Water-Soluble Humic Materials Modulating Metabolism and Triggering Stress Defense in Sinorhizobium fredii.

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.

Plasmid pSfr64a and the symbiotic plasmid pSfr64b of Sinorhizobium fredii GR64 control each other's conjugative transfer through quorum-sensing elements.

Rhizobia are nitrogen-fixing symbionts of plants. Their genomes frequently contain large plasmids, some of which are able to perform conjugative transfer. Plasmid pSfr64a from Sinorhizobium fredii GR64 is a conjugative plasmid, whose transfer is regulated by quorum sensing genes encoded by itself (traR64a, traI64a), in the symbiotic plasmid pSfr64b (traR64b, traI64b), and in the chromosome (ngrI). Also, transfer of pSfr64b requires quorum sensing elements encoded in this plasmid (traR64b, traI64b), in pSfr64a (traR64a), and in the chromosome (ngrI). These results demonstrate that pSfr64a and the symbiotic plasmid depend on each other for conjugative transfer. Plasmid pSfr64a from S. fredii GR64 is unable to transfer from the genomic background of Rhizobium etli CFN42. Our results show that the relaxase of pRet42a is able to process the oriT of pSfr64a, and viceversa, underlining their functional similarity and suggesting that in addition to the external signals, the "cytoplasmic environment" may pose a barrier to plasmid dissemination, even if the plasmids are functional in other aspects.

Sinorhizobium fredii HH103 nolR and nodD2 mutants gain capacity for infection thread invasion of Lotus japonicus Gifu and Lotus burttii.

Sinorhizobium fredii HH103 RifR , a broad-host-range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen-fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 RifR nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen-fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod-factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 RifR . Complementation of the nodD2 and nolR mutants provoked a decrease in Nod-factor production, the incapacity to form nitrogen-fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 RifR nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod-factor overproduction, although other bacterial symbiotic signals could also be involved.

Identification of Soybean Genes Whose Expression is Affected by the Ensifer fredii HH103 Effector Protein NopP.

In some legume⁻rhizobium symbioses, host specificity is influenced by rhizobial nodulation outer proteins (Nops). However, the genes encoding host proteins that interact with Nops remain unknown. We generated an Ensifer fredii HH103 NopP mutant (HH103ΩNopP), and analyzed the nodule number (NN) and nodule dry weight (NDW) of 10 soybean germplasms inoculated with the wild-type E. fredii HH103 or the mutant strain. An analysis of recombinant inbred lines (RILs) revealed the quantitative trait loci (QTLs) associated with NopP interactions. A soybean genomic region containing two overlapping QTLs was analyzed in greater detail. A transcriptome analysis and qRT-PCR assay were used to identify candidate genes encoding proteins that interact with NopP. In some germplasms, NopP positively and negatively affected the NN and NDW, while NopP had different effects on NN and NDW in other germplasms. The QTL region in chromosome 12 was further analyzed. The expression patterns of candidate genes Glyma.12g031200 and Glyma.12g073000 were determined by qRT-PCR, and were confirmed to be influenced by NopP.

Evidence for Phosphate Starvation of Rhizobia without Terminal Differentiation in Legume Nodules.

Phosphate homeostasis is tightly modulated in all organisms, including bacteria, which harbor both high- and low-affinity transporters acting under conditions of fluctuating phosphate levels. It was thought that nitrogen-fixing rhizobia, named bacteroids, inhabiting root nodules of legumes are not phosphate limited. Here, we show that the high-affinity phosphate transporter PstSCAB, rather than the low-affinity phosphate transporter Pit, is essential for effective nitrogen fixation of Sinorhizobium fredii in soybean nodules. Symbiotic and growth defects of the pst mutant can be effectively restored by knocking out PhoB, the transcriptional repressor of pit. The pst homologs of representative rhizobia were actively transcribed in bacteroids without terminal differentiation in nodules of diverse legumes (soybean, pigeonpea, cowpea, common bean, and Sophora flavescens) but exhibited a basal expression level in terminally differentiated bacteroids (alfalfa, pea, and peanut). Rhizobium leguminosarum bv. viciae Rlv3841 undergoes characteristic nonterminal and terminal differentiations in nodules of S. flavescens and pea, respectively. The pst mutant of Rlv3841 showed impaired adaptation to the nodule environment of S. flavescens but was indistinguishable from the wild-type strain in pea nodules. Taken together, root nodule rhizobia can be either phosphate limited or nonlimited regarding the rhizobial differentiation fate, which is a host-dependent feature.

A novel symbiovar (aegeanense) of the genus Ensifer nodulates Vigna unguiculata.

BACKGROUND: Cowpea (Vigna unguiculata) forms nitrogen-fixing root nodules with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium, although a few studies have reported the isolation of fast-growing rhizobia under laboratory and field conditions. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, very limited information is available on cowpea rhizobia in European soils. The aim of this study was to study the genetic and phenotypic diversity of indigenous cowpea-nodulating rhizobia in Greece. RESULTS: The genetic diversity of indigenous rhizobia associated with cowpea was investigated through a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into three groups. Based on the analysis of the 16S rRNA genes, IGS and on the concatenation of six housekeeping genes (recA, glnII, gyrB, truA, thrA and SMc00019), rhizobial isolates were classified within the species Ensifer fredii. However, symbiotic gene phylogenies, based on nodC, nifH and rhcRST genes, showed that the Ensifer isolates are markedly diverged from type and reference strains of E. fredii and formed one clearly separate cluster. The E. fredii strains were able to nodulate and fix nitrogen in cowpea but not in soybean and common bean. CONCLUSION: The present study showed that cowpea is nodulated under field conditions by fast-growing rhizobia belonging to the species E. fredii. Based on the phylogenies, similarity levels of symbiotic genes and the host range, the Ensifer isolates may constitute a new symbiovar for which the name 'aegeanense' is proposed. © 2017 Society of Chemical Industry.

A putative 3-hydroxyisobutyryl-CoA hydrolase is required for efficient symbiotic nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234.

We report that the smb20752 gene of the alfalfa symbiont Sinorhizobium meliloti is a novel symbiotic gene required for full N2 -fixation. Deletion of smb20752 resulted in lower nitrogenase activity and smaller nodules without impacting overall nodule morphology. Orthologs of smb20752 were present in all alpha and beta rhizobia, including the ngr_b20860 gene of Sinorhizobium fredii NGR234. A ngr_b20860 mutant formed Fix- determinate nodules that developed normally to a late stage of the symbiosis on the host plants Macroptilium atropurpureum and Vigna unguiculata. However an early symbiotic defect was evident during symbiosis with Leucaena leucocephala, producing Fix- indeterminate nodules. The smb20752 and ngr_b20860 genes encode putative 3-hydroxyisobutyryl-CoA (HIB-CoA) hydrolases. HIB-CoA hydrolases are required for l-valine catabolism and appear to prevent the accumulation of toxic metabolic intermediates, particularly methacrylyl-CoA. Evidence presented here and elsewhere (Curson et al., , PLoS ONE 9:e97660) demonstrated that Smb20752 and NGR_b20860 can also prevent metabolic toxicity, are required for l-valine metabolism, and play an undefined role in 3-hydroxybutyrate catabolism. We present evidence that the symbiotic defect of the HIB-CoA hydrolase mutants is independent of the inability to catabolize l-valine and suggest it relates to the toxicity resulting from metabolism of other compounds possibly related to 3-hydroxybutyric acid.

Sinorhizobium fredii HH103 Invades Lotus burttii by Crack Entry in a Nod Factor-and Surface Polysaccharide-Dependent Manner.

Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicus.

The Sinorhizobium fredii HH103 MucR1 Global Regulator Is Connected With the nod Regulon and Is Required for Efficient Symbiosis With Lotus burttii and Glycine max cv. Williams.

Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S. fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon.

Bacterial Molecular Signals in the Sinorhizobium fredii-Soybean Symbiosis.

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.

Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants.

The nitrogen phosphotransferase system (PTS(Ntr)) consists of EI(Ntr), NPr, and EIIA(Ntr). The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI(Ntr) and NPr to EIIA(Ntr). Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI(Ntr) and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA(Ntr)) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-ß-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA(Ntr) in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI(Ntr) with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS(Ntr) in symbiosis was also observed when related PTS(Ntr) mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS(Ntr) in addition to the negative role of unphosphorylated EIIA(Ntr).

Sinorhizobium fredii†HH103 bacteroids are not terminally differentiated and show altered O-antigen in nodules of the Inverted Repeat-Lacking Clade legume Glycyrrhiza uralensis.

In rhizobial species that nodulate inverted repeat-lacking clade (IRLC) legumes, such as the interaction between Sinorhizobium meliloti and Medicago, bacteroid differentiation is driven by an endoreduplication event that is induced by host nodule-specific cysteine rich (NCR) antimicrobial peptides and requires the participation of the bacterial protein BacA. We have studied bacteroid differentiation of Sinorhizobium fredii HH103 in three host plants: Glycine max, Cajanus cajan and the IRLC legume Glycyrrhiza uralensis. Flow cytometry, microscopy analyses and viability studies of bacteroids as well as confocal microscopy studies carried out in nodules showed that S. fredii HH103 bacteroids, regardless of the host plant, had deoxyribonucleic acid (DNA) contents, cellular sizes and survival rates similar to those of free-living bacteria. Contrary to S. meliloti, S. fredii HH103 showed little or no sensitivity to Medicago NCR247 and NCR335 peptides. Inactivation of S. fredii HH103 bacA neither affected symbiosis with Glycyrrhiza nor increased bacterial sensitivity to Medicago NCRs. Finally, HH103 bacteroids isolated from Glycyrrhiza, but not those isolated from Cajanus or Glycine, showed an altered lipopolysaccharide. Our studies indicate that, in contrast to the S. meliloti-Medicago model symbiosis, bacteroids in the S. fredii HH103-Glycyrrhiza symbiosis do not undergo NCR-induced and bacA-dependent terminal differentiation.

The Sinorhizobium (Ensifer) fredii HH103 Type 3 Secretion System Suppresses Early Defense Responses to Effectively Nodulate Soybean.

Plants that interact with pathogenic bacteria in their natural environments have developed barriers to block or contain the infection. Phytopathogenic bacteria have evolved mechanisms to subvert these defenses and promote infection. Thus, the type 3 secretion system (T3SS) delivers bacterial effectors directly into the plant cells to alter host signaling and suppress defenses, providing an appropriate environment for bacterial multiplication. Some rhizobial strains possess a symbiotic T3SS that seems to be involved in the suppression of host defenses to promote nodulation and determine the host range. In this work, we show that the inactivation of the Sinorhizobium (Ensifer) fredii HH103 T3SS negatively affects soybean nodulation in the early stages of the symbiotic process, which is associated with a reduction of the expression of early nodulation genes. This symbiotic phenotype could be the consequence of the bacterial triggering of soybean defense responses associated with the production of salicylic acid (SA) and the impairment of the T3SS mutant to suppress these responses. Interestingly, the early induction of the transcription of GmMPK4, which negatively regulates SA accumulation and defense responses in soybean via WRKY33, could be associated with the differential defense responses induced by the parental and the T3SS mutant strain.

The Sinorhizobium fredii HH103 Genome: A Comparative Analysis With S. fredii Strains Differing in Their Symbiotic Behavior With Soybean.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III-secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

Structure and biological roles of Sinorhizobium fredii HH103 exopolysaccharide.

Here we report that the structure of the Sinorhizobium fredii HH103 exopolysaccharide (EPS) is composed of glucose, galactose, glucuronic acid, pyruvic acid, in the ratios 5∶2∶2∶1 and is partially acetylated. A S. fredii HH103 exoA mutant (SVQ530), unable to produce EPS, not only forms nitrogen fixing nodules with soybean but also shows increased competitive capacity for nodule occupancy. Mutant SVQ530 is, however, less competitive to nodulate Vigna unguiculata. Biofilm formation was reduced in mutant SVQ530 but increased in an EPS overproducing mutant. Mutant SVQ530 was impaired in surface motility and showed higher osmosensitivity compared to its wild type strain in media containing 50 mM NaCl or 5% (w/v) sucrose. Neither S. fredii HH103 nor 41 other S. fredii strains were recognized by soybean lectin (SBL). S. fredii HH103 mutants affected in exopolysaccharides (EPS), lipopolysaccharides (LPS), cyclic glucans (CG) or capsular polysaccharides (KPS) were not significantly impaired in their soybean-root attachment capacity, suggesting that these surface polysaccharides might not be relevant in early attachment to soybean roots. These results also indicate that the molecular mechanisms involved in S. fredii attachment to soybean roots might be different to those operating in Bradyrhizobium japonicum.

Isolation and characterization of a gene associated with sulfate assimilation in Sinorhizobium fredii WGF03.

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.

The symbiotic biofilm of Sinorhizobium fredii SMH12, necessary for successful colonization and symbiosis of Glycine max cv Osumi, is regulated by Quorum Sensing systems and inducing flavonoids via NodD1.

Bacterial surface components, especially exopolysaccharides, in combination with bacterial Quorum Sensing signals are crucial for the formation of biofilms in most species studied so far. Biofilm formation allows soil bacteria to colonize their surrounding habitat and survive common environmental stresses such as desiccation and nutrient limitation. This mode of life is often essential for survival in bacteria of the genera Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Rhizobium. The role of biofilm formation in symbiosis has been investigated in detail for Sinorhizobium meliloti and Bradyrhizobium japonicum. However, for S. fredii this process has not been studied. In this work we have demonstrated that biofilm formation is crucial for an optimal root colonization and symbiosis between S. fredii SMH12 and Glycine max cv Osumi. In this bacterium, nod-gene inducing flavonoids and the NodD1 protein are required for the transition of the biofilm structure from monolayer to microcolony. Quorum Sensing systems are also required for the full development of both types of biofilms. In fact, both the nodD1 mutant and the lactonase strain (the lactonase enzyme prevents AHL accumulation) are defective in soybean root colonization. The impairment of the lactonase strain in its colonization ability leads to a decrease in the symbiotic parameters. Interestingly, NodD1 together with flavonoids activates certain quorum sensing systems implicit in the development of the symbiotic biofilm. Thus, S. fredii SMH12 by means of a unique key molecule, the flavonoid, efficiently forms biofilm, colonizes the legume roots and activates the synthesis of Nod factors, required for successfully symbiosis.

RNA sequencing analysis of the broad-host-range strain Sinorhizobium fredii NGR234 identifies a large set of genes linked to quorum sensing-dependent regulation in the background of a traI and ngrI deletion mutant.

The alphaproteobacterium Sinorhizobium fredii NGR234 has an exceptionally wide host range, as it forms nitrogen-fixing nodules with more legumes than any other known microsymbiont. Within its 6.9-Mbp genome, it encodes two N-acyl-homoserine-lactone synthase genes (i.e., traI and ngrI) involved in the biosynthesis of two distinct autoinducer I-type molecules. Here, we report on the construction of an NGR234-ΔtraI and an NGR234-ΔngrI mutant and their genome-wide transcriptome analysis. A high-resolution RNA sequencing (RNA-seq) analysis of early-stationary-phase cultures in the NGR234-ΔtraI background suggested that up to 316 genes were differentially expressed in the NGR234-ΔtraI mutant versus the parent strain. Similarly, in the background of NGR234-ΔngrI 466 differentially regulated genes were identified. Accordingly, a common set of 186 genes was regulated by the TraI/R and NgrI/R regulon. Coregulated genes included 42 flagellar biosynthesis genes and 22 genes linked to exopolysaccharide (EPS) biosynthesis. Among the genes and open reading frames (ORFs) that were differentially regulated in NGR234-ΔtraI were those linked to replication of the pNGR234a symbiotic plasmid and cytochrome c oxidases. Biotin and pyrroloquinoline quinone biosynthesis genes were differentially expressed in the NGR234-ΔngrI mutant as well as the entire cluster of 21 genes linked to assembly of the NGR234 type III secretion system (T3SS-II). Further, we also discovered that genes responsible for rhizopine catabolism in NGR234 were strongly repressed in the presence of high levels of N-acyl-homoserine-lactones. Together with nodulation assays, the RNA-seq-based findings suggested that quorum sensing (QS)-dependent gene regulation appears to be of higher relevance during nonsymbiotic growth rather than for life within root nodules.

Effect of Sinorhizobium fredii strain Sneb183 on the biological control of soybean cyst nematode in soybean.

The soybean cyst nematode (SCN; Heterodera glycines) is a major detriment to soybean production. The endophytic bacterium Sinorhizobium fredii strain Sneb183 is known to inhibit the activity of SCN. In the present study, soybean seedlings were inoculated with Sneb183, to study the penetration juveniles, and their development inside the roots. The number of cysts in the soybean roots was also examined. The induced systemic resistance in soybean was also examined through the split-root system. Our results revealed that the number of juveniles and cysts significantly decreased as a result of Sneb183 inoculation. Sneb183 also prolonged the developmental stage of SCN in the root to 30 days as compared to 27 days in the control. Furthermore, the number of nematodes in each stage was lower in the Sneb183 treated plants than control plants. We also used a split-root system to show that the S. fredii strain Sneb183 induced a systemic resistance to SCN infection in soybean. The repression rate of SCN penetration was 38.75%. Our study showed that Sneb183 can be an effective biocontrol agent for managing SCN infestation in soybean.

The Sinorhizobium fredii HH103 lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules.

In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA, lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA, lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean.