Comparison of gpd genes and their protein products in basidiomycetes

We compared promoters, coding sequences, introns and terminators of glyceraldehyde 3-phosphate dehydrogenase genes (gpd) from various basidiomycetes. Coding regions of these housekeeping genes are highly conserved (between 60 to 99% DNA identity) whilst non-coding regions have DNA identities of around 40%. Amongst all homobasidiomycete promoters, the TATA region and a CT-rich region with the potential transcription start sites are highest conserved. Surprisingly, there are no other conserved motifs common to all promoters. Up to five introns are clustered at the far 5´ ends of the genes, hinting to a potential function in efficient gene expression

Multiple Multi-Copper Oxidase Gene Families in Basidiomycetes – What for?

Genome analyses revealed in various basidiomycetes the existence of multiple genes for blue multi-copper oxidases (MCOs). Whole genomes are now available from saprotrophs, white rot and brown rot species, plant and animal pathogens and ectomycorrhizal species. Total numbers (from 1 to 17) and types of mco genes differ between analyzed species with no easy to recognize connection of gene distribution to fungal life styles. Types of mco genes might be present in one and absent in another fungus. Distinct types of genes have been multiplied at speciation in different organisms. Phylogenetic analysis defined different subfamilies of laccases sensu stricto (specific to Agaricomycetes), classical Fe2+-oxidizing Fet3-like ferroxidases, potential ferroxidases/laccases exhibiting either one or both of these enzymatic functions, enzymes clustering with pigment MCOs and putative ascorbate oxidases. Biochemically best described are laccases sensu stricto due to their proposed roles in degradation of wood, straw and plant litter and due to the large interest in these enzymes in biotechnology. However, biological functions of laccases and other MCOs are generally little addressed. Functions in substrate degradation, symbiontic and pathogenic intercations, development, pigmentation and copper homeostasis have been put forward. Evidences for biological functions are in most instances rather circumstantial by correlations of expression. Multiple factors impede research on biological functions such as difficulties of defining suitable biological systems for molecular research, the broad and overlapping substrate spectrum multi-copper oxidases usually possess, the low existent knowledge on their natural substrates, difficulties imposed by low expression or expression of multiple enzymes, and difficulties in expressing enzymes heterologously

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

Multiple cotransformations in Coprinus cinereus

Plasmids usually integrate ectopically into the genome of the homobasidiomycete Coprinus cinereus in transformations. Often, integration occurs at multiple sites indicating that more than one plasmid copy was incorporated. This feature prompted us to study transformation with mixtures of several different plasmids in several genetic backgrounds. We found multiple cotransformation to be efficient even with four different plasmids. Simultaneous uptake of a second plasmid was between 5-55%, of two additional plasmids between 3-18% and 3% for three additional plasmids. These high frequencies make possible the analysis of interactions between different heterologous genes introduced into the same nucleus

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