31 research outputs found
Glucose counteracts wood-dependent induction of lignocellulolytic enzyme secretion in monokaryon and dikaryon submerged cultures of the white-rot basidiomycete Pleurotus ostreatus
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.Grants numbers AGL2014-55971-R (AEI, FEDER, UE) and RTI2018-099371-B-I00 (MCIU/AEI/FEDER, UE) of the Spanish National Research Plan, and PRO-UPNA18 from the Public University of Navarre have funded this research. Proteomics work in Goettingen was supported within the framework of a Common Lower Saxony-Israel-Project (ZN2043) by the Ministry of Science and Culture of Lower Saxony granted to UK and AM. MA appreciates a grant for research exchanges in Goettingen and a postdoctoral grant by Universidad Pública de Navarra.Grants numbers AGL2014-55971-R (AEI, FEDER, UE) and RTI2018-099371-B-I00 (MCIU/AEI/FEDER, UE) of the Spanish National Research Plan, and PRO-UPNA18 from the Public University of Navarre have funded this research. Proteomics work in Goettingen was supported within the framework of a Common Lower Saxony-Israel-Project (ZN2043) by the Ministry of Science and Culture of Lower Saxony granted to UK and AM. MA appreciates a grant for research exchanges in Goettingen and a postdoctoral grant by Universidad Pública de Navarra. Some of these experiments also featured in the thesis of Manuel Alfaro
Mapping the Pleurotus ostreatus genome
Resumen del poster presentado al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.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
Isolation, molecular characterization and location of telomeric sequences of the basidiomycete Pleurotus ostreatus var. florida
Resumen del poster presentado al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.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)
VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI)
Contribuciones (completas, resúmenes y posters) presentadas al 6th Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra del 3 al 6 de junio de 2005.This volume summarizes the scientific communications presented at the 6th
Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI)
held in Pamplona (Spain) from June 3rd to 6th, 2005. GCBB-VI continues
the tradition of putting together scientist working with basidiomycetes
around the world. Our interest, as organizers, was to strength the communication
between groups working on basic and applied research both in the
field of edible mushrooms and in that of other industrial applications of
these microorganisms.
The scientific program included sessions focused on genetics and breeding
coordinated by Lucy Ramírez and Rick Kerrigan; Genome Analysis
chaired by Allen Gathman; Cellular and Molecular Biology, coordinated by
Regina Kahmann and Erika Kothe, Industrial Applications moderated by
Giovanni Sannia and Kerry Burton; Plant and Animal Pathogens coordinated
by José Pérez-Martín; and Biodiversity coordinated by Philippe Callac. A
complete version of the scientific program can be found at the end en of this
Volume.
In the coffee talks the need of a more active community of scientist working
on basidiomycetes was a recurrent topic. After this meeting took place,
several proposals for the complete sequencing of basidiomycete genomes are
going to be presented for evaluation by groups participating in GCBB-VI.This meeting has been supported by funds of the Spanish National Plan for
Scientific Research (Project No. AGL-2002-04222-C03-01), by a grant of
the Public University of Navarre’s Research Committee, and by the participants’
fees
Molecular characterization of A cellobiohydrolase gene family in the fungus Pleurotus ostreatus
Resumen del poster presentado al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.Cellulose is the most abundant biological polymer on Earth. Its chemical
composition consists of D-glucose units linked by β-1,4- glycosidic bonds
forming linear polymeric chains with a reducing and a non-reducing end.
Cellulose chains may either adhere to each other, via hydrophobic and van
der Waals interactions, forming crystalline structures or remain more loosely
packaged (amorphous cellulose). Consequently, the physical structure and
morphology of native cellulose is complex and not uniform.
Biological degradation of cellulose depends on the action of three types of
enzymes: endoglucanases (E.C.3.2.1.4), cellobiohydrolases (E.C.3.2.1.91) and
β-glucosidases (E.C.3.2.1.21). All them hydrolyse β-1,4-glycosidic bonds but
they differ on the substrate specificity. Endoglucanases hydrolyse the amorphous
regions of the cellulose fibbers generating new reducing and non-reducing
ends, cellobiohydrolases attack the molecule ends yielding cellobiose units,
and β-glucosidases hydrolyse cellobiose molecules yielding glucose.
Cellobiohydrolases can be classified into two groups: type I (CBHI) and
type II (CBHII), each having opposite chain-end specificities. CBHI prefer
the reducing ends while CBHII act at non-reducing ends.
By the screening of a genomic library from the basidiomycete Pleurotus ostreatus
var. florida, we have isolated five cbhI genes, named cbhI1, cbhI2, cbhI3,
cbhI4 and cbhI5, proving the occurrence of a multigenic family coding for this
enzymatic activity. Using this sequences as probe, it has been possible to know
the conditions in which are expressed those genes. This has allowed the synthesis
of the each gene cDNA and, by comparison of this sequence with the
corresponding genomic sequence, the characterization of their structure.
On the other hand, using the RFLP technique and a progeny of 80
monokaryons derived from the dikaryon N001, the five genes have been
mapped on the linkage map of P. ostreatus var. florida mapping the cbhI1 to
the chromosome IV and the others to the chromosome VI
Validation of reference genes for transcriptional analyses in Pleurotus ostreatus by using reverse transcription-quantitative PCR
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
Selection of Pleurotus ostreatus strains in a genetic breeding program
Resumen del poster presentado al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.The basidiomycete Pleurotus ostreatus, commonly known as oyster mushroom,
is the second largest edible mushroom crop behind the white button
mushroom, Agaricus bisporus. It accounts for nearly one-quarter of the total
worldwide mushroom production. Furthermore, P. ostreatus has a high industrial
interest because it is a good source of enzymes and other products with
biotechnological, industrial and medical applications, it is easy to cultivate
and because of its good organoleptic characteristics.
Since of 2003, our group research has carried out genetic breeding programs
based on the determination of QTLs controlling production and quality
in industrial cultures of this fungus. In this breeding program the first test
consisted in putting under fructification conditions 130 strains obtained
from the crossing of protoclon PC21 (P. ostreatus var. ostreatus wild strain) by
a collection of monokarions derived from N001 (P. ostreatus var. florida commercial
strain). For this purpose, 2 kg (3 repetitions per strain) bags of industrial
sustrate were inoculated and cultivated at 21ºC. Mature fruiting bodies
were collected and weighted daily during the fructification period.
The second test was made using the six strains that performed the better
in Test1, but were cultivated at 18ºC and with 15 repetitions per strain were
performed. From this test, three strains were selected and used in Test3. In
this test, other three strains obtained from the crossing between monokarions
descending of N001 and selectioned for their high growth rate were introduced.
In this test the weight of the bags was increased to 5 kg and the
cultures were cultivated at 18ºC.
The strains obtained from PC21 have good charactericts for mushroom
size, with similar behaviour for yield and precocity.
The strains obtained from the crosses between N001 descendants have
better mushroom size and similar yield and precocity than N001, then breeding
was obtained.
The candidate strains for next tests are PC21xMA046 and PC21xMA027
for their high yield and the mushroom good features
Computational prediction of protein-coding gene and annotation of DNA sequences with agronomic interest in Pleurotus ostreatus (Oyster Mushroom)
Resumen del poster presentado al VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI), organizado por y celebrado en la Universidad Pública de Navarra el 3-6 de junio de 2005.Pleurotus ostreatus, commonly known as oyster mushroom, is a commercially
important edible fungus with interesting biotechnological properties. Quantitative
trait loci (QTL) analyses are rare in fungi and little is known about
their number, position, and genetic structure. Previous studies of our group
have allowed the construction of a genetic linkage map of P. ostreatus var.
florida, which has provided the basis for performing an efficient QTL analysis.
In fact, there is a region of the chromosome VII of P. ostreatus where the
most QTLs related to the production and precocity characters have been
mapped. These quantitative traits are presumably under the control of a
polygenic genetic system and could be associated with some chromosomal
regions. The hypothesis of this work is that there is a region in the chromosome
VII of protoclon PC15 (monokaryotic parental of the N001 dikaryotic
strain) where exist genes which are responsible for the QTLs mentioned
above. In order to test this hypothesis, we are developing a molecular QTL
analysis through the sequencing of a region with an approximated size of 320
Kbp in chromosome VII (protoclon PC15). For this purpose, a BAC genomic
library was constructed and two BAC clones spanning the region of
interest are being sequenced. To carry out an efficient computational prediction
of protein-coding genes and its annotation on the partial sequences obtained
up to date, we have used different Internet resources such as
BLASTx, BLASTp, BLASTn, and FGENESH trained on some basidiomycetes
genomic data like Phanerochaete chrysosporium and Cryptococcus
neoformans (SoftBerry). To our knowledge, this is the firs molecular QTL
analysis performed on this edible mushroom
Somatic transposition and meiotically driven elimination of an active helitron family in Pleurotus ostreatus
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
Comparative and transcriptional analysis of the predicted secretome in the lignocellulose-degrading basidiomycete fungus Pleurotus ostreatus
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