VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI)

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Open Access
Selection of Pleurotus ostreatus strains in a genetic breeding program
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Idareta Olagüe, Eneko; Jurado Cabanillas, Javier; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The basidiomycete Pleurotus ostreatus, commonly known as oyster mushroom, is the second largest edible mushroom crop behind the white button mushroom, Agaricus bisporus. It accounts for nearly one-quarter of the total worldwide mushroom production. Furthermore, P. ostreatus has a high industrial interest because it is a good source of enzymes and other products with biotechnological, industrial and medical applications, it is easy to cultivate and because of its good organoleptic characteristics. Since of 2003, our group research has carried out genetic breeding programs based on the determination of QTLs controlling production and quality in industrial cultures of this fungus. In this breeding program the first test consisted in putting under fructification conditions 130 strains obtained from the crossing of protoclon PC21 (P. ostreatus var. ostreatus wild strain) by a collection of monokarions derived from N001 (P. ostreatus var. florida commercial strain). For this purpose, 2 kg (3 repetitions per strain) bags of industrial sustrate were inoculated and cultivated at 21ºC. Mature fruiting bodies were collected and weighted daily during the fructification period. The second test was made using the six strains that performed the better in Test1, but were cultivated at 18ºC and with 15 repetitions per strain were performed. From this test, three strains were selected and used in Test3. In this test, other three strains obtained from the crossing between monokarions descending of N001 and selectioned for their high growth rate were introduced. In this test the weight of the bags was increased to 5 kg and the cultures were cultivated at 18ºC. The strains obtained from PC21 have good charactericts for mushroom size, with similar behaviour for yield and precocity. The strains obtained from the crosses between N001 descendants have better mushroom size and similar yield and precocity than N001, then breeding was obtained. The candidate strains for next tests are PC21xMA046 and PC21xMA027 for their high yield and the mushroom good features.
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
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 Ekoizpena
In 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.
Open Access
Mapping the Pleurotus ostreatus genome
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Castellón Gadea, Jordi; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus is a commercially important edible mushroom commonly known as oyster mushroom which has also important biotechnical applications. Industrial production of P.ostreatus is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. In a previous study a linkage map of P. ostreatus strain N001 was constructed, which provided a basis for performing an efficient QTL (Quantitative trait loci) analysis based in a population of 80 sibling monokaryons. The map is based on the segregation of RAPD markers, RFLP markers, phenotypic characters and cloned genes. Nevertheless the linkage map is just a first step towards the selection of the appropiate parentals for new breeds. In order to organize and improve the access to the data and information accumulated in the previous works mentioned above, a Microsoft® Excel Linkage Map Matrix (MELMM) was designed and created. On this linkage map matrix we could have an easy and functional view of the P. ostreatus linkage map data, such as, recombination frequencies, genotypes information and degree of similarity between monokaryons that will help us in the design of breeding crosses aimed at improving QTLs of agronomic interest of new commercial strains.
Open Access
Computational prediction of protein-coding gene and annotation of DNA sequences with agronomic interest in Pleurotus ostreatus (Oyster Mushroom)
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Palma Dovis, Leopoldo; Peñas Parrila, María Manuela; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus, commonly known as oyster mushroom, is a commercially important edible fungus with interesting biotechnological properties. Quantitative trait loci (QTL) analyses are rare in fungi and little is known about their number, position, and genetic structure. Previous studies of our group have allowed the construction of a genetic linkage map of P. ostreatus var. florida, which has provided the basis for performing an efficient QTL analysis. In fact, there is a region of the chromosome VII of P. ostreatus where the most QTLs related to the production and precocity characters have been mapped. These quantitative traits are presumably under the control of a polygenic genetic system and could be associated with some chromosomal regions. The hypothesis of this work is that there is a region in the chromosome VII of protoclon PC15 (monokaryotic parental of the N001 dikaryotic strain) where exist genes which are responsible for the QTLs mentioned above. In order to test this hypothesis, we are developing a molecular QTL analysis through the sequencing of a region with an approximated size of 320 Kbp in chromosome VII (protoclon PC15). For this purpose, a BAC genomic library was constructed and two BAC clones spanning the region of interest are being sequenced. To carry out an efficient computational prediction of protein-coding genes and its annotation on the partial sequences obtained up to date, we have used different Internet resources such as BLASTx, BLASTp, BLASTn, and FGENESH trained on some basidiomycetes genomic data like Phanerochaete chrysosporium and Cryptococcus neoformans (SoftBerry). To our knowledge, this is the firs molecular QTL analysis performed on this edible mushroom.
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.
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
Nutritional value of protein from vegetative mycelia of edible mushroom Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Parada Albarracín, Julián Andrés; Urdaneta, Elena; Marzo Pérez, Florencio; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
The present work was designed to study the effects of supplementation a control diet with P. ostreatus mycelium for evaluation a nutritional value of mycoprotein and possible cholesterol lowering.
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.
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, Andrea
FTIR 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.
Open Access
Secondary structure of the ribosomal PreRNA ITS2 region as a tool in studies of fungal diversity and phylogeny
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Juuti, J.T.; Salo, V.
The ribosomal RNA gene, and particularly its 18S and 25S sequences, have proven valuable in large-scale phylogenetic analyses. The ITS region of the ribosomal RNA gene, instead, has not been regarded so useful in this respect. The main problem has been that these sequences show significant sequence and length variation and that they have been more or less unalignable beyond small closely related groups. We have determined the common secondary structure of the ITS2 region and used it to align the sequences over the whole fungal kingdom. Significant properties of this structure include a central ring structure and three or four conserved loops – the presence of the ring being the most conserved feature. The core structure has also revealed the most conserved sites that are usable in kingdom-wide phylogenetic analyses. Surprisingly, the tree that is calculated with only the 5.8S ribosomal RNA and the conserved ITS2 sites has a very high correlation with the Fungal Tree of Life that has been calculated with four markers and much longer sequences. Furthermore, finding of the ITS2 secondary structure has revealed a number of group specific sequence signatures and structural RNA elements that can be used for more detailed analyses of different subgroups and their phylogeny. Currently we are (mainly) examining the variation of the loop structures among the basidiomycetes and linking that to the taxonomy of fungi. Some examples of current findings will be shown.
Open Access
Copper in fruiting body development of Coprinus cinereus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Navarro González, Mónica; Kilaru, S.; Majcherczyk, A.; Kües, Ursula
The model homobasidiomycete Coprinopsis cinerea grows best at 37°C, but, normally, it produces fruiting bodies only at moderate temperatures around 25-28°C. Light is needed to induce fruiting and also for fruiting body maturation. Cultures kept after fruiting induction predominantly in the dark form structures with an extended stipe and an underdeveloped cap (so-called “etiolated stipes”). In a day/night rhythm, caps develop further, basidia are formed, in which karyogamy and meiosis occurs and of which the basidiospores bud off. Besides light, fruiting body development in basidiomycetes has been repeatedly linked to enzymes belonging to the group of phenoloxidases, in particular the multi-copper containing laccases. However, their roles in fruiting remain unclear. In attempts to induce laccase production in liquid standing cultures at 37°C, to our surprise we found unusual inititation of fruiting body development. However, the abundantly formed primordia did never develop into mature fruiting bodies but into large-sized etiolated stipes, both in dark and in light. Laccase under these conditions was not detected in the medium but bound to the fruiting initiating mycelium. Moreover, enzyme production and etiolated stipe formation correlated with an increase from pH 5.5 to a slightly alkaline pH. Ammonium was found to be produced and nitrate reductase activity has enzymatically been shown. Under normal fruiting conditions, addition of copper to cultures enhances fruiting initiation in time and number. To further unravel the potential involvement of laccases in fruiting as well as of proteins influencing ammonia secretion, we are studying expression of corresponding genes during vegetative growth and fruiting body development. Work in our laboratory is supported by DBU (Deutsche Bundesstiftung Umwelt). MNG holds a CONACYT (Mexico) PhD studentship.
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
Mitogenic activated protein kinase Kpp6 signaling in the phytopathogenic Fungus Ustilago maydis: identification of downstream elements
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Mendoza Mendoza, Artemio; Vranes, A.; Muller, P.; Schirawski, J.; Kahmann, R.
Pathogenicity of U.maydis is dependent on its ability to mate. Mating requires active cAMP-PKA and MAP kinase cascades and except for the signaling inputs the downstream components are also required during pathogenesis. In addition a MAP kinase, called Kpp6 was described, that works in pathogenicty but not in mating. Kpp6 displays high similarity to Kpp2, the MAP kinase that works in mating. kpp6 mutants were morphologically indistinguishable from wild type but were unable to induce anthocyanin production and were unable to penetrate into the plant, despite the fact that they did produce appressoria. In this work we are studying the downstream elements of Kpp6 and determine their function during the penetration process. To obtain this information we used microarray assays with RNA from Ustilago growing on plant surface. We identified 29 genes which are reduced in expression in Kpp6 mutant with respect the wild type strain. We generated knockout strains for some of these genes in compatible strains of U. maydis and analyzed the phenotypes in pathogenicity. Our preliminary results suggest that Kpp6 regulates the penetration of Ustilago maydis at the stage where lytic enzymes expression is no longer required.
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
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
Molecular analysis of aminopeptidase pumAPE from Ustilago maydis encoded by APEum gene: enzyme purification and differential expression
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Miramón Martínez, P.; Noriega Reyes, M.Y.; Mercado Flores, Y.; Ramírez Zavala, B.; Hernández Rodríguez, C.; Villa Tanaca, L.
Heterobasidiomycete Ustilago maydis is a dimorphic phytopathogenic fungus, causal agent of corn smut, a widespread disease. Recently, proteolytic system of this fungus was described and an aminopeptidase activity, probably involved in pathogenicity, was detected. The aminopeptidase pumAPE was purified from the haploid phase of U. maydis FB1 strain. The purification procedure consisted of ammonium sulphate fractionation and three chromatographic steps, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE respectively. Enzymatic activity was optimal at pH 7.0 and 35 ºC toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10-phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase exhibited a higher specificity for substrates with lysine and arginine in the N-position. The Km value was 54.4 μM and the Vmax value was 408 μmol min-1 mg-1 for Lys-pNA. A pair of primers was designed in order to amplify the gene APEum encoding this activity. In order to determine the number of copies in the genome, a APEum gene fragment was used as probe in a Southern blot. Only one copy of the gene by genome was detected. Also, differential expression of APEum was assessed under different physiological conditions. In brief, high expression levels were detected on media supplemented with corn infusion, proline, and ammonium.
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 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
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
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.