Congresos 2011 y anteriores
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Publication Open Access Biochemistry of volatile compounds synthesis in Agaricus bisporus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Combet, E.; Henderson, J.; Eastwood, D.C.; Griffiths, G.; Burton, K.S.Agaricus bisporus unique flavour is due to the release of a set of eight-carbon volatile compounds, which biosynthetic pathway has not been elucidated yet, despite of the numerous implications of those volatile compounds. Beside their influence on crop quality, they are also important for insect perception and play a part in triggering the switch from vegetative to reproductive growth in mushrooms. 8-carbon volatiles are derived from the oxygenation and the cleavage of the polyunsaturated fatty acid linoleic acid. This reaction has similarities to the plant system, but also major differences. Examination of the enzymic mechanisms and the fatty acid chemistry suggested that the enzyme involved in the oxygenation step could be a lipoxygenase (as found in plants) or a heme-dioxygenase, similar to the recently isolated linoleate diol synthase from Gaeumannomyces graminis. In order to characterise the biochemical pathway leading to eight-carbon volatile production, we investigated fatty acid and lipids distribution in Agaricus bisporus, as well as hydroperoxide and volatile compounds levels. In parallel, we searched for candidate genes susceptible to encode the enzyme responsible for this novel oxidation route in fungi. The combination of analytical methods, such as GC-MS, with a molecular approach based on degenerate PCR and library screening provided us with a broad range of results. These results establish the relation between fatty acids and volatile compounds and enabled us to gain a better understanding of mushroom volatiles biosynthesis and lipid metabolism.Publication Open Access Species identification and detection of fungi in biological materials by FTIR microscopy(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Naumann, Anette; Navarro González, Mónica; Peddireddi, Sudhakar; Schützendübel, Andres; Kües, Ursula; Polle, AndreaFTIR spectroscopy provides the opportunity to simultaneously detect many molecular bonds or functional groups of different polysaccharides, proteins, lipids, aromatic and other compounds. The measurement principle is based on the absorption of infrared light by dipolar molecular bonds. In combination with microscopy, local resolution of the chemical composition is possible. Each absorption point or peak in the spectrum can be integrated to create an image of the distribution of the corresponding compound. We use FTIR-microscopy in order to detect fungi in plant tissues such as in infected wood and in mycorhizal roots. For the development of a fast and inexpensive method for localisation and identification of fungi, differences between FTIR measurements of fungi and plant cells are characterized. In addition, FTIR spectra of different fungi are compared. Beech wood blocks were infected with Trametes versicolor and with Schizophyllum commune and FTIR spectra in sections of the infected wood determined. Cluster analysis revealed major differences between FTIR spectra recorded from wood fibres and empty vessel lumina and spectra from fungal mycelium, irrespectively of whether grown on the surface of wood or inside vessel lumina. Species specific clustering of spectra of fungal mycelium grown on the wood surface and inside vessel lumina demonstrated the potential of FTIR microscopy to identify fungal mycelium in wood. Currently, we are sampling FTIR spectra from various basidiomycetes in order to define species according to their specific FTIR spectra. The work is supported in frame of the Lower Saxony Competence Network for Sustainable Timber Utilisation (NHN) by the Ministry of Culture of Lower Saxony and EFRE. The group of UK is funded by the DBU. MNG holds a PhD studentship from CONACYT, Mexico.Publication Open Access Genetic variability of Flammulina velutipes collections from Armenia(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Badalyan, Suzanna M.; Hughes, Karen W.; Helmbrecht, ElizabethArmenian collections of Flammulina velutipes were examined for DNA sequence variability within the ribosomal ITS1-5.8S-ITS2 region. Of 22 F. velutipes collections examined, several were heterozygous for two indels and were cloned to recover complete ribosomal ITS sequences. Genetic diversity was remarkably high in collections from Armenia when compared to collections from other parts of Eurasia. Haplotypes were assayed by phylogenetic analysis using maximum parsimony. At least 16 haplotypes were recovered (collections were defined as the same haplotype if they differed by no more than two base pairs). Most natural collections were heterozygous with respect to haplotype, suggesting an interbreeding, genetically divergent population of fungi. This high level sequence diversity may be a consequence of survival of ancient genetic variation in the Caucasus and in Armenia during periods of glaciation while in Europe, genetic variation was extirpated. A subset of Armenian genetic diversity is found in Europe.Publication Open Access Isolation, molecular characterization and location of telomeric sequences of the basidiomycete Pleurotus ostreatus var. florida(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza EkoizpenaThe white rot fungus Pleurotus ostreatus is an edible basidiomycete of increasing biotechnological interest due to its ability to degrade both wood and chemicals related to lignin degradation products. Telomeres are specialized structures at the end of all eukaryotic chromosomes. Ensure chromosome stability and protect the ends from degradation and from fusing with other chromosomes. Telomeres sequences are extraordinary highly conserved in evolution. The loss of telomeric repeats triggers replicative senescence in cells. For identification of restriction telomeric fragments in a previously described linkage map of Pleurotus ostreatus var. florida (Larraya et al., 2000), dikaryotic and eighty monokaryotic genomic DNAs were digested with diferents restriction enzymes (BamHI, BglII, HindIII, EcoRI, PstI, SalI, XbaI and XhoI) electrophoresed and transferred to nylon membranes. Numerous polymorphic bands were observed when membranes were hibridized with human telomericd probe (TTAGGG)132 (heterologous probe). Telomeric restriction fragments were genetically mapped to a previously described linkage map of Pleurotus ostreatus var.florida, using RFLPs identified by a human telomeric probe (tandemly repeating TTAGGG hexanucleotide). Segregation of each telomeric restriction fragment was recorded as the presence vs. absence of a hibridizing band. Segregation data for seventy three telomeric restriction fragments was used as an input table to be analysed as described by Ritter et al. (1990) and by Ritter and Salamini (1996) by using the MAPRF program software. Seventeen out of twenty two telomeres were identified. Telomere and telomere-associated (TA) DNA sequences of the basidiomycete Pleurotus ostreatus were isolated by using a modified version of single- specific-primer polymerase chain reaction (SSP-PCR) technique (Sohapal et al., 2000). Telomeres of Pleurotus ostreatus contain at least twenty five copies of non-coding tandemly repeated sequence (TTAGGG).Publication Open Access Sequence analysis and expression of a RecQ gene homologue from Lentinula edodes(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Katsukawa, Shiho; Shishido, KazuoWe cloned and sequenced a recQ gene homologue from Lentinula edodes. This gene, named Le.recQ, was found to have a coding capacity of 945 amino acids (aa). The deduced Le.RECQ protein was clearly smaller than other fungal RecQ proteins such as Neurospora crassa QDE3 (1955 aa), Schizosaccharomyces pombe Rqh1 (1328 aa), and Saccharomyces cerevisiae SGS1 (1447 aa). It exhibited the highest homology to the Arabidopsis thaliana RecQl4A protein (1182 aa) in its size and aa sequence. Northern-blot analysis showed that the Le.recQ gene is transcribed at similar levels during mycelial development in L. edodes fruiting-body formation. The L. edodes dikaryotic mycelial cells were found to contain a clearly larger amount of Le.recQ transcript than the L. edodes two compatible monokaryotic mycelial cells. Results in situ RNA-RNA hybridization showed that subhymenium and outer region of trama contain larger amounts of Le.recQ transcript. Expression of Le.recQ cDNA in S. cerevisiae might partially complement defects associated with the loss of its homologue S. cerevisiae SGS1 gene.Publication Open Access Fungal diversity adds value to biotechnology and agriculture(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Zervakis, Georgios I.Mediterranean countries host rich biological diversity (genetic, population, species, habitats, communities, ecosystems). Until recently research on the fungal diversity was focusing relatively more on phytopathogenic fungi, invertebrate parasites, and saprotrophic and ectomycorrhizal mushrooms (Pezizales, higher Basidiomycetes). For higher Basidiomycetes in particular, detailed inventories and check-lists have been compiled in many western European countries. In the Mediterranean region, however, pertinent data are limited and fragmentary; only recently new information has started to accumulate. Indicative is the case of Greece, where selected ecosystems are studied in respect to their macromycetes diversity, revealing the existence of taxa with significant ecological and economic interest. Prerequisites for the exploitation of biological resources (incl. fungi) is the availability of a large number of individuals with a wide genetic basis, which are correctly identified and suitably evaluated. For example, elucidating taxonomy and clarifying phylogenetic relationships among Pleurotus species has contributed significantly to their widespread use. Large-scale applications related directly (or indirectly) with mushroom resources and their exploitation include the edible mushroom industry, production of medicinal and health-promoting factors, improvement of soil fertility, remediation of soils, enhanced plant growth, suppressiveness of soil-borne pathogens of plants, animal feed, transformation of xenobiotics and antibiotics, biosorption of toxic elements, decolorization of organic pollutants, degradation of industrial and agroforestry wastes, etc. Particular emphasis is given to the upgrade of lignocellulosic wastes and residues through their detoxification and biotransformation into value-added products; among them, soil conditioners and fertilizers generated from spent mushroom substrates conform with the much-sought notion of sustainability in agriculture.Publication Open Access Formation of hyphal loops in xylotrophic coprinoid mushrooms(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Badalyan, Suzanna M.; Kües, UrsulaRecent molecular analysis split the traditional genus Coprinus (Homobasidiomycetes) into four distinct genera: Coprinus, Coprinopsis, Coprinellus and Parasola. Coprinoid mushrooms are usually saprotrophic on soil and/or dung of herbivores. However, more than 60 species are able to grow on wood and straw. Xylotrophic mushrooms are forcing a relatively short supply of nitrogen and phosphorous nutrients. Coprinus comatus has been reported to produce specialized structures (“spiny balls”) to penetrate nematodes for nutrient supply (Luo et al. 2004, Mycologia 96, 1218-1224). Nematode traps of other fungi involve adhesive hyphal network and knobs, hyphal loops and snares. Toxin production may support in nematode immobilisation. Nematode-trapping species belong mainly to the mitosporic Deute romy - ce tes, but some are also found amongst Zygomycetes and Basidiomycetes. We have observed hyphal loops in several wood-decaying basidiomycetes, such as Daedalea quercina, Ganoderma lucidum, Lentinula edodes, Piptoporus betulinus and Pleurotus ostreatus. Furthermore, regular and irregular hyphal loops and/or rings were observed in the four clades of Coprinoid species (Coprinus comatus, Coprinellus angulatus, C. bisporus, C. curtus, C. domesticus, C. disseminatus, C. ellissi, C. micaceus, C. xanthothrix, Coprinopsis cinerea, C. gonophylla, C. radians, C. strossmayeri, C. scobicola, and P. plicatilis). Hyphal loops were particularly often formed in Coprinellus species. Such structures were rare in Coprinopsis atramentaria, C. cothurnata, C. romagnesiana, C. psychromorbida and Coprinus patouillardii (an unclassified isolate). It is not clear yet why Basidiomycetes fungi have these structures. Is it that many species have nematode trapping abilities by formation of such structures? Thanks to the DAAD, NATO and the Deutsche Bundesstiftung Umwelt for financial support.Publication Open Access Fungal laccase: properties and aplications(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Jönsson, Leif J.Laccase (EC 1.10.3.2; benzenediol:oxygen oxidoreductase) was first discovered at the end of the 19th century in the sap of Oriental lacquer trees. Later on, the laccase from the white-rot basidiomycete Trametes versicolor was thoroughly characterized using biochemical and biophysical methods. It is an extracellular blue multicopper glycoprotein. The copper ions are involved in the catalytic process, in which a reducing substrate, typically a phenol, is oxidized and molecular oxygen is reduced to water. Today, a multitude of different laccases and laccase genes from various sources have been characterized. The enzyme seems to have different physiological roles in different types of organisms. Several of the best characterized laccases come from basidiomycete fungi causing white-rot decay of wood. These laccases are generally regarded to be associated with the biodegradation of lignin, although more research is needed to shed light on the fundamental molecular mechanisms. Recent advances with regard to the structural and functional diversity of laccases will be discussed in relation to efforts to clarify the physiological roles of the enzyme and to elucidate its potential in various applications, including detoxification, bleaching, and analysis.Publication Open Access Identification and functional characterisation of ctr1, a Pleurotus ostreatus gene coding for a copper transporter(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Peñas Parrila, María Manuela; Azparren Larraya, María Goretti; Domínguez, A.; Sommer, H.; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza EkoizpenaCopper homeostasis is primordial for life maintenance and especially relevant for ligning-degrading fungi whose phenol-oxidase enzymes depend on this micronutrient for their activity. In this paper we report the identification of a gene (ctr1), coding for a copper transporter in the white rot fungus Pleurotus ostreatus, in a cDNA library constructed from four-days old vegetative mycelium growing in submerged culture. The results presented here indicate that: (1) ctr1 functionally complements the respiratory deficiency of a yeast mutant defective in copper transport supporting the transport activity of the Ctr1 protein; (2) ctr1 transcription is detected in all P. ostreatus developmental stages (with exception of lamellae) and is negatively regulated by the presence of copper in the culture media; (3) ctr1 is a single copy gene that maps to P. ostreatus linkage group III; and (4) the regulatory sequence elements found in the promoter of ctr1 agree with those found in other copper related genes described in other systems. These results provide the first description of a copper transporter in this white rot fungus and open the possibility of further studies on copper metabolism in higher basidiomyetes.Publication Open Access Natural and artificial hybridization of Agaricus subrufescens Peck (= A. Blazei Murrill sensu Heinemann): lessons from the quasi-alleles of the rDNA ITS1+2 region(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Kerrigan, R.Agaricus subrufescens Peck was described from both wild and cultivated specimens in 1893. It has been sporadically cultivated in various countries since that time, and is presently an economically important “nutriceutical” food. It is known by several names, including A. rufotegulis Nauta, A. brasiliensis Wasser et al., and A. blazei Murrill sensu Heinemann. A long-term study of diverse isolates and specimens, emphasizing cultural studies and analysis of rDNA ITS1+2 sequences, strongly indicates that a single phylogenetic entity exists. Some interpopulational interfertility has also been demonstrated. Yet the picture is not simple. The species is amphithallic, with complementary reproductive routes, producing recombinant spores with cryptic karyotic states and some self-fertility. Sequences from the Americas were always highly heteromorphic, while those from Hawaii and the UK were homomorphic. This implies that American isolates may be hybrids between (at least) two formerly isolated populations. To test that idea, ITS1+2 sequences from isolate SBS1, an SSI from a California strain, were amplified, cloned and sequenced. Both allelism and recombination are evident in these 711-713 nt sequences: 4 (3+1) parental and 11 recombinant sequences were recovered. The mechanism of fine-scale recombination is unknown (PCR artifacts have not been ruled out). Recombination events exceeded 1.0 per 700 nt. Physical linkage was apparent among 11 polymorphic characters distributed along the ITS1+2. On this basis the parental allelic sequences were deduced, and a comparison with the homomorphic UK sequence was made. The evidence suggests that a European-like strain may have contributed one ITS1+2 allele to an ancestor of the isolate from California. However, if true, “crossovers” must then occurred prior to the origin of the SBS1 SSI, possibly in the SBRF progenitor (or its progenitor(s)).Publication Open Access Molecular characterization of A cellobiohydrolase gene family in the fungus Pleurotus ostreatus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Eizmendi Goicoechea, Arantza; Sannia, Giovanni; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza EkoizpenaCellulose is the most abundant biological polymer on Earth. Its chemical composition consists of D-glucose units linked by β-1,4- glycosidic bonds forming linear polymeric chains with a reducing and a non-reducing end. Cellulose chains may either adhere to each other, via hydrophobic and van der Waals interactions, forming crystalline structures or remain more loosely packaged (amorphous cellulose). Consequently, the physical structure and morphology of native cellulose is complex and not uniform. Biological degradation of cellulose depends on the action of three types of enzymes: endoglucanases (E.C.3.2.1.4), cellobiohydrolases (E.C.3.2.1.91) and β-glucosidases (E.C.3.2.1.21). All them hydrolyse β-1,4-glycosidic bonds but they differ on the substrate specificity. Endoglucanases hydrolyse the amorphous regions of the cellulose fibbers generating new reducing and non-reducing ends, cellobiohydrolases attack the molecule ends yielding cellobiose units, and β-glucosidases hydrolyse cellobiose molecules yielding glucose. Cellobiohydrolases can be classified into two groups: type I (CBHI) and type II (CBHII), each having opposite chain-end specificities. CBHI prefer the reducing ends while CBHII act at non-reducing ends. By the screening of a genomic library from the basidiomycete Pleurotus ostreatus var. florida, we have isolated five cbhI genes, named cbhI1, cbhI2, cbhI3, cbhI4 and cbhI5, proving the occurrence of a multigenic family coding for this enzymatic activity. Using this sequences as probe, it has been possible to know the conditions in which are expressed those genes. This has allowed the synthesis of the each gene cDNA and, by comparison of this sequence with the corresponding genomic sequence, the characterization of their structure. On the other hand, using the RFLP technique and a progeny of 80 monokaryons derived from the dikaryon N001, the five genes have been mapped on the linkage map of P. ostreatus var. florida mapping the cbhI1 to the chromosome IV and the others to the chromosome VI.Publication Open Access Investigations into the fungal-fungal interaction between Verticillium fungicola and Agaricus bisporus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Collopy, P.D.; Amey, R.; Challen, M.P.; Mills, P.R.; Bailey, A.; Foster, G.D.Plant and animal pathogens section The cultivated button mushroom, Agaricus bisporus, is amenable to number of pathogenic threats including bacteria, viruses, mites, insects and fungi. Currently, the most significant threat to the commercial mushroom industry is the mycoparasite, Verticillium fungicola. Infection by V. fungicola can drastically reduce the yield and value of mushroom crops. The severity of this disease is dependent on the developmental stage of A. bisporus at the time of infection and is manifested in three types of symptoms: spotty cap, stipe blowout and dry bubble. An aim of our research has been to develop molecular tools for V. fungicola that will allow us to study the interaction between this pathogen and A. bisporus. These tools have included transformation methods, marker gene techniques as well as gene-knockout technologies. This has involved the use of Agrobacterium and T-DNA to introduce disruption constructs into V. fungicola as part of a molecular investigation into this fungal-fungal interaction. We have developed an efficient transformation system for V. fungicola that we have now adapted to give high levels of targeted mutagenesis. This technique has successfully generated targeted mutants of a β-1-6 glucanase homologue from Trichoderma harzianum and a Mitogen Activated Protein Kinase homologue (PMK1) from Magnaporthe grisea identified using degenerate PCR primers. We have also developed TDNA tagging technology in a mycology context for random mutagenesis in V. fungicola.Publication Open Access Enzymatic characterization of a monokaryon population of the edible mushroom, Pleurotus ostreatus with a view to genetic improvement(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Terrón, María del Carmen; López, María; Carbajo, José M.; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; González Aldo, E.; Producción Agraria; Nekazaritza EkoizpenaIn this work the lignocellulolytic enzymes produced by the edible mushroom Pleurotus ostreatus var. florida were studied. The objective was to know their relationship with the degradation of the biopolymers present in the cell wall of wheat-straw for the purpose of explaining their influence on the production and quality characters of the fruiting bodies. The following enzymatic activities were studied both in solid and submerged culture: Ligninases (Lignin Peroxidase, Manganese Peroxidase (MnP) and Laccase), Cellulases (Glucohydrolases, Glucosidases) and Hemicellulases from the group Arabinofuran- Xylanases (Xylanase, Xilosidase, Glucoronidase, Arabinofuran-Oxidase and Acetylesterase), cooperating enzymes (Glyoxal Oxidase) and feedback enzymes (Glucose Oxidase (GOD), Aryl Alcohol Oxidase (AAO), Tyrosinase (TYR), Veratryl Alcohol Oxydase (VAO), Cellobiose Dehydrogenase (CDH)). The first studies regarding all the mentioned enzymes were performed using the dikayon (N001) and the parental monokarion strains “fast” (PC9) and “slow” (PC15). The studies on all this whole group of enzymes, which are enough representative of the lignocellulolytic complex, let to conclude that (both in solid or submerged culture) the enzymes of major influence in colonizing the natural substrate and also those whose activity-determination better guarantees their further mapping were Laccases, MnP, AAO and TYR. Subsequently these four activities were measured in the monokaryon population being Laccases and MnP, those yielding the best levels in medium-7 (rich in nitrogen). In addition both enzymes allow the discrimination between “fast-” or “slow-” monokaryon strains both in solid medium with several dyes, or in liquid culture in agitation. The analysis of the enzymatic activities detected in the assayed conditions, in the population of “fast” or “slow” strains let to the observation that they map in different places where the loci corresponding to Laccase (pox) and mnp genes are located. These results open the possibility to design more precise studies that could help to establish a correlation between the contribution of the genes already described and the activity of the different ligninolytic enzymes. In addition the results will contribute to know whether in P. ostreatus genome there are new genes or if they correspond with locations that regulate these enzymatic activities, or it is a gene that has a role in the transport system or a kind of effector in the exportation machinery of the protein to the culture medium.Publication Open Access Expression of mating type genes in heterologous basidiomycetes(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Srivilai, Prayook; James, Timothy Y.; Vilgalys, Rytas; Chaiseana, Wassana; Kües, UrsulaMating processes in basidiomycetes are controlled by genes encoding two types of transcription factors (HD1 and HD2) and by genes encoding pheromones and pheromone receptors. For a successful mating reaction, an HD1 protein and an HD2 protein of different specificity have to interact and a pheromone with a pheromone receptor of different specificity. With now having cloned mating type loci from several different basidiomycetes, evolution of these loci and their genes can be addressed by sequence analysis as well as by transformation of genes into heterologous species. Transformation of cloned mating type genes into strains of the same species with different mating type genes can activate mating type controlled development. The A mating type genes of Coprinopsis scobicola and of Coprinellus disseminatus were found to be functional in Coprinopsis cinerea in combination with the endogenous A mating type genes. Moreover, B genes of C. disseminatus in C. cinerea cause peg formation subapical to septa with A-induced clamps cells and fusion of clamp cells with the subapical peg. In several C. cinerea monokaryons, transformed A genes of Schizophyllum commune were not observed to induce clamp cells by interaction with endogenous A genes. However, in crosses of C. cinerea transformants carrying compatible S. commune A genes, clamp cell production has occasionally been observed. To our surprise, when using S. commune A genes or the homologous b genes of Ustilago maydis to transform a specific C. cinerea monokaryon, colonies of transformants may develop faster growing sectors with hyphae having clamp cells. Our laboratory is supported by Deutsche Bundesstiftung Umwelt (DBU) and scholarships by the Mahasarakham University (to PS) and the Rajamangala Institute of Technology (to WC).Publication Open Access Molecular toolkit development for gene expression and gene silencing technologies in the homobasidiomycete Fungi Agaricus bisporus and Coprinus cinereus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Heneghan, M.N.; Burns, C.; Burton, K.S.; Challen, M.P.; Bailey, A.; Foster, G.D.We have developed a “Molecular Toolkit” comprising interchangeable promoters and marker genes to facilitate transformation of homobasidiomycete mushrooms and subsequent analysis of gene expression. We will describe the testing of a wide range of promoters in both Agaricus bisporus and Coprinus cinereus when linked to a range of selectable and visual marker genes, along with the parameters required to successfully achieve foreign gene expression within these organisms. It has been previously demonstrated that a prerequisite for GFP expression in A. bisporus and C. cinereus is an intron. We describe the construction of an expression vector containing a multiple cloning site linked to an intron thus allowing different genes to be easily expressed in A. bisporus and C. cinereus. We report on the development of gene silencing technologies within A. bisporus and C. cinereus. In particular the serine protease has been targeted for gene silencing in A. bisporus. Serine protease has been implicated in post-harvest and age-related senescence of sporophores. On harvesting, mushrooms degenerate rapidly to give browned caps and loss of texture in the fruit body, and such problems can dramatically reduce sale ability of the mushrooms. Suppression of genes involved in these pathways could increase mushroom shelf-life and profitability for mushroom growers, or help to further elucidate the complex biochemical pathways involved in post-harvest degradation. Progress will also be reported on gene silencing in C. cinereus.Publication Open Access A novel thaumatin-like protein-encoding gene from Lentinula edodes, Tlg1, is involved in lentinan degradation during post-harvest preservation(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Sakamoto, Y.; Nagai, M.; Sato, T.Lentinan, which is a β-1, 3-linked-D-glucan with β-1, 6 branches isolated as anti-tumor active-substance from Lentinula edodes, is purified from fresh fruiting bodies and marketed for clinical use. However, it is known that lentinan content decreases during post-harvest preservation as a result of increased β-1, 3-glucanase activity. We isolated two exo-glucanase encoding genes, exg1 and exg2 from L. edodes. Transcription level of the exg1 and exg2 gene was higher in the stipe than in the pileus of young fruiting bodies. This suggests that the exg1 and exg2 are involved in stipe elongation in L. edodes. We also isolated one endo-glucanase encoding gene, tlg1, from L. edodes. The tlg1 gene had 1.0 kbp cDNA length, and encoded protein was estimated as M.W. of 25 kDa and pI value of 3.48. Putative amino acid sequence of the tlg1 displayed 43% identity to thaumatin-like (TL) proteins from Arabidopsis thaliana. TLG1 had 16 cysteins conserved in TL-proteins. TL-protein is pathogen related protein 5 in plant, and several TL-protein had endo-glucanase activity. Previously, it is considered that TL-protein is unique in plant, however, this research and recent genome sequence project revealed that similar sequences to TL-proteins are conserved in filamentous fungi. We measured β-1, 3-glucanase activity of L. edodes fruiting bodies after harvesting by somogyi-melson method using laminarin as a substrate, and endo-β-1, 3- glucanase activity by using AZCL-pachyman as a substrate. These revealed that glucanase activity increased during post-harvest preservation. Transcription level of the exg1 gene decreased, but the exg2 and tlg1 genes increased during post-harvest preservation. Western blot analysis showed that EXG2 and TLG1 expression increased after harvesting. Purified EXG1 did not degrade lentinan, but EXG2 and TLG1 degraded lentinan, therefore, we concluded that the exg2 and tlg1 genes are involved in lentinan degradation during post-harvest preservation.Publication Open Access Virus related symptoms in crops of the button mushroom Agaricus bisporus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Sonnenberg, Anton S.M.; Lavrijssen, BrianSince more than five years symptoms are seen in Dutch mushroom crops that correlate with the presence of a set of dsRNAs. These dsRNAs are indicative for the presence of viruses. Symptoms are discoloration of white mushrooms varying from cream to brown. Crops can be heavily affected with more than 50% of the mushrooms showing discoloration. Mostly, however, crops show mild symptoms with a few percentages of mushrooms showing an off-white color. Since traiders associate this discoloration with low quality economical damage can be substantial for individual farmers. In affected crops up to 15 dsRNAs are observed with varying intensity. Only the five shortest dsRNAs show a perfect quantitative and qualitative correlation with the symptom. They are always present in discolored mushrooms and the intensity of the bands seen in agarose gels vary with the intensity of the discoloration. Most of the other dsRNAs are also present in affected crops but are also seen in crops without any visual symptoms. We have started a molecular analysis of the dsRNAs in order to design sensitive tests for each dsRNA. This will reveal if all or some dsRNAs are always present in mushrooms or are derived from an unknown infectious source. From a number of dsRNAs 60 to 80% of the sequence has been determined. RT-PCR tests have been designed recently for these dsRNAs and the first result of tests of several crops will be presented.Publication Open Access Rat cytochrome P450-mediated transformation of dichlorodibenzo-Pdioxins by recombinant white-rot basidiomycete Coriolus hirsutus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Shishido, KazuoRat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5’-portion (224-bp of 1st exon-8th base of 4th exon)-rat cyp1a1 cDNALentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1+arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg-, Leu-), obtaining three good Arg+ transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8 (at a ratio of 1:1)-dichlorodibenzo-pdioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.Publication Open Access Verticillium fungicola cell wall glucogactomannan-binding of the lectin from the Pleurotus ostreatus fruit bodies(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Bernardo, D.; Pérez Cabo, A.; García Mendoza, C.The Verticillium fungicola mycoparasitism on Agaricus bisporus fruit bodies appears to be a complex process made up of successive steps in which the recognition and binding between complementary molecules, the A. bisporus fruit body lectin and the V. fungicola cell wall glucogalactomannan, have recently been demonstrated. P. ostreatus fruit bodies have been described as containing a lectin and also presenting the “dry bubble” or the Verticillium disease. The aim of the present work is to purify and characterize the P. ostreatus lectin and compare the properties of both lectins in an attempt to confirm if the specific glucogalactomannan-lectin recognition and binding is the necessary step for the V. fungicola mycoparasitism process in P. ostreatus. The characteristics and properties of the purified P. ostreatus lectin together with those also previously described by us on A. bisporus lectin show that, although both lectins present different chemical structures, they behave very similarly in relation to their glucogalactomannan-binding, thus confirming the existence of the specific recognition and binding step in the Verticillium disease on P. ostreatus fruit bodies.Publication Open Access Development of a sporeless strain of oyster mushroom (Pleurotus ostreatus)(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Baars, Johan; Sonnenberg, Anton S.M.; Hendrickx, PatrickThe enormous amounts of spores produced by oyster mushroom (Pleurotus ostreatus) cause lung-related health problems among employees working in oyster mushroom cultivation. If sporeless varieties are used for large-scale cultivation, these lung problems can be avoided. For development of a commercially attractive strain of sporeless oyster mushroom, strain ATCC 58937, a sporeless strain of oyster mushroom was used as a donor of the trait. Microscopic analysis of basidia showed that meiosis was aborted at an early stage. Both nuclear types that constitute strain ATCC 58937 could be retrieved by protoplasting. Protoplasting commercial strain HK35 yielded only one of its nuclear types. Crosses between the ATCC nuclear types and the HK35 nuclear types (either directly or using the Buller phenomenon) yielded normal sporulating strains, indicating that sporelessness was caused by a recessive trait. Among the offspring of crosses between the ATCC nuclear types and the HK35 nuclear types the sporeless trait segregated in a 1 to 1 ratio. The sporeless trait could be mapped and strongly linked genomic markers were developed. The breeding strategy was to successfully introduce sporelessness into both nuclear types of a commercial variety, to achieve a sporeless variety. In a first cross between a sporeless culture and a commercial strain, not only sporelessness was transferred to the commercial variety. Therefore, repeatedly backcrossing the progeny of the first cross with the commercial variety is used to try to restore the original genetic material from the commercial strain as much as possible. Performance of a number of “prototypes” of a sporeless oyster mushroom was tested on commercial mushrooms farms and proved to be satisfactory.