Amorphae CCNWGS0123 Research Articles

Rhizobial HmuSpSym as a heme-binding factor is required for optimal symbiosis between Mesorhizobium amorphae CCNWGS0123 and Robinia pseudoacacia.

Nitrogen-fixing root nodules are formed by symbiotic association of legume hosts with rhizobia in nitrogen-deprived soils. Successful symbiosis is regulated by signals from both legume hosts and their rhizobial partners. HmuS is a heme degrading factor widely distributed in bacteria, but little is known about the role of rhizobial hmuS in symbiosis with legumes. Here, we found that inactivation of hmuSpSym in the symbiotic plasmid of Mesorhizobium amorphae CCNWGS0123 disrupted rhizobial infection, primordium formation, and nitrogen fixation in symbiosis with Robinia pseudoacacia. Although there was no difference in bacteroids differentiation, infected plant cells were shrunken and bacteroids were disintegrated in nodules of plants infected by the ΔhmuSpSym mutant strain. The balance of defence reaction was also impaired in ΔhmuSpSym strain-infected root nodules. hmuSpSym was strongly expressed in the nitrogen-fixation zone of mature nodules. Furthermore, the HmuSpSym protein could bind to heme but not degrade it. Inactivation of hmuSpSym led to significantly decreased expression levels of oxygen-sensing related genes in nodules. In summary, hmuSpSym of M. amorphae CCNWGS0123 plays an essential role in nodule development and maintenance of bacteroid survival within R. pseudoacacia cells, possibly through heme-binding in symbiosis.

TRAPPC13 Is a Novel Target of Mesorhizobium amorphae Type III Secretion System Effector NopP.

Similar to pathogenic bacteria, rhizobia can inject effector proteins into host cells directly to promote infection via the type III secretion system (T3SS). Nodulation outer protein P (NopP), a specific T3SS effector of rhizobia, plays different roles in the establishment of multiple rhizobia-legume symbiotic systems. Mesorhizobium amorphae CCNWGS0123 (GS0123), which infects Robinia pseudoacacia specifically, secretes several T3SS effectors, including NopP. Here, we demonstrate that NopP is secreted through T3SS-I of GS0123 during the early stages of infection, and its deficiency decreases nodule nitrogenase activity of R. pseudoacacia nodules. A trafficking protein particle complex subunit 13-like protein (TRAPPC13) has been identified as a NopP target protein in R. pseudoacacia roots by screening a yeast two-hybrid library. The physical interaction between NopP and TRAPPC13 is verified by bimolecular fluorescence complementation and coimmunoprecipitation assays. In addition, subcellular localization analysis reveals that both NopP and its target, TRAPPC13, are colocalized on the plasma membrane. Compared with GS0123-inoculated R. pseudoacacia roots, some genes associated with cell wall remodeling and plant innate immunity down-regulated in ΔnopP-inoculated roots at 36 h postinoculation. The results suggest that NopP in M. amorphae CCNWGS0123 acts in multiple processes in R. pseudoacacia during the early stages of infection, and TRAPPC13 could participate in the process as a NopP target.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Nod factor- and type III secretion system-dependent manner for Robinia pseudoacacia to establish symbiosis with Mesorhizobium amorphae CCNWGS0123.

Under nitrogen-limiting conditions, symbiotic nodulation promotes the growth of legume plants via the fixation of atmospheric nitrogen to ammonia by rhizobia in root nodules. The rhizobial Nod factor (NF) and type III secretion system (T3SS) are two key signaling pathways for establishing the legume-rhizobium symbiosis. However, whether NF signaling is involved in the nodulation of Robinia pseudoacacia and Mesorhizobium amorphae CCNWGS0123, and its symbiotic differences compared with T3SS signaling remain unclear. Therefore, to elucidate the function of NF signaling in nodulation, we mutated nodC in M. amorphae CCNWGS0123, which aborted NF synthesis. Compared with the plants inoculated with the wild type strain, the plants inoculated with the NF-deficient strain exhibited shorter shoots with etiolated leaves. These phenotypic characteristics were similar to those of the plants inoculated with the T3SS-deficient strain, which served as a Nod- (non-effective nodulation) control. The plants inoculated with both the NF- and T3SS-deficient strains formed massive root hair swellings, but no normal infection threads were detected. Sections of the nodules showed that inoculation with the NF- and T3SS-deficient strains induced small, white bumps without any rhizobia inside. Analyzing the accumulation of 6 plant hormones and the expression of 10 plant genes indicated that the NF- and T3SS-deficient strains activated plant defense reactions while suppressing plant symbiotic signaling during the perception and nodulation processes. The requirement for NF signaling appeared to be conserved in two other leguminous trees that can establish symbiosis with M. amorphae CCNWGS0123. In contrast, the function of the T3SS might differ among species, even within the same subfamily (Faboideae). Overall, this work demonstrated that nodulation of R. pseudoacacia and M. amorphae CCNWGS0123 was both NF and T3SS dependent.

Type III secretion systems impact Mesorhizobium amorphae CCNWGS0123 compatibility with Robinia pseudoacacia.

Rhizobia and legume plants are famous mutualistic symbiosis partners who provide nitrogen nutrition to the natural environment. Rhizobial type III secretion systems (T3SSs) deliver effectors that manipulate the metabolism of eukaryotic host cells. Mesorhizobium amorphae CCNWGS0123 (GS0123) contains two T3SS gene clusters, T3SS-I and T3SS-II. T3SS-I contains all the basal components for an integrated T3SS, and the expression of T3SS-I genes is up-regulated in the presence of flavonoids. In contrast, T3SS-II lacks the primary extracellular elements of T3SSs, and the expression of T3SS-II genes is down-regulated in the presence of flavonoids. Inoculation tests on Robinia pseudoacacia displayed considerable differences in gene expression patterns and levels among roots inoculated with GS0123 and T3SS-deficient mutant (GS0123ΔrhcN1 (GS0123ΔT1), GS0123ΔrhcN2 (GS0123ΔT2) and GS0123ΔrhcN1ΔrhcN2 (GS0123ΔS)). Compared with the GS0123-inoculated plants, GS0123ΔT1-inoculated roots formed very few infection threads and effective nodules, while GS0123ΔT2-inoculated roots formed a little fewer infection threads and effective nodules with increased numbers of bacteroids enclosed in one symbiosome. Moreover, almost no infection threads or effective nodules were observed in GS0123ΔS-inoculated roots. In addition to evaluations of plant immunity signals, we observed that the coexistence of T3SS-I and T3SS-II promoted infection by suppressing host defense response in the reactive oxygen species defense response pathway. Future studies should focus on identifying rhizobial T3SS effectors and their host target proteins.

Type III secretion systems impact Mesorhizobium amorphae CCNWGS0123 compatibility with Robinia pseudoacacia

Rhizobia and legume plants are famous mutualistic symbiosis partners who provide nitrogen nutrition to the natural environment. Rhizobial type III secretion systems (T3SSs) deliver effectors that manipulate the metabolism of eukaryotic host cells. Mesorhizobium amorphae CCNWGS0123 (GS0123) contains two T3SS gene clusters, T3SS-I and T3SS-II. T3SS-I contains all the basal components for an integrated T3SS, and the expression of T3SS-I genes is up-regulated in the presence of flavonoids. In contrast, T3SS-II lacks the primary extracellular elements of T3SSs, and the expression of T3SS-II genes is down-regulated in the presence of flavonoids. Inoculation tests on Robinia pseudoacacia displayed considerable differences in gene expression patterns and levels among roots inoculated with GS0123 and T3SS-deficient mutant (GS0123ΔrhcN1 (GS0123ΔT1), GS0123ΔrhcN2 (GS0123ΔT2) and GS0123ΔrhcN1ΔrhcN2 (GS0123ΔS)). Compared with the GS0123-inoculated plants, GS0123ΔT1-inoculated roots formed very few infection threads and effective nodules, while GS0123ΔT2-inoculated roots formed a little fewer infection threads and effective nodules with increased numbers of bacteroids enclosed in one symbiosome. Moreover, almost no infection threads or effective nodules were observed in GS0123ΔS-inoculated roots. In addition to evaluations of plant immunity signals, we observed that the coexistence of T3SS-I and T3SS-II promoted infection by suppressing host defense response in the reactive oxygen species defense response pathway. Future studies should focus on identifying rhizobial T3SS effectors and their host target proteins.

Comparative symbiotic plasmid analysis indicates that symbiosis gene ancestor type affects plasmid genetic evolution.

This study compared 24 rhizobial symbiotic plasmids that included four genera and 11 species, illuminating the functional gene composition and symbiosis gene ancestor types of symbiotic plasmids from higher taxonomy. It provides valuable insights into mosaic structures and the evolution of symbiotic plasmids.

Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China

To better understand the diversity of metal resistance genetic determinant from microbes that survived at metal tailings in northwest of China, a highly elevated level of heavy metal containing region, genomic analyses was conducted using genome sequence of three native metal-resistant plant growth promoting bacteria (PGPB). It shows that: Mesorhizobium amorphae CCNWGS0123 contains metal transporters from P-type ATPase, CDF (Cation Diffusion Facilitator), HupE/UreJ and CHR (chromate ion transporter) family involved in copper, zinc, nickel as well as chromate resistance and homeostasis. Meanwhile, the putative CopA/CueO system is expected to mediate copper resistance in Sinorhizobium meliloti CCNWSX0020 while ZntA transporter, assisted with putative CzcD, determines zinc tolerance in Agrobacterium tumefaciens CCNWGS0286. The greenhouse experiment provides the consistent evidence of the plant growth promoting effects of these microbes on their hosts by nitrogen fixation and/or indoleacetic acid (IAA) secretion, indicating a potential in-site phytoremediation usage in the mining tailing regions of China.

Removal of Zinc from Aqueous Solution by Metal Resistant Symbiotic Bacterium Mesorhizobium amorphae

Biosorption of zinc by living biomasses of metal resistant symbiotic bacterium Mesorhizobium amorphae CCNWGS0123 was investigated under optimal conditions at pH 5.0, initial metal concentrations of 100 mg L−1, and a dose of 1.0 g L−1. M. amorphae exhibited an efficient removal of Zn2+ from aqueous solution with maximum biosorption capacity of 120.85 mg g−1. Moreover, more than 70% Zn2+ could be recovered from Zn-loaded biomass at pH 1.0. Both the Langmuir and Freundlich isotherms provided a better fit to experimental data for Zn2+ sorption with correlation coefficients of 0.9885. Kinetics models suggested there was more than one step involved in the Zn2+ sorption process, while a pseudo-second-order model was more suitable to describe the kinetic behavior accurately, indicating a chemisorption process. Carbonyl, amino, carboxyl, and aromatic groups were responsible for the biosorption of Zn2+ by M. amorphae. Cellular deformation, precipitate, and damage were found after Zn2+ treatment. Competitive sorption revealed Cu2+, Cd2+, and Ni2+ were competed with Zn2+ for adsorption sites with the order: Cu2+ > Cd2+ >> Ni2+.

Profiling of Differentially Expressed Genes in Roots of Robinia pseudoacacia during Nodule Development Using Suppressive Subtractive Hybridization

BackgroundLegume-rhizobium symbiosis is a complex process that is regulated in the host plant cell through gene expression network. Many nodulin genes that are upregulated during different stages of nodulation have been identified in leguminous herbs. However, no nodulin genes in woody legume trees, such as black locust (Robinia pseudoacacia), have yet been reported.Methodology/Principal findingsTo identify the nodulin genes involved in R. pseudoacacia-Mesorhizobium amorphae CCNWGS0123 symbiosis, a suppressive subtractive hybridization approach was applied to reveal profiling of differentially expressed genes and two subtracted cDNA libraries each containing 600 clones were constructed. Then, 114 unigenes were identified from forward SSH library by differential screening and the putative functions of these translational products were classified into 13 categories. With a particular interest in regulatory genes, twenty-one upregulated genes encoding potential regulatory proteins were selected based on the result of reverse transcription-polymerase chain reaction (RT-PCR) analysis. They included nine putative transcription genes, eight putative post-translational regulator genes and four membrane protein genes. The expression patterns of these genes were further analyzed by quantitative RT-PCR at different stages of nodule development.ConclusionsThe data presented here offer the first insights into the molecular foundation underlying R. pseudoacacia–M. amorphae symbiosis. A number of regulatory genes screened in the present study revealed a high level of regulatory complexity (transcriptional, post-transcriptional, translational and post-translational) that is likely essential to develop symbiosis. In addition, the possible roles of these genes in black locust nodulation are discussed.

Biosorption and Bioaccumulation of Cu2+ from Aqueous Solution Using Living M. amorphae Isolated from Mine Tailings

Mining generates large amounts of wastewater, which may contain elevated levels of potentially toxic metals. We evaluated the use of live cells of Mesorhizobium amorphae CCNWGS0123 as a new, relatively inexpensive and environment-friendly adsorbent material for removal of toxic metals from aqueous solutions, such as mine tailings leachate. Maximum copper uptake and removal efficiency was attained at a dosage of 0.5 g L−1, a pH of 5.0, an agitation speed of 150 rpm, and at a temperature of 28 °C for an initial Cu2+ concentration of 100 mg L−1; equilibrium was achieved within 30 min. The adsorption isothermal data matched Langmuir and Freundlich isotherms well, with correlation coefficients of 0.931 and 0.932, respectively. FT-IR analysis indicated that many functional groups with a negative charge on the cell surface were involved in the interaction between live CCNWGS0123 and copper ions. Scanning electron microscope results confirmed that there was some copper sediment on the cell surface, causing deformation, aggregation, and cell surface damage. Cu2+ accumulation was related to cell growth, and copper uptake was not only due to cell-surface binding, but also to intracellular accumulation. Live cells of CCNWGS0123 are a new biosorbent that have the potential to be economical when small batches of biomass are used to remove Cu2+ from an aqueous solution.

Biosorption of Copper (II) from Aqueous Solution Using Non-Living <i>Mesorhizobium amorphae</i> Strain CCNWGS0123

The mining industry generates huge amounts of wastewater, containing toxic heavy metals. Treatment to remove heavy metals is necessary and recent work has been focused on finding more environmentally friendly materials for removing heavy metals from wastewater. Biosorption can be an effective process for heavy metal removal from aqueous solutions. Our objectives were to investigate the removal of copper (II) from aqueous solutions using dead cells of Mesorhizobium amorphae CCNWGS0123 under differing levels of pH, agitation speed, temperature, initial copper concentration, biosorbent dose and contact time using flame atomic absorption spectroscopy for metal estimation. The maximum copper removal rate was achieved at pH 5.0, agitation speed 150×g, temperature 28°C and initial Cu (II) concentration of 100 mg L−1. Maximum biosorption capacity was at 0.5 g L−1 and equilibrium was attained within 30 min. Langmuir and Freundlich isotherms showed correlation coefficients of 0.958 and 0.934, respectively. Fourier transform-infrared spectroscopy (FT-IR) analysis indicated that many functional groups, such as O-H, N-H, C-H, C=O, -NH, -CN, C-N, C-O, amide -I, -II, -III and unsaturated alkenes, alkyls and aromatic groups on the cell surface were involved in the interaction between CCNWGS0123 and Cu. Scanning electron microscope and energy dispersive X-ray scanning results showed deformation, aggregation, and cell-surface damage due to the precipitation of copper on the cell surface. Dead cells of CCNWGS0123 showed potential as an efficient biosorbent for the removal of Cu2+ from aqueous solutions.