Lcc1 and Lcc5 are the main laccases secreted in liquid cultures of Coprinopsis cinerea strains

The litter-degrading dung fungus Coprinopsis cinerea has the high number of seventeen different laccase genes. In this work, ten different monokaryons were compared in their ability to produce laccases in two different complete media at different temperatures. Few strains showed laccase activity at the optimal growth temperature of 37 °C. Nine of the strains gave laccase activities between 0.2 and 5.9 U mL(−1) at the suboptimal temperature of 25 °C in mKjalke medium. Laccase activities in YMG/T medium were detected for only three strains (0.5–4.5 U mL(−1)). Zymograms of supernatants from mKjalke medium resulted in total in 10 different laccase bands but strains differed in distribution. LC–MS/MS analysis with Mascot searches of the annotated C. cinerea genome identified isoenzymes from five different genes (Lcc1, Lcc2, Lcc5, Lcc9 and Lcc10) and of Lcc1 three and of Lcc5 two distinct electrophoretical forms. Lcc1 and Lcc5 were expressed in all laccase positive strains, but not all forms were found in all of the strains. Lcc2, Lcc9 and Lcc10 occurred only in three strains as minor laccases, indicating that Lcc1 and Lcc5 are the main laccases of C. cinerea secreted in liquid mKjalke medium. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10482-013-9883-7) contains supplementary material, which is available to authorized users

Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptoms

<p>Abstract</p> <p>Background</p> <p><it>Verticillium longisporum </it>is one of the most important pathogens of <it>Brassicaceae </it>that remains strictly in the xylem during most stages of its development. It has been suggested that disease symptoms are associated with clogging of xylem vessels. The aim of our study was to investigate extracellular defence reactions induced by <it>V. longisporum </it>in the xylem sap and leaf apoplast of <it>Brassica napus </it>var. <it>napus </it>in relation to the development of disease symptoms, photosynthesis and nutrient status.</p> <p>Results</p> <p><it>V. longisporum </it>(strain VL43) did not overcome the hypocotyl barrier until 3 weeks after infection although the plants showed massive stunting of the stem and mild leaf chlorosis. During this initial infection phase photosynthetic carbon assimilation, transpiration rate and nutrient elements in leaves were not affected in VL43-infected compared to non-infected plants. Proteome analysis of the leaf apoplast revealed 170 spots after 2-D-protein separation, of which 12 were significantly enhanced in response to VL43-infection. LS-MS/MS analysis and data base searches revealed matches of VL43-responsive proteins to an endochitinase, a peroxidase, a PR-4 protein and a β-1,3-glucanase. In xylem sap three up-regulated proteins were found of which two were identified as PR-4 and β-1,3-glucanase. Xylem sap of infected plants inhibited the growth of <it>V. longisporum</it>.</p> <p>Conclusion</p> <p><it>V. longisporum </it>infection did not result in drought stress or nutrient limitations. Stunting and mild chlorosis were, therefore, not consequences of insufficient water and nutrient supply due to VL43-caused xylem obstruction. A distinct array of extracellular PR-proteins was activated that might have limited <it>Verticillium </it>spreading above the hypocotyl. In silico analysis suggested that ethylene was involved in up-regulating VL43-responsive proteins.</p

Signal peptide replacement resulted in recombinant homologous expression of laccase Lcc8 in Coprinopsis cinerea

Abstract Although the model agaricomycete Coprinopsis cinerea possess 17 different laccase genes, up to now only four C. cinerea laccases have been purified and characterized to some degree. By exchanging the nucleotide sequence of the deduced signal peptide of Lcc8 it was possible to homologously express lcc8 in C. cinerea under control of the Agaricus bisporus gdpII promoter and the C. cinerea lcc1 terminator. The purified Lcc8 showed two bands in the SDS-PAGE with a molecular weight of 64 kDa and 77 kDa, respectively. The IEF determined pI values of 3.3 and 3.4 for both bands. The optimal pH for oxidation of the substrates ABTS, 2,6-dimethoxyphenol, guaiacol and syringaldazine was pH 4.0, pH 5.0, pH 4.5 and pH 5.0, respectively. Best pH for enzyme storage was pH 8.0. The optimal temperature for oxidation of ABTS was 63 °C, while Lcc8 showed activity of at least 50% over 300 min at 50 °C. The comparable high stability of Lcc8 at alkaline pH and higher temperatures can be of interest for biotechnical applications

BMC Plant Biology BioMed Central

Research article Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptom

New Armenian Wood-Associated Coprinoid Mushrooms: Coprinopsis strossmayeri and Coprinellus aff. radians

Coprinoid mushrooms grown on wood of broad-leaf species were collected for the first time in Armenia and dikaryotic mycelial cultures were established. ITS (internal transcribed spacer) sequences identified one species as Coprinopsis strossmayeri and the other as a species closely related to Coprinellus radians. Mycelial growth and morphological features on different media are described. The pearl-white-silky colonies of C. strossmayeri are characterized by mycelial strands and by a light-yellow agar colorization. The species forms chlamydospores intercalary in its hyphae. Some hyphal ends form hyphal loops. Colonies of C. aff. radians have a characteristic yellow pigmentation and stain the agar yellowish. Hyphae are mostly clampless but at some septa, pseudoclamps are seen from which side-branches develop growing along the parental hyphae. In the mycelium of C. aff. radians, hyphal loops, hyphal swellings, cystidia and typical allocysts were observed. Both new species from Armenia show growth optima at temperatures of 25 to 30 °C and pHs of 6.0 to 7.0. Both grow in alkaline conditions up to pH 12.0 but not at pHs 3.0 and 4.0, classifying them with other coprinoid mushrooms as “ammonia fungi”. Both species grew on a variety of lignocellulosic substrates and both show polyphenol oxidase activities

Fungal DNA, stunting of the rosette, and relative transcript abundance of genes encoding apoplastic proteins from <i>Arabidopsis thaliana</i> that were either induced or repressed by treatment with <i>Verticillium longisporum</i> (VL43).

<p>(A) Typical time course of stunting white symbols) and VL43 DNA (black symbols) in Arabidopsis rosettes (n≥4 biological replicates ± SE, except 10 dpi, where pooled samples were analyzed due limited material), T = traces of VL43 DNA detected. Stunting was determined as projected rosette area of VL infected plants/projected rosette area of mock inoculated plants. (B) Transcript levels of <i>CILLP</i>, <i>SCPL20</i>, <i>AGAL2</i> and <i>GLP3</i> and (C) transcript levels of the peroxidases <i>PRX52</i> (right axis), <i>PRX34</i>, <i>P37</i> and <i>PA2</i> (left axis). Transcript levels were normalized to actin and expressed as transcript abundance in VL-infected plants/transcript abundance in mock-inoculated plants (n≥6 biological replicates ± SE). *indicate significant differences between mock and VL-infected plants.</p

Metabolic fingerprinting of AWFs of mock- and <i>Verticillium longisporum</i> VL43-infected <i>Arabidopsis thaliana</i> plants.

<p>AWF was obtained from <i>A. thaliana</i> leaves 21 days post infection and analyzed by UPLC-TOF-MS. (A) PCA plot of compounds in extracted AWFs measured in negative ionization mode. Samples from mock treated plants are framed in black whereas samples from infected plants are framed in red. (B) 1D-SOM matrix after metabolite-based clustering of the 1775 infection markers with <i>p</i><5×10⁻⁴. Data sets from positive and negative ionisation mode were combined. Red framed prototypes indicate markers with increased intensities after VL infection. The data set includes three biological replicates with three technical replicates per sample. The vertical axis represents the two experimental conditions. The horizontal axis corresponds to the cluster numbers. The intensities were normalized and colour coded according to the indicated scale.</p