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Open Access
Molecular characterization of A cellobiohydrolase gene family in the fungus Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Eizmendi Goikoetxea, María Arantzazu; Sannia, Giovanni; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Cellulose 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.
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 Ekoizpena
The 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).
Open Access
Formation of hyphal loops in xylotrophic coprinoid mushrooms
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Badalyan, Suzanna M.; Kües, Ursula
Recent 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.
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 Ekoizpena
Copper 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.
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)).
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.
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.
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, Kazuo
We 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.
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.
Open Access
Ectopic expression of constitutively activated small GTPase cdc42 alters the morphology of haploid and dikaryotic hyphae in the filamentous homobasidiomycete Schizophyllum commune
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Raudaskoski, M.; Salo, V.; Weber, M.; Uuskallio, M.
Cloning of the Cdc42 gene from Schizophyllum commune enabled investigation of the role of ScCdc42 in the regulation of vegetative growth and sexual reproduction in this fungus, which has a well-characterized hyphal cell structure, cytoskeleton and mating system. Ectopic expression of the constitutively active Sccdc42G12V or Sccdc42Q61L allele from native or inducible ScCel1 promoters had dramatic effects on hyphal morphology, cytoskeletal structure and Cdc42 localization, while ectopic over-expression of the wt or the dominant negative ScCdc42D118A allele had no detectable effect. For transformants with constitutively active Sccdc42 tip growth of apical cells in the leading hyphae was normal but polar tip growth in side branches was altered implying different regulation of polarity establishment in the two groups of apical cells. The S. commune genome also contains a gene encoding RacGTPase. Rac1 might regulate the polarized growth of leading hyphae while ScCdc42 regulates the development of side branches in S. commune. In transformants with constitutively active Sccdc42 branch emergence at exceptional sites and isotropic growth next to the septum proved that the branch site selection and subsequent hyphal development are under ScCdc42 control. Poor dikaryotization along with irregular clamp connections in mates with Sccdc42G12V or Sccdc42Q61L allele suggested that Cdc42 also contributes to efficient mating in S. commune. Our results provide strong evidence that Cdc42 is involved in the control of hyphal morphogenesis in filamentous homobasidiomycetes.
Open Access
The Coprinus cinereus genome project
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Gathman, A.; Lilly, W.; Stajich, Jason E.; Carlson, M.; Murphy, B.; Smith, A.; Fargo, D.; Dietrich, F.; Pukkila, P.J.
Coprinus cinereus is an increasingly attractive basidiomycete model system. Its genome has been sequenced and is publicly available; it is readily cultured in the laboratory on defined media, it has highly synchronous meiosis, and numerous laboratory techniques have been adapted for use with it. The 10X shotgun sequence released by the Whitehead Institute comprises 36 Mb of the 37 Mb of the genome, which have been assembled into 106 supercontigs containing 431 contigs. cDNA libraries have been constructed from two meiotic stages, and 1432 candidate genes have been identified from them. Another set of cDNA libraries has been constructed from vegetative Coprinus cinereus Okayama 7 grown under different environmental conditions, including heat shock, rapamycin treatment, minimal medium, rich medium, and complex carbon and nitrogen sources. 5000 ESTs are being sequenced from these libraries. The EST sequences have been aligned with the genomic sequence, as have known C. cinereus genes from GenBank. Data from known ascomycete gene sequences have been used to train SNAP software to predict a total of 11,340 genes from the remainder of the genome. BlastX and Pfam have been used to assign tentative functions to predicted genes as well as ESTs. tRNA genes have also been identified in the genome. All genomic information is available online via our Gbrowse server.
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, Ursula
Mating 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).
Open Access
Study of two acidic proteinases, probably involved in the dimorphism and pathogenicity of Ustilago maydis, basidiomycete of the corn smut disease
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Noriega Reyes, M.Y.; Miramón Martínez, P.; Mercado Flores, Y.; Ramírez Zavala, B.; Hernández Rodríguez, C.; Villa Tanaca, L.
Ustilago maydis is a dimorphic phythopathogenic fungus and the causal agent of the corn smut disease. In this work, the purification and biochemical characterization of the acid proteinases pumAe (extracellular) and pumAi (intracellular) of U. maydis were performed. Also, identity of the gene that encodes for pumAi (PRAum) was explored in the genome of the fungus. The proteases were purified and biochemically characterized. The molecular masses of pumAe and pumAi were 72 and 35.3 kDa respectively. The optimal pH of activity of proteinases was 4.0. The pumAe Km value was of 3.5 μM and a Vmax of 11430 μmol h-1 mg-1 when Suc-R-P-F-H-L-L-V-Y-MCA was used as substrate. The protease pumAi was inhibited by pepstatine A. Yeast-tomycelium transition was inhibited by Pepstatine A in the culture medium. The hypothetical gene that encodes for protease pumAi (PRAum gene) was located in the U. maydis genome project and was amplified by PCR and cloned into TOPO-TA 2.1 plasmid and pNMT-1, a Schizosaccharomyces pombe expression vector. In the U. maydis genome one copy of the gene by Southern blot analyses was detected. In brief, the expression of this gene (PRAum), performed by RT-PCR assays, was regulated by the source of nitrogen. The heterologous expression experiments in S. pombe allowed a fast purification and confirmed that pumAi enzymatic activity was encoded by PRAum gene.
Open Access
QTL mapping of pathogenicity in Heterobasidion annosum sensu lato
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Olson, Ake; Lind, Mårten; Stenlid, Jan
Heterobasidion annosum (Fr.) Bref. sensu lato, casual agent of annosum root rot in conifers, is the economical most devastating forest pathogen in the northern hemisphere. A genetic linkage map of H. annosum, was constructed from a compatible mating between isolates from the North American S and P intersterility groups. The linkage analysis of 358 AFLP markers in 102 progeny isolates generated 19 linkage groups containing 6 or more markers that covered 1468 cM. Two distinct methods were used to analyse the segregation of pathogenicity. 1 year old pine seedlings grown in the greenhouse were infected with H. annosum progeny isolates. Pathogenicity was measured as mean necrosis lengths caused in 10 pine plants. One QTL for pathogenicity was found on linkage group 15 with a LOD peak of 2.95, spanning a 31.2 cM large area. This QTL explained 14.3% of the variation. Another QTL were found in a small linkage group containing 5 markers and spanning 36.8 cM, with a LOD peak of 4.40 at marker paacts02. 19.1% of the variation could be explained by this QTL. The heritability of pathogenicity on pine was estimated to be 0.21 in this study. The disease increase rate values from an in vitro test were used as another estimate of pathogenicity and used for the QTL-analysis. From the in vitro pathogenicity test, a large area of 26.5 cM on linkage group 11 between the markers acgcs5 and paacgp08 contains a high QTL probability of LOD 3.09. This QTL explained 16.4% of the total variation in this experiment. The heritability of pathogenicity on pine was estimated to be 0.088 in this study. Successfully localization of the two intersterility genes (S and P) on the map were carried out through mating of the progeny isolates with three tester strains carrying known intersterility genotypes.
Open Access
Evidence for a 200 gene ribosome and rRNA biosynthesis (rrb) regulon in fungi
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Wade, Christopher; Umbarger, Mark; McAlear, Michael A.
Two challenges of the post genomic era are 1) the need to assign functions to as yet uncharacterized gene products, and 2) the requirement to understand how the expression profiles of large sets of genes are regulated in response to changing environments. Towards these aims, we have used transcriptional profiling analysis to identify and characterize a large set (over 200 genes) of transcriptionally co-regulated genes whose products are involved in rRNA and ribosome biosynthesis. Many of the genes within this set were previously unknown with regards to their function. This RRB regulon is distinct from the ribosomal protein (RP) regulon, and is characterized by a unique pair of conserved promoter motifs. The organization of the RRB regulon appears to be evolutionarily conserved at least from S. cerevisiae to S. pombe. The strategies used to identify and characterize this gene set can be widely used in other organisms to help fulfill the two needs outlined above.
Open Access
Ras module function is involved in regulation of sexual development Schizophyllum commune
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Schubert, D.; Kothe, E.
The white rot fungus Schizophyllum commune is used as a model to investigate sexual development in hymenomycetes. We isolated the gene gap1 encoding a GTPase-activating protein for Ras. Disruption of gap1 should therefore lead to strains accumulating Ras in its activated, GTP-bound state and to constitutive Ras signaling. Mating behavior was not altered in Δgap1 monokaryons whereas growth rate in Δgap1 monokaryons was reduced about 25%. Dikaryotic Δgap1/Δgap1 strains displayed 50% growth reduction. Hyphal growth was disturbed showing a wavy growth pattern. In dikaryons, clamp formation was severly disturbed as hook cells failed to fuse with the penultimate cell at the site that in wildtype cells is marked by a peg formed from the mother cell. Instead, the dikaryotic character of the hyphae was rescued by fusion of the hooks with nearby developing branches. The mating type genes of the B factors encoding a pheromone receptor system are known to be required for clamp cell fusion. A role for Ras in the same process in discussed. Fruitbody formation was observed in homozygous Δgap1/Δgap1 dikaryons which, however, formed increased numbers of fruit body primordia, whereas the amount of fruit bodies was not raised. Mature fruit bodies formed no or abnormal gills. No production of spores could be observed. Similar phenotypes in fruitbody development had been previously described for elevated intracellular cAMP levels. Thus, the signalling of Ras is discussed with respect to cAMP signalling.
Open Access
Genetic analysis of Coprinopsis cinerea mutants with defects in fruiting body development
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Srivilai, Prayook; Chaiseana, Wassana; Kües, Ursula
Few genes have so far been cloned and characterized in fruiting of the heterothallic mushroom Coprinopsis cinerea. Fruiting body development normally occurs on the dikaryon. However, the binucleate state of the mycelium hinders easy access of genes. Self-compatible mutants with defects in the mating type pathways can form fruiting bodies without prior fusion to another strain. Uninucleate haploid oidia of such mutants can easily be mutagenized and germinated mycelia tested for defects in fruiting. Mutants can be produced from oidia by classical techniques such as UV treatment or by modern REMI (restriction enzyme-mediated integration) mutagenesis via transformation. Such mutants of self-compatible strains have now been successfully appointed in cloning genes acting in sexual development. Co-isogenic strains of compatible mating types support in genetic characterisation of the mutants.
Open Access
Molecular characterisation and expression analysis of developmentally regulated genes in Agaricus bisporus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Sreenivasaprasad, S.; Molloy, S.; Fleming Archibald, C.; West, D.; Herman, B.; Eastwood, D.C.; Burton, K.S.; Henderson, J.
Analysis of cDNA transcripts, PCR based methods and genomic library screening have been used to clone and characterise developmentally regulated genes in the cultivated white button mushroom Agaricus bisporus. Up-regulated genes identified during the rapid expansion phase of the sporophore include sugar transporter gene sut1, putative riboflavin-aldehyde-formingenzyme gene (raf) and three novel morphogenes mag2-mag4. Further, a hexose transporter gene sut2 and lectin genes abl1 and abl2, among others have been cloned from A. bisporus using PCR based strategies. Northern analysis indicated their up-regulation during sporophore differentiation and development. Sugar transporter gene sut1 transcripts increased abundantly during sporophore development and although sut1 showed varying levels of homology to other sugar transporters, its substrate preference could not be identified based on homology. Interestingly, analysis of basidiomycete genome sequences revealed the presence of a putative sut1 homolog in the white rot fungus Phanerocheate chrysosporium. On the other hand, Ab sut2 showed strong homology to fungal glucose/hexose transporters and its homologs also appear to be present in A. bitorquis and Coprinus cinereus suggesting a generic role. Analysis of the genomic cosmid clones revealed that the lectin genes abl1 and abl2 are present in close proximity to each other and further characterisation is on-going.
Open Access
Expression of GFP and DsRed in the homobasidiomycete Schizophyllum commune
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Vinck, Arman; Wösten, Han A. B.; Lugones, Luis G.
Expression of GFP and DsRed was studied in the homobasidiomycete Schizophyllum commune. CGFP or sGFP fused to the SC3 promoter resulted in similar steady state mRNA levels. These levels were considerably lower than that of SC3. Despite the low mRNA levels, both GFP variants resulted in fluorescent hyphae. The sGFP expressing strains showed stronger fluorescence than the CGFP expressers. When CGFP was fused to the N-terminal or C-terminal part of the mature SC3 protein, no fluorescence was observed. However, accumulation of GFP
Open Access
Wild strains of Agaricus bisporus: a source of tolerance to dry bubble disease: resumen de póster
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Largeteau, M.L.; Baars, Johan; Regnault Roger, C.; Savoie, J.M.
Agaricus bisporus is susceptible to various pests and diseases. Dry bubble, caused by Verticillium fungicola, is currently the most serious disease and is distributed worldwide. All cultivars are susceptible and the pathogen develops resistance towards the very few fungicides admitted. Breeding for resistance is necessary and wild strains of A. bisporus are putative sources of tolerance. We present results on the susceptibility (severity of the disease, ability to develop the various symptoms) of some wild strains of the INRA-CTC and the PPO MRU collections. A commercial strain revealed significant variability in agressiveness among isolates of V. fungicola var. fungicola responsible for the disease in Europe at present. Isolate VCTC, which induced severe symptoms revealed interesting tolerance among five wild A. bisporus strains and hybrids between wild strains. A cross test was performed with two cultivars and seven wild stains of A. bisporus contaminated with five isolates, two of var. fungicola and three of var. aleophilum, the latter responsible for the disease in USA and Canada. The wild strains screened were far more tolerant (3-9% of diseased mushrooms) than the cultivars (20-22%). All the strains were more susceptible to the var. aleophilum than to the var. fungicola isolates. These experiments showed that very tolerant material exists in collection and can be used as parents to breed for resistance. The greater susceptibility of A. bisporus to V. fungicola var. aleophilum must be taken into consideration in breeding programmes, this variety being present in North America and being isolated in Europe in the past.