A nodule endophytic plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> and its effects on growth,nodulation and metal uptake in <Emphasis Type="Italic">Medicago lupulina</Emphasis> under copper stress

The aim of this study was to determine the plant growth-promoting potential of the nodule endophytic Pseudomonas brassicacearum strain Zy-2-1 when used as a co-inoculant of Medicago lupulina with Sinorhizobium meliloti under copper (Cu) stress conditions. Strain Zy-2-1 was capable of producing ACC deaminase activity, IAA and siderophores, and was able to grow in the presence of Cu²⁺ up to 2.0 mmol/L. Co-inoculation of S. meliloti with Zy-2-1 enhanced M. lupulina root fresh weight, total plant dry weight, number of nodules, nodule fresh weight and nitrogen content in the presence of 100 or 300 mg/kg Cu²⁺. In the presence of 500 mg/kg Cu²⁺, co-inoculation with S. meliloti and strain Zy-2-1 increased plant height, number of nodules, nodule fresh weight and nitrogen content in comparison to S. meliloti inoculation alone. Furthermore, a higher amount of Cu accumulation in both shoots and roots and a higher level of Cu translocation to shoots were observed in co-inoculated plants. These results demonstrate that co-inoculation of M. lupulina with S. meliloti and P. brassicacearum Zy-2-1 improves plant growth, nitrogen nutrition and metal extraction potential. This can be of practical importance in the remediation of heavy metal-contaminated soils.     

HmuS and HmuQ of <Emphasis Type="Italic">Ensifer</Emphasis>/<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> degrade heme in vitro and participate in heme metabolism in vivo

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a K_d value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.     

Characterization of surface motility in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>: regulation and role in symbiosis

Sinorhizobium meliloti can exhibit diverse modes of surface translocation whose manifestation depends on the strain. The mechanisms involved and the role played by the different modes of surface motility in the establishment of symbiosis are largely unknown. In this work, we have characterized the surface motility shown by two S. meliloti reference strains (Rm1021 and GR4) under more permissive conditions for surface spreading and analyzed the symbiotic properties of two flagella-less S. meliloti mutants with different behavior on surfaces. The use of Noble agar in semisolid minimal medium induces surface motility in GR4, a strain described so far as non-motile on surfaces. The motility exhibited by GR4 is swarming as revealed by the non-motile phenotype of the flagella-less flaAB mutant. Intriguingly, a flgK mutation which also abolishes flagella production, triggers surface translocation in GR4 through an as yet unknown mechanism. In contrast to GR4, Rm1021 moves over surfaces using mostly a flagella-independent motility which is highly reliant on siderophore rhizobactin 1021 production. Surprisingly, this motility is absent in a flagella-less flgE mutant. In addition, we found that fadD loss-of-function, known to promote surface motility in S. meliloti, exerts different effects on the two reference strains: while fadD inactivation promotes a flagella-independent type of motility in GR4, the same mutation interferes with the surface translocation exhibited by the Rm1021 flaAB mutant. The symbiotic phenotypes shown by GR4flaAB and GR4flgK, non-flagellated mutants with opposite surface motility behavior, demonstrate that flagella-dependent motility positively influences competitiveness for nodule occupation, but is not crucial for optimal infectivity.     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.     

Structural Polymorphism of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> Genes Related to Virulence and Salt Tolerance

Analysis of the structural polymorphism of eight genes in Sinorhizobium meliloti (nodA, nodB, nodC, and nodH, as well as betA, betB, betC, and betB2) involved in virulence control and salt tolerance, respectively, was carried out in native populations from two geographically distant areas of alfalfa diversity. These areas are located in the North Caucasian gene center of cultivated plants (NCG) and in the modern center of introgressive hybridization of alfalfa located next to the Aral Sea area (PAG) subjected to salinization. RFLP types (alleles) of the nod and bet genes, similar to those in the reference strain Rm1021 (A-type) and different from them (divergent, or D-type alleles) were revealed. The combinations for A- and D-type alleles of the aforementioned genes (analysis of the linkage disequilibrium, LD) were studied in both populations. It was shown that D-type alleles of the nod genes were two times more frequent in the NCG population, while D-type alleles of the bet genes were predominantly identified in the PAG population. At the same time, different combinations of D-type alleles of both the nod and bet genes prevailed in populations. For instance, in the case of the glycine betaine metabolism pathway, these were the betC and betB2 genes in NCG population and betB and betA genes in PAG population. The state of linkage disequilibrium was shown for 60.7% of combinations of alleles of the nod and bet genes in the S. meliloti strains from NCG and more than twice less in strains from the PAG population. It was concluded that clonal lines prevailed in NCG, while the PAG population of S. meliloti had a panmictic structure with revealed single clonal lines.     

A metabolomic approach to characterize the acid-tolerance response in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

Introduction

Sinorhizobium meliloti establishes a symbiosis with Medicago species where the bacterium fixes atmospheric nitrogen for plant nutrition. To achieve a successful symbiosis, however, both partners need to withstand biotic and abiotic stresses within the soil, especially that of excess acid, to which the Medicago-Sinorhizobium symbiotic system is widely recognized as being highly sensitive.

Objective

To cope with low pH, S. meliloti can undergo an acid-tolerance response (ATR(+)) that not only enables a better survival but also constitutes a more competitive phenotype for Medicago sativa nodulation under acid and neutral conditions. To characterize this phenotype, we employed metabolomics to investigate the biochemical changes operating in ATR(+) cells.

Methods

A gas chromatography/mass spectrometry approach was used on S. meliloti 2011 cultures showing ATR(+) and ATR(?) phenotypes. After an univariate and multivariate statistical analysis, enzymatic activities and/or reserve carbohydrates characterizing ATR(+) phenotypes were determined.

Results

Two distinctive populations were clearly defined in cultures grown in acid and neutral pH based on the metabolites present. A shift occurred in the carbon-catabolic pathways, potentially supplying NAD(P)H equivalents for use in other metabolic reactions and/or for maintaining intracellular-pH homeostasis. Furthermore, among the mechanisms related to acid resistance, the ATR(+) phenotype was also characterized by lactate production, envelope modification, and carbon-overflow metabolism.

Conclusions

Acid-challenged S. meliloti exhibited several changes in different metabolic pathways that, in specific instances, could be identified and related to responses observed in other bacteria under various abiotic stresses. Some of the observed changes included modifications in the pentose-phosphate pathway (PPP), the exopolysaccharide biosynthesis, and in the myo-inositol degradation intermediates. Such modifications are part of a metabolic adaptation in the rhizobia that, as previously reported, is associated to improved phenotypes of acid tolerance and nodulation competitiveness.

     

Use of RNA Immunoprecipitation Method for Determining <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> RNA<Emphasis Type="Italic">-</Emphasis>Hfq Protein Associations In Vivo

Background

Soil bacterium Sinorhizobium meliloti (S. meliloti) forms an endosymbiotic partnership with Medicago truncatula (M. truncatula) roots which results in root nodules. The bacteria live within root nodules where they function to fix atmospheric N₂ and supply the host plant with reduced nitrogen. The bacterial RNA-binding protein Hfq (Hfq) is an important regulator for the effectiveness of the nitrogen fixation. RNA immunoprecipitation (RIP) method is a powerful method for detecting the association of Hfq protein with specific RNA in cultured bacteria, yet a RIP method for bacteria living in root nodules remains to be described.

Results

A modified S. meliloti gene encoding a His-tagged Hfq protein (Hfq^His) was placed under the regulation of the native Hfq gene promoter (P_hfqsm). The trans produced Hfq^His protein was accumulated at its nature levels during all stages of the symbiosis, allowing RNAs that associated with the given protein to be immunoprecipitated with the anti-His antibody against the protein from root nodule lysates. RNAs that associated with the protein were selectively enriched in the immunoprecipitated sample. The RNAs were recovered by a simple method using heat and subsequently analyzed by RT-PCR. The nature of PCR products was determined by DNA sequencing. Hfq association with specific RNAs can be analyzed at different conditions (e. g. young or older root nodules) and/or in wild-type versus mutant strains.

Conclusions

This article describes the RIP method for determining Sinorhizobium meliloti RNA-Hfq associations in vivo. It is also applicable to other rhizobia living in planta, although some tissue-specific modification related to sample disruption and homogenization may be needed.

     

Heterozygosity for mutations affecting coat pigmentation in the American mink (<Emphasis Type="Italic">Neovison</Emphasis> vison) enhances structural stability of adrenal cortex under stress conditions

The results of the study of the effects of heterozygosity for mutations affecting coat pigmentation on the response to the environmental stress caused by extreme feeding conditions are provided. The animals with the following genotypes were taken into the study: homozygotes standard (+/+), hedlund white (h/h), and aleutian (a/a) and heterozygotes hedlund white (h/+) and aleutian (a/+). The animals homozygous for the aleutian mutation (a/a) showed a statistically lower growth rate than the animals of other genotypes both in the control and in the experiment (p < 0.05). Under the control conditions, the animals homozygous for both the wild type standard allele (+/+) and the mutant hedlund white (h/h) and aleutian (a/a) alleles showed the evident tendency for the zona fasciculata and zona reticularis of the adrenal cortex broadening compared to the experimental conditions. At the same time, in the animals heterozygous for the hedlund white (h/+) and the aleutian (a/+) mutations, a clear tendency for increasing size of the zona fasciculata and zona reticularis under the experimental conditions was observed. In the heterozygous animals, although we observed single destructive changes in the adrenal cortex under stress conditions, they were much less profound than in the homozygous ones. This may be related to the broader range of morphological adaptation in the heterozygotes, which gives them the possibility of more significant enlargement of the secreting zone to provide for its adequate functioning.     

<Emphasis Type="Italic">Trichoderma</Emphasis> Modulates Stomatal Aperture and Leaf Transpiration Through an Abscisic Acid-Dependent Mechanism in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Trichoderma species are widespread phytostimulant fungi that act through biocontrol of root pathogens, modulation of root architecture, and improving plant adaptation to biotic and abiotic stress. With the major challenge to better understand the contribution of Trichoderma symbionts to plant adaptation to climate changes and confer stress tolerance, we investigated the potential of Trichoderma virens and Trichoderma atroviride in modulating stomatal aperture and plant transpiration. Arabidopsis wild-type (WT) seedlings and ABA-insensitive mutants, abi1-1 and abi2-1, were co-cultivated with either T. virens or T. atroviride, and stomatal aperture and water loss were determined in leaves. Arabidopsis WT seedlings inoculated with these fungal species showed both decreased stomatal aperture and reduced water loss when compared with uninoculated seedlings. This effect was absent in abi1-1 and abi2-1 mutants. T. virens and T. atroviride induced the abscisic acid (ABA) inducible marker abi4:uidA and produced ABA under standard or saline growth conditions. These results show a novel facet of Trichoderma-produced metabolites in stomatic aperture and water-use efficiency of plants.     

Characteristics and expression of genes encoding two small heat shock protein genes lacking introns from <Emphasis Type="Italic">Chilo suppressalis</Emphasis>

Small heat shock proteins (sHSPs) constitute a large, diverse, and functionally uncharacterized family of heat shock proteins. To gain insight regarding the function of sHSPs in insects, we identified genes encoding two sHSPs, Cshsp22.9b and Cshsp24.3, from the rice pest Chilo suppressalis. The cDNAs of Cshsp22.9b and Cshsp24.3 encoded proteins of 206 and 216 amino acids with isoelectric points of 5.79 and 9.28, respectively. Further characterization indicated that both Cshsp22.9b and Cshsp24.3 lacked introns. Real-time quantitative PCR indicated that Cshsp22.9b and Cshsp24.3 were expressed at higher levels within the fat body as compared to other tissues (head, epidermis, foregut, midgut, hindgut, Malpighian tubules, and hemocytes). Expression of Cshsp22.9b and Cshsp24.3 was lowest in the hindgut and Malpighian tubules, respectively. Cshsp22.9b and Cshsp24.3 showed identical patterns in response to thermal stress from ?11 to 43 °C, and both genes were up-regulated by hot and cold temperatures. The mRNA (messenger ribonucleic acid) expression levels of Cshsp22.9b (KY701308) and Cshsp24.3 (KY701309) were highest after a 2-h exposure at 39 °C and started to decline at 42 °C. In response to cold temperatures, both Cshsp22.9b and Cshsp24.3 showed maximal expression after a 2-h exposure to ?3 °C. The two Cshsps were more responsive to hot than cold temperature stress and were not induced by mildly cold or warm temperatures. In conclusion, Cshsp22.9b and Cshsp24.3 could play a very important role in the regulation of physiological activities in C. suppressalis that are impacted by environmental stimuli.