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Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus Podospora anserina. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate.

Filamentous fungi frequently present degenerative processes, whose molecular basis is very often unknown. Here, we present three mutant screens that result in the identification of 29 genes that directly or indirectly control Crippled Growth (CG), an epigenetic cell degeneration of the filamentous ascomycete Podospora anserina. Two of these genes were previously shown to encode a MAP kinase kinase kinase and an NADPH oxidase involved in a signal transduction cascade that participates in stationary phase differentiations, fruiting body development and defence against fungal competitors. The numerous genes identified can be incorporated in a model in which CG results from the sustained activation of the MAP kinase cascade. Our data also emphasize the complex regulatory network underlying three interconnected processes in P. anserina: sexual reproduction, defence against competitors, and cell degeneration.

Background: Bistability and hysteresis are increasingly recognized as major properties of regulatory networks governing numerous biological phenomena, such as differentiation and cell cycle progression. The full scope of the underlying molecular mechanisms leading to bistability and hysteresis remains elusive. Nectria haemaotcocca, a saprophytic or pathogenic fungus with sexual reproduction, exhibits a bistable morphological modification characterized by a reduced growth rate and an intense pigmentation. Bistability is triggered by the presence or absence of sigma, a cytoplasmic determinant. This determinant spreads in an infectious manner in the hyphae of the growing margin, insuring hysteresis of the differentiation., Results: Seven mutants specifically affected in the generation of sigma were selected through two different screening strategies. The s1 and s2 mutations completely abolish the generation of sigma and of its morphological expression, the Secteur. The remaining five mutations promote its constitutive generation, which determines an intense pigmentation but not growth alteration. The seven mutations map at the same locus, Ses (for 'Secteur-specific'). The s2 mutant was obtained by an insertional mutagenesis strategy, which permitted the cloning of the Ses locus. Sequence and transcription analysis reveals that Ses is composed of two closely linked genes, SesA, mutated in the s1 and s2 mutant strains, and SesB, mutated in the s* mutant strains. SesB shares sequence similarity with animal and fungal putative proteins, with potential esterase/lipase/thioesterase activity, whereas SesA is similar to proteins of unknown function present only in the filamentous fungi Fusarium graminearum and Podospora anserina., Conclusions: The cloning of Ses provides evidence that a system encoded by two linked genes directs a bistable and hysteretic switch in a eukaryote. Atypical regulatory relations between the two proteins may account for the hysteresis of Secteur differentiation.

Morphological plasticity is a hallmark of eumycetes. In addition to genes and environment, epigenetic factors control cell, colony and thallus forms in many species, by creating reversible switches. Current knowledge indicates that the different shapes are due to structural or regulatory heritable states of cytoplasmic components. Cellular physiology differs in the various forms, permitting adaptation to fluctuation in the environment. These switches are part of the adaptation repertoire that fungi exhibit to colonize most niches.

eEF1A is encoded by a unique gene in the filamentous fungus Podospora anserina. We show here that (1) this gene is essential for vegetative growth, (2) readthrough at UGA stop codon level is positively correlated with eEF1A level, (3) eEF1A level is regulated in P. anserina. (4) Increasing eEF1A gene dosage does not modify P. anserina life cycle parameters, especially longevity is not changed. These data confirm and extend those previously obtained in yeast and Drosophila.

In an attempt to decipher their role in the life history and senescence process of the filamentous fungus Podospora anserina, we have cloned the su1 and su2 genes, previously identified as implicated in cytosolic translation fidelity. We show that these genes are the equivalents of the SUP35 and SUP45 genes of Saccharomyces cerevisiae, which encode the cytosolic translation termination factors eRF3 and eRF1, respectively. Mutations in these genes that suppress nonsense mutations may lead to drastic mycelium morphology changes and sexual impairment but have little effect on life span. Deletion of su1, coding for the P. anserina eRF3, is lethal. Diminution of its expression leads to a nonsense suppressor phenotype whereas its overexpression leads to an antisuppressor phenotype. P. anserina eRF3 presents an N-terminal region structurally related to the yeast eRF3 one. Deletion of the N-terminal region of P. anserina eRF3 does not cause any vegetative alteration; especially life span is not changed. However, it promotes a reproductive impairment. Contrary to what happens in S. cerevisiae, deletion of the N terminus of the protein promotes a nonsense suppressor phenotype. Genetic analysis suggests that this domain of eRF3 acts in P. anserina as a cis-activator of the C-terminal portion and is required for proper reproduction.

We have cloned and sequenced the gene encoding the translation elongation factor eEF1A from two filamentous fungi, Podospora curvicolla and Sordaria macrospora. These fungi are close relatives of Podospora anserina and also show senescence syndromes. Comparison of the sequences of the deduced proteins with that of P. anserina reveals that the three proteins differ in several positions. Replacement of the P. anserina gene by either of the two exogenous genes does not entail any modification in P. anserina physiology; the longevity of the fungus is not affected. No alteration of in vivo translational accuracy was detected; however, the exogenous proteins nonetheless promoted a modification of the resistance to the aminoglycoside antibiotic paromomycin. These data suggest that optimization of life span between these closely related fungi has likely not been performed during evolution through modifications of eEF1A activity, despite the fact that mutations in this factor can drastically affect longevity., (Copyright 1997 Academic Press.)

Two genetic methods were used to estimate the number of genes that potentially modulate longevity in the filamentous fungus Podospora anserina. First, life span of strains carrying mutations selected on criteria unrelated to senescence was measured. Second, strains bearing random mutations were generated by insertional mutagenesis. Life span of these strains was then measured. Surprisingly, both methods lead to the conclusion that a large number of genes (between 600 and 3000) can modulate life span. Among, the mutations that affect longevity, 50% increase life span and 50% diminish it.

Various translation initiation and elongation factors seem to participate in the control of the cellular proliferation and the ageing process in higher eukaryotes. Studies indicate that EF-1 alpha, one of the translation elongation factors, may be one of the major components involved. We here present the cloning of the filamentous fungus P. anserina EF-1 alpha encoding gene and show that strains bearing high fidelity mutations in the EF-1 alpha gene have a drastically increased longevity as well as an impairment in sporulation. This suggests that EF-1 alpha involved in the sexual and senescence processes in lower eukaryotes, through the control of translational errors.

We have constructed new shuttle vectors to facilitate the screening of recombinant plasmids after direct transformation of yeast cells. The vectors are pBluescript-based shuttle vectors in which the lacZ marker has been replaced by an analogous system based on the Saccharomyces cerevisiae URA3 gene. DNA fragments are inserted in a polylinker located after the beginning of the URA3 coding sequence. Transformants are selected either by Trp or Leu prototrophy. Plasmids bearing an insert are selected by growth on 5-fluoro-orotic acid (5-FOA), a uracil analog toxic to cells containing a functional URA3+ gene (thus, this method requires the recipient strain to be ura3-); only cells containing a plasmid with an insert that disrupts the functional continuity of the URA3 gene can grow on medium containing 5-FOA. Using these plasmids, we were able to directly reclone the ACE1 gene from genomic DNA by directly transforming a strain deleted for ACE1. These vectors can be used for a variety of purposes including rapid cloning of genes by complementation or expression of fusion genes driven from the URA3 promoter.