Genome-Wide Gene Expression Profiling of Fertilization Competent Mycelium in Opposite Mating Types in the Heterothallic Fungus Podospora anserina

are the major regulators of fertilization, and this study presents a genome-wide view of their target genes and analyzes their target gene regulation. strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. Interspecies comparisons of mating-type target genes revealed significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species.This study represents the first comprehensive genome-wide analysis of mating-type direct and indirect target genes in a heterothallic filamentous fungus. Mating-type transcription factors have many more target genes than are found in yeasts and exert a much greater diversity of regulatory actions on target genes, most of which are not directly related to mating

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

<p>Abstract</p> <p>Background</p> <p>The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of <it>de novo </it>gene prediction programs, and annotation up-dating. We present a novel <it>in silico </it>procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the <it>in silico </it>outcome.</p> <p>Findings</p> <p>We used four criteria for <it>in silico </it>probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus <it>Podospora anserina </it>and the selection of a single 60-mer probe for each of the 10,556 <it>P. anserina </it>CDS.</p> <p>Conclusions</p> <p>A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.</p

Tracing the Origin of the Fungal α1 Domain Places Its Ancestor in the HMG-Box Superfamily: Implication for Fungal Mating-Type Evolution

BACKGROUND: Fungal mating types in self-incompatible Pezizomycotina are specified by one of two alternate sequences occupying the same locus on corresponding chromosomes. One sequence is characterized by a gene encoding an HMG protein, while the hallmark of the other is a gene encoding a protein with an α1 domain showing similarity to the Matα1p protein of Saccharomyces cerevisiae. DNA-binding HMG proteins are ubiquitous and well characterized. In contrast, α1 domain proteins have limited distribution and their evolutionary origin is obscure, precluding a complete understanding of mating-type evolution in Ascomycota. Although much work has focused on the role of the S. cerevisiae Matα1p protein as a transcription factor, it has not yet been placed in any of the large families of sequence-specific DNA-binding proteins. METHODOLOGY/PRINCIPAL FINDINGS: We present sequence comparisons, phylogenetic analyses, and in silico predictions of secondary and tertiary structures, which support our hypothesis that the α1 domain is related to the HMG domain. We have also characterized a new conserved motif in α1 proteins of Pezizomycotina. This motif is immediately adjacent to and downstream of the α1 domain and consists of a core sequence Y-[LMIF]-x(3)-G-[WL] embedded in a larger conserved motif. CONCLUSIONS/SIGNIFICANCE: Our data suggest that extant α1-box genes originated from an ancestral HMG gene, which confirms the current model of mating-type evolution within the fungal kingdom. We propose to incorporate α1 proteins in a new subclass of HMG proteins termed MATα_HMG

Systematic Deletion of Homeobox Genes in Podospora anserina Uncovers Their Roles in Shaping the Fruiting Body

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures

Wood Utilization Is Dependent on Catalase Activities in the Filamentous Fungus Podospora anserina

Catalases are enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. They are implicated in various physiological and pathological conditions but some of their functions remain unclear. In order to decipher the role(s) of catalases during the life cycle of Podospora anserina, we analyzed the role of the four monofunctional catalases and one bifunctional catalase-peroxidase genes present in its genome. The five genes were deleted and the phenotypes of each single and all multiple mutants were investigated. Intriguingly, although the genes are differently expressed during the life cycle, catalase activity is dispensable during both vegetative growth and sexual reproduction in laboratory conditions. Catalases are also not essential for cellulose or fatty acid assimilation. In contrast, they are strictly required for efficient utilization of more complex biomass like wood shavings by allowing growth in the presence of lignin. The secreted CATB and cytosolic CAT2 are the major catalases implicated in peroxide resistance, while CAT2 is the major player during complex biomass assimilation. Our results suggest that P. anserina produces external H2O2 to assimilate complex biomass and that catalases are necessary to protect the cells during this process. In addition, the phenotypes of strains lacking only one catalase gene suggest that a decrease of catalase activity improves the capacity of the fungus to degrade complex biomass

Podospora anserina bibliography n° 10 - Additions

Podospora anserina is a coprophilous fungus growing on herbivore dung. It is a pseudohomothallic species in which ascus development results, as in Neurospora tetrasperma but through a different process, in the formation of four large ascospores containing nuclei of both mating types

Mise au point d'un systeme de transformation du champignon filamenteux Podospora anserina et recherche de sequences susceptibles d'assurer l'autonomie de replication

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Podospora anserina bibliography n° 10 - Additions

Podospora anserina is a coprophilous fungus growing on herbivore dung. It is a pseudohomothallic species in which ascus development results, as in Neurospora tetrasperma but through a different process, in the formation of four large ascospores containing nuclei of both mating types

Les évènements d'inactivation génique chez le champignon filamenteux Podospora anserina (description et relation avec le développement sexué)

Le gène ami1 impliqué dans la distribution nucléaire a été cloné et délété chez P. anserina. Le croisement homozygote pour une délétion d'ami1 a montré des anomalies de distribution nucléaire et un effet sur la fréquence de perte de fonction de transgènes durant le cycle sexué. J'ai montré que le RIP (un mécanisme d'inactivation génique décrit chez N. crassa) et les recombinaisons préméiotiques augmentent dans un croisement homozygote pour une délétion d'ami1. Pour expliquer cette observation, nous avons proposé une relation entre l'efficacité des mécanismes d'inactivations et les processus cellulaires au cours du développement. Le RIP agit entre la fécondation et la réplication préméiotique. Plusieurs gènes intervenant à des étapes déterminées au cours de cette période ont été étudiés chez P. anserina, des mutations dans ces gènes conduisent à des phénotypes caractéristiques. La perte de fonction de ces gènes par RIP, à l'état de transgène, avant ou après leur intervention dans le développement sexué, devrait permettre de déterminer avec précision à quel moment se produit ce phénomène d'inactivation par rapport à l'étape qu'ils contrôlent. Les expériences sont en cours. Le gène SMR2 dupliqué en répétition inverse introduit dans P. anserina est capable de s'auto-inactiver et d'induire une inactivation épigénétique réversible du gène SMR2 résident. Certaines propriétés de cette inactivation suggèrent que le mécanisme responsable pourrait être apparenté aux phénomènes d'extinction génique agissant au niveau post-transcriptionnel.ami1, a gene involved in nuclear distribution, was cloned and deleted in P. anserina. The ami1 alteration led to abnormal nuclear distribution and affected loss of transgenes functions during the sexual phase. This observation prompted us to study more precisely the homology-dependent gene silencing mechanisms and their relationships with the sexual development in P. anserina. We show that RIP (a gene silencing mechanism described in N. crassa) and premeiotic recombinations are highly increased in deleted ami1 homozygous cross. To explain this observation, we proposed a relationship between silencing mechanisms efficiency and cellular process during the development. In fact, a sexual development slow-down was observed in deleted ami1 homozygous cross. Because of this slow-down, the nuclei would be much more exposed to gene silencing mechanisms deleted ami1 context than in ami1+ context.RIP acts between fertilisation and premeiotic replication. Several genes, which control developmental events between these two stages, were characterised in P. anserina. Mutations in these genes display specific phenotypes. RIP induced loss of function of these genes (introduced as duplicated transgenes) before or after their intervention in sexual development, should allow precise determination of the timing of RIP relative to stages controlled by these genes. The experiences are in progress.Inverted repeat of SMR2 gene introduced in P. anserina is able to induce self-silencing and reversible epigenetic inactivation of the resident SMR2 gene. Several lines of evidence suggest that gene silencing of SMR2 occurred at post-transcriptional level.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Recherche des gènes impliqués dans le développement sexué du champignon Podospora anserina

Le champignon filamenteux, Podospora anserina, possède deux types sexuels, mat+ et mat-, caractérisés chacun par une séquence spécifique. La séquence mat+ contient un seul gène FPR1; la séquence mat- contient trois gènes : FMR1, SMR1 et SMR2. La fonction moléculaire de SMR1 est inconnue, les autres gènes codent des facteurs de transcription qui contrôlent la fécondation (reconnaissance intercellulaire), et le passage d un syncytium à un hyphe spécialisé binucléé contenant un noyau mat+ et un noyau mat- (reconnaissance internucléaire). Il n y a pas eu d analyse exhaustive des gènes impliqués dans la reconnaissance intercellulaire et le mécanisme de la reconnaissance internucléaire est encore inconnu. Afin de déterminer les cibles de FPR1 et FMR1, et les différents mécanismes impliqués, nous avons utilisé une approche microarray. Le profil transcriptomique des souches mat+ et mat- compétentes pour la fécondation a permis d identifier 157 gènes cibles, et l analyse transcriptomique des souches mutantes fpr1- et fmr1- a révélé que ces cibles peuvent être soit réprimées, soit activées par FMR1 ou FPR1, ou être sous le contrôle de ces deux facteurs. Ces expériences ont aussi détecté l existence de 10 gènes activés ou réprimés au même niveau dans mat+ et mat-. La délétion de 32 gènes choisis parmi ces 167 gènes cibles n a permis de mettre en évidence que deux gènes impliqués dans la fécondation. Les comparaisons des gènes cibles des facteurs de transcription MAT de Gibberella moniliformis et Sordaria macrospora avec ceux de P. anserina révèlent un nombre significatif de gènes cibles communs entre ces espèces, mais ces gènes ont des profils transcriptomiques différents, soulevant la question du rôle de ces gènes cibles. La recherche des gènes cibles de FPR1, FMR1 et SMR2 impliqués dans la reconnaissance internuléaire a été effectuée en comparant le transcriptome des périthèces issus de deux croisements, l un n exprimant que les gènes spécifiques mat+, l autre que les gènes spécifiques mat-. Les résultats ont été interprétés selon le modèle d identité nucléaire et le modèle de ségrégation aléatoire. Le premier modèle a conduit à l identification de 27 gènes cibles, tandis que 154 gènes cibles ont été identifiés en appliquant le deuxième modèle. Au total 46 souches mutantes ont été construites. Cependant aucune délétion n a affecté le développement sexué. En parallèle de ces expériences transcriptomiques, nous avons invalidé tous les gènes à HMG-box de P. anserina. Les résultats montrent que ces derniers ont un rôle très important dans le développement sexué, particulièrement Pa_1_13940 qui code un régulateur des gènes des types sexuels, le premier identifié chez les Pezizomycotina.The filamentous fungus, Podospora anserina, has two mating-type idiomorphs, mat+ and mat-. The mat+ sequence contains one gene FPR1, while mat- contains three genes: FMR1, SMR1 and SMR2. The molecular function of SMR1 is unknown, FPR1, FMR1 and SMR2 encode transcriptional regulators which control the fertilization (intercellular recognition) and the transition from a syncytium to a specialized dikaryotic hypha which contains one mat+ and one mat- nucleus (internuclear recognition). No exhaustive analysis is available for the genes involved in the intercellular recognition, while the mechanism of the internuclear recognition is unknown. In order to understand the mechanism of these events and to identify the target genes of mating-type transcription factors, we used a microarray approach. The transcriptomic profiles of the mat+ and mat- strains that are competent for fertilization revealed 157 differentially transcribed genes, and transcriptomic analysis of fmr1- and fpr1- mutant strains was used to determine the regulatory actions exerted by FMR1 and FPR1 on these differentially transcribed genes. All possible combinations of transcription repression and/or activation by FMR1 and/or FPR1 were observed. Furthermore, 10 additional mating-type target genes were identified that were up- or down-regulated to the same level in mat+ and mat- strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. A comparison with similar data set from the two ascomycetes, Gibberella moniliformis and Sordaria macrospora, reveals significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species, questioning the function of these target genes. Internuclear recognition was investigated by the transcriptomic analysis of perithecia from two crosses expressing mat+ and mat- genes, respectively. The tow internuclear recognition models: nuclear identity and random segregation, were used to interpret our results. According to the former model, 27 target genes have been identified, while 154 target genes were identified with the latter model. A total of 46 mutant strains were constructed. However, these strains showed no defects in sexual development. Besides this microarray experiences, we have invalidated all HMG-box genes of P. anserina. The results show that the HMG-box genes have a very important role in sexual development, especially Pa_1_13940 which encodes the first identified regulator of Pezizomycotinan mating-type genes.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF