Open Access
Genomics and transcriptomics characterization of genes expressed during postharvest at 4 degrees C by the edible basidiomycete Pleurotus ostreatus
(Viguera Editores, S. L., 2011) Ramírez Nasto, Lucía; Oguiza Tomé, José Antonio; Pérez Garrido, María Gumersinda; Lavín Trueba, José Luis; Omarini, Alejandra; Santoyo Santos, Francisco; Alfaro Sánchez, Manuel; Castanera Andrés, Raúl; Parenti, Alejandra; Muguerza Domínguez, Elaia; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus is an industrially cultivated basidiomycete with nutritional and environmental applications. Its genome, which was sequenced by the joint Genome Institute, has become a model for lignin degradation and for fungal genomics and transcriptomics studies. The complete P. ostreatus genome contains 35 Mbp organized in 11 chromosomes, and two different haploid genomes have been individually sequenced. In this work, genomics and transcriptomics approaches were employed in the study of P. ostreatus under different physiological conditions. Specifically, we analyzed a collection of expressed sequence tags (EST) obtained from cut fruit bodies that had been stored at 4 degrees C for 7 days (postharvest conditions). Studies of the 253 expressed clones that had been automatically and manually annotated provided a detailed picture of the life characteristics of the self-sustained fruit bodies. The results suggested a complex metabolism in which autophagy, RNA metabolism, and protein and carbohydrate turnover are increased. Genes involved in environment sensing and morphogenesis were expressed under these conditions. The data improve our understanding of the decay process in postharvest mushrooms and highlight the use of high-throughput techniques to construct models of living organisms subjected to different environmental conditions.
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
Ligninolytic peroxidase gene expression by Pleurotus ostreatus: differential regulation in lignocellulose medium and effect of temperature and pH
(Elsevier, 2014) Fernández Fueyo, Elena; Castanera Andrés, Raúl; Ruiz Dueñas, Francisco J.; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25 C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25 C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn2+ (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3 > vp2/vp3/mnp1/mnp2/mnp6 > mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10 C or 37 C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10 C treatment, while many of them were alternatively upregulated (often 6 h after the thermal shock) and downregulated (12 h) at 37 C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion.
Open Access
Transposon-associated epigenetic silencing during Pleurotus ostreatus life cycle
(Oxford University Press, 2018) Borgognone, Alessandra; Castanera Andrés, Raúl; Morselli, Marco; López Varas, Leticia; Rubbi, Liudmilla; Pisabarro de Lucas, Gerardo; Pellegrini, Matteo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Transposable elements constitute an important fraction of eukaryotic genomes. Given their mutagenic potential, host-genomes have evolved epigenetic defense mechanisms to limit their expansion. In fungi, epigenetic modifications have been widely studied in ascomycetes, although we lack a global picture of the epigenetic landscape in basidiomycetes. In this study, we analysed the genome-wide epigenetic and transcriptional patterns of the white-rot basidiomycete Pleurotus ostreatus throughout its life cycle. Our results performed by using high-throughput sequencing analyses revealed that strain-specific DNA methylation profiles are primarily involved in the repression of transposon activity and suggest that 21 nt small RNAs play a key role in transposon silencing. Furthermore, we provide evidence that transposon-associated DNA methylation, but not sRNA production, is directly involved in the silencing of genes surrounded by transposons. Remarkably, we found that nucleus-specific methylation levels varied in dikaryotic strains sharing identical genetic complement but different subculture conditions. Finally, we identified key genes activated in the fruiting process through the comparative analysis of transcriptomes. This study provides an integrated picture of epigenetic defense mechanisms leading to the transcriptional silencing of transposons and surrounding genes in basidiomycetes. Moreover, our findings suggest that transcriptional but not methylation reprogramming triggers fruitbody development in P. ostreatus.
Open Access
Comparative genomics of Coniophora olivacea reveals different patterns of genome expansion in Boletales
(BioMed Central, 2017) Castanera Andrés, Raúl; Pérez Garrido, María Gumersinda; López Varas, Leticia; Amselem, Joëlle; LaButti, Kurt; Singan, Vasanth; Lipzen, Anna; Haridas, Sajeet; Barry, Kerrie; Grigoriev, Igor V.; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Background: Coniophora olivacea is a basidiomycete fungus belonging to the order Boletales that produces brown-rot decay on dead wood of conifers. The Boletales order comprises a diverse group of species including saprotrophs and ectomycorrhizal fungi that show important differences in genome size. Results: In this study we report the 39.07-megabase (Mb) draft genome assembly and annotation of C. olivacea. A total of 14,928 genes were annotated, including 470 putatively secreted proteins enriched in functions involved in lignocellulose degradation. Using similarity clustering and protein structure prediction we identified a new family of 10 putative lytic polysaccharide monooxygenase genes. This family is conserved in basidiomycota and lacks of previous functional annotation. Further analyses showed that C. olivacea has a low repetitive genome, with 2.91% of repeats and a restrained content of transposable elements (TEs). The annotation of TEs in four related Boletales yielded important differences in repeat content, ranging from 3.94 to 41.17% of the genome size. The distribution of insertion ages of LTRretrotransposons showed that differential expansions of these repetitive elements have shaped the genome architecture of Boletales over the last 60 million years. Conclusions: Coniophora olivacea has a small, compact genome that shows macrosynteny with Coniophora puteana. The functional annotation revealed the enzymatic signature of a canonical brown-rot. The annotation and comparative genomics of transposable elements uncovered their particular contraction in the Coniophora genera, highlighting their role in the differential genome expansions found in Boletales species.
Open Access
Genetic linkage map of the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2000) Larraya Reta, Luis María; Pérez Garrido, María Gumersinda; 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
We have constructed a genetic linkage map of the edible basidiomycete Pleurotus ostreatus (var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (est1); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of P. ostreatus, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of P. ostreatus.
Open Access
Glucose counteracts wood-dependent induction of lignocellulolytic enzyme secretion in monokaryon and dikaryon submerged cultures of the white-rot basidiomycete Pleurotus ostreatus
(Nature Research, 2020) Alfaro Sánchez, Manuel; Majcherczyk, A.; Kües, Ursula; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The secretome complexity and lignocellulose degrading capacity of Pleurotus ostreatus monokaryons mkPC9 and mkPC15 and mated dikaryon dkN001 were studied in submerged liquid cultures containing wood, glucose, and wood plus glucose as carbon sources. The study revealed that this white-rot basidiomycete attacks all the components of the plant cell wall. P. ostreatus secretes a variety of glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases, especially when wood is the only carbon source. The presence of wood increased the secretome complexity, whereas glucose diminished the secretion of enzymes involved in cellulose, hemicellulose and pectin degradation. In contrast, the presence of glucose did not influence the secretion of redox enzymes or proteases, which shows the specificity of glucose on the secretion of cellulolytic enzymes. The comparison of the secretomes of monokaryons and dikaryons reveals that secretome complexity is unrelated to the nuclear composition of the strain.
Open Access
Metatranscriptomics sheds light on the links between the functional traits of fungal guilds and ecological processes in forest soil ecosystems
(Blackwell Scientific Publications Ltd, 2023) Auer, Lucas; Buée, Marc; Fauchery, Laure; Lombard, Vincent; Barry, Kerrie; Clum, Alicia; Copeland, Alex; Daum, Chris; LaButti, Kurt; Singan, Vasanth; Yoshinaga, Yuko; Martineau, Christine; Castillo Martínez, Federico; Alfaro Sánchez, Manuel; Imbert Rodríguez, Bosco; Ramírez Nasto, Lucía; Castanera Andrés, Raúl; Pisabarro de Lucas, Gerardo; Finlay, Roger; Lindahl, Björn D.; Olson, Ake; Séguin, Armand; Kohler, Annegret; Henrissat, Bernard; Grigoriev, Igor V.; Martin, Francis; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB
Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. We showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.
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 Ekoizpena
Efficient 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.
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).