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 Publikoa
Mycelium 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.
Open Access
Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion
(National Academy of Sciences, 2009) Martínez, Diego; Challacombe, Jean; Morgenstern, Ingo; Hibbett, David; Schmoll, Monika; Kubicek, Christian P.; Ferreira, Patricia; Pisabarro de Lucas, Gerardo; Lavín Trueba, José Luis; Oguiza Tomé, José Antonio; Producción Agraria; Nekazaritza Ekoizpena
Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative β -1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also upregulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.
Open Access
Strain degeneration in pleurotus ostreatus: a genotype dependent oxidative stress process which triggers oxidative stress, cellular detoxifying and cell wall reshaping genes
(MDPI, 2021) Pérez Garrido, María Gumersinda; Lopez-Moya, Federico; Chuina Tomazeli, Emilia; Ibañez Vea, María; Garde Sagardoy, Edurne; López Llorca, Luis V.; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Strain degeneration has been defined as a decrease or loss in the yield of important commercial traits resulting from subsequent culture, which ultimately leads to Reactive Oxygen Species (ROS) production. Pleurotus ostreatus is a lignin-producing nematophagous edible mushroom. Mycelia for mushroom production are usually maintained in subsequent culture in solid media and frequently show symptoms of strain degeneration. The dikaryotic strain P. ostreatus (DkN001) has been used in our lab as a model organism for different purposes. Hence, different tools have been developed to uncover genetic and molecular aspects of this fungus. In this work, strain degeneration was studied in a full-sib monokaryotic progeny of the DkN001 strain with fast (F) and slow (S) growth rates by using different experimental approaches (light microscopy, malondialdehyde levels, whole-genome transcriptome analysis, and chitosan effect on monokaryotic mycelia). The results obtained showed that: (i) strain degeneration in P. ostreatus is linked to oxidative stress, (ii) the oxidative stress response in monokaryons is genotype dependent, (iii) stress and detoxifying genes are highly expressed in S monokaryons with symptoms of strain degeneration, (iv) chitosan addition to F and S monokaryons uncovered the constitutive expression of both oxidative stress and cellular detoxifying genes in S monokaryon strains which suggest their adaptation to oxidative stress, and (v) the overexpression of the cell wall genes, Uap1 and Cda1, in S monokaryons with strain degeneration phenotype indicates cell wall reshaping and the activation of High Osmolarity Glycerol (HOG) and Cell Wall Integrity (CWI) pathways. These results could constitute a hallmark for mushroom producers to distinguish strain degeneration in commercial mushrooms.
Open Access
Transcriptional and enzymatic profiling of Pleurotus ostreatus laccase genes in submerged and solid-state fermentation cultures
(American Society for Microbiology, 2012) Castanera Andrés, Raúl; Pérez Garrido, María Gumersinda; Omarini, Alejandra; Alfaro Sánchez, Manuel; Pisabarro de Lucas, Gerardo; Faraco, Vicenza; Amore, Antonella; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription- quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.
Open Access
Non-additive transcriptional profiles underlie dikaryotic superiority in Pleurotus ostreatus laccase activity
(Public Library of Science, 2013) Castanera Andrés, Raúl; Omarini, Alejandra; Santoyo Santos, Francisco; Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Background: The basidiomycete Pleurotus ostreatus is an efficient producer of laccases, a group of enzymes appreciated for their use in multiple industrial processes. The aim of this study was to reveal the molecular basis of the superiority of laccase production by dikaryotic strains compared to their parental monokaryons. Methodology/Principal Findings: We bred and studied a set of dikaryotic strains starting from a meiotic population of monokaryons. We then completely characterised the laccase allelic composition, the laccase gene expression and activity profiles in the dikaryotic strain N001, in two of its meiotic full-sib monokaryons and in the dikaryon formed from their mating. Conclusions/Significance: Our results suggested that the dikaryotic superiority observed in laccase activity was due to nonadditive transcriptional increases in lacc6 and lacc10 genes. Furthermore, the expression of these genes was divergent in glucose- vs. lignocellulose-supplemented media and was highly correlated to the detected extracellular laccase activity. Moreover, the expression profile of lacc2 in the dikaryotic strains was affected by its allelic composition, indicating a putative single locus heterozygous advantage.
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 - IMAB
Pleurotus 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.
Open Access
Mapping of genomic regions (quantitative trait loci) controlling production and quality in industrial cultures of the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2003) Larraya Reta, Luis María; Alfonso Esquíroz, Mikel; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua
Industrial production of the edible basidiomycete Pleurotus ostreatus (oyster mushroom) 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. Traditionally, fungal breeding has been carried out by an empirical trial and error process. In this study, we used a different approach by mapping quantitative trait loci (QTLs) controlling culture production and quality within the framework of the genetic linkage map of P. ostreatus. Ten production traits and four quality traits were studied and mapped. The production QTLs identified explain nearly one-half of the production variation. More interestingly, a single QTL mapping to the highly polymorphic chromosome VII appears to be involved in control of all the productivity traits studied. Quality QTLs appear to be scattered across the genome and to have less effect on the variation of the corresponding traits. Moreover, some of the new hybrid strains constructed in the course of our experiments had production or quality values higher than those of the parents or other commercial strains. This approach opens the possibility of marker-assisted selection and breeding of new industrial strains of this fungus.
Open Access
Comparative and transcriptional analysis of the predicted secretome in the lignocellulose-degrading basidiomycete fungus Pleurotus ostreatus
(Wiley, 2016) Alfaro Sánchez, Manuel; Castanera Andrés, Raúl; Lavín Trueba, José Luis; Oguiza Tomé, José Antonio; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Fungi interact with their environment by secreting proteins to obtain nutrients, elicit responses and modify their surroundings. Because the set of proteins secreted by a fungus is related to its lifestyle, it should be possible to use it as a tool to predict fungal lifestyle. To test this hypothesis, we bioinformatically identified 538 and 554 secretable proteins in the monokaryotic strains PC9 and PC15 of the white rot basidiomycete Pleurotus ostreatus. Functional annotation revealed unknown functions (37.2%), glycosyl hydrolases (26.5%) and redox enzymes (11.5%) as the main groups in the two strains. When these results were combined with RNA‐seq analyses, we found that the relative importance of each group was different in different strains and culture conditions and the relevance of the unknown function proteins was enhanced. Only a few genes were actively expressed in a given culture condition in expanded multigene families, suggesting that family expansi on could increase adaptive opportunities rather than activity under a specific culture condition. Finally, we used the set of P. ostreatus secreted proteins as a query to search their counterparts in other fungal genomes and found that the secretome profiles cluster the tested basidiomycetes into lifestyle rather than phylogenetic groups.
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
Genetic breeding of edible mushrooms: from the genome to the production of new varieties of Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pisabarro de Lucas, Gerardo; Peñas Parrila, María Manuela; Pérez Garrido, María Gumersinda; Park, Sang-Kyu; Eizmendi Goicoechea, Arantza; Parada Albarracín, Julián Andrés; Palma Dovis, Leopoldo; Idareta Olagüe, Eneko; Jurado Cabanillas, Javier; Castellón Gadea, Jordi; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The breeding of new varieties of industrially cultivated edible mushrooms must proceed in the framework defined by the breeding objectives, the biological characteristics of the material and the legal and cultural constraints imposed to the breeding technology to be used. This last aspect is of the greatest importance in the case of a food that is considered in European countries as high quality and closer to nature than other industrially produced foods. This fact prevents the use of genetic-engineering based technologies for breeding, as the consumers would hardly accept genetically modified mushrooms. Consequently, mushroom breeding should be based on time-consuming processes of classic breeding. Molecular biology, however, can offer to the breeders useful tools for speeding up the selection process, for evaluating the new bred lines and, last but not least, to identify and eventually protect legally the outcome of their breeding programs.