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
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
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
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
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
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
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
Expansion of the global RNA virome reveals diverse clades of bacteriophages
(Elsevier, 2022) Neri, Uri; Wolf, Yuri I.; Roux, Simon; Camargo, Antonio Pedro; Lee, Benjamin; Kazlauskas, Darius; Chen, I. Min; Ivanova, Natalia; Zeigler Allen, Lisa; Paez-Espino, David; Bryant, Donald A.; Bhaya, Devaki; Krupovic, Mart; Dolja, Valerian V.; Kyrpides, Nikos C.; Koonin, Eugene V.; Gophna, Uri; RNA Virus Discovery Consortium; Narrowe, Adrienne B.; Ramírez Nasto, Lucía; Wang, Zhong; Nusslein, Klaus; Meredith, Laura K.; Buée, Marc; Huntemann, Marcel; Kalyuzhnaya, Marina G.; Waldrop, Mark P.; Sullivan, Matthew B.; Schrenk, Matthew O.; Hess, Matthias; Vega, Michael A.; O’Malley, Michelle A.; Medina, Mónica; Gilbert, Naomi E.; Delherbe, Nathalie; Mason, Olivia U.; Probst, Alexander J.; Sczyrba, Alexander; Kohler, Annegret; Séguin, Armand; Shade, Ashley; Campbell, Barbara J.; Lindahl, Björn D.; Reese, Brandi Kiel; Roque, Breanna M.; DeRito, Christopher; Averill, Colin; Cullen, Daniel; Beck, David A.C.; Walsh, David A.; Ward, David M.; Wu, Dongying; Eloe-Fadrosh, Emiley; Brodie, Eoin L.; Dijkstra, Paul; Chuckran, Peter F.; Baldrian, Petr; Constant, Philippe; Stepanauskas, Ramunas; Daly, Rebecca A.; Lamendella, Regina; Gruninger, Robert J.; McKay, Robert M.; Hylander, Samuel; Lebeis, Sarah L.; Esser, Sarah P.; Acinas, Silvia G.; Young, Erica B.; Lilleskov, Erik A.; Castillo, Federico J.; Martin, Francis; LeCleir, Gary R.; Attwood, Graeme T.; Cadillo-Quiroz, Hinsby; Simon, Holly M.; Hewson, Ian; Grigoriev, Igor V.; Tiedje, James M.; Jansson, Janet K.; Lee, Janey; VanderGheynst, Jean S.; Dangl, Jeff; Bowman, Jeff S.; Blanchard, Jeffrey L.; Bowen, Jennifer L.; Xu, Jiangbing; Banfield, Jillian F.; Deming, Jody W.; Kostka, Joel E.; Gladden, John M.; Rapp, Josephine Z.; Sharpe, Joshua; McMahon, Katherine D.; Treseder, Kathleen K.; Bidle, Kay D.; Wrighton, Kelly C.; Thamatrakoln, Kimberlee; Wilhelm, Steven S.; Singer, Steven W.; Tringe, Susannah S.; Woyke, Tanja; Reddy, T.B.K.; Bell, Terrence H.; Mock, Thomas; McAllister, Tim; Thiel, Vera; Denef, Vincent J.; Liu, Wen-Tso; Martens-Habbena, Willm; Liu, Xiao-Jun Allen; Cooper, Zachary S.; Agronomía, Biotecnología y Alimentación; Agronomia, Bioteknologia eta Elikadura; Institute for Multidisciplinary Research in Applied Biology - IMAB
High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.
Open Access
Genomic analysis enlightens agaricales lifestyle evolution and increasing peroxidase diversity
(Oxford University Press, 2021) Ruiz Dueñas, Francisco J.; Barrasa, José M.; Sánchez-García, Marisol; Camarero, Susana; Miyauchi, Shingo; Linde, Dolores; Babiker, Rashid; Drula, Elodie; Ayuso-Fernández, Iván; Pacheco, Remedios; Padilla, Guillermo; Ferreira, Patricia; Barriuso, Jorge; Kellner, Harald; Castanera Andrés, Raúl; Alfaro Sánchez, Manuel; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Riley, Robert; Kuo, Alan; Andreopoulos, William; LaButti, Kurt; Pangilinan, Jasmyn; Tritt, Andrew; Lipzen, Anna; He, Guifen; Yan, Mi; Vivian, Ng; Grigoriev, Igor V.; Cullen, Daniel; Martin, Francis; Rosso, Marie-Noëlle; Henrissat, Bernard; Hibbett, David; Martínez, Ángel T.; Institute for Multidisciplinary Research in Applied Biology - IMAB
As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates-namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases-we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestralsequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree.