Gα and Gβ Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen Mycosphaerella graminicola

We identified and functionally characterized genes encoding three Gα proteins and one Gβ protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Gα_i and Gα_s families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.Signal transduction pathways are important for sensing and responding to different environmental stimuli in both lower and higher eukaryotes. The highly conserved heterotrimeric guanine nucleotide-binding proteins (G proteins) belong to a family of regulatory proteins that are crucial for the transduction of signals, which are perceived by a distinct family of cell surface receptors (4). Heterotrimeric G proteins contain three subunits (α, β, and γ) that are linked in the inactive state. Activation of a Gα subunit by a transmembrane receptor leads to exchange of bound GDP with GTP on the Gα subunit, resulting in dissociation of the Gα and the Gβγ dimeric subunits, which can now interact with downstream effectors that subsequently generate changes in cellular responses (for a review, see reference 10).Filamentous fungi have one Gβ- and usually three Gα-encoding genes that belong to three major groups. Encoded proteins in groups I and III are related to the mammalian Gα_i and Gα_s families, respectively, but group II fungal Gα proteins have no mammalian counterpart (1, 4, 14, 22, 33, 53). Interestingly, the corn smut fungus Ustilago maydis contains a unique fourth Gα-encoding gene, and Saccharomyces cerevisiae contains only two Gα proteins (10, 57). Irrespective of the observed numerical variation, Gα proteins regulate a variety of cellular and developmental responses (4). For plant-pathogenic fungi, Gβ-encoding genes have been characterized functionally (9, 14, 22, 27, 31, 48, 52). Apart from the fact that individual Gα-encoding genes and the Gβ-encoding gene have been demonstrated to regulate growth, reproduction, and virulence, comparative functional characterization of all Gα-encoding genes has been reported only for a few plant-pathogenic fungi, including Magnaporthe grisea, Cryphonectria parasitica, and U. maydis (5, 41, 57).Mycosphaerella graminicola (anamorph Septoria tritici) causes septoria tritici blotch disease in bread and durum wheat in areas with high rainfall during the growing season, particularly in Western Europe, where it is considered to be the most important wheat disease (30). It is a ubiquitous phytopathogen with a lifestyle completely different from that of the aforementioned plant-pathogenic fungi. It is a dimorphic pathogen, and therefore the transition from a yeast-like to a filamentous form is important for initiation of infection (45). M. graminicola does not form appressoria but penetrates the leaves through stomata without forming specific infection structures. Furthermore, as a hemibiotroph, it has a biotrophic phase of about 10 days that is followed by a rapid switch to necrotrophy. The necrotic foliar lesions bear anamorphic and teleomorphic fructifications. M. graminicola is the model fungus for the Mycosphaerellaceae and even for the order Dothideales, an extremely large and diverse class of fungi with over 1,000 named species, including major plant pathogens such as the banana leaf streak fungus Mycosphaerella fijiensis (12, 21). Large expressed sequence tag (EST) libraries and the recently released genome sequence have been instrumental for the identification and characterization of genes involved in the development and pathogenicity of M. graminicola (http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html). Recently, we reported that genes encoding mitogen-activated protein kinases (MAPKs) (MgFus3, MgSlt2, and MgHog1) and the catalytic (MgTpk2) and regulatory (MgBcy1) subunits of protein kinase A (PKA) are essential pathogenicity factors and regulate specific steps during the infection process (8, 43-45). To extend our knowledge about the role of G proteins in the development and pathogenicity of M. graminicola, we functionally analyzed three Gα-encoding genes and one Gβ-encoding gene of M. graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Our results show the requirement of MgGpa1, MgGpa3, and MgGpb1 for pathogenicity, whereas the latter also negatively regulates cell fusion and anastomosis. Among the G protein-encoding genes characterized in this study, MgGpa3 and MgGpb1 positively regulate the cyclic AMP (cAMP) pathway. MgGpa1 seems to be dispensable for cAMP regulation, whereas MgGpa2 appears to be redundant, for none of the assays rendered altered phenotypes. Our results open new perspectives for studying the regulatory machinery of the cAMP pathway in M. graminicola and other plant-pathogenic fungi.     

Molecular genetic diversity of Satureja bachtiarica

Fifty-seven genotypes from eight population of Satureja bachtiarica was evaluated using fifteen ISSR and eleven RAPD markers. DNA profiling using RAPD primers amplified 84 loci, among which 81 were polymorphic with an average of 7.36 polymorphic fragments per locus. Also, using RAPD markers maximum and minimum polymorphic bands observed for Semyrom (77.38 %) and Farsan (40.48 %) populations, respectively. Semyrom population recorded the highest unbiased expected heterozygosity (0.259) and Shannon’s Indices (0.38). While, the lowest values of unbiased expected heterozygosity (0.172) and Shannon’s Index (0.245) were recorded for Eghlid and Farsan populations, respectively. On the other hand, ISSR primers produced 136 bands, from which 134 were polymorphic with an average of 9.06 polymorphic fragments per primer (98.52 %). The ISSR markers evaluation revealed that maximum and minimum polymorphic bands observed for Semyrom (66.18 %) and Farsan (31.62 %), respectively. Shahrekorud population recorded the highest unbiased expected heterozygosity (0.211) and Shannon’s Indices (0.301). While, the lowest value of unbiased expected heterozygosity (0.175) observed for Farsan and Yazd populations and the lowest Shannon’s Index (0.191) recorded by Farsan population. The overall results of the study revealed that both ISSR and RAPD markers were effective for evaluation of genetic variation of S. bachtiarica.     

Study on the physiology of diapause, cold hardiness and supercooling point of overwintering pupae of the pistachio fruit hull borer, Arimania comaroffi

The pistachio fruit hull borer, Arimania comaroffi (Ragonot) (Lepidoptera: Pyralidae), is a key pest of pistachio orchards in Iran. This pest passes the winter as diapausing pupae. In this study, some physiological changes in relation to environmental temperature were investigated in field collected pupae. The relationship between supercooling point, cold hardiness and physiological changes of a wild population of this pest was also investigated. The glycogen content decreased with decrease in environmental temperature. Decrease in glycogen content was proportional to increase in total body sugar, trehalose, myo-inositol and sorbitol contents. In January with mean ambient temperature of 5.4°C, glycogen (5 mg/g fresh body weight) content was at the lowest level whereas total body sugar (10.3 mg/g fresh body weight), trehalose (8.6 mg/g fresh body weight), myo-inositol (5.3 mg/g fresh body weight) and sorbitol (2.6 mg/g fresh body weight) were at the highest levels. Total body sugar, trehalose, myo-inositol and sorbitol contents increased as mean temperature decreased from 22.7°C in October to 5.4°C in January. Total body lipid decreased during overwintering and reached to the lowest level at the end of March. Supercooling points were decreased from October to January and reached to the lowest level (-16°C) in January with minimum ambient temperature of -10°C. Survival at low temperature after 24 h was also greatest in January with 72% survival at -10°C, 39% survival at -15°C and 0% survival at -20°C. Increase in temperature from February onward, was proportional with increase in supercooling points and decrease in survival rate. Regardless of sampling date, all pupae died after 24 h at -20°C, whereas none pupae died after 24 h at -5°C. This indicates that this insect is freeze-intolerant.     

Mathematical modeling and study of mass transfer parameters in supercritical fluid extraction of fatty acids from Trout powder

Mathematical modeling of fatty acids (FAs) extraction from Trout powder by supercritical carbon dioxide was performed in the present work. Trout powder with its low cost contains high amount of essential FAs and it is commonly available as a proper source of FAs. The effect of process parameters, such as pressure (25, 28, 31, 34 and 37 MPa) and temperature (310, 318 and 326 K) of extraction and void fraction of the bed (0.25, 0.35 and 0.45, v/v) on the yield of FAs extraction was examined in a series of experiments conducted in a laboratory scale apparatus. The results indicated a significant increase of extraction yield with an increase of pressure from 25 to 34 MPa, but working at the higher pressure (37 MPa) caused reduction of the extract. Increasing the temperature higher than 318 K revealed significant reduction of the FAs yield and increasing the bed void fraction from 0.25 to 0.45 showed enhancement of the extraction.

The mathematical model was developed considering diffusion-controlled regime in the particle and film mass transfer resistance around the particle with axial dispersion of the bulk phase at dynamic conditions. Henry law was used to describe the equilibrium state of solid and fluid phases. The proposed mass balance equations were numerically solved using implicit finite difference method and the model parameters were correlated using the experimental results of the outlet FAs concentration in the oil extracted at dynamic conditions. Well-known Nelder–Mead method was applied to estimate the four parameters of the model, namely, mass transfer coefficient (k_f), axial dispersion coefficient (D_ax) in the bulk phase, effective diffusivity (D_eff) into the pores and Henry coefficient (H). In the range of studied conditions, the higher extraction efficiency with higher pressure resulted lower correlated H, although the temperature increasing which showed a retrograde phenomena in the FAs yield, revealed H passing though a minimum.     

Identification and characterization of Rhodopseudomonas spp., a purple, non-sulfur bacterium from microbial mats

A species of facultative photo-organotrophic, purple, non-sulfur bacterium was isolated from mixed-species microbial mats, characterized and examined for metal tolerance and bioremediation potential. Contributing mats were natural consortia of microbes, dominated by cyanobacteria and containing several species of bacteria arranged in a laminar structure, stabilized within a gel matrix. Constructed microbial mats were used for bioremediation of heavy metals and organic chemical pollutants. Purple, non-sulfur bacteria are characteristically found in lower strata of intact mats, but their contributing function in mats survival and function by mediating the chemical environment has not been explored. The gram-negative rod-shaped bacterium, reported here, produced a dark red culture under phototrophic conditions, reproduced by budding and formed a lamellar intracytoplasmic membrane (ICM) system parallel to cytoplasmic membrane, which contained bacteriochlorophyll a and carotenoids. This strain was found to have multiple metal resistances and to be effective in the reductive removal of Cr(VI) and the degradation of 2,4,6-trichlorophenol. Based on the results obtained from morphology, nutrient requirements, major bacteriochlorophyll content, GC content, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) profile and 16S-rDNA phylogenetic analysis, this member of the microbial mats may be identified as a new strain of the genus Rhodopseudomonas.     

Ectophoma iranica sp. nov. and new hosts and records of Allophoma spp. in Iran

A survey was conducted to identify fungi associated with leaf diseases of ornamental herbs, including Christ plant, dracaena, rose geranium, rose periwinkle and treasure flower, in Ahvaz, southwestern Iran. Twenty‐five symptomatic herbs were collected and studied, which led to the isolation of seven Phoma‐like strains belonging to four taxa. These fungi were characterised based on DNA sequence data for the partial large subunit 28S nrDNA (LSU‐D1/D2), the internal transcribed spacer 1 and 2 and 5.8S nrDNA (ITS) and part of the β‐tubulin (tub2) gene regions. A multi‐locus‐based phylogeny, in combination with morphology, allowed for the identification of Allophoma hayatii, A. labilis, A. tropica and a novel species of the genus Ectophoma. Ectophoma iranica sp. nov. is morphologically and genetically distinctive from previously described species. In pathogenicity tests, the two strains of E. iranica (CBS 144681 and IRAN 3354C) caused leaf spot symptoms on leaves of Catharanthus roseus. New host plant species and records for A. hayatii, A. labilis and A. tropica are reported, plus further emended characterization of A. hayatii.     

Hydrogen-halide versus alkyl-halide oxidative addition in dimethyl platinum(II) complexes: Crystal structure of [PtBr2(bpy)]

Dimethyl platinum(II) complexes [PtMe₂(NN)] {NN = bu₂bpy (4,4′-di-tert-butyl-2,2′-bipyridine) (1a), bpy (2,2′-bipyridine) (1b), phen (1,10-phenanthroline) (1c)} reacted with commercial 3-bromo-1-propanol in the presence of 1,3-propylene oxide to afford cis, trans- [PtBrMe₂{(CH₂)₃OH}(NN)] (NN = bu₂bpy (2a), bpy (2b), phen (2c)). On the other hand, [PtMe₂(NN)] (1a)-(1b) reacted with the trace of HBr in commercial 3-bromo-1-propanol to give [PtBr₂(NN)] (NN = bu₂bpy (3a), bpy (3b)). The reaction pathways were monitored by ¹H NMR at various temperatures. Treatment of 1a-1b with a large excess of 3-bromo-1-propanol at −80 °C gave the corresponding methyl(hydrido)platinum(IV) complexes [PtBr(H)Me₂(NN)] (NN = bu₂bpy (4a), bpy (4b)) via the oxidative addition of dimethyl platinum(II) complexes with HBr. The complexes [PtBr(H)Me₂(NN)] decomposed by reductive elimination of methane above −20 °C for bu₂bpy and from −20 to 0 °C for bpy analogue to give methane and platinum(II) complexes [PtBrMe(NN)] (5a)-(5b) and then decomposed at about 0 °C to yield [PtBr₂(NN)] and methane. When the reactions were performed at a molar ratio of Pt:RX/1:10, the corresponding complexes [PtBrMe(NN)] (5a)-(5b) were also obtained. The crystal structure of the complex 3b shows that platinum adopts square planar geometry with a twofold axis through the platinum atom. The Pt…Pt distance (5.164 Å) is considerably larger than the interplanar spacing (3.400 Å) and there is no platinum-platinum interaction.     

Astaxanthin Modulates Autophagy,Apoptosis, and Neuronal Oxidative Stress in a Rat Model of Compression Spinal Cord Injury

The effects of astaxanthin (AST) were evaluated on oxidative mediators, neuronal apoptosis, and autophagy in functional motor recovery after spinal cord injury (SCI). Rats were divided into three groups of sham, SCI?+?DMSO (dimethyl sulfoxide), and SCI?+?AST. Rats in the sham group only underwent a laminectomy at thoracic 8–9. While, the SCI?+?DMSO and SCI?+?AST groups had a compression SCI with an aneurysm clip. Then, this groups received an intrathecal (i.t.) injection of 5% DMSO and AST (10 μl of 0.005 mg/kg), respectively. The rat motor functions were assessed weekly until the 28th day using a combined behavioral score (CBS). Total antioxidant capacity (TAC), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were measured in spinal tissue to evaluate oxidative stress-related parameters. Besides, autophagy-related proteins (P62, LC3B, and Beclin1) and apoptosis-associated proteins (Bax and Bcl2) were determined using western blotting on the 1st and 7th days after surgery. Hematoxylin–eosin and Fluoro-Jade B staining were performed to detect the histological alterations and neuronal degeneration. As the result, treatment with AST potentially attenuated rat CBS scores (p?<?0.001) towards a better motor performance. AST significantly reduced the spinal level of oxidative stress by increasing TAC, SOD, and GPx, while decreasing MDA (p?<?0.001). Furthermore, AST treatment remarkably upregulated expression of LC3B (p?<?0.001), and Beclin1 (p?<?0.05) in the spinal cord, but downregulated P62 (p?<?0.05) and the Bax/Bcl2 ratio (p?<?0.001). Consequently, AST reduced SCI-induced histological alterations and neuronal degeneration (p?<?0.001). In conclusion, AST can improve motor function after SCI by reducing oxidative stress/apoptosis and increasing neuronal autophagy.

     

Molecular characterization of the wild relatives of wheat using CAAT-box derived polymorphism

A molecular assessment of genetic diversity was performed on a set of 228 selected accessions belonging to the different Aegilops and Triticum species using CAAT-box derived polymorphism (CBDP) markers. Fifteen CBDP primers generated 141 polymorphic fragments with an average of 9.40 per primer. The average of polymorphic information content and resolving power revealed a high efficiency of CBDP markers in analyzing genetic diversity among different wheat genotypes. The diversity indexes including polymorphic loci percent, number of observed and effective alleles, Shannon’s index and gene diversity among different populations were 76.24%, 1.67, 1.49, 0.42 and 0.28, respectively. These essentially coincided with the analysis of molecular variance results, indicating that 86, 68, and 59.46% of genetic variation were found within two Aegilops and Triticum genera and their populations, respectively. Genetic relationships inferred from cluster analysis was matched with STRUCTURE analysis, disclosing the accessions were grouped based on their genomic constitution. Furthermore, these results were confirmed by principal coordinate analysis (PCoA). Taken together, our results suggest that CBDP markers will be useful for genetic diversity assessment in the domesticated and wild relatives of wheat.