QTL analysis of nodule traits and the identification of loci interacting with the type III secretion system in soybean.

Symbiotic nitrogen fixation is the main source of nitrogen for soybean growth. Since the genotypes of rhizobia and soybean germplasms vary, the nitrogen-fixing ability of soybean after inoculation also varies. A few studies have reported that quantitative trait loci (QTLs) control biological nitrogen fixation traits, even soybean which is an important crop. The present study reported that the Sinorhizobium fredii HH103 gene rhcJ belongs to the tts (type III secretion) cluster and that the mutant HH103ΩrhcJ can clearly decrease the number of nodules in American soybeans. However, few QTLs of nodule traits have been identified. This study used a soybean (Glycine max (L.) Merr.) 'Charleston' × 'Dongnong 594' (C × D, n = 150) recombinant inbred line (RIL). Nodule traits were analysed in the RIL population after inoculation with S. fredii HH103 and the mutant HH103ΩrhcJ. Plants were grown in a greenhouse with a 16-h light cycle at 26 °C and an 8-h dark cycle at 18 °C. Then, 4 weeks after inoculation, plants were harvested for evaluation of nodule traits. Through QTL mapping, 16 QTLs were detected on 8 chromosomes. Quantitative PCR (qRT-PCR) and RNA-seq analysis determined that the genes Glyma.04g060600, Glyma.18g159800 and Glyma.13g252600 might interact with rhcJ.

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.

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).

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.

The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea.

In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysaccharides (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ- and rkpU-encoded products are orthologous to Escherichia coli proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium-legume interaction.

Isolation and characterization of an operon involved in sulfate and sulfite metabolism in Sinorhizobium fredii.

A gene cluster ORFabcd from a Sinorhizobium fredii HN01 mutant strain HSMRalpha was isolated. We showed that it was an operon involved in sulfur metabolism. Functional studies revealed that, except for ORFb, the three genes ORFa, ORFc and ORFd were involved in sulfite reduction. ORFa and ORFc were similar to the cysG and cysI from Sinorhizobium meliloti 1021 and Rhizobium etli CFN 42, respectively. ORFd encodes a conserved hypothetical protein in other bacteria. We demonstrate here, for the first time, that it was a new locus involved in sulfate assimilation in S. fredii HN01 and we designated it as cysII.

Identification of fast and slow growing rhizobia nodulating soybean (Glycine max [L.] Merr) by a multiplex PCR reaction.

Two DNA fragments, a 730-bp and a 900-bp fragment, one homologous to host cultivar specificity genes nolBT of Sinorhizobium fredii and the other one homologous to RSalpha, an insertion-like sequence present in Bradyrhizobium japonicum, were generated by polymerase chain reaction (PCR) with two pairs of primers. The amount of each fragment generated by the multiplex PCR was proportional to the amount of template DNA present. The amplification of the 900-bp RSalpha fragment was more sensitive, since it was amplified from a smaller amount of template DNA than the 730-bp nolBT fragment. By running the multiplex reaction in the presence of template DNA isolated from different sources, we confirmed that the reaction can discriminate between S. fredii, Bradyrhizobium japonicum and Sinorhizobium xinjiangensis.

Salt-tolerance genes involved in cation efflux and osmoregulation of Sinorhizobium fredii RT19 detected by isolation and characterization of Tn5 mutants.

Salt-tolerance genes of Sinorhizobium fredii RT19 were identified by the construction and screening of a transposon Tn5-1063 library containing over 30,000 clones. Twenty-one salt-sensitive mutants were obtained and five different genes were identified by sequencing. Eight mutants were found with disruptions in the phaA2 gene, which encodes a cation efflux system protein, while mutations in genes encoding other cation effux system proteins were found in seven (phaD2), two (phaF2) and two (phaG2) mutants. A mutation in the metH gene, encoding 5' methyltetrahydrofolate homocysteine methyltransferase, was found in two of the salt sensitive strains. Growth experiments showed that phaA2, phaD2, phaF2 and phaG2 mutants were hypersensitive to Na+/Li+ and slightly sensitive to K+ and not sensitive to sucrose and that metH mutants were highly sensitive to any of Na+, Li+, K+ and sucrose. Na+ intracellular content measurements established that phaA2, phaD2, phaF2 and phaG2 are mainly involved in the Na+ efflux in S. fredii RT19. Recovery of growth of the metH mutants incubated with different concentrations of NaCl could be obtained by additions of methionine, choline and betaine, which showed that the metH gene is probably involved in osmoregulation in S. fredii RT19.

[A sinorhizobium fredll strain that effectively nodulate Medicago sativa].

Sinorhizobium fredii 042BS was isolated from root nodules of alfalfa (Medicago sativa) from Xinjiang Region. Nodulation experiments showed that both soybean and alfalfa were nodulated by 042BS effectively. The 16S rDNA PCR-RFLP analysis was carried out by four restriction endonucleases, and the restriction maps of strain 042BS were identical with those of S. fredii USDA205. The DNA G + C mol% of strain 042BS was 60.0. The DNA homology between 042BS and S. fredii USDA205 and USDA191 were 84.9% and 89.6%, respectively. To prove the capability of 042BS to nodulate both soybean and alfalfa, constitutively expressed green fluorescence protein gene(gfp) was introduced to 042BS, and the recombinant strain 042BSG was obtained. The reisolates from nodules of the soybean and alfalfa inoculated with 042BSG were observed using the confocal laser-scanning microscope, and the expressions of gfp were detected, respectively. 042BS showed various nodulation capacities with different alfalfa cultivars used.

A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes.

Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5 - monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution.

The effect of FITA mutations on the symbiotic properties of Sinorhizobium fredii varies in a chromosomal-background-dependent manner.

nodD1 of Sinorhizobium fredii HH103, which is identical to that of S. fredii USDA257 and USDA191, repressed its own expression. Spontaneous flavonoid-independent transcription activation (FITA) mutants of S. fredii HH103 M (=HH103 RifR pSym::Tn 5-Mob) showing constitutive expression of nod genes were isolated. No differences were found among soybean cultivar Williams plants inoculated with FITA mutants SVQ250 or SVQ253 or with the parental strain HH103M. Soybean plants inoculated with mutant SVQ255 formed more nodules, and those inoculated with mutant SVQ251 had symptoms of nitrogen starvation. Sequence analyses showed that all of the FITA mutants carried a point mutation in their nodD1 coding region. Mutants SVQ251 and SVQ253 carried the same mutation, but only the former was symbiotically impaired, which indicated the presence of an additional mutation elsewhere in the genome of mutant SVQ251. Mutants SVQ251 and SVQ255 were outcompeted by the parental strain for nodulation of soybean cultivar Williams. The symbiotic plasmids of mutants SVQ251 and SVQ255 (pSym251 and pSym255, respectively) and that (pSymHH103M) of the parental strain were transferred to pSym-cured derivatives of S. fredii USDA192 and USDA193 (USDA192C and USDA193C, respectively). Soybean responses to inoculation with S. fredii USDA192C and USDA193C transconjugants carrying pSym251 and pSymHH103M were not significantly different, whereas more nodules were formed after inoculation with transconjugants carrying pSym255. Only transconjugant USDA192C(pSym255) produced a significant increase in soybean dry weight.