Stem Nodules Research Articles

Occurrence and diversity of stem nodulation in Aeschynomene and Sesbania legumes from wetlands of Madagascar

Legumes have the ability to establish a nitrogen-fixing symbiosis with soil rhizobia that they house in specific organs, the nodules. In most rhizobium-legume interactions, nodulation occurs on the root. However, certain tropical legumes growing in wetlands possess a unique trait: the capacity to form rhizobia-harbouring nodules on the stem. Despite the originality of the stem nodulation process, its occurrence and diversity in waterlogging-tolerant legumes remains underexplored, impeding a comprehensive analysis of its genetics and biology. Here, we aimed at filling this gap by surveying stem nodulation in legume species-rich wetlands of Madagascar. Stem nodulation was readily observed in eight hydrophytic species of the legume genera, Aeschynomene and Sesbania, for which significant variations in stem nodule density and morphology was documented. Among these species, A. evenia, which is used as genetic model to study the rhizobial symbiosis, was found to be frequently stem-nodulated. Two other Aeschynomene species, A. cristata and A. uniflora, were evidenced to display a profuse stem-nodulation as occurs in S. rostrata. These findings extend our knowledge on legumes species that are endowed with stem nodulation and further indicate that A. evenia, A. cristata, A. uniflora and S. rostrata are of special interest for the study of stem nodulation. As such, these legume species represent opportunities to investigate different modalities of the nitrogen-fixing symbiosis and this knowledge could provide cues for the engineering of nitrogen-fixation in non-legume crops.

Evaluation of the Co-inoculation Effect of Rhizobium and Plant Growth Promoting Non-rhizobial Endophytes on Vigna radiata

A unique feature of leguminous plants is the establishment of symbiotic bacterial genera inside root or stem nodules that is being recently re-evaluated for investigating the micro-flora discrete to nitrogen fixation. The present research was carried out to evaluate non-rhizobial endophytes and Rhizobium from root nodules of Vigna radiata and ascertain their co-inoculation effect in pot and field conditions. Each strain displayed one or more plant growth-promoting behaviors in varying degrees. The ability to fix nitrogen was observed in all strains; however, a noticeable enhancement in nitrogen fixation was observed when all three strains were co-inoculated. All three strains were found to possess the nifH gene, which plays a key role in the nitrogen fixation process. However, only Rhizobium sp. AAU B3 also had the nodD gene present. Furthermore, combinations of all three strains produced the highest levels of phosphate solubilization, potash mobilisation, Indole Acetic Acid (IAA), and the stress-relieving enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. Interestingly, the succession of the root nodule formation within root hairs seedlings was observed under a fluorescence microscope and two NRE were found to be located inside the root nodules, indicating that they are endophytic. Additionally, a pot and field investigation revealed that the combination of chosen Rhizobium and NRE strains had a favorable impact on the growth and yield characteristics of a green gram. Selected bio-inoculants can reduce the utilizationof synthetic fertilizers by 75%, which might lead to the restoration of the soil's health. Therefore, these bio-inoculants might be explored commercially for sustainable agriculture production.

Nutritional and microbiological effects of vermicompost tea in hydroponic cultivation of maple peas (Pisum sativum var. arvense L.).

Hydroponics receives considerable attentions due to population expansion, soil pollution, and farmland scarcity. However, one significant problem is that its residual effluents are detrimental to the surrounding ecosystem. There is a dire need to find an organic, alternative, biodegradable substrate. Vermicompost tea (VCT) was investigated on its suitability as a hydroponic substrate to provide both nutritional and microbiological benefits. It was found VCT increased the biomass of maple peas (Pisum sativum var. arvense L.), increased stem length, raised the potassium ion content, and promoted the uptake of nitrogen by the roots. Meanwhile, the microorganisms associated with earthworm guts were detected in the maple peas root system, namely the inter-rhizosphere of maple peas, including Enterobacteriaceae, Pseudomonadaceae, and Flavobacteriaceae. The presence of these microorganisms in large number indicated the ability for VCT to retain earthworm intestinal microbes via intestinal tract movement, excreting, and other vital activities. In addition, Rhizobia spp., such as Burkholderiaceae and Rhizobiaceae were also identified in VCT. They are critical for legumes as they can form root or stem nodule symbioses to produce growth hormone, vitamins, nitrogen fixation, and protection against plant stress. These findings are consistent with our chemical analysis that VCT-treated maple peas had increased nitrate and ammonium nitrogen content relative to the control in roots, stems, and leaves, hence increasing maple peas' biomass. The abundance and species of the inter-root bacterial population were found to change during the experimental period, indicating the importance of microbial balance to the growth and nutrient uptake of maple peas.

Systematic Analysis of Lysine Acetylation Reveals Diverse Functions in Azorhizobium caulinodans Strain ORS571.

Protein acetylation can quickly modify the physiology of bacteria to respond to changes in environmental or nutritional conditions, but little information on these modifications is available in rhizobia. In this study, we report the lysine acetylome of Azorhizobium caulinodans strain ORS571, a model rhizobium isolated from stem nodules of the tropical legume Sesbania rostrata that is capable of fixing nitrogen in the free-living state and during symbiosis. Antibody enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used to characterize the acetylome. There are 2,302 acetylation sites from 982 proteins, accounting for 20.8% of the total proteins. Analysis of the acetylated motifs showed the preferences for the amino acid residues around acetylated lysines. The response regulator CheY1, previously characterized to be involved in chemotaxis in strain ORS571, was identified as an acetylated protein, and a mutation of the acetylated site of CheY1 significantly impaired the strain's motility. In addition, a Zn+-dependent deacetylase (AZC_0414) was characterized, and the construction of a deletion mutant strain showed that it played a role in chemotaxis. Our study provides the first global analysis of lysine acetylation in ORS571, suggesting that acetylation plays a role in various physiological processes. In addition, we demonstrate its involvement in the chemotaxis process. The acetylome of ORS571 provides insights to investigate the regulation mechanism of rhizobial physiology. IMPORTANCE Acetylation is an important modification that regulates protein function and has been found to regulate physiological processes in various bacteria. The physiology of rhizobium A. caulinodans ORS571 is regulated by multiple mechanisms both when free living and in symbiosis with the host; however, the regulatory role of acetylation is not yet known. Here, we took an acetylome-wide approach to identify acetylated proteins in A. caulinodans ORS571 and performed clustering analyses. Acetylation of chemotaxis proteins was preliminarily investigated, and the upstream acetylation-regulating enzyme involved in chemotaxis was characterized. These findings provide new insights to explore the physiological mechanisms of rhizobia.

Characterization of rhizobia isolated from leguminous plants and their impact on the growth of ICCV 2 variety of chickpea (Cicer arietinum L.)

Six rhizobia-like-bacterial strains in total, secluded from the root and stem nodules of various leguminous plants were characterized for growth promoting ability on ICCV 2 variety of chickpea. Bacterial strains showed production of IAA, NH3, siderophore, HCN, ACC deaminase, hydrolytic enzyme production such as chitinase, amylase, protease, lipase, β-1, 3-glucanase and solubilization of nutrients such as phosphate, zinc and potassium. However the performance of PGP traits characterized in-vitro varied among the six bacterial strains. The sequences of 16S rRNA gene of bacterial strains IHSR, IHRG, IHAA, IHGN-3, IHCP-1 and IHCP-2 showed maximum identity with Rhizobium sp., Rhizobium tropici, Rhizobium multihospitium, Mesorhizobium sp., Burkholderia cepacia and Rhizobium pusense. In plate culture conditions the bacterial strains changed the colour of media (NFB) from green to blue and showed amplification of nifH gene by PCR, and also enhanced nodule formation in chickpea under greenhouse conditions, which explains their nitrogen fixing ability. Scanning electron microscopy studies of chickpea roots showed colonization by all the six bacterial strains in solo and by consortium (IHRG + IHGN-3). Under greenhouse conditions, chickpea plants inoculated with different strains showed improvement in plant height, number of branches, total chlorophyll, nodule number, nodule weight, shoot weight, root weight, root volume and root surface area at 30 and 45 days after sowing (DAS) over the uninoculated control plants. It was also observed at the crop maturity stage all the bacterial strains inoculated separately enhanced pod number, seed number and total NPK compared to uninoculated control plants. This study suggests that bacteria associated with root and stem nodules can be a promising resource to enhance nodulation, PGP and crop yields in chickpea.

Ecology of pitaya stem rot caused by Gilbertella persicaria and its biological control by phyllosphere bacteria

AbstractGilbertella persicaria stem rot is a disease that causes rotting in the stem nodules and fruits of pitaya. Here, we aimed to study the ecology of G. persicaria and its biological control using several Bacillus subtilis strains isolated from the phyllosphere. Sporangiospores of G. persicaria were detected on different parts of the plant, with maximum density (c.239.9 propagules/cm2) found on the petals after flowering, and spore germination was observed to be considerably affected by humidity. Furthermore, the study revealed that an incision wound of at least 2 mm depth was necessary for infection by the pathogen to the stem nodule, and exposure to high temperatures (50°C) accelerated disease incidence, by disrupting resistance to the spread of the pathogen. Additionally, biological control of G. persicaria was effectively achieved by inoculation with B. subtilis strains isolated from pitaya stem nodules. Intriguingly, treatment with antagonistic bacteria reduced stem rot incidence to zero (bacterial suspension: PNB‐5, PNB‐7, and PNB‐93; culture filtrate: PNB‐7, PNB‐93, and PNB‐95), equivalent to that achieved by fungicide application in both prophylactic and simultaneous treatments. Collectively, this is the first study to demonstrate the ecology of G. persicaria and to show the potential applicability of phyllosphere bacterial strains for biological control of G. persicaria in pitaya, which could help develop effective integrated disease management strategies for the control of pitaya stem rot.

The importance of Rhizobium, Agrobacterium, Bradyrhizobium, Herbaspirillum, Sinorhizobium in sustainable agricultural production

Rhizobia which are soil bacteria capable of symbiosis with legume plants in the root or stem nodules and perform nitrogen fixation. Rhizobial genera include Agrobacterium, Allorhizobium, Aminobacter, Azorhizobium, Bradyrhizobium, Devosia, Mesorhizobium, Methylobacterium, Microvirga, Ochrobacterum, Phyllobacterium, Rhizobium, Shinella and Ensifer (Sinorhizobium). Review of the literature was carried out using the keywords Rhizobium, Agrobacterium, Bradyrhizobium, Herbaspirillum and Sinorhizobium. Rhizobial nodulation symbioses steps are included flavonoid signaling, Nod factor induction, and Nod factor perception, root hair responses, rhizobial infection, cell division and formation of nitrogen-fixing nodule. Rhizobium improves sustainable production by boosting organic nitrogen content.

Culture-independent assessment of the diazotrophic Bradyrhizobium communities in the Pampa and Atlantic Forest Biomes localities in southern Brazil

The isolation of rhizobial strains from the root and stem nodules remains a commonly used method despite its limitations as it enables the identification of mainly dominant symbiotic groups within rhizobial communities. To overcome these limitations, we used genus-specific nifD primers in a culture-independent assessment of Bradyrhizobium communities inhabiting soils in southern Brazil. The majority of nifD sequences were generated from DNA isolated from tropical-lowland pasture soils, although some soil samples originated from the Campos de Cima da Serra volcanic plateau. In the nifD tree, all the bradyrhizobial sequences comprised 38 clades, including 18 new clades. The sequences generated in this study were resolved into 22 clades and 21 singletons. The nifD bradyrhizobial assemblage contained Azorhizobium and α-proteobacterial methylotrophic genera, suggesting that these genera may have acquired their nif loci from Bradyrhizobium donors. The most common in the lowland pasture soils subclade III.3D branch comprises the isolates of mainly an American origin. On the other hand, subclade III.4, which was earlier detected in Brazil among Bradyrhizobium isolates nodulating native lupins, appears more common in the Campos de Cima da Serra soils. The second-largest group, Clade XXXVIII, has not yet been reported in culture-dependent studies, while another common group called Clade I represents a symbiovar predominating in Australia. The identification of the diverse nifD Clade I haplotypes in the tropical-lowland pastures infested by Australian Acacia spp implies that the introduction of these legumes to southern Brazil has resulted in the dissemination of their bradyrhizobial symbionts.

Genetics of nodulation in Aeschynomene evenia uncovers mechanisms of the rhizobium\u2013legume symbiosis

Among legumes (Fabaceae) capable of nitrogen-fixing nodulation, several Aeschynomene spp. use a unique symbiotic process that is independent of Nod factors and infection threads. They are also distinctive in developing root and stem nodules with photosynthetic bradyrhizobia. Despite the significance of these symbiotic features, their understanding remains limited. To overcome such limitations, we conduct genetic studies of nodulation in Aeschynomene evenia, supported by the development of a genome sequence for A. evenia and transcriptomic resources for 10 additional Aeschynomene spp. Comparative analysis of symbiotic genes substantiates singular mechanisms in the early and late nodulation steps. A forward genetic screen also shows that AeCRK, coding a receptor-like kinase, and the symbiotic signaling genes AePOLLUX, AeCCamK, AeCYCLOPS, AeNSP2, and AeNIN are required to trigger both root and stem nodulation. This work demonstrates the utility of the A. evenia model and provides a cornerstone to unravel mechanisms underlying the rhizobium–legume symbiosis.

Recent development and new insight of diversification and symbiosis specificity of legume rhizobia: mechanism and application

Currently, symbiotic rhizobia (sl., rhizobium) refer to the soil bacteria in α- and β-Proteobacteria that can induce root and/or stem nodules on some legumes and a few of nonlegumes. In the nodules, rhizobia convert the inert dinitrogen gas (N2 ) into ammonia (NH3 ) and supply them as nitrogen nutrient to the host plant. In general, this symbiotic association presents specificity between rhizobial and leguminous species, and most of the rhizobia use lipochitooligosaccharides, so called Nod factor (NF), for cooperating with their host plant to initiate the formation of nodule primordium and to inhibit the plant immunity. Besides NF, effectors secreted by type III secretion system (T3SS), exopolysaccharides and many microbe-associated molecular patterns in the rhizobia also play important roles in nodulation and immunity response between rhizobia and legumes. However, the promiscuous hosts like Glycine max and Sophora flavescens can nodulate with various rhizobial species harbouring diverse symbiosis genes in different soils, meaning that the nodulation specificity/efficiency might be mainly determined by the host plants and regulated by the soil conditions in a certain cases. Based on previous studies on rhizobial application, we propose a '1+n-N' model to promote the function of symbiotic nitrogen fixation (SNF) in agricultural practice, where '1' refers to appreciate rhizobium; '+n' means the addition of multiple trace elements and PGPR bacteria; and '-N' implies the reduction of chemical nitrogen fertilizer. Finally, open questions in the SNF field are raised to future think deeply and researches.

Growth Promotion of ICCV 2 Variety of Chickpea ( <i>Cicer arietinum</i> L.) by Diazotrophic Bacteria Isolated From Root and Stem Nodules of Leguminous Plants

Six rhizobia-like-bacterial strains in total, secluded from the root and stem nodules of various leguminous plants were characterized for growth promoting ability and nitrogen fixation potential on ICCV 2 variety of chickpea. All the six bacteria, were found to produce PGP traits including IAA, NH 3 , protease, lipase, cellulase, β-1, 3-glucanase, siderophore, ACC deaminase (except IHSR), chitinase (except IHSR, IHAA and IHCP-1), amylase (except IHSR, IHRG and IHAA), hydrocyanic acid (except IHGN-3), solubilize phosphate, zinc (except IHSR), rock phosphate (except IHSR and IHCP-2) and potassium (except IHSR, IHRG, IHGN-3 and IHCP-2). The sequences of 16S rRNA gene of IHSR, IHRG, IHAA, IHGN-3, IHCP-1 and IHCP-2 showed maximum identity with Rhizobium sp., Rhizobium tropici , Rhizobium multihospitium , Mesorhizobium sp., Burkholderia cepacia and Rhizobium pusense . All the isolated bacteria showed nodule formation with chickpea, amplified nifH gene and fixed nitrogen. SEM study of chickpea roots showed a significant degree of colonization by all the six bacterial strains in solo and by consortium. Seeds of chickpea, when treated with the isolated bacteria, increased the plant height, number of branches, total chlorophyll, nodule number, nodule weight, shoot weight, root weight, root volume and root surface area respectively at 30 and 45 DAS, over the control plants. At the crop maturity, the diazotrophic bacteria treated pots exhibited enhanced pod number, shoot weight, seed number, total NPK respectively, over the control plants. This study suggests that bacteria associated with root and stem nodules can be a promising resource for enhancing nodulation, nitrogen fixation, PGP and crop yields in chickpea.

Application of molibidenum seed treatment (TS)

Due to the advancement of soybean production, alternatives are sought to improve the ability to biologically fix nitrogen, however, the effectiveness of this procedure may be impaired by the lack of micronutrients, such as molybdenum. The objective of this study was to evaluate the productive and developmental aspects and the nodulation of soybean, submitted to different inoculation doses and varied application techniques of Molybdenum. The experiment was conducted with the application of Mo in 4 ways, seed treatment, leaf pathway, added seed pathway treatment and sowing furrow. Height and stem diameter, nodulation, dry matter and plant biomass were analyzed. Significant data were observed for plant height and biomass, where the best application was in the treatment of seeds added from the leaf pathway. The presence of inoculant provided characteristics very close to the treatment where no inoculant was used.

CheY1 and CheY2 of Azorhizobium caulinodans ORS571 Regulate Chemotaxis and Competitive Colonization with the Host Plant.

The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37 kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the ΔcheY1 mutant exhibited decreased frequency of direction reversal, whereas the ΔcheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ΔcheY1 and ΔcheY2 mutants was lower than that in the WT, but the ΔcheY2 mutant had more obvious EPS defects that were similar to those of the ΔcheY1 ΔcheY2 and Δeps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ΔcheY1 and ΔcheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ΔcheY2 mutant was severely impaired compared to that of the ΔcheY1 mutant. Taken together, the ΔcheY2 phenotypes are more severe than the ΔcheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ΔcheY2 single-mutant phenotype. These defects of ΔcheY1 and ΔcheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions.IMPORTANCEAzorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.

Ohr and OhrR Are Critical for Organic Peroxide Resistance and Symbiosis in Azorhizobium caulinodans ORS571.

Azorhizobium caulinodans is a symbiotic nitrogen-fixing bacterium that forms both root and stem nodules on Sesbania rostrata. During nodule formation, bacteria have to withstand organic peroxides that are produced by plant. Previous studies have elaborated on resistance to these oxygen radicals in several bacteria; however, to the best of our knowledge, none have investigated this process in A. caulinodans. In this study, we identified and characterised the organic hydroperoxide resistance gene ohr (AZC_2977) and its regulator ohrR (AZC_3555) in A. caulinodans ORS571. Hypersensitivity to organic hydroperoxide was observed in an ohr mutant. While using a lacZ-based reporter system, we revealed that OhrR repressed the expression of ohr. Moreover, electrophoretic mobility shift assays demonstrated that OhrR regulated ohr by direct binding to its promoter region. We showed that this binding was prevented by OhrR oxidation under aerobic conditions, which promoted OhrR dimerization and the activation of ohr. Furthermore, we showed that one of the two conserved cysteine residues in OhrR, Cys11, was critical for the sensitivity to organic hydroperoxides. Plant assays revealed that the inactivation of Ohr decreased the number of stem nodules and nitrogenase activity. Our data demonstrated that Ohr and OhrR are required for protecting A. caulinodans from organic hydroperoxide stress and play an important role in the interaction of the bacterium with plants. The results that were obtained in our study suggested that a thiol-based switch in A. caulinodans might sense host organic peroxide signals and enhance symbiosis.

Effectiveness of nitrogen fixation in rhizobia.

SummaryBiological nitrogen fixation in rhizobia occurs primarily in root or stem nodules and is induced by the bacteria present in legume plants. This symbiotic process has fascinated researchers for over a century, and the positive effects of legumes on soils and their food and feed value have been recognized for thousands of years. Symbiotic nitrogen fixation uses solar energy to reduce the inert N2 gas to ammonia at normal temperature and pressure, and is thus today, especially, important for sustainable food production. Increased productivity through improved effectiveness of the process is seen as a major research and development goal. The interaction between rhizobia and their legume hosts has thus been dissected at agronomic, plant physiological, microbiological and molecular levels to produce ample information about processes involved, but identification of major bottlenecks regarding efficiency of nitrogen fixation has proven to be complex. We review processes and results that contributed to the current understanding of this fascinating system, with focus on effectiveness of nitrogen fixation in rhizobia.

Aeschynomene indica-Nodulating Rhizobia Lacking Nod Factor Synthesis Genes: Diversity and Evolution in Shandong Peninsula, China.

Aeschynomene indica is a semiaquatic legume that forms both stem and root nodules with rhizobia. Some A. indica rhizobia (AIRs) have been reported to nodulate the host using a Nod factor-independent pathway and possess photosynthetic abilities. To investigate the diversity and community structure of AIRs in China, a total of 300 rhizobial isolates were acquired from the root and stem nodules of A. indica grown at 4 sites in Shandong Peninsula, China. Nineteen representative strains were selected according to their recA phylogeny. With further classification in comparison with reference strains, 10 Bradyrhizobium genospecies were defined based on the 16S rRNA gene phylogeny and multilocus sequence analysis (MLSA) of housekeeping genes (HKGs) recA, atpD, glnII, dnaK, gyrB, and rpoB In addition, 6 genospecies were found only in China. No nodulation gene (nodA, nodB, nodC, or nodZ) was detected in the AIRs isolates by PCR amplification and Southern blotting. Phylogenetic analysis of nifH and the photosynthesis-related gene pufLM revealed their common origins. All representative strains formed root nodules, but only 9 representative strains for 4 genospecies formed stem nodules on A. indica, indicating that the stem nodulation process of A. indica is limited to some strains. The nucleotide diversity and recombination events of the HKGs, as well as nifH and pufLM genes, showed that mutation contributes more than recombination in evolution. The distribution of dominant AIR genospecies was mainly affected by available nitrogen, organic carbon, total nitrogen, and pH. Our study helps to characterize the diversity and evolution of AIRs.IMPORTANCEAeschynomene indica rhizobia (AIRs) can form both root and stem nodules via Nod factor-independent processes, which distinguishes them from other rhizobia. This study systematically uncovered the diversity and community composition of A. indica rhizobia distributed in eastern China. Our results reclassified all the A. indica rhizobia across the world and represent a useful contribution to evaluating the diversity and distribution of the symbiont. The presence of novel genospecies specifically distributed in China enriched the A. indica rhizobia resources and provided insight into the geographic distribution of rhizobia. The phylogenetic relationship between nifH and pufLM of A. indica rhizobia across the world provides insight into the evolution of their nitrogen fixation and photosynthetic abilities.

Overexpression of the Chromosome Partitioning Gene parA in Azorhizobium caulinodans ORS571 Alters the Bacteroid Morphotype in Sesbania rostrata Stem Nodules.

Azorhizobium caulinodans ORS571 is a diazotroph that forms N2-fixing nodules on the roots and stems of the tropical legume Sesbania rostrata. Deletion of the parA gene of this bacterium results in cell cycle defects, pleiomorphic cell shape, and formation of immature stem nodules on its host plant. In this study, we constructed a parA overexpression mutant (PnptII-parA) to complement a previous study and provide new insights into bacteroid formation. We found that overproduction of ParA did not affect growth, cell morphology, chromosome partitioning, or vegetative nitrogen fixation in the free-living state. Under symbiosis, however, distinctive features, such as a single swollen bacteroid in one symbiosome, relatively narrow symbiosome space, and polyploid cells were observed. The morphotype of the PnptII-parA bacteroid is reminiscent of terminal differentiation in some IRLC indeterminate nodules, but S. rostrata is not thought to produce the NCR peptides that induce terminal differentiation in rhizobia. In addition, the transcript patterns of many symbiosis-related genes elicited by PnptII-parA were different from those elicited by the wild type. Accordingly, we propose that the particular symbiosome formation in PnptII-parA stem-nodules is due to cell cycle disruption caused by excess ParA protein in the symbiotic cells during nodulation.

Inactivation of the Phosphatase CheZ Alters Cell-Surface Properties of Azorhizobium caulinodans ORS571 and Symbiotic Association with Sesbania rostrata.

Azorhizobium caulinodans can form root and stem nodules with the host plant Sesbania rostrata. The role of the CheZ phosphatase in the A. caulinodans chemotaxis pathway was previously explored using the nonchemotactic cheZ mutant strain (AC601). This mutant displayed stronger attachment to the root surface, enhancing early colonization; however, this did not result in increased nodulation efficiency. In this study, we further investigated the role of CheZ in the interaction between strain ORS571 and the roots of its host plant. By tracking long-term colonization dynamic of cheZ mutant marked with LacZ, we found a decrease of colonization of the cheZ mutant during this process. Furthermore, the cheZ mutant could not spread on the root surface freely and was gradually outcompeted by the wild type in original colonization sites. Quantitative reverse-transcription PCR analyses showed that exp genes encoding exopolysaccharides synthesis, including oac3, were highly expressed in the cheZ mutant. Construction of a strain carrying a deletion of both cheZ and oac3 resulted in a mutant strain defective in the colonization process to the same extent as found with the oac3 single-mutant strain. This result suggested that the enhanced colonization of the cheZ mutant may be achieved through regulating the formation of exopolysaccharides. This shows the importance of the chemotactic proteins in the interaction between rhizobia and host plants, and expands our understanding of the symbiosis interaction between rhizobium and host plant.

Alkyl hydroperoxide reductase is important for oxidative stress resistance and symbiosis in Azorhizobium caulinodans.

Reactive oxygen species (ROS) are not only toxic products of oxygen from aerobic metabolism or stress but also signalling molecules involved in the development of the legume-Rhizobium symbiosis. To assess the importance of alkyl hydroperoxide reductase (AhpCD) in the nitrogen-fixating bacterium Azorhizobium caulinodans, we investigated the phenotypes of the ∆ahpCD strain with regards to ROS resistance and symbiotic interactions with Sesbania rostrata. The ∆ahpCD strain was notably more sensitive than its parent strain to hydrogen peroxide (H2O2) but not to two organic peroxides, in the early log phase. The expression of ahpCD was not controlled by a LysR-type transcriptional activator either in vitro or in vivo. The catalase activity of the ∆ahpCD strain was affected at a relatively low level of H2O2 stress. Furthermore, the ∆ahpCD strain induced a reduced number of stem nodules in S. rostrata with lowering of nitrogenase activity. These data suggest that A. caulinodans AhpCD is not only important for H2O2 detoxification in vitro but also critical for symbiosis with S. rostrata. Functional analysis of AhpCD is worth investigating in other rhizobia to gain a comprehensive view of its contributions to ROS defence and symbiotic association with legumes.

Localization of the reb operon expression is inconsistent with that of the R-body production in the stem nodules formed by Azorhizobium caulinodans mutants having a deletion of praR.

Azorhizobium caulinodans, a kind of rhizobia, has a reb operon encoding pathogenic R-body components, whose expression is usually repressed by a transcription factor PraR. Mutation on praR induced a high expression of reb operon and the formation of aberrant nodules, in which both morphologically normal and shrunken host cells were observed. Histochemical GUS analyses of praR mutant expressing reb operon-uidA fusion revealed that the bacterial cells within the normal host cells highly expressed the reb operon, but rarely produced R-bodies. On the other hand, the bacterial cells within the shrunken host cells frequently produced R-bodies but rarely expressed the reb operon. This suggests that R-body production is not only regulated at the transcriptional level, but by other regulatory mechanisms as well.