Characterisation of the mating-type locus in the genus Xanthoria (lichen-forming ascomycetes, Lecanoromycetes)

Conserved regions of mating-type genes were amplified in four representatives of the genus Xanthoria (X. parietina, X. polycarpa, X. flammea, and X. elegans) using PCR-based methods. The complete MAT locus, containing one ORF (MAT1-2-1) coding for a truncated HMG-box protein, and two partial flanking genes, were cloned by screening a genomic lambda phage library of the homothallic X. parietina. The flanking genes, a homologue of SLA2 of Saccharomyces cerevisiae and a DNA lyase gene, served to amplify the two idiomorphs of the X. polycarpa MAT locus. Each idiomorph contains a single gene: MAT1-2-1 codes for a HMG-box protein, MAT1-1-1 encodes an alpha domain protein. The occurrence of mating-type genes in eight single spore isolates derived from one ascus was studied with a PCR assay. In the homothallic X. parietina a HMG fragment, but no alpha box fragment was found in all isolates, whereas in X. elegans, another homothallic species, all tested isolates contained a fragment of both idiomorphs. Conversely, isolates of the heterothallic X. polycarpa contained either a HMG or an alpha box fragment, but never both.     

Chromosomal location of a race-specific resistance gene to <Emphasis Type="Italic">Mycosphaerella graminicola</Emphasis> in the spring wheat ST6

Septoria tritici blotch, caused by Mycosphaerella graminicola, is a serious foliar disease of wheat worldwide. Qualitative, race-specific resistance sources have been identified and utilized for resistant cultivar development. However, septoria tritici blotch resistant varieties have succumbed to changes in virulence of M. graminicola on at least three continents. The use of resistance gene pyramids may slow or prevent the breakdown of resistance. A clear understanding of the genetics of resistance and the identification of linked PCR-based markers will facilitate the recovery of wheat lines carrying multiple septoria tritici blotch resistance genes. The resistance gene in ST6 to isolate MG2 of M. graminicola was mapped with microsatellite markers in two populations, ST6/Erik and ST6/Katepwa. Bulk segregant analysis identified a marker on chromosome 4AL putatively linked to the resistance gene. A large linkage group was identified in each population using additional microsatellite markers mapping to chromosome 4AL. The resistance gene in ST6 mapped to the distal end of chromosome 4AL in each mapping population and was designated Stb7. Three of the microsatellite loci, Xwmc313, Xwmc219 and Xgwm160, mapped within 3.5 cM of Stb7; however, none flanked Stb7. Xwmc313 was the closest and mapped 0.3 and 0.5 cM from Stb7 in the crosses ST6/Katepwa and ST6/Erik, respectively. WMC313 will be very useful for marker-assisted selection of Stb7 in Canadian breeding programs because the ST6 allele of Xwmc313 was not identified in any of the Canadian common wheat cultivars tested.Communicated by P. Langridge     

Molecular mapping and physical location of major gene conferring seedling resistance to Septoria tritici blotch in wheat

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola (anamorph: Septoria tritici), is one of the most important foliar diseases of wheat. We assessed three doubled-haploid (DH) populations derived from Chara (STB-susceptible)/WW2449 (STB-resistant), Whistler (STB-susceptible)/WW1842 (STB-resistant) and Krichauff (STB susceptible)/WW2451 (STB-resistant) for resistance to a single-pycnidium isolate 79.2.1A of M. graminicola at the seedling stage. STB resistance in each of the three DH populations was conditioned by a single major gene designated as StbWW2449, StbWW1842 and StbWW2451. Linkage analyses and physical mapping indicated that the StbWW loci were located on the short arm of chromosome 1B (IBS). Four simple sequence repeat (SSR) markers linked with STB resistance: Xwmc230, Xbarc119b, Xksum045 and Xbarc008 were located to the distal bin of 1BS.sat1BS-4 (FL: 0.52–1.00) in the 1BS physical map. Xwmc230, Xbarc119b and Xksum045 markers, mapped within 7 cM from StbWW were validated for their linkage and predicted the STB resistance with over 94% accuracy in the 79 advanced breeding lines having WW2449 as one of the parents. The marker interval Xwmc230/Xksum045-Xbarc119b also explained up to 38% of the phenotypic variance at the adult plant stage in all three DH mapping populations. These results have proven that SSR markers are useful in monitoring STB resistance both at seedling and adult plant stages and hence are suitable for routine marker-assisted selection in the wheat breeding programs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Construction and characterization of a bacterial artificial chromosome library of the causal agent of Black Sigatoka fungal leaf spot disease of banana and plantain, <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>

A bacterial artificial chromosome library of the causal agent of the Black Sigatoka leaf spot disease of banana and plantain, Mycosphaerella fijiensis, has been constructed using a non-sphaeroplasting technique and characterized using both homologous and heterologous probes. After first and a second size selection of PFGE-fractionated DNA, a ligation was obtained using a 1:4 molar ratio (insert:vector). One hundred random clones were analyzed, and the mean insert size was estimated to be 90 kb. The range of the insert sizes was between 40 and 160 kb. The highest percentage of inserts belonged to the range between 80 and 100 kb; 32% of the inserts had 2 or 3 internal NotI sites. This library consists of 1920 clones, if the genomic size is at least 35 Mb, then this represents 4.9× genome equivalents, which was supported by hybridization results with homologous and heterologous probes. Blondy Canto-Canché and Diana Karina Guillén-Maldonado contributed equally to this work and should be regarded as co-first authors.     

Isolation and characterization of bacteria from the rhizosphere of wheat

In this study, we isolated bacteria from rhizosphere and endorhizophere of wheat crops of the central region of Argentina. The isolates were phenotypically characterized and the restriction patterns of 16S rDNA (ARDRA) using endonuclease AluI were analysed. Representative isolates were used to evaluate the effect of the inoculation on the growth of wheat under greenhouse conditions. The effects of plant growth-promoting bacteria on wheat plants were studied by evaluating shoot fresh and dry weights and root fresh and dry weights. One native strain increased the shoot and root dry biomass by 23% and 45% respectively. Other strains increased the shoot dry biomass. A 1.5 kb fragment of the 16S rRNA gene of one isolate was sequenced. This isolate showed high identity with different species of Pseudomonas.     

The differential effect of Eriobotrya japonica hydrophilic leaf extract on cytokines production and modulation

Stimulating or modulating the release of cytokines by immunomodulators or immunostimulating agents is an attractive mode for treating several diseases such as viral infections. For instance, patients with viral infections may be in need of increasing or inducing T helper 1 (Th1) or proinflammatory cytokines, which ultimately activate T cytotoxic and Natural killer lymphocytes to kill virally infected cells. Of these agents, we found that Eriobotrya japonica hydrophilic leaf extract (EJHE) can induce and modulate cytokines in dose-dependent manner. Twenty-four hour exposure of increasing concentrations of EJHE increased significantly (p < 0.001) the production of IFN-γ and TNF-α, from PHA+LPS-stimulated whole blood. However, the production of IFN-γ and TNF-α plateaued at high EJHE concentrations (10–100 μg/ml). No significant changes in the production of IL-10 were seen. In addition, EJHE at 1 and 10 μg/ml reversed significantly (p < 0.01) the inhibitory effect of hydrocortisone on the IL-12 p70, IFN-γ and TNF-α production from PHAS+LPS stimulated whole blood. Without PHA and LPS, EJHE was found to induce significantly (p < 0.001) IFN-γ, IL-12 p70, TNF-α, and IL-10 from whole blood culture in concentration dependent manner. The maximum induction of IFN-γ, IL-12 p70, and TNF-α by EJHE was at 1 and 10 μg/ml. On the other hand, IL-10 induction kept increasing even at the highest concentration used (100 μg/ml) of EJHE. Furthermore, intra-peritoneal injection of EJHE into mice increased significantly serum cytokines level mainly at 10 and 100 μg/ml. Two-hour post i.p. injection, EJHE increased serum IFN-γ, TNF-α, and IL-10 to 750, 1000, and 250 pg/ml, respectively. However, 24 h post i.p. injection, the levels of TNF-α, and IL-10 were similar to basal levels but IFN-γ levels were 200 pg/ml. These results indicate that EJHE induces proinflammatory and Th1 cytokines in concentration dependent manner and the effect of this induction should be studied further in viral models to check the efficacy of such cytokine induction.     

Isolation and partial characterization of chromoprotein from the larval hemolymph of the Japanese oak silkworm (Antheraea yamamai)

A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.     

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.     

Mycosphaerella chaenomelis sp. nov.: The teleomorph ofCercosporella sp., the causal fungus of frosty mildew inChaenomeles sinensis, and its role as the primary infection source

A leaf spot disease called frosty mildew was observed onChaenomeles sinensis throughout Japan. Small brown spots with white tufts occurred followed by successive defoliation. On the fallen leaves, minute black dots are formed. The causal fungus was regarded as a new species ofMycosphaerella, M. chaenomelis, andCercosporella chaenomelis in anamorph. Pathogenicity of the fungus was confirmed only inC. sinensis by inoculation experiments. Colonies of the fungus grew well on potato sucrose agar, and grew at 10–30°C with an optimum temperature of 25°C. The fungus overwintered on the fallen diseased leaves in the form of pseudothecia, and ascospores served as the primary infection source from April to June.     

Cloning, characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii

The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.     

Isolation of matrices from maize leaf nuclei: identification of a matrix-binding site adjacent to theAdh1 gene

Nuclear matrices were isolated from maize leaves by the two conventional methods usually employed for the preparation of the corresponding structures of animal origin. It is demonstrated that functionally competent matrices, recognizing and specifically binding the MAR-containing DNA of the mousek-immunoglobulin gene may be prepared by both 2 M NaCl and LIS extractions of maize nuclei.A DNA region with a high affinity for the nuclear matrix was identified at the 5 end of the maizeAdh1-S gene, distal to the promoter region. The presence of sites of reported altered chromatin structure in this particular region is discussed. While the proximity and the cohabitation of MARs with different regulatory elements is a common feature of matrix association regions in animal systems, this is the first plant MAR identified in a region of known significance for gene regulation.     

一株产耐热右旋糖酐酶真菌的筛选、鉴定及其酶学性质

【目的】从土壤中筛选得到1株产耐热右旋糖酐酶的真菌。【方法】采用营养缺陷型培养基,结合稀释涂布法和平板透明圈法分离筛选出产耐热右旋糖酐酶的菌株。通过观察菌落形态、菌体形态和培养特征,结合ITS r DNA序列分析对菌株进行鉴定。研究菌株所产右旋糖酐酶的酶学性质。【结果】通过筛选得到1株产耐热右旋糖酐酶的菌株DG001,经鉴定为淡紫拟青霉(Paecilomyces lilacinus)。菌株DG001所产右旋糖酐酶的最佳催化条件为55°C,p H 5.0;最适底物为5%Dextran T70。酶在60°C以下和p H 4.0–7.0之间稳定。urea、Mn~(2+)和Mg~(2+)对酶活均有促进作用,低浓度的Mn~(2+)和urea可使酶活分别提高到116.91%和110.14%,而Cu~(2+)则对其有强烈抑制作用。该酶水解右旋糖酐T2000的产物主要是异麦芽糖和异麦芽三糖,被确定为内切右旋糖酐酶。酶对底物的亲和性随底物分子量的增加而增强。【结论】成功筛选获得1株产耐热右旋糖酐酶的菌株DG001,所产酶在较宽温度范围内具有较高活力,热稳定性好。该酶在制糖工业及不同分子量右旋糖酐的制备中具有很好的应用前景。     

大熊猫源肺炎克雷伯菌生物学特性

【背景】肺炎克雷伯菌是仅次于大肠杆菌的常见条件致病菌之一,严重时可导致大熊猫发生出血性肠炎、全身性败血症等。【目的】明确大熊猫源肺炎克雷伯菌的生物学特性,对防控该病作出科学指导。【方法】分别采用结晶紫染色法、拉丝实验、K-B纸片法和PCR技术对46株大熊猫源肺炎克雷伯菌的生物被膜形成能力、高黏性表型、耐药表型和15种常见毒力基因等生物学特性进行研究,并根据以上生物学特性选择一株可能具有致病性的分离菌pneumoniae-X-5,研究其对小鼠的致病性。【结果】46株肺炎克雷伯菌均可形成荚膜;12株为高黏性表型肺炎克雷伯菌;能形成生物被膜的菌株占比为65%(30/46);分离出的46株菌中多重耐药菌株占58%(27/46),对氨苄西林、苯唑西林、青霉素、万古霉素呈100%耐药;毒力基因检出率最高的为ureA(91.30%,42/46)。pneumoniae-X-5菌株对小鼠的LD₅₀为8.9×10⁴CFU/mL;该菌株攻毒小鼠肺泡间隔增厚,炎性细胞浸润,肝细胞变性坏死,脾充血,十二指肠黏膜上皮和固有层分离,固有层部分细胞坏死。死亡小鼠脾脏含细菌量最多,其次为肝脏。【结论】本试验阐明了部分大熊猫源肺炎克雷伯菌的多重耐药性、能形成生物被膜、具有高黏表型等病原生物学特性,为大熊猫肺炎克雷伯杆菌病的防控及临床治疗提供了科学依据。     

Cloning, heterologous gene expression and biochemical characterization of the alpha-1,3-glucanase from the filamentous fungus Penicillium purpurogenum

There has been much recent interest in α-1,3-glucanases (mutanases) as they have the potential to be used in the treatment of dental caries. Mutanases have been reported in a number of bacteria, yeast and fungi but remain a relatively uncharacterised family of enzymes. In this study we heterologously expressed the mutanase gene from the filamentous fungus Penicillium purpurogenum to enable further characterization of its enzymatic activity. The mutanase cDNA was cloned and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was specific for mutan and could not cleave a variety of other polysaccharides demonstrating a specificity for α-1,3-glucosidic linkages. The pH and temperature optima were pH 4.6 and 45 °C, respectively. Synthetic compounds were also tested as substrates to assess whether the P. purpurogenum mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an endo-hydrolytic mode of action. The type of mechanism was confirmed since mutanase activity was not suppressed in the presence of inhibitors of exo-type enzymes.     

Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica

Temporins are small antimicrobial peptides isolated from North American and Eurasian ranid frogs that are particularly active against Gram-positive bacteria. To date, no temporins have been characterized from North African frog species. We isolated three novel members of the temporin family, named temporin-1Sa (FLSGIVGMLGKLF(amide)), -1Sb (FLPIVTNLLSGLL(amide)), and -1Sc (FLSHIAGFLSNLF(amide)), from the skin of the Sahara frog Pelophylax (Rana) saharica originating from Tunisia. These temporins were identified by a combined mass spectrometry/molecular cloning approach. Temporin-1Sa was found to be highly active against Gram-positive and Gram-negative bacteria, yeasts and fungi (MIC=2-30muM). To our knowledge, this is the first 13-residue member of the temporin family with a net charge of +2 that shows such broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC=10muM) and Pseudomonas aeruginosa (MIC=31muM). Moreover, temporin-1Sa displays significant antiparasitic activity (IC(50) approximately 20muM) against the promastigote and amastigote stages of Leishmania infantum.