Fungal Model System Research Articles

Chapter Four - The Filamentous Fungus Sordaria macrospora as a Genetic Model to Study Fruiting Body Development

Filamentous fungi are excellent experimental systems due to their short life cycles as well as easy and safe manipulation in the laboratory. They form three-dimensional structures with numerous different cell types and have a long tradition as genetic model organisms used to unravel basic mechanisms underlying eukaryotic cell differentiation. The filamentous ascomycete Sordaria macrospora is a model system for sexual fruiting body (perithecia) formation. S. macrospora is homothallic, i.e., self-fertile, easily genetically tractable, and well suited for large-scale genomics, transcriptomics, and proteomics studies. Specific features of its life cycle and the availability of a developmental mutant library make it an excellent system for studying cellular differentiation at the molecular level. In this review, we focus on recent developments in identifying gene and protein regulatory networks governing perithecia formation. A number of tools have been developed to genetically analyze developmental mutants and dissect transcriptional profiles at different developmental stages. Protein interaction studies allowed us to identify a highly conserved eukaryotic multisubunit protein complex, the striatin-interacting phosphatase and kinase complex and its role in sexual development. We have further identified a number of proteins involved in chromatin remodeling and transcriptional regulation of fruiting body development. Furthermore, we review the involvement of metabolic processes from both primary and secondary metabolism, and the role of nutrient recycling by autophagy in perithecia formation. Our research has uncovered numerous players regulating multicellular development in S. macrospora. Future research will focus on mechanistically understanding how these players are orchestrated in this fungal model system.

Enhanced glycosyl hydrolase production in Aspergillus nidulans using transcription factor engineering approaches

Background: Engineered fungi are attractive platforms for the production of plant cell wall hydrolytic enzymes which, among other biotechnological applications, are required for the efficient conversion of biomass to glucose and other fermentable sugars. As a fungal model system, Aspergillus nidulans provides genetic tools that are of relevance in this context and potentially applicable to industrially important filamentous fungi. The goal of this study is to assess the utility of A. nidulans as a host for recombinant protein production. Results: We have successfully applied a transcription factor engineering approach to improve the efficiency of the A. nidulans xylanolytic XlnR-xlnp expression system. Specifically, endo-1,4-β-xylanases and an α-L-rhamnosidase were chosen as representatives of endogenous and heterologous glycosyl hydrolases involved in plant cell wall deconstruction. By deregulating the expression of the xylanolytic transcriptional activator XlnR and modulating the activity of the pH regulator PacC we improved protein production and reduced production times. Xylanase activity was about 200-fold greater in gpdAp::xlnR strains compared to controls 4 hours after transfer to inducing conditions, and 10-fold greater after 24 hours. Remarkably, 75% of the xylanase activity was present in the engineered strains within 4 hours. Engineering XlnR expression also had a considerable impact on foreign protein production, especially when the promoter of the 'acidic' xlnB gene was used to express the transgene. α-L-rhamnosidase activity in xlnBp::rhaA, gpdAp::xlnR strains was about 19-fold greater than that of controls 72 hours after transfer to xylan (about 85% of the total activity produced), and 10-fold greater at later times (120 hours). The performance of these strains was further enhanced by impeding the proteolytic activation of PacC; introduction of the palA1 allele in xlnBp::rhaA, gpdAp::xlnR strains resulted in an additional 2.7-fold increase in α-L-rhamnosidase activity by 48 hours (about 87% of the total activity produced) and a 1.7-fold increase at later times. Conclusions: Our results show that the XlnR-xlnp expression system is a valuable tool for manipulating the production of plant cell wall degrading enzymes in A. nidulans and establish the biotechnological potential of the transcription factors XlnR and PacC to boost and control the strength of xylanolytic promoters.

Lipid composition is a determinant for human defensin hnp1 selectivity

Human neutrophilpeptide 1 (HNP1) is a human defensin with antimicrobial activity against different bacteria (both Gram-positive and negative), fungi, and viruses. HNP1 is stored in the cytoplasmic azurophilic granules of neutrophils. To elucidate the mode of action of this antimicrobial peptide, studies based on its lipid selectivity were carried out. Large unilamellar vesicles with different lipid compositions were used as biomembranes model systems (mammal, fungal, and bacterial models). Changes on the intrinsic fluorescence of HNP1 upon membrane binding/insertion show that HNP1 has quite distinct preferences for mammalian and fungal membrane model systems. HNP1 showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a higher partition for ergosterol-containing membranes (as fungal membranes) and lower interaction with cholesterol-containing membranes (as in mammalian cells). These results reveal that lipid selectivity is a determinant step for HNP1 action. Fluorescence quenching data obtained using acrylamide indicate that HNP1 interacts with membranes without a full insertion in the lipid bilayer. Generalized polarization of laurdan indicates a change in membrane fluidity in the presence of HNP1 for POPC membranes but not for ergosterol-enriched membranes.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XlnR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-d-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xlnA and xlnD mRNA accumulation. The ΔfbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi.

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.

Transient binding of dynein controls bidirectional long-range motility of early endosomes

In many cell types, bidirectional long-range endosome transport is mediated by the opposing motor proteins dynein and kinesin-3. Here we use a fungal model system to investigate how both motors cooperate in early endosome (EE) motility. It was previously reported that Kin3, a member of the kinesin-3 family, and cytoplasmic dynein mediate bidirectional motility of EEs in the fungus Ustilago maydis. We fused the green fluorescent protein to the endogenous dynein heavy chain and the kin3 gene and visualized both motors and their cargo in the living cells. Whereas kinesin-3 was found on anterograde and retrograde EEs, dynein motors localize only to retrograde organelles. Live cell imaging shows that binding of retrograde moving dynein to anterograde moving endosomes changes the transport direction of the organelles. When dynein is leaving the EEs, the organelles switch back to anterograde kinesin-3-based motility. Quantitative photobleaching and comparison with nuclear pores as an internal calibration standard show that single dynein motors and four to five kinesin-3 motors bind to the organelles. These data suggest that dynein controls kinesin-3 activity on the EEs and thereby determines the long-range motility behavior of the organelles.

Editorial [Hot Topic: Fungal Model Systems to Understand the Mechanisms Unravelling the Oxidative Stress Response (Guest Editor: Maria Angeles de la Torre Ruiz)

Editorial [Hot Topic: Fungal Model Systems to Understand the Mechanisms Unravelling the Oxidative Stress Response (Guest Editor: Maria Angeles de la Torre Ruiz)

Mitochondrial pathways governing stress resistance, life, and death in the fungal aging model Podospora anserina

Work from more than 50 years of research has unraveled a number of molecular pathways that are involved in controlling aging of the fungal model system Podospora anserina. Early research revealed that wild-type strain aging is linked to gross reorganization of the mitochondrial DNA. Later it was shown that aging of P. anserina does also take place, although at a slower pace, when the wild-type specific mitochondrial DNA rearrangements do not occur. Now it is clear that a network of different pathways is involved in the control of aging. Branches of these pathways appear to be connected and constitute a hierarchical system of responses. Although cross talk between the individual pathways seems to be fundamental in the coordination of the overall system, the precise underlying interactions remain to be unraveled. Such a systematic approach aims at a holistic understanding of the process of biological aging, the ultimate goal of modern systems biology.

Modelling and mutational evidence identify the substrate binding site and functional elements in APC amino acid transporters

The Amino acid-Polyamine-Organocation (APC) superfamily is the main family of amino acid transporters found in all domains of life and one of the largest families of secondary transporters. Here, using a sensitive homology threading approach and modelling we show that the predicted structure of APC members is extremely similar to the crystal structures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities. All of these proteins, despite having no primary amino acid sequence similarity, share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. Based on this model, we reviewed available data on functional mutations in bacterial, fungal and mammalian APCs and obtained novel mutational data, which provide compelling evidence that the amino acid binding pocket is located in the vicinity of the unwound part of two broken helices, in a nearly identical position to the structures of similar transporters. Our analysis is fully supported by the evolutionary conservation and specific amino acid substitutions in the proposed substrate binding domains. Furthermore, it allows predictions concerning residues that might be crucial in determining the specificity profile of APC members. Finally, we show that two cytoplasmic loops constitute important functional elements in APCs. Our work along with different kinetic and specificity profiles of APC members in easily manipulated bacterial and fungal model systems could form a unique framework for combining genetic, in-silico and structural studies, for understanding the function of one of the most important transporter families.

CD8– dendritic cells preferentially cross‐present Saccharomyces cerevisiae antigens

Mouse splenic dendritic cell (DC) subsets possess distinct antigen-presentation abilities. CD8(+) DC are specialized in cross-presentation of antigens to CD8(+) T cells, whereas CD8(-) DC are more efficient in antigen presentation to CD4(+) T cells. In this study, we examined the capacity of CD8(+) and CD8(-) DC subsets to present fungal antigens in MHC class I and II molecules to CD8(+) and CD4(+) T cells, respectively. We used ovalbumin-expressing Saccharomyces cerevisiae (yeast-OVA) as a fungal model system. Both CD8(+) and CD8(-) DC subsets phagocytosed yeast in equal amounts and uptake was mediated via dectin-1. In addition, both DC subsets induced similar OVA-specific CD4(+) T cell proliferation after incubation with yeast-OVA. However, the induction of OVA-specific CD8(+) T cell activation was largely restricted to the CD8(-) DC subset. Furthermore, only CD8(-) DC produced cytokines such as IL-10 and TNF-alpha and increased IL-23p19 and IL-23p40 mRNA levels in response to yeast. Our results strongly suggest that DC subsets have different functions in the elicitation of adaptive immune responses in vivo.

Effects of Propagule Density and Survival Strategies on Establishment and Growth: Further Investigations in the Phylloplane Fungal Model System

This work builds on an earlier culture study where we determined that species diversity of competing saprotrophic phyllpolane fungi had only a negligible effect on the establishment and coexistence of a target fungus, Pestalotia vaccinii. Here, we explore preliminary evidence suggesting that spore density is a more important contributing factor to colonization and coexistence. We examine the influence of propagule density in vitro on establishment and growth of select members of the phylloplane of Vaccinium macrocarpon (American cranberry). To evaluate the response of the weak pathogen P. vaccinii to changes in competitors spore density, we chose saprotrophs from the previous investigation that had the greatest inhibitory effect on the establishment of P. vaccinii (Curvularia lunata), an intermediate inhibitory effect (Alternaria alternata) and the least inhibitory effect (Penicillium sp.). A constant target spore concentration of 50 viable spores of P. vaccinii was pit against densities of the three individual competitors ranging between 12 and 200 spores. As viable propagule density increased, establishment and coexistence of P. vaccinii significantly decreased, with C. lunata and A. alternata decreasing the growth of P. vaccinii more than Penicillium sp. Concomitantly, both C. lunata and Penicillium sp. were not significantly affected by overall spore density but were significantly affected by the presence of P. vaccinii. A. alternata, on the other hand, was not significantly influenced by the presence of P. vaccinii but was significantly affected by overall spore density. An in vitro investigation into the effect of interspecific competition on mycelial growth suggests how different survival strategies and community assembly rules might influence both growth and development. Growth of P. vaccinii was significantly less when interacting with C. lunata than when interacting with either A. alternata or Penicillium sp. Conversely, P. vaccinii had the greatest effect on the growth of C. lunata, less of an effect on the growth of A. alternata, and the least effect on Penicillium sp.

Model systems for studying the biology of filamentous fungi: Rumors of their death should be postponed

If judged by the almost doubling of the number of publications concerning fungalrelated topics during the past decade, fungal research is ourishing. The motivation that fuels the study of lamentous fungi ranges from the use of fungi as model systems for the study of eukaryote biology in general and fungal biology specically , to facing plant, animal and human fungal-based health threats and to the harnessing of fungi (or their traits) for industrial applications (19). The availability of the complete genome sequence of an organism provides an invaluable tool for hypothesis-driven research as well as setting the basis for comparative analyses. Coupled with the technological advances that allow for genetic and environmental manipulation of a growing number of fungi, a genome database can be used for functional analysis of almost any conceivable fungal trait. Furthermore, with the proper efforts invested, almost any fungal species can be rendered amenable (with various limitations) to genetic manipulations. One of the outcomes of the technological progress made is the raising of doubts concerning the future validity, potential contribution and, subsequently, justication for continued support for research of so-called ‘fungal model systems’ (and, more specically , lamentous fungi). Model systems are usually chosen on the basis of amenability to growth, manipulation and analysis (including the capability to

A synchrotron FTIR microspectroscopy investigation of fungal hyphae grown under optimal and stressed conditions

Synchrotron FTIR can provide high spatial resolution (<10 microm pixel size) in situ biochemical analyses of intact biotissues, an area of increasing importance in the post-genomic era, as gene functions and gene networks are coming under direct scrutiny. With this technique, we can simultaneously assess multiple aspects of cell biochemistry and cytoplasmic composition. In this paper, we report the first results of our synchrotron FTIR examination of hyphae of three important fungal model systems, each with sequenced genomes and a wealth of research: Aspergillus, Neurospora, and Rhizopus. We have analyzed the FTIR maps of Aspergillus nidulans cells containing the hypA1 allele, a well-characterized single-gene temperature-sensitive morphogenetic mutation. The hypA1 cells resemble wildtype at 28 degrees C but have growth defects at 42 degrees C. We have also investigated Neurospora and Rhizopus cultures grown in media with optimal or elevated pH. Significant differences between the spectra of the three fungi are likely related to differences in composition and structure. In addition, high spatial resolution synchrotron FTIR spectroscopy provides an outstanding method for monitoring subtle subcellular changes that accompany environmental stress.

Cryptococcus neoformans: the yeast that likes it hot

The ability of fungi to grow well at mammalian body temperatures is a fundamental characteristic of invasive human fungal pathogens. Cryptococcus neoformans, with its genetics, molecular biology, robust animal models and clinical importance, has become a premier fungal model system for molecular fungal pathogenesis studies. There has been over a half century of study into how C. neoformans grows at high temperatures. However, recently the understanding of high-temperature growth at a molecular level has dramatically accelerated. Many strategies have been used to identify genes and over a dozen genes have already been identified to be necessary for high-temperature growth. It is likely that there are many more to discover. It is clear that, as further studies understand how this encapsulated yeast is able to withstand the stresses of high temperature at a genetic and molecular basis, we will also know more about how it and other fungal pathogens have evolved into well-established human pathogens.

Impact of ROS on ageing of two fungal model systems: Saccharomyces cerevisiae and Podospora anserina

To provide a foundation for the development of effective interventions to counteract various age-related diseases in humans, ageing processes have been extensively studied in various model organisms and systems. However, the mechanisms underlying ageing are still not unravelled in detail in any system including rather simple organisms. In this article, we review some of the molecular mechanisms that were found to affect ageing in two fungal models, the unicellular ascomycete Saccharomyces cerevisiae and the filamentous ascomycete Podospora anserina. A selection of issues like retrograde response, genomic instability, caloric restriction, mtDNA reorganisation and apoptosis is presented and discussed with special emphasis on the role reactive oxygen species (ROS) play in these diverse molecular pathways.

The HSP90 chaperone complex, an emerging force in plant development and phenotypic plasticity

The essential cellular functions of the molecular chaperone HSP90 have been intensively investigated in fungal and mammalian model systems. Several recent publications have highlighted the importance of this chaperone complex in plant development and responsiveness to external stimuli. In particular, HSP90 is crucial for R-protein-mediated defense against pathogens. Other facets of HSP90 function in plants include its involvement in phenotypic plasticity, developmental stability, and buffering of genetic variation. Plants have emerged as powerful tools that complement other model systems in attempts to extend our knowledge of the myriad impacts of protein folding and chaperone function.

A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to γ-glutamylcysteine and lacks phytochelatin synthase activity

Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to γ-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC–MSMS analysis was unequivocally identified as γ-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to γ-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd 2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.

Effects of Species Diversity on Establishment and Coexistence: A Phylloplane Fungal Community Model System

A model system was devised, evaluating the influence that species diversity (species richness) has on fungal establishment and coexistence. Seven members of the fungal phylloplane community of Vaccinium macrocarpon (American cranberry) were selected to assess how species diversity affected development and coexistence of another community member, Pestalotia vaccinii. Pestalotia was engaged in competitive interactions on 1% Malt Extract Agar (MEA) petri dishes with each of the seven individual saprotrophs (two-way interaction), in random combinations with three of the seven saprotrophs (four-way interaction), and in random combinations with five of the seven saprotrophs (six-way interaction). The saprotrophic fungi used in this study were Aspergillus sp., Alternaria alternata, Cladosporium cladosporoides, Curvularia lunata, Epicoccum purpuracens, Penicillium sp., and Pithomyces chartarum. We hypothesized that species diversity would have a significant impact on the establishment and coexistence of Pestalotia vaccinii in culture. In an effort to minimize density-dependent effects, the number of viable spores employed in the three types of interactions was kept constant. Target spore concentrations of 50 viable spores of P. vaccinii and 50 saprotroph spores were used, regardless of the number of species involved in the interaction. This proved to be a very important factor in the experiment. As our results show, species diversity had little or no effect on the establishment and coexistence of Pestalotia vaccinii; however, spore density played an extremely important role in the establishment and development of fungal propagules in our model.

Use of the Black Yeast Phaeococcomyces Fungal Melanin Model System for Preparation of 1,3,6,8‐Tetrahydroxynaphthalene and the Other Components of the DHN Fungal Melanin Pathway

Compounds of the dihydroxynaphthalene (DHN) fungal melanin pathway are in short supply worldwide, especially 1,3,6,8‐tetrahydroxynaphthalene, the chemically unstable starting point of the pathway, and are not commercially available. This is becoming an impediment to physiological survey work in the DHN pathway. We describe the use of our black yeast Phaeococcomyces DHN melanin model system to produce sufficient amounts of 1,3,6,8‐tetrahydroxynaphthalene for physiological studies, using a simple modified culture method that uses an oxygen‐scavenging growth medium containing cysteine and thioglycollate, which is also sealed with an oxygen‐excluding layer of the black yeast. The medium also contains the DHN pathway reductase enzyme inhibitor tricyclazole, which causes excretion of 1,3,6,8‐tetrahydroxynaphthalene and 1,3,8‐dihydroxynaphthalene. The presence of oxygen scavengers and the thick yeast growth prevents oxidation of the hydroxynaphthalenes. Most of the other components of the DHN fungal melanin pathway can be produced using the black yeast or using a scytalone dehydratase negative mutant of the black yeast that accumulates scytalone.

Conidiation induction in Penicillium

Asexual spores or conidia are dispersive propagules produced as an alternative to vegetative growth by a diverse group of filamentous fungi. The cellular development programmes which govern conidiation have been intensely studied in the last few decades, although important gaps stand in the way of our understanding of this phenomenon, namely in the areas of the environmental sensing mechanisms and signal transduction pathways. The aim of this review is to summarize the current advances in conidiation induction in the genus Penicillium, and to put them into context with the state of our knowledge stemming from work in widely studied fungal model systems.