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 - IMAB
Truffles 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.
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 - IMAB
Dothideomycetes 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.
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 Publikoa
Lignocellulose, 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.
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
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
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
Effect of nutritional factors and copper on the regulation of laccase enzyme production in Pleurotus ostreatus
(MDPI, 2022) Durán Sequeda, Dinary Eloísa; Suspes, Daniela; Maestre, Estibenson; Alfaro Sánchez, Manuel; Pérez Garrido, María Gumersinda; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Sierra Ramírez, Rocío; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
This research aimed to establish the relationship between carbon–nitrogen nutritional factors and copper sulfate on laccase activity (LA) by Pleurotus ostreatus. Culture media composition was tested to choose the nitrogen source. Yeast extract (YE) was selected as a better nitrogen source than ammonium sulfate. Then, the effect of glucose and YE concentrations on biomass production and LA as response variables was evaluated using central composite experimental designs with and without copper. The results showed that the best culture medium composition was glucose 45 gL−1 and YE 15 gL−1, simultaneously optimizing these two response variables. The fungal transcriptome was obtained in this medium with or without copper, and the differentially expressed genes were found. The main upregulated transcripts included three laccase genes (lacc2, lacc6, and lacc10) regulated by copper, whereas the principal downregulated transcripts included a copper transporter (ctr1) and a regulator of nitrogen metabolism (nmr1). These results suggest that Ctr1, which facilitates the entry of copper into the cell, is regulated by nutrient-sufficiency conditions. Once inside, copper induces transcription of laccase genes. This finding could explain why a 10–20-fold increase in LA occurs with copper compared to cultures without copper when using the optimal concentration of YE as nitrogen sources.
Open Access
Transposable elements versus the fungal genome: impact on whole-genome architecture and transcriptional profiles
(Public Library of Science, 2016) Castanera Andrés, Raúl; López Varas, Leticia; Borgognone, Alessandra; LaButti, Kurt; Lapidus, Alla; Schmutz, Jeremy; Grimwood, Jane; Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Grigoriev, Igor V.; Stajich, Jason E.; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
Transposable elements (TEs) are exceptional contributors to eukaryotic genome diversity. Their ubiquitous presence impacts the genomes of nearly all species and mediates genome evolution by causing mutations and chromosomal rearrangements and by modulating gene expression. We performed an exhaustive analysis of the TE content in 18 fungal genomes, including strains of the same species and species of the same genera. Our results depicted a scenario of exceptional variability, with species having 0.02 to 29.8% of their genome consisting of transposable elements. A detailed analysis performed on two strains of Pleurotus ostreatus uncovered a genome that is populated mainly by Class I elements, especially LTR-retrotransposons amplified in recent bursts from 0 to 2 million years (My) ago. The preferential accumulation of TEs in clusters led to the presence of genomic regions that lacked intra- and inter-specific conservation. In addition, we investigated the effect of TE insertions on the expression of their nearby upstream and downstream genes. Our results showed that an important number of genes under TE influence are significantly repressed, with stronger repression when genes are localized within transposon clusters. Our transcriptional analysis performed in four additional fungal models revealed that this TE-mediated silencing was present only in species with active cytosine methylation machinery. We hypothesize that this phenomenon is related to epigenetic defense mechanisms that are aimed to suppress TE expression and control their proliferation.
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
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.