BUSCADOR RECOLECTA

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., This work was supported by the PEROXICATS (KBBE-2010-4-265397; www.peroxicats.org) and INDOX (KBBE-2013-7-613549; www.indoxproject.eu) EU projects, and by the BIO2011-26694 and AGL2011-30495 projects of the Spanish Ministry of Economy and Competitiveness (MINECO). EF-F acknowledges a Junta de Ampliación de Estudios fellowship of the CSIC, co-funded by the European Social Fund, and FJR-D acknowledges a MINECO Ramón y Cajal contract.

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., This work was supported by funds obtained from AGL2011-30495 of the Spanish National Research Plan; Bioethanol-Euroinnova, the Autonomous
Government of Navarre; and by additional institutional support from the Public University of Navarre.

Incluye 8 ficheros de datos, Background: Helitrons are class-II eukaryotic transposons that transpose via a rolling circle mechanism. Due to their
ability to capture and mobilize gene fragments, they play an important role in the evolution of their host genomes.
We have used a bioinformatics approach for the identification of helitrons in two Pleurotus ostreatus genomes using
de novo detection and homology-based searching. We have analyzed the presence of helitron-captured genes as
well as the expansion of helitron-specific helicases in fungi and performed a phylogenetic analysis of their
conserved domains with other representative eukaryotic species.
Results: Our results show the presence of two helitron families in P. ostreatus that disrupt gene colinearity and
cause a lack of synteny between their genomes. Both putative autonomous and non-autonomous helitrons were
transcriptionally active, and some of them carried highly expressed captured genes of unknown origin and function.
In addition, both families contained eukaryotic, bacterial and viral domains within the helitron’s boundaries. A
phylogenetic reconstruction of RepHel helicases using the Helitron-like and PIF1-like helicase conserved domains
revealed a polyphyletic origin for eukaryotic helitrons.
Conclusion: P. ostreatus helitrons display features similar to other eukaryotic helitrons and do not tend to capture
host genes or gene fragments. The occurrence of genes probably captured from other hosts inside the helitrons
boundaries pose the hypothesis that an ancient horizontal transfer mechanism could have taken place. The viral
domains found in some of these genes and the polyphyletic origin of RepHel helicases in the eukaryotic kingdom
suggests that virus could have played a role in a putative lateral transfer of helitrons within the eukaryotic kingdom.
The high similarity of some helitrons, along with the transcriptional activity of its RepHel helicases indicates that
these elements are still active in the genome of P. ostreatus., This work was supported by funds from the AGL2011-30495 project of the
Spanish National Research Plan and by additional institutional support from
the Public University of Navarre.

Most prokaryotic and eukaryotic life forms have to deal with the presence of repetitive DNA sequences called transposable elements (TEs), whose ability to mobilize through the genome and insert at random position has an impact on genome stability and functionality. For a long time, TEs were described as ‘selfish’ DNA fragments, owing to their proliferation within the host genome without conferring any benefit or inducing detrimental effects when inserted in gene coding regions. Over time, however, this view was changed by the discovery of the contribution of transposons in genome integrity and evolution.
Recent genome-wide characterizations have focused on the distribution of these mobile elements and the effects of active transposon copies within closely related genomes. Given their mutagenic potential, host genomes have evolved endogenous mechanisms to limit the mobilization and repress the transcriptional activity of transposons. In higher organisms, repeat sequences are transcriptionally silenced through epigenetic modifications, which modulate gene expression without producing permanent modifications along the nucleotide sequence. The integrated and dynamic nature of epigenetic pathways, including DNA methylation and RNA-silencing systems, can be regulated at different levels, leading to the targeted silencing of specific genomic regions. Thus, the revolutionary advent of high-throughput sequencing led to an unprecedented opportunity to generate genome-wide epigenetic profiles and extend the understanding of the contribution of TEs in genome evolution.
The filamentous fungi, in particular, Neurospora crassa and other ascomycetes, have provided fundamental advances in many of the aforementioned areas. These organisms possess complex epigenetic pathways that are also conserved in other higher eukaryotes to efficiently shut down transposon activity. Despite their importance, the occurrence of epigenetic events as well as the impact of transposon activity in basidiomycetes have been poorly analyzed so far.
Recent studies in Pleurotus ostreatus uncovered that the genome of this basidiomycete model is populated by a diverse set of TE families, providing a detailed picture of the distribution and importance of helitrons, which are a group of DNA transposons that mobilize through a rolling-circle mechanism.
Therefore, the principal aim of this work is to investigate the inheritance of helitron transposons and profile the epigenetic and transcriptomic landscape of P. ostreatus at different growing stages. Both objectives have been performed through the use of molecular techniques integrated with subsequent Sanger or Next-Generation sequencing data analysis. This PhD dissertation is comprised of four main chapters.
Chapter I describes the current state-of-the-art of genome sequencing, transposon identification and epigenetic mechanisms (DNA methylation and RNA silencing pathways); it also provides an introductory overview on the fungal kingdom and the model system P. ostreatus.
Chapter II presents the inheritance patterns of HELPO1 and HELPO2 helitron families in the meiotically-derived progeny of P. ostreatus. Our results report the distorted segregation patterns of HELPO2 helitrons that lead to a strong under-representation of these elements in the progeny. Further analyses of the HELPO2 flanking sites showed that the meiotic process of gene conversion may contribute to the elimination of such repetitive elements, favoring the presence of HELPO2 vacant loci. Moreover, the analysis of HELPO2 content in a reconstructed pedigree of subclones maintained under different culture conditions revealed an event of helitron somatic transposition. Additional investigations of genome and transcriptome data indicated that P. ostreatus carries active RNAi machinery that could be involved in the control of transposable element proliferation. These findings provide the first evidence of helitron mobilization in the fungal kingdom and highlight the interaction between genome defense mechanisms and invasive DNA using P. ostreatus as a model.
Chapter III describes the occurrence of epigenetic defense strategies in this fungal model. The analyses report a picture of genome-wide epigenetic (DNA methylation and small RNAs) and transcriptional (mRNA) patterns in P. ostreatus throughout its life cycle. High-throughput sequencing analyses (BS-seq, sRNA-seq and mRNA-seq) performed in monokaryon and dikaryon samples revealed epigenetic differences among strains but not within developmental stages. Our results uncovered strain-specific DNA methylation profiles (~ 2 to 7 % global methylation levels) and 21-22nt small RNA production primarily involved in the repression of transposon activity. Furthermore, our findings provide evidence that TE-associated DNA methylation—but not small RNA production—is directly involved in the silencing of genes surrounded by transposons. Finally, these findings also report key genes that are activated in the fruiting process through a comparative analysis of transcriptomes.
A general discussion on findings presented in this thesis is given in Chapter IV. This last part reports a critical outlook on the epigenetic and transcriptional state of the two helitron families of the P. ostreatus strains that are differentially subcultured during several years, as well as nucleus-specific methylation variations in dikaryotic strains that share an identical genetic complement but different subculture conditions., Alessandra Borgognone obtained a PhD scholarship from the Public University of Navarre (May 2013). This work has been supported by Spanish National Research Plan: Effect of helitrons in genome structure and transcriptional profile of Pleurotus ostreatus, AGL2011-30495, funded by the Spanish Ministry of Economy, Department of Research, Development and Innovation; Study of the interactions between transcriptome and methylome to explain differences in growth rate and mushroom yield in the edible fungus Pleurotus ostreatus, AGL2014-55971-R, funded by the Spanish Ministry of Economy, Department of Research, Development and Innovation., Programa Oficial de Doctorado en Biotecnología (RD 1393/2007), Bioteknologiako Doktoretza Programa Ofiziala (ED 1393/2007)

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., This work was supported by funds from the project AGL2011‐30495 of the Spanish National Research Plan, by additional institutional support from the Public University of Navarre, and by the Office of Science of the U.S. Department of Energy under Contract No. DE‐AC02‐05CH11231 (work conducted by the U.S. Department of Energy Joint Genome Institute). JLL is supported by the Basque Country Government (Etortek Research Programs 2011/2014) and from the Innovation Technology Dept. of Bizkaia.

Recently, the lignin-degrading basidiomycete Pleurotus ostreatus has become a widely used model organism for fungal genomic
and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional
regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time
PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we
tested the suitability of 13 candidate genes for their use as reference genes in P. ostreatus time course cultures for enzyme production.
We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization
of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of
transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase
(lacc10 and mnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state
fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal
controls leads to biased results and misinterpretations of the biological responses underlying expression changes., This work was supported by funds from the AGL2011-30495 project of
the Spanish National Research Plan and by additional institutional support
from the Public University of Navarre. R.C. holds an FPI Ph.D. studentship.

Helitrons constitute a superfamily of DNA transposons that were discovered in silico and are
widespread in most eukaryotic genomes. They are postulated to mobilize through a “rollingcircle”
mechanism, but the experimental evidence of their transposition has been described
only recently. Here, we present the inheritance patterns of HELPO1 and HELPO2 helitron families
in meiotically derived progeny of the basidiomycete Pleurotus ostreatus. We found distorted
segregation patterns of HELPO2 helitrons that led to a strong under-representation of
these elements in the progeny. Further investigation of HELPO2 flanking sites showed that
gene conversion may contribute to the elimination of such repetitive elements in meiosis, favouring
the presence of HELPO2 vacant loci. In addition, the analysis of HELPO2 content in a reconstructed
pedigree of subclones maintained under different culture conditions revealed an
event of helitron somatic transposition. Additional analyses of genome and transcriptome data
indicated that P. ostreatus carries active RNAi machinery that could be involved in the control
of transposable element proliferation. Our results provide the first evidence of helitron mobilization
in the fungal kingdom and highlight the interaction between genome defence mechanisms
and invasive DNA., This work was supported by Spanish National Research Plan (Projects
AGL2011-30495 and AGL2014-55971-R) and FEDER funds; Public
University of Navarre (http://www.unavarra.es); AB holds a PhD scholarship
from the Public University of Navarre and RC holds a FPI-PhD scholarship
from the Ministry of Economy and Competitiveness.

Background: Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification
applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available,
and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose
cultures.
Results: Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the
extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography
coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified.
Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar
cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two
lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the
poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only
slightly overproduced (14–16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes
(10–14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3
(MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins,
but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes
were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less
abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic
substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative
to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an
increase of sugar reducing ends.
Conclusions: Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic
substrates. One laccase occupied the first position in both secretomes, and three more were overproduced
together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal
of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results., This work was supported by the INDOX (KBBE-2013-613549) EU project, the BIO2014-56388-R, AGL2014-53730-R, and AGL2011-30495 projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the ProteoRed platform of the Spanish Institute of Health Carlos III (ISCIII). The work conducted by the US DOE JGI is supported by the Office of Science of the US DOE under contract number DE-AC02-05CH11231. FJR-D thanks a Ramón y Cajal contract of the Spanish MINECO, and JR thanks a contract of the CSIC project 201440E097.