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Edamame is a special soybean ( Glycine max (L.) Merr .) harvested as a vegetable when the seeds are immature (R6 and R7 stage) and have expanded to fill 80 to 90 percent of the pod width. The study was conducted in the experimental field of Federal Scientific Vegetable Center in the Moscow Region in 2020-2022 and Agro-biotechnological department, Agrarian and Technological Institute, RUDN University. The object of research was 3 accessions of soybean ( Glycine max L.): accession A, accession F, Hidaka and Lira. Before sowing, soybean seeds were inoculated with Optimays 400 ( Bradyrhizobium japonicum ) and Biobesta ( Sinorhizobium fredii ). In the field experiment, 10 soybean samples were taken from each plot at the stage of biological ripeness to determine plant height (cm), number of pods per plant, number of stems per plant, stem width (mm), average number of seeds per plant, weight of 1000 seeds (g) and yield (t/ha). The experiment was designed in a complete random block with a factorial arrangement. Thus, we had 4 accessions and 3 treatments (C - control, V1 - Bradyrhizobium japonicum and V2 - Sinorhizobium fredii ) with 3 replacations. The study showed that accession A had the highest average yield per hectare (2.70 t/ha) and weight of 1000 seeds (159 g). Under conditions of the Moscow region, variant V2 ( Sinorhizobium fredii ) had the highest average yield (2.57 t/ha) and weight of 1000 seeds (100.25 g). Therefore, more than 90 % of the results obtained for these two indicators are associated with the use of V2 treatment. For the rest of the studied indicators, no significant differences were found, and they did not affect the efficiency indicators. The combination of accession A and treatment V2 led to the highest average yield (2.99 t/ha). The product based on Sinorhizobium fredii showed the best indicators for all the studied samples.

Simple Summary: Rhizobia are soil proteobacteria able to establish nitrogen-fixing symbiosis with host legumes. This symbiotic interaction, which is highly important from ecological and agronomical points of view since it allows growth of legumes in soils poor in nitrogen, requires a complex interchange of molecular signals between both symbionts. The production of rhizobial molecular signals is elicited by flavonoids, which are phenolic compounds exuded by legume roots. Recent work has shown that osmotic stress can also promote the formation of symbiotic signals in some rhizobia. In this work, we show that this is also the case for Sinorhizobium fredii HH103, a rhizobial strain able to establish symbiosis with hundreds of legumes, including the very important crop, soybean. Non-ionic osmotic stress, which can be encountered by the bacterium in the rhizosphere or inside the legume host, affected the expression of hundreds of bacterial genes and, consequently, influenced diverse bacterial traits, including the production of symbiotic signals and certain characteristics that may be relevant for successful interaction with the host: motility, production of the phytohormone indole acetic acid, and production of molecules involved in bacterial cell-to-cell communication. Thus, our work provides new evidence of how stress can promote rhizobia-legume symbiosis. (1) Background: Some rhizobia, such as Rhizobium tropici CIAT 899, activate nodulation genes when grown under osmotic stress. This work aims to determine whether this phenomenon also takes place in Sinorhizobium fredii HH103. (2) Methods: HH103 was grown with and without 400 mM mannitol. β-galactosidase assays, nodulation factor extraction, purification and identification by mass spectrometry, transcriptomics by RNA sequencing, motility assays, analysis of acyl-homoserine lactones, and indole acetic acid quantification were performed. (3) Results: Non-ionic osmotic stress induced the production of nodulation factors. Forty-two different factors were detected, compared to 14 found in the absence of mannitol. Transcriptomics indicated that hundreds of genes were either activated or repressed upon non-ionic osmotic stress. The presence of 400 mM mannitol induced the production of indole acetic acid and acyl homoserine lactones, abolished swimming, and promoted surface motility. (4) Conclusions: In this work, we show that non-ionic stress in S. fredii HH103, caused by growth in the presence of 400 mM mannitol, provokes notable changes not only in gene expression but also in various bacterial traits, including the production of nodulation factors and other symbiotic signals. [ABSTRACT FROM AUTHOR]

Bacteria can spread on surfaces to colonize new environments and access more resources. Rhizobia, a group of α- and β-Proteobacteria, establish nitrogen-fixing symbioses with legumes that rely on a complex signal interchange between the partners. Flavonoids exuded by plant roots and the bacterial transcriptional activator NodD control the transcription of different rhizobial genes (the so-called nod regulon) and, together with additional bacterial regulatory proteins (such as TtsI, MucR or NolR), influence the production of different rhizobial molecular signals. In Sinorhizobium fredii HH103, flavonoids and NodD have a negative effect on exopolysaccharide production and biofilm production. Since biofilm formation and motility are often inversely regulated, we have analysed whether flavonoids may influence the translocation of S. fredii HH103 on surfaces. We show that the presence of nod gene-inducing flavonoids does not affect swimming but promotes a mode of surface translocation, which involves both flagella-dependent and -independent mechanisms. This surface motility is regulated in a flavonoid-NodD1-TtsI-dependent manner, relies on the assembly of the symbiotic type 3 secretion system (T3SS), and involves the participation of additional modulators of the nod regulon (NolR and MucR1). To our knowledge, this is the first evidence indicating the participation of T3SS in surface motility in a plant-interacting bacterium. Interestingly, flavonoids acting as nod-gene inducers also participate in the inverse regulation of surface motility and biofilm formation, which could contribute to a more efficient plant colonisation. [ABSTRACT FROM AUTHOR]

The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-component signal transduction systems (TCSTS), in which the signaling domain is fused to an SLC5 solute symporter domain through a STAC domain. Only two members of this family have been characterized experimentally, the CrbS/R system that regulates acetate utilization in Vibrio and Pseudomonas , and the CbrA/B system that regulates the utilization of histidine in Pseudomonas and glucose in Azotobacter. In an attempt to expand the characterized members of this family beyond the Gammaproteobacteria, we identified two putative TCSTS in the Alphaproteobacterium Sinorhizobium fredii NGR234 whose sensor histidine kinases belong to the SLC5/STAC family. Using reverse genetics, we were able to identify the first TCSTS as a CrbS/R homolog that is also needed for growth on acetate, while the second TCSTS, RpuS/R, is a novel system required for optimal growth on pyruvate. Using RNAseq and transcriptional fusions, we determined that in S. fredii the RpuS/R system upregulates the expression of an operon coding for the pyruvate symporter MctP when pyruvate is the sole carbon source. In addition, we identified a conserved DNA sequence motif in the putative promoter region of the mctP operon that is essential for the RpuR-mediated transcriptional activation of genes under pyruvate-utilizing conditions. Finally, we show that S. fredii mutants lacking these TCSTS are affected in nodulation, producing fewer nodules than the parent strain and at a slower rate. [ABSTRACT FROM AUTHOR]

The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-component signal transduction systems (TCSTS), in which the signaling domain is fused to an SLC5 solute symporter domain through a STAC domain. Only two members of this family have been characterized experimentally, the CrbS/R system that regulates acetate utilization in Vibrio and Pseudomonas, and the CbrA/B system that regulates the utilization of histidine in Pseudomonas and glucose in Azotobacter. In an attempt to expand the characterized members of this family beyond the Gammaproteobacteria, we identified two putative TCSTS in the Alphaproteobacterium Sinorhizobium fredii NGR234 whose sensor histidine kinases belong to the SLC5/STAC family. Using reverse genetics, we were able to identify the first TCSTS as a CrbS/R homolog that is also needed for growth on acetate, while the second TCSTS, RpuS/R, is a novel system required for optimal growth on pyruvate. Using RNAseq and transcriptional fusions, we determined that in S. fredii the RpuS/R system upregulates the expression of an operon coding for the pyruvate symporter MctP when pyruvate is the sole carbon source. In addition, we identified a conserved DNA sequence motif in the putative promoter region of the mctP operon that is essential for the RpuR-mediated transcriptional activation of genes under pyruvate-utilizing conditions. Finally, we show that S. fredii mutants lacking these TCSTS are affected in nodulation, producing fewer nodules than the parent strain and at a slower rate.

(1) Background: Some rhizobia, such as Rhizobium tropici CIAT 899, activate nodulation genes when grown under osmotic stress. This work aims to determine whether this phenomenon also takes place in Sinorhizobium fredii HH103. (2) Methods: HH103 was grown with and without 400 mM mannitol. β-galactosidase assays, nodulation factor extraction, purification and identification by mass spectrometry, transcriptomics by RNA sequencing, motility assays, analysis of acyl-homoserine lactones, and indole acetic acid quantification were performed. (3) Results: Non-ionic osmotic stress induced the production of nodulation factors. Forty-two different factors were detected, compared to 14 found in the absence of mannitol. Transcriptomics indicated that hundreds of genes were either activated or repressed upon non-ionic osmotic stress. The presence of 400 mM mannitol induced the production of indole acetic acid and acyl homoserine lactones, abolished swimming, and promoted surface motility. (4) Conclusions: In this work, we show that non-ionic stress in S. fredii HH103, caused by growth in the presence of 400 mM mannitol, provokes notable changes not only in gene expression but also in various bacterial traits, including the production of nodulation factors and other symbiotic signals.

ABSTRACT Bacteria have evolved a series of mechanisms to maintain their survival and reproduction in changeable and stressful environments. In-depth understanding of these mechanisms can allow for better developing and utilizing of bacteria with various biological functions. In this study, we found that water-soluble humic materials (WSHM), a well-known environment-friendly plant growth biostimulant, significantly promoted the free-living growth and survival of Sinorhizobium fredii CCBAU45436 in a bell-shaped, dose-dependent manner, along with more-efficient carbon source consumption and relief of medium acidification. By using RNA-Seq analysis, a total of 1,136 genes significantly up-/downregulated by external addition of WSHM were identified under test conditions. These differentially expressed genes (DEGs) were enriched in functional categories related to carbon/nitrogen metabolism, cellular stress response, and genetic information processing. Further protein-protein interaction (PPI) network analysis and reverse genetic engineering indicated that WSHM might reprogram the transcriptome through inhibiting the expression of key hub gene rsh, which encodes a bifunctional enzyme catalyzing synthesis and hydrolysis of the “magic spot” (p)ppGpp. In addition, the root colonization and viability in soil of S. fredii CCBAU45436 were increased by WSHM. These findings provide us with new insights into how WSHM benefit bacterial adaptations and demonstrate great application value to be a unique inoculant additive. IMPORTANCE Sinorhizobium fredii CCBAU45436 is a highly effective, fast-growing rhizobium that can establish symbiosis with multiple soybean cultivars. However, it is difficult to maintain the high-density effective viable cells in the rhizobial inoculant for the stressful conditions during production, storage, transport, and application. Here, we showed that WSHM greatly increased the viable cells of S. fredii CCBAU45436 in culture, modulating metabolism and triggering stress defense. The root colonization and viability in soil of S. fredii CCBAU45436 were also increased by WSHM. Our results shed new insights into the effects of WSHM on bacteria and the importance of metabolism and stress defense during the bacteria’s whole life. In addition, the functional mechanism of WSHM may provide candidate genes for improving environmental adaptability and application potential of bacteria through genetic engineering.

Bacteria can spread on surfaces to colonize new environments and access more resources. Rhizobia, a group of α- and β-Proteobacteria, establish nitrogen-fixing symbioses with legumes that rely on a complex signal interchange between the partners. Flavonoids exuded by plant roots and the bacterial transcriptional activator NodD control the transcription of different rhizobial genes (the so-called nod regulon) and, together with additional bacterial regulatory proteins (such as TtsI, MucR or NolR), influence the production of different rhizobial molecular signals. In Sinorhizobium fredii HH103, flavonoids and NodD have a negative effect on exopolysaccharide production and biofilm production. Since biofilm formation and motility are often inversely regulated, we have analysed whether flavonoids may influence the translocation of S. fredii HH103 on surfaces. We show that the presence of nod gene-inducing flavonoids does not affect swimming but promotes a mode of surface translocation, which involves both flagella-dependent and -independent mechanisms. This surface motility is regulated in a flavonoid-NodD1-TtsI-dependent manner, relies on the assembly of the symbiotic type 3 secretion system (T3SS), and involves the participation of additional modulators of the nod regulon (NolR and MucR1). To our knowledge, this is the first evidence indicating the participation of T3SS in surface motility in a plant-interacting bacterium. Interestingly, flavonoids acting as nod-gene inducers also participate in the inverse regulation of surface motility and biofilm formation, which could contribute to a more efficient plant colonisation.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, poses a serious threat to soybean production worldwide. Biological control agents have become eco-friendly candidates to control pathogens. Our previous study indicated that the biocontrol agent, Sinorhizobium fredii strain Sneb183, may induce soybean resistance to SCN. To study the mechanisms underlying induced disease resistance in the plant by Sneb183, an iTRAQ (isobaric tag for relative and absolute quantitation)-based proteomics approach was used to identify proteomic changes in SCN-infected soybean roots derived from seeds coated with the Sneb183 fermentation broth or water. Among a total of 456 identified differentially expressed proteins, 212 and 244 proteins were upregulated and downregulated, respectively, in Sneb183 treated samples in comparison to control samples. Some identified differentially expressed proteins are likely to be involved in the biosynthesis of phenylpropanoid, flavone, flavanol, and isoflavonoid and have a role in disease resistance and adaptation to environmental stresses. We used quantitative real-time PCR (qRT-PCR) to analyze key genes, including GmPAL (phenylalanine ammonia-lyase), GmCHR (chalcone reductase), GmCHS (chalcone synthase), and GmIFS (isoflavone synthase), that are involved in isoflavonoid biosynthesis in Sneb183-treated and control samples. The results showed that these targeted genes have higher expression levels in Sneb183-treated than in control samples. High performance liquid chromatography (HPLC) analysis further showed that the contents of daidzein in Sneb183-treated samples were 7.24 times higher than those in control samples. These results suggested that the Sinorhizobium fredii strain Sneb183 may have a role in inducing isoflavonoid biosynthesis, thereby resulting in enhanced resistance to SCN infection in soybean.

Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved.

In the framework of the comprehensive research of Far Eastern natural populations of soybean nodule bacteria, laboratory experiments have been conducted at the All-Russia Scientific Research Institute of Soybean (Blagoveshchensk) with the purpose to identify distinctive features of rhizobia species Sinorhizobium fredii (Scholla and Elkan, 1984) and Bradyrhizobium japonicum (Jordan, 1982) whose pure cultures were isolated from soils of Far Eastern regions practicing soybean cultivation. It is established that B. japonicum strains start growing in Petri dishes on the seventh to tenth and even on the 20th day after the inoculation, assimilate a limited number of carbon nutrition sources, release mostly alkaline metabolic products, and feature a relatively low osmotic resistance. Representatives of this species are susceptible to extreme environmental conditions; their growth sharply slows down on acidic and alkaline nutrient media and stops at high (37–42°C) temperatures. However, under the optimal conditions, this rhizobia species dominates in the nodulation of soybean plants due to its high and persistent virulence. The restriction analysis of the studied B. japonicum strains confirmed their identity. S. fredii strains start growing in Petri dishes on the second to fourth day after the inoculation, assimilate well a broad spectrum of carbon nutrition sources, and release acidic metabolic products. Most strains of this species feature high osmotic resistance. Cultures retaining universal growth capacity under extreme environmental conditions (high temperatures and low and high pH values) have been identified in the group of S. fredii strains. This rhizobia species can predominate in the formation of symbiotic mechanisms in years featuring extreme weather conditions. Enzymatic fermentation of gene 16S rRNA in the studied S. fredii strains was performed using restriction enzyme HaeIII; the analysis of the fermentation results confirmed the identity of these strains. The RAPD-PCR analysis has demonstrated the intraspecific specificity of the studied B. japonicum and S. fredii strains: these species feature high degrees of polymorphism reflecting their population heterogeneity. [ABSTRACT FROM AUTHOR]

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, poses a serious threat to soybean production worldwide. Biological control agents have become eco-friendly candidates to control pathogens. Our previous study indicated that the biocontrol agent, Sinorhizobium fredii strain Sneb183, may induce soybean resistance to SCN. To study the mechanisms underlying induced disease resistance in the plant by Sneb183, an iTRAQ (isobaric tag for relative and absolute quantitation)-based proteomics approach was used to identify proteomic changes in SCN-infected soybean roots derived from seeds coated with the Sneb183 fermentation broth or water. Among a total of 456 identified differentially expressed proteins, 212 and 244 proteins were upregulated and downregulated, respectively, in Sneb183 treated samples in comparison to control samples. Some identified differentially expressed proteins are likely to be involved in the biosynthesis of phenylpropanoid, flavone, flavanol, and isoflavonoid and have a role in disease resistance and adaptation to environmental stresses. We used quantitative real-time PCR (qRT-PCR) to analyze key genes, including GmPAL (phenylalanine ammonia-lyase), GmCHR (chalcone reductase), GmCHS (chalcone synthase), and GmIFS (isoflavone synthase), that are involved in isoflavonoid biosynthesis in Sneb183-treated and control samples. The results showed that these targeted genes have higher expression levels in Sneb183-treated than in control samples. High performance liquid chromatography (HPLC) analysis further showed that the contents of daidzein in Sneb183-treated samples were 7.24 times higher than those in control samples. These results suggested that the Sinorhizobium fredii strain Sneb183 may have a role in inducing isoflavonoid biosynthesis, thereby resulting in enhanced resistance to SCN infection in soybean. [ABSTRACT FROM AUTHOR]

• Isolation of a true symbiont from root nodules of Sesbania sesban , a medicinal and forage legume. • Isolation of an endophyte from root nodules of Cajans Cajan. • Plant growth promoting activities of the true symbiont and the endophyte. • Fertilizer adaptive bacterial variants in chemotaxis and growth promotion of pigeon pea. • Integrated nutrient management using the endophyte and cross nodulating symbiont. Plant growth-promoting (Indole acetic acid producing, phosphate solubilizing and siderophore producing) and chemical fertilizer adaptive variants of endophyte Acidovorax valerianellae CCR1 (JQ424873) with cross nodulating Sinorhizobium fredii SSR1 (JQ424873) were blended with a half dose of fertilizers for integrated nutrient management (INM) to enhance the productivity of pigeon pea (Cajanus cajan). Plant attributes showed enhancement in the growth of pigeon pea when used with lower concentration of urea, diammonium phosphate (DAP), muriate of potash (MOP) and gypsum, termed as inducer doses determined by sub-lethal concentration (LC 50). Plant parameters described as early and late phase of growth (days after sowing; DAS) were enhanced significantly as compared to control and found to be statistically significant. The enhanced plant growth and yield parameters revealed integrated effect of SSR1 and CCR1 when blended with a half dose of chemical fertilizers. The recommended dose of chemical fertilizers attained about 20% increase in yield over control whereas, blend of SSR1 and CCR1 reduced (half) dose of chemical fertilizers enhanced the average yield of the plant by 49–40% over the control. Thus, it is propounded that the increase in the pigeon pea production with the use of fertilizer adaptive endophyte and rhizobia was highly positive. The chemotaxis phenomenon proved both the bacteria as a voracious root colonizer, thereby improving the plant growth directly, hence, these were redefined as fertilizer adaptive bacteria (FAB). [ABSTRACT FROM AUTHOR]

The broad-host-range bacterium Sinorhizobium fredii HH103 cannot nodulate the model legume Lotus japonicus Gifu. This bacterium possesses a type III secretion system (T3SS), a specialized secretion apparatus used to deliver effector proteins (T3Es) into the host cell cytosol to alter host signaling and/or suppress host defence responses to promote infection. However, some of these T3Es are recognized by specific plant receptors and hence trigger a strong defence response to block infection. In rhizobia, T3Es are involved in nodulation efficiency and host-range determination, and in some cases directly activate host symbiosis signalling in a Nod factor-independent manner. In this work, we show that HH103 RifR T3SS mutants, unable to secrete T3Es, gain nodulation with L. japonicus Gifu through infection threads, suggesting that plant recognition of a T3E could block the infection process. To identify the T3E involved, we performed nodulation assays with a collection of mutants that affect secretion of each T3E identified in HH103 RifR so far. The nopC mutant could infect L. japonicus Gifu by infection thread invasion and switch the infection mechanism in Lotus burttii from intercellular infection to infection thread formation. Lotus japonicus gene expression analysis indicated that the infection-blocking event occurs at early stages of the symbiosis. [ABSTRACT FROM AUTHOR]

Split-root systems (SRS) constitute an appropriate methodology for studying the relevance of both local and systemic mechanisms that participate in the control of rhizobia-legume symbioses. In fact, this kind of approach allowed to demonstrate the autoregulation of nodulation (AON) systemic response in soybean in the 1980s. In SRS, the plant main root is cut and two lateral roots that emerge from the seedlings after root-tip removal are confined into separate compartments. After several days of growth, these plants have two separate roots that can be inoculated with the same or with different bacteria, at the same or at different times. In this work, we have used a non-destructive SRS to study two different competitiveness relations between rhizobial strains in soybean roots. One of them is the competition for nodulation between two soybean-nodulating rhizobia: the slow-grower Bradyrhizobium japonicum USDA110 and the fast-grower Sinorhizobium fredii HH103. The second is the competitive blocking of S. fredii 257DH4 nodulation in the American soybean Osumi by Sinorhizobium fredii USDA257, which is unable to nodulate American soybeans. Our results showed that the competitiveness relationships studied in this work are mitigated or even avoided when the competitive strains are spatially separated in different compartments containing half-roots from the same plant, suggesting that competitive relations are more related to local plant responses. In our opinion, split-root systems are an appropriate approach to further study competitive relations among rhizobial strains.

Sinorhizobium fredii is a dominant rhizobium on alkaline-saline land that can induce nitrogen-fixing symbiotic root nodules in soybean. Two S. fredii strains, CCBAU25509 and CCBAU45436, were used in this study to facilitate in-depth analyses of this species and its interactions with soybean. We have previously completed the full assembly of the genomes and detailed transcriptomic analyses for these two S. fredii strains, CCBAU25509 and CCBAU45436, that exhibit differential compatibility toward some soybean hosts. In this work, we performed high-throughput Orbitrap analyses of the whole proteomes and secretomes of CCBAU25509 and CCBAU45436 at different growth stages. Our proteomic data cover coding sequences in the chromosome, chromid, symbiotic plasmid, and other accessory plasmids. In general, we found higher levels of protein expression by genes in the chromosomal genome, whereas proteins encoded by the symbiotic plasmid were differentially accumulated in bacteroids. We identified secreted proteins from the extracellular medium, including seven and eight Nodulation Outer Proteins (Nops) encoded by the symbiotic plasmid of CCBAU25509 and CCBAU45436, respectively. Differential host restriction of CCBAU25509 and CCBAU45436 is regulated by the allelic type of the soybean Rj2(Rfg1) protein. Using sequencing data from this work and available in public databases, our analysis confirmed that the soybean Rj2(Rfg1) protein has three major allelic types (Rj2/rfg1, rj2/Rfg1, rj2/rfg1) that determine the host restriction of some Bradyrhizobium diazoefficiens and S. fredii strains. A mutant defective in the type 3 protein secretion system (T3SS) in CCBAU25509 allowed this strain to nodulate otherwise-incompatible soybeans carrying the rj2/Rfg1 allelic type, probably by disrupting Nops secretion. The allelic forms of NopP and NopI in S. fredii might be associated with the restriction imposed by Rfg1. By swapping the NopP between CCBAU25509 and CCBAU45436, we found that only the strains carrying NopP from CCBAU45436 could nodulate soybeans carrying the rj2/Rfg1 allelic type. However, no direct interaction between either forms of NopP and Rfg1 could be observed.

Conventional systems used to tag and transfer symbiotic plasmids (pSyms) of rhizobial strains are based in mutagenesis with transposons. In those processes, numerous clones must be analyzed to find one of them with the transposon inserted in the pSym. Following this strategy, the insertion might interrupt a gene that can affect the symbiotic phenotype of the bacteria tagged. Here, we have developed a new system based in homologous recombination that generates Sinorhizobium fredii strains with pSyms tagged by the insertion of a suicide vector which harbor a truncated copy of S. fredii HH103 nodZ gene, a mob site, and a kanamycin-resistant gene. When it is introduced by conjugation in a S. fredii strain, the vector integrates in pSym by only one recombination event. This pSym tagged can be transferred in matting experiments to other strains in the presence of a helper plasmid. Following this method, we have tagged several strains and transferred their pSyms to a recipient strain demonstrating the potential of this new system., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Pigeon pea (Cajanus cajan (L.) Millspaugh) is cultivated widely in semiarid agricultural regions in over 90 countries around the world. This important legume can enter into symbiotic associations with a wide range of rhizobia including Bradyrhizobium and fast-growing rhizobia. In comparison with other major legumes such as soybean and common bean, only limited information is available on the symbiotic interaction of pigeon pea with rhizobia. In this study, we investigated the ability of two classical soybean symbionts—S. fredii USDA191 and B. diazoefficiens USDA110—and their type 3 secretion system (T3SS) mutants, to nodulate pigeon pea. Both S. fredii USDA191 and a T3SS mutant S. fredii RCB26 formed nitrogen-fixing nodules on pigeon pea. Inoculation of pigeon pea roots with B. diazoefficiens USDA110 and B. diazoefficiens Δ136 (a T3SS mutant) resulted in the formation of Fix− and Fix+ nodules, respectively. Light and transmission electron microscopy of Fix- nodules initiated by B. diazoefficiens USDA110 revealed the complete absence of rhizobia within these nodules. In contrast, Fix+ nodules formed by B. diazoefficiens Δ136 revealed a central region that was completely filled with rhizobia. Ultrastructural investigation revealed the presence of numerous bacteroids surrounded by peribacteroid membranes in the infected cells. Analysis of nodule proteins by one- and two-dimensional gel electrophoresis revealed that leghemoglobin was absent in B. diazoefficiens USDA110 nodules, while it was abundantly present in B. diazoefficiens Δ136 nodules. Results of competitive nodulation assays indicated that B. diazoefficiens Δ136 had greater competitiveness for nodulation on pigeon pea than did the wild type strain. Our results suggest that this T3SS mutant of B. diazoefficiens, due to its greater competitiveness and ability to form Fix+ nodules, could be exploited as a potential inoculant to boost pigeon pea productivity.

Background: Rhizobial surface polysaccharides are important molecular determinants required for successful symbiosis with legumes. In Sinorhizobium ( Ensifer) meliloti Rm41, the rkp-2 region is involved in the biosynthesis of K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS). This region is composed of two genes, lpsL and rkpK, which are respectively responsible for the production of galacturonic and glucuronic acid. Results: In this work, we show that in S. ( Ensifer) fredii HH103 these genes do not form a transcriptional unit and that the transcriptional rate of rkpK is much higher than that of lpsL. Inactivation of each of these genes resulted in alterations in LPS, but did not affect KPS production, which is in agreement with the lack of uronic acids in S. fredii HH103 KPS. Mutation of rkpK also impaired HH103 exopolysaccharide (EPS) production, most probably due to the presence of glucuronic acid in HH103 EPS, as well as increased bacterial autoaggregation and osmosensitivity and decreased biofilm formation on plastic surfaces. Inactivation of rkpK affected negatively symbiosis with cowpea but not with soybean. Mutation of lpsL led to a complete symbiotic impairment with cowpea, whereas soybean plants inoculated with this mutant only formed pseudonodules. In both plants, the lpsL mutant showed defects in root infection. Conclusion: These results confirm the symbiotic importance of HH103 LPS in symbiosis with legumes. [ABSTRACT FROM AUTHOR]

The type III secretion system (T3SS) is a specialized secretion apparatus that is commonly used by many plant and animal pathogenic bacteria to deliver proteins, termed effectors, to the interior of the host cells. These effectors suppress host defenses and interfere with signal transduction pathways to promote infection. Some rhizobial strains possess a functional T3SS, which is involved in the suppression of host defense responses, host range determination, and symbiotic efficiency. The analysis of the genome of the broad-host-range rhizobial strain Sinorhizobium fredii HH103 identified eight genes that code for putative T3SS effectors. Three of these effectors, NopL, NopP, and NopI, are Rhizobium specific. In this work, we demonstrate that NopI, whose amino acid sequence shows a certain similarity with NopP, is secreted through the S. fredii HH103 T3SS in response to flavonoids. We also determined that NopL can be considered an effector since it is directly secreted to the interior of the host cell as demonstrated by adenylate cyclase assays. Finally, the symbiotic phenotype of single, double, and triple nopI, nopL, and nopP mutants in soybean and cowpea was assayed, showing that NopI plays an important role in determining the number of nodules formed in both legumes and that the absence of both NopL and NopP is highly detrimental for symbiosis. [ABSTRACT FROM AUTHOR]

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signalling cascades.

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signaling cascades. [ABSTRACT FROM AUTHOR]

Sinorhizobium (Ensifer) fredii (S. fredii) is a rhizobial species exhibiting a remarkably broad nodulation host-range. Thus, S. fredii is able to effectively nodulate dozens of different legumes, including plants forming determinate nodules, such as the important crops soybean and cowpea, and plants forming indeterminate nodules, such as Glycyrrhiza uralensis and pigeon-pea. This capacity of adaptation to different symbioses makes the study of the molecular signals produced by S. fredii strains of increasing interest since it allows the analysis of their symbiotic role in different types of nodule. In this review, we analyze in depth different S. fredii molecules that act as signals in symbiosis, including nodulation factors, different surface polysaccharides (exopolysaccharides, lipopolysaccharides, cyclic glucans, and K-antigen capsular polysaccharides), and effectors delivered to the interior of the host cells through a symbiotic type 3 secretion system. [ABSTRACT FROM AUTHOR]

Sulfur is an essential element for rhizobia, such as sulfated modified Nod factors and nitrogenase. To investigate the role of sulfur metabolism in Rhizobium-Soybean symbiosis, a transponson random insertional mutants' library was constructed and a sulfur assimilation-related gene was isolated and characterized. A mutant strain unable to utilized sulfate was screened from 11,000 random insertional mutants of Sinorhizobium fredii WGF03. Sequencing analysis showed that a sulfate assimilation-related gene ( cysDN) was inserted by the Tn transponson. Mutants inactivated in cysD and cysN (SMcysDF and SMcysNF) were constructed by homologous recombination using the suicide plasmid pK18mob. The mutants SMcysDF and SMcysNF could no longer utilize sulfate as sulfur source. Phenotype analysis revealed that mutation of cysDN had multiple effects on S. fredii WGF03. Root hair deformation assay showed that the activity of Nod factors secreted by mutants SMcysDR and SMcysNR elicited minimal hair initiation only. Soybean plant tests indicated that the mutant strains delayed 1-2 days to nodulate and exhibited lower nodulation efficiency and symbiotic efficiency than the wild-type strain. The complementary strain of cysD and cysN (HcysDF and HcysNF) could restore the nodulation efficiency. [ABSTRACT FROM AUTHOR]

Sinorhizobium (Ensifer) fredii (S. fredii) is a rhizobial species exhibiting a remarkably broad nodulation host-range. Thus, S. fredii is able to effectively nodulate dozens of different legumes, including plants forming determinate nodules, such as the important crops soybean and cowpea, and plants forming indeterminate nodules, such as Glycyrrhiza uralensis and pigeon-pea. This capacity of adaptation to different symbioses makes the study of the molecular signals produced by S. fredii strains of increasing interest since it allows the analysis of their symbiotic role in different types of nodule. In this review, we analyze in depth different S. fredii molecules that act as signals in symbiosis, including nodulation factors, different surface polysaccharides (exopolysaccharides, lipopolysaccharides, cyclic glucans, and K-antigen capsular polysaccharides), and effectors delivered to the interior of the host cells through a symbiotic type 3 secretion system.

5-Enopyruvylshikimate-3-phosphate synthase (EPSP synthase) is an important enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants and microorganisms. A novel class II EPSP synthase AroA from Sinorhizobium fredii NGR234 was overexpressed in Escherichia coli BL21. It was purified to homogeneity and its catalytic properties were studied. The enzyme exhibited optimum catalytic activity at pH 8.0 and 50 °C. It was stable below 40 °C, and over a broad range of pH 5.0-9.0. The EPSP synthase was increasingly activated by 100 mM of the chlorides of NH, K, Na and Li. Kinetic analysis of AroA suggested that the enzyme exhibited a high glyphosate tolerance and high level of affinity for phosphoenolpyruvate, which indicates the enzyme with a high potential for structural and functional studies and its potential usage for the generation of transgenic crops resistant to the herbicide. [ABSTRACT FROM AUTHOR]