Congresos 2011 y anteriores

Permanent URI for this community

http://hdl.handle.net/2454/27011

Browse

Now showing 1 - 20 of 124

Open Access
Overexpression of laccases in Coprinopsis cinerea
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Kilaru, S.; Rühl, M.; Saathoff, A.; Dwivedi, R.C.; Zomorrodi, M.; Lange, K.; Majcherczyk, A.; Hoegger, P.J.; Kües, Ursula
Laccases are versatile redox-enzymes that oxidize various phenolic compounds and aromatic amines. Because of the broad substrate specificity, these enzymes are of interest for many different biotechnological applications. White-rot fungi are the major source of laccases. Although basidiomycete species give rise to relatively high enzyme yields, these might not be optimal for applications. Basidiomycetous laccases have been reported to show hyper- or hypo-glycosylation when expressed in ascomycetes. In consequence, enzyme characteristics were found to be altered. Therefore, we use the basidiomycete Coprinopsis cinerea as an organism for laccase overproduction. We present a vector system for easy and rapid cloning of promoters and/or genes of interest and show that such constructs can be functional in laccase production.
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
Mushroom breeding program in Iran
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Gordan, H.R.; Farsi, M.
Mushroom cultivation is a newly established, but growing industry in Iran. There are about 120 producers with a total of about 20,000 tones per year in the country. Agaricus bisporus consists of more than 85 percents of the total production of all mushrooms produced in Iran. Its yield average is about 12-15 kg/m2, while in the global production it is about 27-30 kg/m2. This is mainly due to using strains of genetically weak performance. Since ten years ago a breeding program was started with emphasis on breeding high yielding strains in Mashhad. The short-term effort consisted of selection among single spore isolates and multispores cultures with a better performance in yield. The long-term effort consisted of employing of heterosis in hybrid strains. To reach the aim, more than 350 homokaryone isolates were selected through RAPD markers followed by yield trials from commercial and domestic strains, and crosses were made in many combinations using diallel method. Selection among spores of commercial strains could somehow recover their potential genetic capacity, so that an average of 22 Kg/m2 was recorded in the selected strains. Using growth type as a marker, it was possible to decrease the number of isolates in final stages of selection for homokaryones in solid medium or spawn. Cluster analysis based on average of band numbers emerged by RAPD markers, could separate homokaryotic and heterokaryotic isolates in two distinct groups. Some hybrids showed a better mycelia growth and a considerable higher yield than their parents. Efforts are now being made to collect wild strains of Agaricus bisporus in Iran and evaluating them for desirable genes including resistance to pest and diseases.
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
Characterisation of Agaricus bisporus response genes to Verticillium fungicola infection
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Costa, A.; Thomas, D.J.I.; Bailey, A.; Foster, G.D.; Challen, M.P.; Mills, P.R.
The mycoparasite Verticillium fungicola is a persistent threat to the cultivation of the mushroom Agaricus bisporus. Mushroom “dry bubble” is characterised by an undifferentiated mass of cells and can result in major crop losses. During the establishment of “dry bubble” substantial changes occur in the biochemistry and physiology of both partners. To enable new insights to be made into the molecular events underlying the disease, work is in progress to identify genes expressed during pathogen infection. Subtractive Suppressive Hybridisation (SSH) has enabled recovery of 65 expressed sequenced tags (ESTs) differentially expressed during infection. After database searches 27 of the genes were identified as most likely from V. fungicola, 25 from A. bisporus and 13 unknown. Bioinformatic analysis suggested that the response genes identified were involved in a range of biological functions that included stress, signalling, protein synthesis and cell wall structure and function. Specific full-length genes will be recovered using cDNA library constructed from lesions of A. bisporus infected with V. fungicola, enabling silencing approaches to be used to further investigate the role of the identified genes in disease. An alternative higher-throughput method of gene function analysis, RNA interference (RNAi) using A. bisporus model genes (URA3, CBX), is also being developed. Silencing constructs expressing RNAi hairpin were transformed into A. bisporus using Agrobacterium tumefaciens and hygromycin resistance. Screening of the transformants by PCR confirmed integration of the silencing construct in 24 transformants. RT-PCR is being used to confirm transcription of the RNAi hairpin. Quantitative PCR will be used to analyse levels of target gene transcripts post RNAi transformation. The role of A. bisporus genes identified, in the infection process, will be determined through infection trails with A. bisporus silenced lines.
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.
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
Monstruosities under the inkcap mushrooms
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Navarro González, Mónica; Domingo Martínez, A.; Navarro González, S. S.; Beutelmann, P.; Kües, Ursula
Four different Inkcaps were isolated from horse dung and tested for growth on different medium. In addition to normal-shaped mushrooms, three of the isolates formed fruiting body-like structures resembling the anamorphs of Rhacophyllus lilaceus, a species originally believed to be asexual. Teleomorphs of this species were later found and are known as Coprinus clastophyllus, respectively Coprinopsis clastophylla. The fourth of our isolates also forms mushrooms but most of them are of crippled shape. Well-shaped umbrella-like mushrooms assigns this Inkcap to the clade Coprinellus. ITS sequencing confirmed that the first three strains and the Rhacophyllus type strain belong to the genus Coprinopsis and that the fourth isolate belongs to the genus Coprinellus.
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
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
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
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
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
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
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
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
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
Agaricus devoniensis complex comprises a group of heterothallic isolates constituting a basis for breeding
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Callac, P.; Spataro, C.; Lataillade, E.; Blasi, P.; Guinberteau, J.
A recent phylogenetic reconstruction of Agaricus section Duploannulati revealed that A. devoniensis and A. subfloccosus are two complexes of species close to A. bisporus. The A. subfloccosus complex comprises two homothallic entities, while the A. devoniensis complex was never studied until now. A sample of 26 isolates, some being unreliably determined, were examined to (i) confirm their identity using a PCR-RFLP marker revealing a characteristic A. devoniensis ITS polymorphism, and (ii) for their ability to fruit in standard conditions used for A. bisporus cultivation. Twenty one isolates were confirmed as A. devoniensis, and only two collections from USA were unable to fruit. The five remaining isolates were excluded from the complex and were unable to fruit; their ITS1+2 regions were sequenced and alignments indicated that four of them were similar to A. campestris and that one belonged to a new entity close to A. bitorquis and A. cappellianus. For the 19 fructifying isolates of the complex, we attempted intrastock and interstock mating tests with single spore isolates: for three isolates, we did not get spore germination; and for seven isolates, we observed partial to complete intersterility between strains. The nine remaining isolates exhibited a unifactorial system of sexual incompatibility for which eight different mating type alleles were detected. Within this group, the heterothallic and presumably interfertile isolates differed in their origin (Greece, France), their habitat (dune, coniferous trees), and their morphology (mean spore length: 5.6 to 6.6 μm); they constitute a diversified genetic basis usable to select smooth white and attractive cultivars for this tasteful edible and cultivable species.
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.