper-treated soils was linked to its higher sensitivity to copper as compared to the higher tolerance of isolates belonging to the other rhizobial lineage. The new lineage was functionally efficient for symbiotic nitrogen fixation with P. vulgaris. Our results suggest that functional redundancy among species exhibiting variability for copper tolerance preserved the size of Phaseolus-nodulating communities. In contrast, the abundance of the vetch-nodulating rhizobia, which was a monospecific functional group mainly constituted by copper-sensitive genotypes, was adversely affected by repeated application of copper sulfate.