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.

A Methionine Sulfoxide Reductase B Is Required for the Establishment of Astragalus sinicus-Mesorhizobium Symbiosis.

Methionine sulfoxide reductase B (MsrB) is involved in oxidative stress or defense responses in plants. However, little is known about its role in legume-rhizobium symbiosis. In this study, an MsrB gene was identified from Astragalus sinicus and its function in symbiosis was characterized. AsMsrB was induced under phosphorus starvation and displayed different expression patterns under symbiotic and nonsymbiotic conditions. Hydrogen peroxide or methyl viologen treatment enhanced the transcript level of AsMsrB in roots and nodules. Subcellular localization showed that AsMsrB was localized in the cytoplasm of onion epidermal cells and co-localized with rhizobia in nodules. Plants with AsMsrB-RNAi hairy roots exhibited significant decreases in nodule number, nodule nitrogenase activity and fresh weight of the aerial part, as well as an abnormal nodule and symbiosome development. Statistical analysis of infection events showed that plants with AsMsrB-RNAi hairy roots had significant decreases in the number of root hair curling events, infection threads and nodule primordia compared with the control. The content of hydrogen peroxide increased in AsMsrB-RNAi roots but decreased in AsMsrB overexpression roots at the early stage of infection. The transcriptome analysis showed synergistic modulations of the expression of genes involved in reactive oxygen species generation and scavenging, defense and pathogenesis and early nodulation. In addition, a candidate protein interacting with AsMsrB was identified and confirmed by bimolecular fluorescence complementation. Taken together, our results indicate that AsMsrB plays an essential role in nodule development and symbiotic nitrogen fixation by affecting the redox homeostasis in roots and nodules.

Microbial cooperation in the rhizosphere improves liquorice growth under salt stress.

Liquorice (Glycyrrhiza uralensis Fisch.) is one of the most widely used plants in food production, and it can also be used as an herbal medicine or for reclamation of salt-affected soils. Under salt stress, inhibition of plant growth, nutrient acquisition and symbiotic interactions between the medicinal legume liquorice and rhizobia have been observed. We recently evaluated the interactions between rhizobia and root-colonizing Pseudomonas in liquorice grown in potting soil and observed increased plant biomass, nodule numbers and nitrogen content after combined inoculation compared to plants inoculated with Mesorhizobium alone. Several beneficial effects of microbes on plants have been reported; studies examining the interactions between symbiotic bacteria and root-colonizing Pseudomonas strains under natural saline soil conditions are important, especially in areas where a hindrance of nutrients and niches in the rhizosphere are high. Here, we summarize our recent observations regarding the combined application of rhizobia and Pseudomonas on the growth and nutrient uptake of liquorice as well as the salt stress tolerance mechanisms of liquorice by a mutualistic interaction with microbes. Our observations indicate that microbes living in the rhizosphere of liquorice can form a mutualistic association and coordinate their involvement in plant adaptations to stress tolerance. These results support the development of combined inoculants for improving plant growth and the symbiotic performance of legumes under hostile conditions.

Mesorhizobium soli sp. nov., a novel species isolated from the rhizosphere of Robinia pseudoacacia L. in South Korea by using a modified culture method.

Strain NHI-8(T) was isolated from a forest soil sample, collected in South Korea, by using a modified culture method. Comparative analysis of its nearly full-length 16S rRNA gene sequence showed that strain NHI-8(T) belongs to the genus Mesorhizobium and to be closely related to Mesorhizobium chacoense PR5(T) (97.32 %). The levels of DNA-DNA relatedness between strain NHI-8(T) and reference type strains of the genus Mesorhizobium were 32.28-53.65 %. SDS-PAGE of total soluble proteins and the sequences of the housekeeping genes recA, glnII, and atpD were also used to support the clade grouping in rhizobia. The new strain contained summed feature 8 (57.0 %), cyclo-C19:0ω8c (17.3 %), and C18:0 (11.0 %) as the major fatty acids, as in genus Mesorhizobium. The strain contained cardiolipin, phosphatidylglycerol, ornithine-containing lipid, phosphatidylethanolamine, phosphatidyl-N-dimethylethanolamine, and phosphatidylcholine. Morphological and physiological analyses were performed to compare the characteristics of our strain with those of the reference type strains. Based on the results, strain NHI-8(T) was determined to represent a novel member of the genus Mesorhizobium, and the name Mesorhizobium soli is proposed. The type strain is NHI-8(T) (=KEMB 9005-153(T) = KACC 17916(T) = JCM 19897(T)).

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.

Diverse nodule bacteria were associated with Astragalus species in arid region of northwestern China.

The legume species of Astragalus as traditional Chinese medicine source and environmental protection plants showed an extensive distribution in the arid region of northwestern China. However, few rhizobia associating with Astragalus have been investigated in this region so far. In this study, 78 endophytic bacteria were isolated from root nodules of 12 Astragalus species and characterized by the PCR-RFLP of 16S rRNA gene and symbiotic genes together with the phylogenetic analysis. Results showed that the majority (53%) of isolates are non-nodulating Agrobacterium sp. and the rest are Mesorhizobium genomic species (41%), Ensifer spp. and Rhizobium gallicum (6%), respectively. Mesorhizobium genomic species are broadly distributed in the Astragalus symbioses and most of them share similar symbiotic genes. It seems that horizontal gene transfer occurred frequently among different genomic species independent of their original hosts and sites. Astragalus adsurgens is nodulated by a widely range of rhizobial species in the nodulation test, revealing that it could play an important role in diversification of Astragalus symbionts and that might be a reason for its wide adaptation to diverse environments.

Profiling of differentially expressed genes in roots of Robinia pseudoacacia during nodule development using suppressive subtractive hybridization.

BACKGROUND: Legume-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 FINDINGS: To 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. CONCLUSIONS: The 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.