Person: Ramírez Nasto, Lucía
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Ramírez Nasto
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Lucía
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Agronomía, Biotecnología y Alimentación
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IMAB. Research Institute for Multidisciplinary Applied Biology
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0000-0002-0023-4240
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425
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Publication Open Access Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences(Springer, 2024) Nakazawa, Takehito; Kawauchi, Moriyuki; Otsuka, Yuitsu; Han, Junxian; Koshi, Daishiro; Schiphof, Kim; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Honda, Yoichi; Institute for Multidisciplinary Research in Applied Biology - IMABPleurotus ostreatus, also known as the oyster mushroom, is a popular edible mushroom cultivated worldwide. This review aims to survey recent progress in the molecular genetics of this fungus and demonstrate its potential as a model mushroom for future research. The development of modern molecular genetic techniques and genome sequencing technologies has resulted in breakthroughs in mushroom science. With efficient transformation protocols and multiple selection markers, a powerful toolbox, including techniques such as gene knockout and genome editing, has been developed, and numerous new findings are accumulating in P. ostreatus. These include molecular mechanisms of wood component degradation, sexual development, protein secretion systems, and cell wall structure. Furthermore, these techniques enable the identification of new horizons in enzymology, biochemistry, cell biology, and material science through protein engineering, fluorescence microscopy, and molecular breeding.Publication Open Access Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus(American Society for Microbiology, 2002) Larraya Reta, Luis María; Idareta Olagüe, Eneko; Arana, Dani; Ritter, Enrique; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaMycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.Publication 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 ZientziakThe 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.Publication Open Access In silico analysis of the expression profile of AA9 Lytic Polysaccharide Monooxygenases (LPMOs) and the CDH Cellobiose Dehydrogenase enzyme in wood-degrader Agaricomycetes. The Pleurotus ostreatus case(Elsevier, 2024-08-22) Jiménez Miguel, Idoia; Roscales, Gabriel; Garde Sagardoy, Edurne; Chuina Tomazeli, Emilia; Honda, Yoichi; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Barry, Kerrie; Grigoriev, Igor V.; Ramírez L.; Ramírez Nasto, Lucía; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaLignocellulose, the Earth's most abundant biopolymer, is degraded by wood-decaying fungi, specifically white rot fungi (WRF) and brown rot fungi (BRF), which use different strategies. This study examines the expression profiles of the AA9 and CDH enzymes of three WRF species (Heterobasidion annosum, Phanerochaete chrysosporium, and Pleurotus ostreatus) and two BRF species (Fomitopsis pinicola and Rhodonia placenta) from the Agaricomycetes class, grown on poplar wood or glucose as the sole carbon source. Mycelia were collected between days 10 and 12, revealing distinct lignocellulose degradation strategies between WRF and BRF, evidenced by the upregulation of AA9 LPMO (lytic polysaccharide monooxygenases) and AA3_1 (Cellobiose Dehydrogenase) genes, with the co-occurrence of both types of transcripts at the time of mycelial collection. The genome analysis showed variability in the number of AA9LPMO genes between WRF and BRF, which were differentially regulated depending on the carbon source. WRF exhibited a significant upregulation of AA9 LPMO genes,. In Phanerochaete chrysosporium, only one AA9LPMO gene was homologous to Pleurotus ostreatus, which had the highest number of AA9LPMO genes among the WRF studied. Some AA9 LPMO genes in Pleurotus ostreatus were associated to transposable elements (TEs, mainly footprints of LTRs) and grouped in clustered. LTRs were found either in the flanking or within the gene coding regions with no effect on gene transcription. In silico analysis of the AA9LPMO proteins in WRF uncovered distinct features at their C-terminal ends. Most of them lacked an appended module, but those with a CBM1 were highly induced in poplar wood media. The proportion of AA9 proteins with a CBM1 module was similar in Phanerochaete chrysosporium and Heterobasidion irregulare, but lower in Pleurotus ostreatus, which contained more AA9LPMO genes overall. In Pleurotus ostreatus, AA9LPMO proteins were grouped into three clades based on their C oxidizing type, with each clade containing proteins with specific features. The abundance (redundancy) of AA9LPMO genes in WRF especially associated to footprints LTRs in Pleurotus ostreatus suggests these genes may have other roles beyond lignocellulose degradation.Publication Open Access Enzymatic characterization of a monokaryon population of the edible mushroom, Pleurotus ostreatus with a view to genetic improvement(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Terrón, María del Carmen; López, María; Carbajo, José M.; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; González Aldo, E.; Producción Agraria; Nekazaritza EkoizpenaIn this work the lignocellulolytic enzymes produced by the edible mushroom Pleurotus ostreatus var. florida were studied. The objective was to know their relationship with the degradation of the biopolymers present in the cell wall of wheat-straw for the purpose of explaining their influence on the production and quality characters of the fruiting bodies. The following enzymatic activities were studied both in solid and submerged culture: Ligninases (Lignin Peroxidase, Manganese Peroxidase (MnP) and Laccase), Cellulases (Glucohydrolases, Glucosidases) and Hemicellulases from the group Arabinofuran- Xylanases (Xylanase, Xilosidase, Glucoronidase, Arabinofuran-Oxidase and Acetylesterase), cooperating enzymes (Glyoxal Oxidase) and feedback enzymes (Glucose Oxidase (GOD), Aryl Alcohol Oxidase (AAO), Tyrosinase (TYR), Veratryl Alcohol Oxydase (VAO), Cellobiose Dehydrogenase (CDH)). The first studies regarding all the mentioned enzymes were performed using the dikayon (N001) and the parental monokarion strains “fast” (PC9) and “slow” (PC15). The studies on all this whole group of enzymes, which are enough representative of the lignocellulolytic complex, let to conclude that (both in solid or submerged culture) the enzymes of major influence in colonizing the natural substrate and also those whose activity-determination better guarantees their further mapping were Laccases, MnP, AAO and TYR. Subsequently these four activities were measured in the monokaryon population being Laccases and MnP, those yielding the best levels in medium-7 (rich in nitrogen). In addition both enzymes allow the discrimination between “fast-” or “slow-” monokaryon strains both in solid medium with several dyes, or in liquid culture in agitation. The analysis of the enzymatic activities detected in the assayed conditions, in the population of “fast” or “slow” strains let to the observation that they map in different places where the loci corresponding to Laccase (pox) and mnp genes are located. These results open the possibility to design more precise studies that could help to establish a correlation between the contribution of the genes already described and the activity of the different ligninolytic enzymes. In addition the results will contribute to know whether in P. ostreatus genome there are new genes or if they correspond with locations that regulate these enzymatic activities, or it is a gene that has a role in the transport system or a kind of effector in the exportation machinery of the protein to the culture medium.Publication Open Access Transcriptome metabolic characterization of tuber borchii SP1-A new spanish strain for in vitro studies of the bianchetto truffle(MDPI, 2023) Chuina Tomazeli, Emilia; Alfaro Sánchez, Manuel; Zambonelli, Alessandra; Garde Sagardoy, Edurne; Pérez Garrido, María Gumersinda; Jiménez Miguel, Idoia; Ramírez Nasto, Lucía; Salman, Hesham; Pisabarro de Lucas, Gerardo; Institute for Multidisciplinary Research in Applied Biology - IMABTruffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.Publication Open Access 101 Dothideomycetes genomes: a test case for predicting lifestyles and emergence of pathogens(Westerdijk Fungal Biodiversity Institute, 2020) Haridas, Sajeet; Castanera Andrés, Raúl; Culley, D. E.; Daum, C.; Ramírez Nasto, Lucía; Alfaro Sánchez, Manuel; Institute for Multidisciplinary Research in Applied Biology - IMABDothideomycetes is the largest class of kingdom Fungi and comprises an incredible diversity of lifestyles, many of which have evolved multiple times. Plant pathogens represent a major ecological niche of the class Dothideomycetes and they are known to infect most major food crops and feedstocks for biomass and biofuel production. Studying the ecology and evolution of Dothideomycetes has significant implications for our fundamental understanding of fungal evolution, their adaptation to stress and host specificity, and practical implications with regard to the effects of climate change and on the food, feed, and livestock elements of the agro-economy. In this study, we present the first large-scale, whole-genome comparison of 101 Dothideomycetes introducing 55 newly sequenced species. The availability of whole-genome data produced a high-confidence phylogeny leading to reclassification of 25 organisms, provided a clearer picture of the relationships among the various families, and indicated that pathogenicity evolved multiple times within this class. We also identified gene family expansions and contractions across the Dothideomycetes phylogeny linked to ecological niches providing insights into genome evolution and adaptation across this group. Using machine-learning methods we classified fungi into lifestyle classes with >95 % accuracy and identified a small number of gene families that positively correlated with these distinctions. This can become a valuable tool for genome-based prediction of species lifestyle, especially for rarely seen and poorly studied species.Publication Open Access Isolation, molecular characterization and location of telomeric sequences of the basidiomycete Pleurotus ostreatus var. florida(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza EkoizpenaThe white rot fungus Pleurotus ostreatus is an edible basidiomycete of increasing biotechnological interest due to its ability to degrade both wood and chemicals related to lignin degradation products. Telomeres are specialized structures at the end of all eukaryotic chromosomes. Ensure chromosome stability and protect the ends from degradation and from fusing with other chromosomes. Telomeres sequences are extraordinary highly conserved in evolution. The loss of telomeric repeats triggers replicative senescence in cells. For identification of restriction telomeric fragments in a previously described linkage map of Pleurotus ostreatus var. florida (Larraya et al., 2000), dikaryotic and eighty monokaryotic genomic DNAs were digested with diferents restriction enzymes (BamHI, BglII, HindIII, EcoRI, PstI, SalI, XbaI and XhoI) electrophoresed and transferred to nylon membranes. Numerous polymorphic bands were observed when membranes were hibridized with human telomericd probe (TTAGGG)132 (heterologous probe). Telomeric restriction fragments were genetically mapped to a previously described linkage map of Pleurotus ostreatus var.florida, using RFLPs identified by a human telomeric probe (tandemly repeating TTAGGG hexanucleotide). Segregation of each telomeric restriction fragment was recorded as the presence vs. absence of a hibridizing band. Segregation data for seventy three telomeric restriction fragments was used as an input table to be analysed as described by Ritter et al. (1990) and by Ritter and Salamini (1996) by using the MAPRF program software. Seventeen out of twenty two telomeres were identified. Telomere and telomere-associated (TA) DNA sequences of the basidiomycete Pleurotus ostreatus were isolated by using a modified version of single- specific-primer polymerase chain reaction (SSP-PCR) technique (Sohapal et al., 2000). Telomeres of Pleurotus ostreatus contain at least twenty five copies of non-coding tandemly repeated sequence (TTAGGG).Publication Open Access Identification and functional characterisation of ctr1, a Pleurotus ostreatus gene coding for a copper transporter(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Peñas Parrila, María Manuela; Azparren Larraya, María Goretti; Domínguez, A.; Sommer, H.; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza EkoizpenaCopper homeostasis is primordial for life maintenance and especially relevant for ligning-degrading fungi whose phenol-oxidase enzymes depend on this micronutrient for their activity. In this paper we report the identification of a gene (ctr1), coding for a copper transporter in the white rot fungus Pleurotus ostreatus, in a cDNA library constructed from four-days old vegetative mycelium growing in submerged culture. The results presented here indicate that: (1) ctr1 functionally complements the respiratory deficiency of a yeast mutant defective in copper transport supporting the transport activity of the Ctr1 protein; (2) ctr1 transcription is detected in all P. ostreatus developmental stages (with exception of lamellae) and is negatively regulated by the presence of copper in the culture media; (3) ctr1 is a single copy gene that maps to P. ostreatus linkage group III; and (4) the regulatory sequence elements found in the promoter of ctr1 agree with those found in other copper related genes described in other systems. These results provide the first description of a copper transporter in this white rot fungus and open the possibility of further studies on copper metabolism in higher basidiomyetes.Publication Open Access Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis(National Academy of Sciences, 2012) Fernández Fueyo, Elena; Ruiz Dueñas, Francisco J.; Ferreira, Patricia; Floudas, Dimitrios; Lavín Trueba, José Luis; Oguiza Tomé, José Antonio; Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Santoyo Santos, Francisco; Producción Agraria; Nekazaritza EkoizpenaEfficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.