Gúmer Pérez

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis

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.

Genetic Linkage Map of the Edible Basidiomycete Pleurotus ostreatus

ABSTRACT 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 ofP. 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.

Transcriptional and enzymatic profiling of Pleurotus ostreatus laccase genes in submerged- and solid-state fermentation cultures.

ABSTRACT The genome of the white rot basidiomycete Pleurotus ostreatus includes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription-quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) the Lacc2 and Lacc10 genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and the Lacc gene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.

Identification of incompatibility alleles and characterisation of molecular markers genetically linked to the A incompatibility locus in the white rot fungus Pleurotus ostreatus.

Pleurotus ostreatus is a hetertothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Although this mechanism is well accepted, there is a lack of knowledge about its molecular basis, as the incompatibility loci have not been cloned and sequenced. As a first step towards the elucidation of the molecular structure of the A-type incompatibility locus, molecular markers have been isolated which correspond to genomic sequences present in different strains of P. ostreatus but not in other higher basidiomycetae. These markers reveal single-copy genetic regions in which some degree of genetic variability can be detected.

Relationship between Monokaryotic Growth Rate and Mating Type in the Edible Basidiomycete Pleurotus ostreatus

ABSTRACT The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at thematB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matBlocus that allowed us to dissect it into two genetically linked subunits (matBα and matBβ) and to correlate the presence of specific matBα and matAalleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.

Transposable Elements versus the Fungal Genome: Impact on Whole-Genome Architecture and Transcriptional Profiles

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.

Genomics and transcriptomics characterization of genes expressed during postharvest at 4°C by the edible basidiomycete Pleurotus ostreatus

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°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.

Telomere organization in the ligninolytic basidiomycete Pleurotus ostreatus.

ABSTRACT Telomeres are structural and functional chromosome regions that are essential for the cell cycle to proceed normally. They are, however, difficult to map genetically and to identify in genome-wide sequence programs because of their structure and repetitive nature. We studied the telomeric and subtelomeric organization in the basidiomycete Pleurotus ostreatus using a combination of molecular and bioinformatics tools that permitted us to determine 19 out of the 22 telomeres expected in this fungus. The telomeric repeating unit in P. ostreatus is TTAGGG, and the numbers of repetitions of this unit range between 25 and 150. The mapping of the telomere restriction fragments to linkage groups 6 and 7 revealed polymorphisms compatible with those observed by pulsed field gel electrophoresis separation of the corresponding chromosomes. The subtelomeric regions in Pleurotus contain genes similar to those described in other eukaryotic systems. The presence of a cluster of laccase genes in chromosome 6 and a bipartite structure containing a Het-related protein and an alcohol dehydrogenase are especially relevant; this bipartite structure is characteristic of the Pezizomycotina fungi Neurospora crassa and Aspergillus terreus. As far as we know, this is the first report describing the presence of such structures in basidiomycetes and the location of a laccase gene cluster in the subtelomeric region, where, among others, species-specific genes allowing the organism to adapt rapidly to the environment usually map.

Genetic networks for the functional study of genomes

The high-throughput analytical techniques used in genome, proteome and metabolome studies produce large sets of data that must be studied using appropriate tools. The construction of networks linking different genetic elements and/or functions makes it possible to obtain an integrated view of the cell molecular biology and will eventually help us to predict complex phenotypes from molecular data. Genetic networks can be constructed using different types of data such as genes involved in the control of complex phenotypic traits, genes controlling global gene expression, genetic elements involved in the same metabolic process, gene products interacting physically between them. The connections linking these genetic elements in the network reflect the genetic, physical and/or functional interaction among them. All these networks share common properties and reflect the different layers of the cells complexity. In this review, we will study how different types of networks can be constructed, how the different networks complement each other and how this information can be used to obtain an integrated picture of the cell.

Highly expressed captured genes and cross-kingdom domains present in Helitrons create novel diversity in Pleurotus ostreatus and other fungi

BackgroundHelitrons 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.ResultsOur 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.ConclusionP. 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.