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Cellular and Molecular Biology of Filamentous Fungi
Editors: Katherine A. Borkovich1, Daniel J. Ebbole2
Affiliations: 1: Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521; 2: Department of Plant Pathology and Microbiology, Program for the Biology of Filamentous Fungi, Texas A&M University, College Station, TX 77843-2132
Content Type: Monograph
Format: Electronic, Hardcover
Category: Microbial Genetics and Molecular Biology; Fungi and Fungal Pathogenesis
Print ISBN : 9781555814731
e-ISBN : 9781555816636
Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase
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Incorporating the latest findings from such disciplines as physiology, taxonomy, genomics, molecular biology and cell biology, this publication is an ideal starting point for any research study of filamentous fungi. Its extensive use of illustrations and tables also makes it an excellent textbook for both graduate and undergraduate students, helping them easily grasp and apply new concepts.
Cellular and Molecular Biology of Filamentous Fungi begins with an historical perspective, cell morphology and taxonomy, and then moves on to such topics as cell growth, development, metabolism, and pathogenesis. The book covers model organisms in detail as well as a broad variety of dimorphic and "obligate" filamentous fungal species. You'll learn how individual species have adapted themselves to highly diverse ecological niches. In addition, comparisons to studies of the yeasts Saccharomyces cerevisiae and Schizosaccharomycespombe help you better understand the nature of fungi and the underlying evolution that leads to diversity in this fascinating kingdom.
Each chapter has been written by one or more leading experts. These authors, representing a "Who's Who" in the field of fungal biology, offer you the latest genomic-based approaches alongside a careful evaluation of the current literature. Presenting the full range of the fungal kingdom and covering such important topics as saprophytes, pathogens and endophytes, this publication captures our current understanding of fungal biology, all in one volume.
Hardcover, 568 pages, full-color insert, ullustrations, index.
I INTRODUCTION TO THE FILAMENTOUS FUNGI
Chapter 1 : History and Importance to Human Affairs
Authors: Joan W. Bennett, Rowland Davis, Carlene Raper
Scientifically, fungi are studied primarily for their impact on human affairs and for what they can tell us about fundamental biological principles. Fungi possess all the basic attributes of eukaryotes, including membrane-bound nuclei, chromatin, mitochondria, vacuoles, and cytoskeleton. While few fungi are obligate animal pathogens, some facultative species cause severe illness in humans, particularly in those who are immunocompromised. Among these are Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum. Up-to-date research on these animal pathogens as well as several plant pathogens is presented in this chapter on filamentous fungi. In addition to their ability to cause disease, many fungi damage materials useful to mankind by invading stored foods, fabrics, lumber, cellulose, and even plaster and cement. Both fungi (S. cerevisiae and the filamentous N. crassa) have served significantly in our understanding of vacuolar function and the role that this organelle plays in nitrogen and phosphate storage and amino acid metabolism. Since the earlier classical studies, fungal research has progressed rapidly with the advent of molecular biology. It is now possible to extract, manipulate, and amplify genes for reintroduction into most model fungi and thus determine cause and effect from genotype to phenotype. Research with the model species S. commune and Coprinopsis cinerea provides unique opportunities for understanding how so many different versions of these genes function at the molecular level to detect compatible mates. This volume is a testament to the continuing robustness of fungal systems as a source of insight into all of the life sciences.
Chapter 2 : Hyphal Structure
Authors: Robert W. Roberson, Maritza Abril, Meredith Blackwell, Peter Letcher, David J. McLaughlin, Rosa R. Mouri ÑO-PÉrez, Meritxell Riquelme, Maho Uchida
The ability to understand cytoplasmic structure can provide powerful insights into the biology of cells and organisms. This chapter has briefly reviewed the diversity of hyphal structures and presented examples of how bioimaging has contributed to a broader understanding of hyphal biology and phylogenetic relationships between fungal taxa. At the heart of polarized growth is the secretory pathway in which vesicles are targeted to sites of growth and subsequently fuse with the plasma membrane. In mature hyphae of the septate fungi, these events have given rise to the Spitzenkörper, a complex and dynamic structure that clearly influences hyphal growth and morphogenesis. The presence or absence of certain morphological characters (e.g., septa and Woronin bodies) already has been useful in defining higher taxa, especially since evolutionary polarity often can be established using stable phylogenetic trees based on DNA sequences. Ever-enlarging molecular databases, especially those of whole genomes, are allowing us to look for the genetic basis of many structural features, such as the presence or absence of Woronin body matrix proteins. This capability will allow us to understand the basis of these features not only in an evolutionary sense but also in a functional one. Collaboration among different types of fungal biologists including systematists is essential to understanding structure and how it applies to the study of the Fungi.
Chapter 3 : A Top-Down Systems Biology Approach for the Identification of Targets for Fungal Strain and Process Development
Authors: Machtelt Braaksma, Robert A. van den Berg, Mariët J. van der Werf, Peter J. Punt
This chapter discusses approaches to select targets for improvement of production processes, with a special focus on the application of functional genomics technologies as an unbiased approach towards target selection. The development of a fungal production process starts with the selection of a strain that produces the compound of interest or with the construction of such a strain. Often only the obvious targets for metabolic engineering are addressed. In this chapter such a systems biology approach, based on the information gathered with functional genomics technologies and in combination with multivariate data analysis tools, is discussed as a method to achieve unbiased selection and ranking of targets for both strain improvement and bioprocess optimization. Besides classical approaches for strain development, such as screening for protease mutants and targeted disruption of known protease genes, a top-down systems biology approach was applied to further elucidate the proteolytic system and its regulation in A. niger. The ultimate goal is to identify new targets for further improvement of the fungal cell factory for heterologous protein production. The available selection methods for relevant targets for fungal strain and process development, or for that matter any microbial production process, have been very successful in numerous cases. Recently introduced functional genomics technologies in combination with multivariate data analysis (MVDA) tools enable an open and comprehensive top-down systems biology approach towards target selection. Due to its unbiased nature, a successful top-down systems biology approach will provide a new boost in the ongoing cycle of bioprocess optimization.
Chapter 4 : Phylogenetics and Phylogenomics of the Fungal Tree of Life
Authors: Joseph W. Spatafora, Barbara Robbertse
Phylogenetic trees, once restricted to studies on systematics, are now used throughout all disciplines of fungal biology and provide the evolutionary context for a broad suite of studies that include understanding the evolution of major life forms, description of complex biotic communities, and predictive experimental biology. This is especially true in the genomic era, where a rapid convergence of phylogenetics and genomics is occurring and is resulting in the emerging field of phylogenomics. This chapter provides a review of (i) the current status of fungal phylogenetics based on multigene phylogenies, (ii) current evolutionary hypotheses on the evolutionary relationships of organisms that are classified in the Kingdom Fungi, and (iii) the use of genome-scale sampling to infer evolutionary relationships of the fungi. The goal of the Deep Hypha Research Coordination Network was to accelerate the collection of multigene sequence data across the Fungal Tree of Life. By the Fungal Tree of Life, we explicitly refer to the monophyletic Kingdom Fungi (Fungi) and all of its subgroups. One of the more elusive areas of research in fungal phylogenetics has been the calibration of the Fungal Tree of Life to geologic time. Here we focus on the use of genome-scale data sets in phylogenetic analyses. Multigene analyses have had a major impact on phylogenetic studies of the Fungi, resulting in our most thorough understanding of evolutionary relationships of the Kingdom to date.
Chapter 5 : Signal Transduction Pathways
Authors: Gyungsoon Park, Carol A. Jones, Katherine A. Borkovich
Most filamentous fungi are exquisitely sensitive to changes in their environment. Sensing and integration of signals from multiple sources require a complex web of signal transduction pathways. This chapter covers major signal transduction pathways that have been characterized in multiple species of filamentous fungi. The signaling pathways included are monomeric and heterotrimeric GTP-binding proteins, mitogen-activated protein kinases (MAPKs), protein kinase A/cyclic AMP (PKA/cAMP) signaling, two-component regulatory systems, calcium signaling, target of rapamycin (Tor) pathways and pH regulatory mechanisms. With the exception of two-component systems, related pathways are found in animals, where they also play fundamental roles. In general, the elements of these systems are found in all fungal species that have been sequenced; however, the number of genes representing each signaling protein class often varies. In spite of this conservation, several interesting variations in how pathway components are arranged or regulated have also emerged. Furthermore, accumulating evidence indicates that multiple pathways often cooperate to regulate the same function in the same species, resulting in more complex signal transduction networks. The response regulators from two-component regulatory systems have also been shown to regulate Hog1p-like cascades in filamentous fungi. Two-component regulatory systems are major signal transduction pathways in filamentous fungi.
II REPLICATION AND EXPRESSION OF THE GENETIC CODE
Chapter 6 : Mitotic Cell Cycle Control
Authors: Colin P. C. De Souza, Stephen A. Osmani
This chapter focuses on the current understanding of how control of the mitotic phase of the cell cycle is achieved in filamentous fungi. Survival of filamentous fungi requires exploration by rapid polarized growth to find nutritional requirements, as well as the production of large numbers of asexual and/or sexual spores that can lie dormant until suitable conditions trigger germination. In fungi, the mitotic microtubule organizing center is the spindle pole body, while in mammalian cells the centrosomes perform this function. As suggested by the fact that filamentous fungi often maintain many nuclei in a common cytoplasm, cytokinesis does not accompany every nuclear division. The process of cytokinesis during filamentous growth is generally achieved by the formation of a cross wall called a septum at a specific point in hyphae. The chapter talks about the biochemical activities that regulate mitotic entry, which are conserved in different filamentous fungi. It discusses some of the genes identified in the extragenic suppressor screens and highlights one particular extragenic suppressor screen. This screen, performed by Berl Oakley's lab, was for extragenic suppressors of the benA33β-tubulin mutant and led to the discovery of mipB, which encoded a new type of tubulin now known by its universal name, γ-tubulin. The chapter also discusses in detail how NIMA regulates changes in mitotic nuclear transport. Filamentous fungi provide rich and varied experimental opportunities to further our understanding of mitosis and its regulation. Until relatively recently, A. nidulans has been the workhorse of the filamentous fungi for studies of mitosis.
Chapter 7 : Meiosis
Authors: Claire Burns, Patricia J. Pukkila, Miriam E. Zolan
This chapter discusses the hallmarks of meiosis, crossover (chiasma) distribution, genes necessary for meiosis, and the transcriptional program of meiosis, with a focus on recent studies. A recent review, Neurospora as a model fungus for studies in cytogenetics and sexual biology at Stanford, is highly recommended as a complement to this chapter. Filamentous fungi display the interesting characteristic of maintaining haploid nuclei throughout mycelial development until nuclear fusion (karyogamy), which begins meiosis. Filamentous fungi have several advantages for the study of meiosis. First, the meiotic program and chromosome behavior in filamentous fungi are similar to those of more complex organisms. The genetic and cytological tractability of fungal systems allows genes to be well characterized, shedding light on the functions of conserved proteins. Second, unique aspects of development in filamentous fungi provide ideal conditions for analysis of certain meiotic events. Third, the chromosomes of some filamentous fungi are particularly accessible for meiotic study, within intact cells or by surface spreads of meiotic nuclei. Studies of mutants in filamentous fungi have made substantial contributions to the analysis of the two core features of meiosis: the structure of meiotic chromosomes and the role of interhomolog crossovers in meiotic chromosome structure and behavior. Regulation of meiosis at the epigenetic level, manipulation of recombination hot spots, and targeted disruption of cohesion complex components are also exciting targets for future analyses of the meiotic process in filamentous fungi.
Chapter 8 : DNA Repair and Recombination
Authors: P. Jane Yeadon, Hirokazu Inoue, Frederick J. Bowring, Keiichiro Suzuki, David E. A. Catcheside
Investigations using filamentous fungi have made major contributions to the understanding of the core biological processes of DNA repair and recombination, as certain model species provide particularly favorable opportunities for insight. The Ascomycete fungi, including the filamentous fungus Neurospora crassa, conveniently provide a full set of the products of a single meiosis packaged in spores inside a single ascus. The study of DNA repair began with the discovery of photoreactivation and UV-sensitive mutants in bacteria, followed by isolation and characterization of mutants with sensitivity to UV, IR, and chemical mutagens in both Escherichia coli and the yeast S. cerevisiae. The segregation patterns of parental DNA in recombinant chromosomes from yeasts and filamentous fungi as considered stimulated Whitehouse and Holliday to formulate models of meiotic recombination, and aspects of both models remain valid to this day. Research into DNA repair and recombination can now proceed apace by deletion of each annotated gene with a predicted role in these processes, enabling to move beyond understanding individual repair systems to an understanding of more complex networks. Epigenetic control of DNA metabolism undoubtedly plays a part in DNA repair and recombination. The isolation of mutants sensitive to mutagens remains as an indispensable tool if a comprehensive understanding of DNA repair and recombination is developed. The filamentous fungi have greater genetic complexity than yeast and are more similar to complex organisms such as mammals. The filamentous fungi will continue to provide efficient model systems for such investigation.
Chapter 9 : Chromatin Structure and Modification
Authors: Zachary A. Lewis, Eric U. Selker
Histones and chromatin modifiers are quite conserved, but there is some variation, even among the fungi. This chapter discusses what is known about chromatin structure in the filamentous fungi. The information is compared to what is known for other eukaryotes and exciting areas for future research are highlighted. In budding yeast, transcription of the core histones is tightly regulated and is primarily restricted to the S phase of the cell cycle. Posttranslational modification of histone proteins by acetylation and methylation was first described in 1964. Acetylated histones supported higher rates of RNA synthesis than did unacetylated histones, suggesting that posttranslational modification of the histone proteins could produce functionally distinct chromatin domains. Histone modifications can be loosely grouped into "active marks," which facilitate processes such as transcription, recombination, and DNA repair, and "repressive marks," which tend to inhibit these processes. Changes in chromatin structure from a closed to an open conformation may result from transcription. The chromatin remodelers that catalyze the dramatic changes in nucleosome organization at the Aspergillus promoters are also described in the chapter. The investigation of chromatin structure and function in the filamentous fungi has been fruitful, but much remains unknown. It will be interesting to determine how histone variants, chromatin remodeling, and chromatin modifications impact additional biological processes in filamentous fungi. The complete genome sequences available for many fungi allow high-resolution mapping of the distributions of modified histones, histone variants, and nonhistone chromatin proteins, which should provide some clues to their functions.
Chapter 10 : Transposable Elements and Repeat-Induced Point Mutation
Authors: A. John Clutterbuck, Eric U. Selker
This chapter concentrates on the impact of repeat-induced point mutation (RIP) on transposable elements (TEs). It is clear that RIP does not follow an identical pattern in all fungi: whereas RIP in Neurospora crassa is intense enough to reduce the C+G content of the most affected elements to below 30% and widespread enough for unmutated TEs to be absent from the sequenced genome, in Magnaporthe grisea and Podospora anserina RIP is described as "light" or "mild", and in P. anserina it was observed only in late-maturing ascospores. The genomes of Neurospora and other filamentous fungi show a large variety of sequences homologous to TEs found in other organisms. The distribution of TEs in fungal genomes suggests that there are a limited number of innocuous genomic locations. A striking feature of some of the TE sequences is illustrated. Like other genomic components, TEs provide a record of the evolutionary processes to which they were subjected, but unlike most genomic components, their turnover is rapid and the record they leave is largely one of decay, including defeat by the defense mechanisms of the host. RIP is the most potent genome defense system known in eukaryotes. It is perhaps surprising that RIP is apparently unique to filamentous ascomycetes, but genome defense mechanisms appear to be unusually labile in evolution perhaps because they have to be retailored to meet each new emergency encountered by the host.
Chapter 11 : Meiotic trans-Sensing and Silencing in Neurospora
Authors: Rodolfo Aramayo, Robert J. Pratt
Before any genetic exchange can take place between the chromosomes participating in meiosis, the molecules must first physically pair. The process of meiotic long-distance pairing must involve trans-sensing between chromosomal segments, but the relationship between these kinds of sensing and that of the trans-sensing that activates meiotic silencing is unknown. This chapter considers the trans-sensing that is involved in the early evaluation of every chromosomal DNA segment and the one involved in meiotic silencing as being the same. The trans-sensing between the genomes participating in meiosis occurs concomitantly with the coalignment of homologous chromosomes and requires extensive searching and satisfaction of stringent molecular homology criteria before recombination is allowed. Understanding meiotic silencing in Neurospora requires knowledge about the life cycle of N. crassa. The pairs of homologous chromosomes of N. crassa will first undergo premeiotic DNA replication, generating sister chromatids attached along their length by cohesin. The participating parental nuclei will then fuse at karyogamy, generating a diploid nucleus. In Neurospora, testing the involvement of genes in meiotic silencing is not straightforward. The diploid nature of the meiotic cell makes it difficult to identify recessive alleles in genes involved in the process. The ancient origins of meiotic silencing in all of its current manifestations are likely grounded in RNAi-mediated genome defense mechanisms. The study of meiotic trans-sensing and meiotic silencing is important not only from a mechanistic perspective but also from an evolutionary point of view.
Chapter 12 : Mycoviruses
Author: Donald L. Nuss
This chapter provides an overview of viruses associated with filamentous fungi while highlighting recent developments in mycovirus molecular biology that illustrate the potential utility of mycoviruses for fundamental research and practical applications. Hypoviruses were originally classified as double-stranded RNA (dsRNA) viruses due to the prominence of dsRNA found in extracts of infected C. parasitica. While the six taxonomic families described in this chapter accommodate the majority of mycoviruses, a growing list of recently characterized mycoviruses remains unclassified. Mycovirus infections share several features that are distinctive from plant and animal viruses. Perhaps the most significant difference is that the life cycle of mycoviruses is not punctuated by an extracellular transmission phase. The major taxonomic classes of mycoviruses are represented among the viruses that are associated with the hypovirulence phenotype, which also includes a number of interesting unclassified mycoviruses. Antiviral defense mechanisms currently identified in fungi include a self/nonself recognition system that presents barriers to the major mode of mycovirus transmission and an RNA recognition system that targets mycovirus RNA for destruction. The concept of engineering mycoviruses to manipulate the phenotypic traits of the fungal host has, in fact, been reduced to practice. Mycoviruses are now known to be widely distributed throughout the kingdom Fungi. Advances made with the hypovirus-C. parasitica experimental system have demonstrated that, similar to viruses of plants and animals, mycoviruses have utility for elucidating host function and manipulating host phenotype.
III ORGANELLES
Chapter 13 : Mitochondria and Respiration
Authors: Frank E. Nargang, John C. Kennell
This chapter covers the functions of mitochondria in filamentous fungi. Mitochondrial respiration occurs via transfer of electrons from reduced electron carriers to molecular oxygen. This is accomplished using an electron transport chain housed in the mitochondrial inner membrane (MIM). Fungal mitochondrial genomes are AT-rich DNAs that map as circular molecules. However, several studies suggest that fungal mtDNAs are actually long, concatenated linear molecules. The study of fungal mitochondrial introns has been an active area of research that has yielded a number of significant findings. There are numerous reports of RNA elements being associated with mitochondria. Though they are formally classified as mitoviruses, they can also be considered plasmids based on their structural similarities to linear mt plasmids and lack of an extracellular form. The large number of different proteins that are found in mitochondria (probably about 1,000 for fungi) and the limited coding capacity of mtDNA emphasize the fact that most mitochondrial proteins are encoded by nuclear genes, translated in the cytosol, and imported into the organelle. The chapter attempts to define the basic import process and summarizes the literature while pointing out contributions that have involved filamentous fungi-almost entirely N. crassa. Filamentous fungi are generally considered immortal. Podospora anserina is an exception to this rule, as it has a defined vegetative- growth life span.
Chapter 14 : Vacuoles in Filamentous Fungi
Authors: Emma Jean Bowman, Barry J. Bowman
Using the ectomycorrhizal fungus Pisolithus tinctorius, four zones of vacuolar morphology from the growing hyphal tip were observed: (i) the apical zone, which has few or no vacuoles; (ii) the subapical zone with small ovoid-spherical vacuoles; (iii) the nuclear zone, where tubular vacuoles predominate; and (iv) the basal zone, where large spherical vacuoles are most common. This vacuolar structure is the product of live-cell imaging in microscopes utilizing fluorescent dyes that accumulate in vacuoles and green fluorescent protein (GFP)or red fluorescent protein (RFP)-tagged proteins targeted to vacuoles or vacuolar membranes. For biochemical studies in vitro, vacuoles can be isolated from filamentous fungi as a pure organellar fraction of round vesicles, approximately 0. 2 to 2 μ in diameter. For filamentous fungi, the study of formation and biogenesis of the vacuole is a nascent research area. Fungal vacuoles serve as storage reservoirs for high levels of phosphorus and nitrogen in the form of basic amino acids and polyphosphate (polyPi), respectively. The vacuolar system is highly variable in appearance at different locations within the mycelium and in response to different growth conditions. Several labs are introducing new approaches to study the dynamic behavior of the vacuolar system of filamentous fungi. Homologs of S. cerevisiae genes with known functions in vacuolar biogenesis, autophagy, and ion transport are excellent candidates for identifying the genes involved in these processes in filamentous fungi. Investigations performed in recent years demonstrate that the vacuole is a dynamic organelle with many important roles in metabolism, growth, and development.
Chapter 15 : Peroxisomes in Filamentous Fungi
Authors: Leonardo Peraza-Reyes, Eric Espagne, Sylvie Arnaise, Véronique Berteaux-Lecellier*
This chapter presents an overview summarizing the current knowledge of the function of peroxisomes in filamentous fungi. Peroxisome formation among diverse eukaryotic organisms shares a common basic biogenetic process mediated by a number of conserved proteins known as peroxins. At the start of photosynthesis, peroxisome metabolism plays an important role in photorespiration, and peroxisomes have thus been described as "leaf peroxisomes". The chapter mentions the main metabolic peroxisomal functions of fungi, with emphasis on those that are particular to, and have been characterized more fully in, filamentous fungi. Plants, yeasts, and filamentous fungi display a wide spectrum of peroxisomal activities, mainly due to the existence of peroxisome-specific function. In addition to the fruiting-body constitution, peroxisomes also participate in differentiation processes taking place in the fertile portion (centrum) of these structures. P. anserina strains lacking the peroxisome-targeting signal (PTS) receptors peroxisomal matrix 5 (PEX5) and PEX7 exhibit abnormal formation of asci, resulting in ascospores with uneven numbers of nuclei or spores with no nuclei. The second centrum developmental event in which peroxisomes are involved in P. anserina is the transition from the prekaryogamy mitotic phase to the karyogamy and meiotic phase. In spite of the progress made in understanding peroxisome function in fungi, a detailed picture of how peroxisomes affect several other metabolic and developmental processes remains elusive. Further innovative approaches are required to fully understand the function of this organelle in fungi.
IV HYPHAL GROWTH
Chapter 16 : The Cytoskeleton in Filamentous Fungi
Authors: Xin Xiang, Berl Oakley
This chapter focuses on the microtubule and actin cytoskeletons of filamentous fungi, including the motor proteins that are integral to cytoskeletal function. Relevant results from yeasts are discussed to provide background and context. The emphasis is on more recent data from live-cell imaging as well as genetic and molecular genetic studies. It has been shown that both the microtubule and the actin cytoskeletons play roles in polarized growth of hyphae, and how these cytoskeletal elements function to support hyphal growth and organelle distribution in elongated hyphae is a topic of great interest. Dynein in filamentous fungi also participates in organizing the microtubule network by regulating microtubule dynamics and by providing force for transporting microtubules. In filamentous fungi, the actin cytoskeleton and its myosin motors are important for the delivery of cell membrane and cell wall components to the growing hyphal tip and to the septum. Myosins are a diverse superfamily of actin motor proteins that play various cellular roles. In filamentous fungi such as A. nidulans and N. crassa, four families of myosins have been found, including myosin-I, myosin-II, myosin-V, and the fungus-specific chitin synthases with myosin motor domains. Hyphal growth in filamentous fungi needs both microtubule and actin cytoskeletons, and thus, it would be important to understand how these two systems interact to coordinate vesicle transport towards the hyphal tip.
Chapter 17 : The Cell Wall of Filamentous Fungi
Authors: Nir Osherov, Oded Yarden
This chapter describes the molecular composition and organization of the cell wall in the filamentous fungi. The major differences between the cell wall of the filamentous fungi and that of the better-studied yeast are stressed. The main fibrous component of the cell wall is glucan, a polymer of glucose. Chitin provides tensile strength to the cell wall and composes ~2% of the total cell wall dry weight in yeast, and 10 to 15% in filamentous fungi. Differences in the cell wall composition of yeast and filamentous fungi may have arisen because of differing evolutionary pressures; the filamentous fungal cell wall is adapted to extremely rapid deposition and growth at the hyphal tip and an ability to penetrate hard surfaces. The fungal cell wall in both yeasts and filamentous fungi is studied by various techniques that are addressed in this chapter. They include (i) the chemical analysis of carbohydrate composition; (ii) microscopic analysis (electron, light, and immunofluorescence); (iii) biochemical separation and identification of cell wall proteins (CWPs); (iv) use of cell wall biosynthesis inhibitors and cell wall mutants; and (v) genomics, transcriptomics, and proteomics. The chapter focuses on compounds affecting specific constituents of the fungal cell wall. The significance of the cell wall as a target for antifungals has long been appreciated, yet the number of successful drugs developed is very small. Important progress has also been made in the analysis of the cell wall.
Chapter 18 : Hyphal Growth and Polarity
Author: Steven D. Harris
This chapter summarizes the progress achieved toward understanding the organization of fungal hyphae and the cellular systems involved in hyphal morphogenesis. Particular attention is paid to the mechanisms that have been implicated in the regulation of polarized growth and septum formation in filamentous fungi. Finally, the intriguing question of how morphogenetic regulatory systems may have evolved in the fungal kingdom is briefly addressed. Hyphal growth encompasses several different morphogenetic processes. Foremost among these is the establishment and maintenance of a stable axis of polarized growth. As a result, cell surface expansion and cell wall deposition are confined to a discrete location that ultimately becomes the hyphal tip. Genetic analyses demonstrate that Bni1 is absolutely essential for the establishment of hyphal polarity in A. gossypii. The importance of understanding the molecular mechanisms underlying polarized hyphal growth cannot be understated. The genetic tractability of filamentous fungi such as A. nidulans and N. crassa affords a tremendous opportunity to elucidate these mechanisms and to acquire insight that might be relevant to neurological disorders and other motor diseases. The use of increasingly sophisticated microscopy techniques has revealed the subcellular organization of hyphal tip cells and, in particular, emphasized the role of the Spitzenkörper in polarized hyphal growth. Future experiments that exploit genomic and proteomic tools will undoubtedly provide new insights that test the validity of this hypothesis and reveal the key symmetry-breaking event(s) that lead to polarized hyphal growth.
Chapter 19 : Hyphal Fusion
Authors: Nick D. Read, André Fleißner, M. Gabriela Roca, N. Louise Glass
This chapter focuses on hyphal fusion in filamentous ascomycete and basidiomycete species, with emphasis on the model ascomycete fungus, Neurospora crassa. It reviews the different types of hyphal fusion, its mechanistic basis, and the varied functions that it serves, and it compares hyphal fusion with processes of cell fusion in fungi and other eukaryotic species. Hyphal fusion between spores and spore germlings during colony initiation is very common. In members of the Ascomycota and Basidiomycota, hyphal fusion occurs during mating-cell fusion and during the formation and maintenance of the dikaryon during the sexual phase of the life cycle. Future comparison of different types of hyphal fusion at different stages during the fungal life cycle will be important to distinguish molecular components universally involved in cell fusion from those that are specific to individual cell fusion pathways. Many of the processes required for hyphal fusion in filamentous fungi during vegetative growth are also required during cell fusion processes in general, including signaling by diffusible substances, directed cell growth or movement towards each other, attachment of the two cell types to one another, production and targeting of enzymes to the attachment site, and fusion of the plasma membranes of the interacting cells. Understanding the molecular basis of hyphal fusion during vegetative growth in filamentous fungi may provide a paradigm for self-signaling and self-fusion mechanisms in eukaryotic microbial species, as well as provide a useful model for somatic cell fusion events in complex, multicellular species.
Chapter 20 : Biology and Genetics of Vegetative Incompatibility in Fungi
Authors: Duur K. Aanen, Alfons J. M. Debets, N. Louise Glass, Sven J. Saupe
Filamentous fungi have the ability to undergo somatic cell fusion. When somatic cell fusion occurs between distinct natural isolates of a given species, the fusion cell is adversely affected to various extents. The adverse reaction ranges from a simple growth impairment to an acute cell death reaction. This phenomenon is known as vegetative (or heterokaryon) incompatibility (VI). VI can be envisioned as a conspecific somatic self/nonself recognition process analogous to other somatic allorecognition processes described in other phyla. The VI reaction is triggered by genetic differences between fungal individuals and is defined by precise gene-to-gene interactions. The plant pathogen Pseudomonas syringae may have acquired a het gene as a potential virulence factor to trigger the VI reaction in N. crassa and utilize the fungus as a sole nutrient source. Two types of heterokaryons have been reported in filamentous ascomycete fungi. Genes involved in VI have been identified so far in only two species: N. crassa and P. anserina. Two categories of genes involved in VI have been characterized. The first category includes het genes that encode recognition function and are polymorphic between individuals. The second category includes downstream or upstream effector genes, in which mutations suppress or attenuate phenotypes associated with VI. VI in fungi as a paradigm for allorecognition in genetically tractable simple eukaryotic species holds great promise to gain a better understanding of the general principles that govern the evolution of nonself recognition systems.
V METABOLISM
Chapter 21 : Glucose
Authors: Margaret E. Katz, Joan M. Kelly
This chapter reviews the regulation of glucose uptake and metabolism in filamentous fungi and highlights the similarities with and differences from mechanisms in yeast. Fungal glucose transporters are classified as high-affinity transporters if the Km for glucose is in the micromolar range and low-affinity transporters if Km for glucose is in the millimolar range. S. cerevisiae contains a large number of proteins that can transport glucose across the yeast cell membrane, 17 of which (Hxt1 through Hxt11p, Hxt13 through Hxt17p, and Gal2p) belong to the yeast glucose transporter family. The use of glucose analogues has been used to determine whether glucose sensing in fungi requires uptake and/or further metabolism of glucose. The role of hexokinases in glucose sensing was confirmed in studies using strains carrying mutations in the three genes encoding sugar-phosphorylating enzymes: HXK1, HXK2, and GLK2. Given the central role of glucose in carbon metabolism and the diverse nutrient sources used by different fungi, it is not surprising that differences in glucose transport, glucose metabolism, glucose signaling, and carbon catabolite repression have arisen through selection.
Chapter 22 : Gluconeogenesis
Author: Michael J. Hynes
The major pathways of carbon metabolism are glycolytic breakdown of sugars and the tricarboxylic acid (TCA) cycle for energy generation and the synthesis of biosynthetic intermediates. Growth on carbon compounds metabolized via TCA cycle intermediates requires the net formation of sugars from TCA cycle intermediates in the process of gluconeogenesis-a reversal of glycolysis, in which TCA cycle intermediates are converted to sugars. It is likely that mycelia undergoing carbon starvation in the wild are common, and survival depends on the breakdown of cellular components resulting in carbon sources requiring gluconeogenesis. In asexual spores, mRNAs for gluconeogenic, glyoxylate cycle, and β-oxidation enzymes as well as peroxisomes are present, indicating that gluconeogenesis may be significant for spore survival and germination via the use of stored lipids. The shuttling of metabolites between mitochondria, cytosol, and peroxisomes is crucial for gluconeogenesis. The enzymatic generation of the appropriate reducing power in the form of NADH/NADPH in the different compartments is an important factor in the ability to use carbon sources. In Saccharomyces cerevisiae, there are three genes encoding NADP-dependent isocitrate dehydrogenases, each of which is located in just one of the three compartments. This raises the possibility that other genes encoding the reversible enzymes of glycolysis/ gluconeogenesis are subject to dual control.
Chapter 23 : Nitrogen Metabolism in Filamentous Fungi
Authors: Meryl A. Davis, Koon Ho Wong
Among the filamentous fungi, the genetic basis of nitrogen metabolism has been most intensively studied in the model ascomycetes Aspergillus nidulans and Neurospora crassa by utilizing the excellent classical and molecular genetic systems provided by these species. Much of one's current knowledge is based on classical genetic analysis of mutants affected in specific aspects of the enzymology or the regulation of nitrogen metabolism. There are also instances where significant differences across species provide fascinating insights into the evolutionary divergence of nitrogen metabolism within the filamentous fungi. In this chapter, the molecular genetics of the ammonium assimilatory pathways is considered as the starting point for the biosynthesis of complex nitrogenous macromolecules. The switch from anabolism to catabolism requires the relief of nitrogen metabolite repression, a global control system that modulates the expression of large sets of nitrogen-catabolic enzymes. Recent studies suggest some diversity in the complex molecular mechanisms underlying this regulation among different fungal groups. Details of several nitrogen-catabolic systems are reviewed to illustrate the metabolic and regulatory strategies employed by fungi in the acquisition of nitrogen metabolites. The catabolism of certain amino acids, such as proline and arginine, provides a good source of nitrogen metabolites and supports strong growth in A. nidulans, whereas other amino acids, such as histidine and leucine, are very poor sources of nitrogen for the wild-type organism.
Chapter 24 : Amino Acids and Polyamines: Polyfunctional Proteins, Metabolic Cycles, and Compartmentation
Author: Rowland H. Davis
This chapter focuses on three unusual aspects of certain of these pathways: polyfunctional proteins, metabolic cycles, and compartmentation. The study of aromatic amino acid biosynthesis in Neurospora crassa provided some of the earliest insights into unusual genetic relationships among related metabolic enzymes. Most fungi, including yeasts, display a global response to amino acid deprivation. In N. crassa and other filamentous fungi, it is referred to as cross-pathway control, with many of the genes carrying the cpc designation. In S. cerevisiae, the system is called general amino acid control, with gene names starting with GC. It is referred to here as cross-pathway control and is described briefly to fill out the physiological aspects of amino acid synthesis and control. Polyamine metabolism in fungi has been studied mainly in S. cerevisiae and N. crassa. The chapter focuses on features of fungi that organize and optimize metabolic systems. The management of polyamine pools has revealed a complex physiological landscape in which enzyme regulation, metabolic flow, small-molecule binding, and vacuolar activity converge. Very little catabolism of polyamines takes place in N. crassa, even at high external amine concentrations. The ionic binding of spermidine and basic amino acids to polyphosphate in the vacuole explains why the intact organelle becomes so dense upon isolation in sucrose gradients, where they lose water as they sediment. Indeed, it is likely that in fungi, as in plants, they have important roles in fine-tuning and otherwise managing metabolic and ionic traffic, varying widely with the species and the environment.
Chapter 25 : Sulfur, Phosphorus, and Iron Metabolism
Author: John V. Paietta
This chapter focuses on the varied fungal responses to limitation for iron, phosphorus, and sulfur. Common threads include the versatility and resourcefulness of fungi in the acquisition of these nutrients. Further, transcriptional upregulation of high-affinity transporters is a repeated theme that allows for the scavenging under low and growth limiting nutrient element levels. Storage and macronutrient homeostasis, including regulatory aspects, are also briefly discussed. While Aspergillus and Neurospora are considered overall in greater depth in the chapter, details of the metabolism of iron, phosphorus, and sulfur from other filamentous fungal species are included throughout. Release of cysteine and methionine from exogenous protein can also serve as a source of sulfur for many fungi. The uptake of sulfate into fungal cells is carried out by sulfate permeases, providing a primary sulfur source for the subsequent assimilation pathway. Most studies on sulfur transport in fungi have focused on sulfate permeases in Saccharomyces cerevisiae, Neurospora crassa, and A. nidulans. Among the filamentous fungi, N. crassa and A. nidulans have served as primary model systems for sulfur metabolism. Siderophore-mediated nonreductive iron uptake represents the second type of high-affinity system for iron acquisition. Siderophores, which can be classified as catecholates, phenolates, carboxylates, hydroxylates, and mixed types, are synthesized under iron limiting conditions to chelate ferric (Fe3+) iron.
Chapter 26 : Secondary Metabolism
Authors: B. Gillian Turgeon, Kathryn E. Bushley
In this chapter, several contemporary questions are considered that have arisen as genome sequencing and genomic resources have propelled the field of secondary metabolism to the forefront of fungal biology. The approach is to use case studies to illustrate areas currently under consideration. There are four main classes of fungal compounds considered to be secondary metabolites: polyketides (PKs), nonribosomal peptides (NRPs), terpenoids, and alkaloids. The focus in subsequent sections is mainly on PKs and NRPs, as these constitute the two most prominent classes. The structure of each NRPS in each fungus is usually unique, and both monomodular and multimodular NRPSs are found. PKs are synthesized enzymatically by PK synthases (PKSs). Fungal PKSs are closely related to fatty acid synthetases (FASs). All terpenes are polymers of repeating isopentyl units built by prenyltransferases. Monoterpenes are derived from geranyl diphosphate (GPP), sesquiterpenes are derived from farnesyl diphosphate, and diterpenes are derived from geranylgeranyldiphosphate (GGPP) by the action of terpene synthases or cyclases. Ergot alkaloid toxins are assembled from prenylated tryptophan and include clavines, lysergic acid, and derivatives thereof. The study of epipolythiodioxopiperazine (ETP) clusters indicates that cluster genes share closest relationships with paralogous genes elsewhere in the genomes. The dung of herbivores is an attractive habitat for diverse species of coprophilous fungi, which appear to have adapted to this specific niche by evolving mechanisms to compete with other fungi.
Chapter 27 : Plant Cell Wall and Chitin Degradation
Authors: Christian P. Kubicek, Verena Seidl, Bernhard Seiboth
A large group of fungi has specialized in the degradation of the complex plant cell walls. The natural resistance of plant cell walls to microbial and enzymatic decomposition is largely responsible for the high cost of lignocellulose conversion. A polymer that is structurally related to the plant cell wall polysaccharide cellulose but does not occur in plants is chitin. Due to practical applications, most strategies to use plant cell walls in biotechnological processes exploit the cellulose and hemicellulose sugars following depolymerization. Most of the plant cell wall polysaccharides occur in the form of lignocelluloses. Xyloglucan is quantitatively the predominant hemicellulosic polysaccharide of dicotyledons and nongraminaceous monocotyledons, comprising up to 20% of the plant cell wall. Degradation and catabolism of the individual carbon sources present in complex mixtures follow a mainly energy-driven hierarchy, but adaptation of saprobic and plant pathogenic fungi to their habitats has resulted in species-specific carbon source priorities. A list of fungal glycoside hydrolases (GH) and carbohydrate esterases (CE) that are involved in the degradation of the side chains of plant cell wall polysaccharides is provided. Fungi depolymerize pectin by using not only hydrolytic enzymes (PGAs) but also enzymes that cleave polysaccharide chains via a β-elimination mechanism, resulting in the formation of a Δ-4,5-unsaturated bond at the newly formed, nonreducing end. Many aspects of chitin degradation resemble that of cellulose and have potential impacts on the development of second-generation ("lignocellulosic") bioethanol. N-acetylglucosamine, the monomer of chitin, is an excellent carbon source for T. atroviride but only induces N-acetylglucosaminidases.
V PHOTOBIOLOGY AND CIRCADIAN RHYTHMS
Chapter 28 : Light Sensing
Authors: Luis M. Corrochano, Javier Avalos
This chapter summarizes the current knowledge of the mechanism of light sensing in fungi, including a description of fungal photoreceptors and their mechanism of action, and describes the fungal responses that are mediated by these photoreceptors. Blue-light responses in Neurospora include the induction of sporulation, sexual development, synthesis of mycelial carotenoids, and the regulation of the circadian clock; all of these responses require the products of the wc-1 and wc-2 genes. WC- 1 and WC-2 interact through their PAS domains to form a WC complex that binds the promoter of light-inducible genes. In order to understand the molecular mechanism of light-dependent gene regulation, most research has focused on the behavior and activity of the WC complex during and after exposure to light. Light transduction seems to be reduced to a minimum in Neurospora and other fungi using WC-type photoreceptors. The regulation by light of fungal development (photomorphogenesis) can be measured precisely, allowing the determination of useful parameters, such as thresholds. The carotenoid pathways in filamentous fungi coincide in the first steps, namely, the formation of the colorless phytoene from the condensation of two geranylgeranyl pyrophosphate (GGPP) molecules, a reaction catalyzed by phytoene synthase. Fungal photobiology provides a unique opportunity to investigate the effect of light on a wide group of microbial eukaryotes without the complexities related to photosynthesis and other energy-oriented light perception mechanisms.
Chapter 29 : Circadian Rhythms
Authors: Michael W. Vitalini, Jay C. Dunlap, Christian Heintzen, Yi Liu, Jennifer Loros, Deborah Bell-Pedersen
There are three cardinal properties that define circadian rhythms. First, the period length of circadian rhythms is, by definition, close to 24 h. The second characteristic of circadian rhythms is that the rhythms are entrained to environmental signals such as light and temperature cycles. Thirdly, circadian rhythms are temperature compensated such that the period length of the rhythm is relatively constant at different physiologically relevant temperatures. In the FRQ/WCC molecular cycle, degradation and posttranslational modifications of FRQ play an essential role in period length determination and the overall function of the circadian negative-feedback loop. The study of mutations that altered the pattern of rhythmic development was critical for the initial identification of FRQ/WCC oscillator components in Neurospora, and the identification of genes that are regulated by the circadian clock has supported the existence of FRQ-independent oscillators in the Neurospora cell. Circadian rhythms have been observed in several different fungal species but are primarily limited to field documentation of rhythms in spore development and liberation. Solving the mechanisms of the circadian clock has become an important goal, mainly because of their ubiquity, their adaptive value, and their significance for health and disease in many organisms. The past several years have seen significant advances in one's understanding of the mechanisms of circadian rhythmicity, with the molecular genetic analysis of clocks in Neurospora continuing to provide major insights into the molecular bases of circadian rhythmicity.
VII NUTRIENT, pH, AND STRESS SENSING
Chapter 30 : How Fungi Sense Sugars, Alcohols, and Amino Acids
Authors: Chaoyang Xue*, Daniel J. Ebbole, Joseph Heitman
G-protein-coupled receptors (GPCRs) are an important receptor gene family and play important roles in sensing sugars in eukaryotic organisms, including fungi. Recent studies on the interactions between Cryptococcus and plants, a major environmental niche, reveal that myo-inositol, produced and secreted by plants, is sensed by the fungus and promotes fungal sexual reproduction, providing a potential explanation for how this organism completes its life cycle in nature. Several fusel alcohols, such as 1-butanol and isoamyl alcohol, stimulate filamentous growth of haploid cells. A recent study showed that aromatic alcohols (such as tryptophol and phenylethanol) secreted by yeast cells function as quorum-sensing molecules and stimulate filamentous growth through a Flo11-dependent mechanism. Amino acids are important nutrients for fungi and are detected by specialized sensor systems, which include the general amino acid permease Gap1, the Ssy1-Ptr3-Ssy5 (SPS) system, GPCRs, and the target of rapamycin (TOR). In Neurospora crassa, three transport systems have been described based on the analysis of the kinetics of amino acid uptake and the patterns of competitive inhibition between amino acids. The Gap1 homolog in Neurospora crassa is encoded by the PMG locus, which can transport all L-amino acids except proline. Besides this system, two other transport systems have also been identified. One is encoded by the MTR gene and transports neutral and aromatic amino acids. The other is encoded by the PMB gene and transports basic amino acids, such as arginine and lysine.
Chapter 31 : Regulation of Gene Expression by Ambient pH
Authors: Joan Tilburn, Herbert N. Arst, Jr., Miguel A. Peñalva
Ambient pH regulation of gene expression is a transcriptional regulatory system that enables the presence of gene products appropriate to the pH of the environment and prevents those that are inappropriate to the environmental pH. Much of the progress in elucidating the mechanism of ambient pH gene regulation has been made using Aspergillus nidulans, and there are a number of reasons why this organism is particularly favorable for the study of pH regulation. This chapter discusses some of these reasons and presents a brief description of the current model of fungal pH regulation. Mutations resulting in gene expression appropriate to acidic environments confer intense staining for acid phosphatase activity even with growth at alkaline pH but prevent staining for alkaline phosphatase activity. The product of the signaling proteolysis, PacC53, is thought to be accessible to the processing protease due to an open conformation assumed in the absence of interacting region C, and the processing proteolysis occurs in a pH-independent manner, removing a further ~250 C-terminal residues to form PacC27. In Aspergillus nidulans and in unicellular yeasts, the pH signaling pathway involves six dedicated proteins. Significant advances in one's current understanding of pH signaling have come from research using budding yeast as an experimental model. Compelling evidence obtained in studies of Saccharomyces cerevisiae and A. nidulans strongly supports the existence of a second pH signaling complex associated to the endosomal sorting complex required for transport (ESCRT) complexes on endosomal membranes.
Chapter 32 : Heat Shock Response
Author: Nora Plesofsky
Increased synthesis of heat shock proteins (hsps) was seen in response to physical and chemical stresses and during developmental transitions. Other stresses, particularly oxidative stress and osmotic stress, elicit characteristic changes in gene expression that overlap with one another and with heat stress. The same regulatory factors may be involved in multiple stress responses. These include the heat shock transcription factor and the stress mitogen-activated protein kinases (MAPKs) Hog1 and Slt2 of Saccharomyces cerevisiae and their orthologs in other organisms. The importance of ubiquitin-dependent proteolysis to the heat shock response is shown by the restorative effect of over-expressing UBI4 on survival of cells that are deficient in hsp synthesis. Heat shock transcription factor (HSF) binds as a trimer to heat shock elements (HSEs) within target gene promoters. The fundamental unit of the HSE is nGAAn repeated in tandem on alternating DNA strands (perfect HSE), with a minimum of three pentanucleotides being required in S. cerevisiae for activity and a five-nucleotide gap between two of the pentanucleotides still supporting induction (gapped HSE). If stress responses were unregulated, they would be detrimental, rather than helpful, as seen when there is a buildup of trehalose, particular sphingolipids, Hsp90, or a hyperactivated Hog1 MAPK.
VIII SEXUAL DEVELOPMENT
Chapter 33 : Mating Systems and Sexual Morphogenesis in Ascomycetes
Authors: Robert Debuchy, Véronique Berteaux-Lecellier*, Philippe Silar
This chapter provides an up-to-date review of complex cellular transition steps in Pezizomycotina, from the differentiation of reproductive cells to the development of the fructification. Initiation of sexual reproduction in the sordariomycete Arachniotus albicans, as it is for several ascomycetes, is clearly not dependent on a particular pH range. In many fungi, light is one of the prominent physical factors controlling sexual reproduction, either by stimulating or inhibiting the formation of reproductive structures. Although most Pezizomycotina can be classified as self-compatible or self-incompatible, some species present peculiar mating characteristics. There are several studies showing that pheromone and pheromone receptors are essential for the fusion of the trichogyne with the male cell in self-incompatible filamentous ascomycetes. Recent data have begun to shed light on the various molecular processes occurring during sexual reproduction in filamentous Ascomycetes. In addition to the well-known variety of body plan exhibited by Pezizomycotina fruiting bodies, sequencing has uncovered a large set of mating-type structures, all based on a common pattern. Indeed, it is now well established that MAT1-1-1 and MAT1-1-2 genes control fertilization by using pheromone/receptor genes in self-incompatible Pezizomycotina, but little is known about their roles after fertilization. The few indications that come from Podospora anserina, N. crassa, and C. heterostrophus indicate that they control the formation of biparental ascogenous hyphae and meiosis.
Chapter 34 : Mating and Sexual Morphogenesis in Basidiomycete Fungi
Authors: Lorna Casselton, Michael Feldbrügge
The mating pathways of filamentous ascomycete and basidiomycete fungi are clearly similar, relying on pheromones and cognate receptors for cell communication as well as specific transcription factors to regulate gene expression. Fungal mating-type (MAT) genes encode critical transcription factors, but in basidiomycetes, unlike ascomycetes, they also encode the pheromones and receptors. The reason for this difference between the two major divisions of the Dikarya will become evident when specific aspects of the basidiomycete lifestyle are considered. Understanding the role that these MAT genes play in cell-specific gene expression and mate attraction is particularly relevant to understanding mating in all other members of the Dikarya, even though the lifestyles of these fungi and the actual genes at the MAT loci may differ. The pheromones encoded at the MAT loci of all basidiomycete fungi studied belong to the Saccharomyces cerevisiae a lipopeptide pheromone family, and the receptors are correspondingly members of the S. cerevisiae Ste3p family. Signal transduction processes during mating are initiated by the binding of pheromones to their cognate receptors. Thereby, specific signaling cascades that trigger defined cellular events such as increased pheromone secretion or cell cycle arrest are elicited. Besides pheromones and cognate receptors, genes encoding the basidiomycete homologues of the a1 and α2 proteins of S. cerevisiae are found at the second MAT locus, called MATb in Ustilago maydis and MATA in hymenomycetes. Isolating mutants defective in morphogenesis has been simplified by the use of special strains with self-compatible mutations in both sets of MAT genes.
IX ASEXUAL SPORULATION
Chapter 35 : Regulation of Aspergillus Conidiation
Authors: Min Ni, Na Gao, Nak-Jung Kwon, Kwang-Soo Shin, Jae-Hyuk Yu
This chapter discusses our current understanding of how conidiation in Aspergillus is regulated, with emphasis on the model fungus A. nidulans. The three genes (brlA, abaA, and wetA) have been proposed to define a central regulatory pathway that acts in concert with other genes to control conidiation-specific gene expression and determine the order of gene activation during conidiophore development and spore maturation. Identification and characterization of the developmental activators greatly enhanced our understanding of the molecular mechanisms for upstream regulation of conidiation in Aspergillus. Mutational inactivation of flbB, flbC, flbD, or flbE results in a third, distinct class of developmental defects classified as delayed conidiation. Importantly, while mutational inactivation of any one of these G protein components restores conidiation of the ΔflbA mutant to a certain level, no mutation can bypass the need for FluG during conidiation. Although asexual sporulation is the most common reproductive mode of many filamentous fungi, little is known about the regulatory mechanisms controlling this process. VosA plays two principal roles: (i) coupling sporogenesis and trehalose biogenesis to complete spore maturation and (ii) exerting negative-feedback regulation of developmental specific genes by repressing the expression of brlA encoding the key activator of conidiation in Aspergillus. VosA and related velvet proteins are found to be crucial global regulators for fungal development and metabolism.
Chapter 36 : The Conidium
Author: Daniel J. Ebbole
A discussion of hyphal growth and polarity determination, cell cycle and signal transduction, is integral to understanding the cell biology of development. In Saccharomyces cerevisiae Tup1p can alter chromatin structure as a mechanism to regulate gene expression. The rco-1 ortholog in Aspergillus nidulans, rcoA, has been shown to affect chromatin structure at some promoters. Heterotrimeric G proteins positively regulate adenylate cyclase in Neurospora crassa, and mutation of gna-3 has the most dramatic effect of the three G-alpha subunit mutations in derepressing conidiation, suggesting that GNA-3 plays the greatest role in stimulating cAMP levels under vegetative growth conditions. Thus, signals that downregulate cAMP levels are likely to stimulate conidiophore morphogenesis. A dominant activated G-alpha subunit stimulates conidiation. Although G-protein and cAMP signal transduction is used as part of the overall pathway controlling conidiation in all A. nidulans, N. crassa, and Magnaporthe grisea, the wiring of the circuit differs among them, just as the effect of light on conidiation can differ (stimulatory in A. nidulans and N. crassa and inhibitory in M. grisea). The central regulatory pathway controlling conidiation in A. nidulans involves BrlA, AbaA (for "abacus"), and WetA (for "wet-white") to regulate production of vesicles, sterigmata (metulae and phialide cells), and conidia. Principles gained from defining the evolution of conidiation pathways are likely to be informative for understanding the origins of other novel developmental pathways, such as pathogenesis.
X INTERACTIONS OF FUNGI WITH PLANTS OR OTHER FUNGI
Chapter 37 : Magnaporthe oryzae and Rice Blast Disease
Authors: Chang Hyun Khang, Barbara Valent
The Magnaporthe grisea species complex includes pathogens of more than 50 grass species. Magnaporthe oryzae was recently segregated as a distinct species from M. grisea based on a multilocus phylogenetic analysis and on mating properties of the strains. M. grisea isolates are pathogenic on crabgrass, Digitaria sanguinalis, and related grasses, and M. oryzae is associated with pathogens of diverse grasses with agricultural significance. Evolution of host-specific populations is an important topic that can be addressed within the M. grisea species complex. The abundance of transposable elements in the rice isolates from the field suggests that M. oryzae lacks the repeat-induced point mutation (RIP) mechanism described in the related pyrenomycete Neurospora crassa. Fluorescent effectors remained localized to the biotrophic interfacial complex (BIC) region as long as invasive hyphae (IH) continued to grow in the rice cell. Secreted effector fusions partially colocalized with an aggregation of plant endocytotic membranes that labeled with FM4-64. Some of the blast fungal metabolites, such as tenuazonic acid (TA) and picolinic acid, were demonstrated to be hypersensitive-response elicitors, inducing resistance responses in rice. For rice blast disease, the increasing numbers of avirulence (AVR)-like genes that control host specificity and the large number of R proteins that are predicted to be localized in the rice cytoplasm are consistent with the hypothesis that M. oryzae translocates many effectors into the host cytoplasm.
Chapter 38 : Fusarium Genetics and Pathogenicity
Authors: John F. Leslie, Jin-Rong Xu
Various members of the Fusarium genus are associated with plant diseases and the production of various classes of mycotoxins, with many secondary metabolites still only poorly characterized. The conditionally dispensable chromosome of F. solani appears to contain many pea pathogenicity genes that are important for host range determinants but are dispensable for normal growth in culture. Several well-conserved signal transduction pathways have been studied in some Fusarium species. Mitogen-activated protein kinase (MAPK) genes homologous to the M. grisea PMK1 have been characterized in F. oxysporum (FMK1) and F. graminearum (GPMK1/MAK1). All of the sequenced Fusarium genomes contain multiple copies of genes encoding cutinases, xylanases, polygalacturonases (PG), and other cell wall-degrading enzyme (CWDE) genes, indicating the importance of these hydrolytic enzymes. Fusarium head scab has been linked to deoxynivalenol (DON) in two ways. First there were mechanistic studies conducted by researchers showing that strains that produced more DON were more aggressive than strains that produced nivalenol as an alternative to DON or that produced no trichothecenes whatsoever. Second, in a quantitative trait locus analysis, another group of researchers showed that the cluster of genes responsible for trichothecene biosynthesis was at the heart of the only major quantitative trait locus in a cross between fungal strains that differed in their ability to cause fusarium head scab. Genetics- and genomics-based approaches will certainly further improve our understanding of molecular mechanisms and evolution of Fusarium pathogenesis.
Chapter 39 : Ustilago maydis and Maize: a Delightful Interaction
Author: Flora Banuett
Understanding the molecular mechanisms of the interaction of Ustilago maydis with maize entails understanding the molecular mechanisms that regulate its life cycle. This chapter presents an overview of the current knowledge regarding the interaction of Ustilago maydis with maize. First, useful features that have facilitated analysis of the life cycle are described, followed by a brief synopsis of the morphological transitions that characterize the life cycle and their control by the mating-type loci and the mitogen-activated protein kinase (MAPK) and cyclic AMP (cAMP) signal transduction pathways. Lastly, specific genes known to be required or to be expressed at different stages of the infectious cycle are described. Infection of anthers at various developmental stages not only induces tumors but can also cause aberrant development of different parts of the anther. Thus, infections with U. maydis may provide important insights about floral development in maize. Hyphal fragmentation occurs within the tumors, though it has been reported that this process can occur between cells. Studies that utilized different maize lines suggested that the host can modulate the course of infection. Analysis of mutants in combination with sophisticated imaging techniques and the application of expression profiling of individual infected versus noninfected cells will allow a more precise dissection of the infectious process and identification of the genes that are differentially regulated in the partners of this "apparent" harmonious interaction.
Chapter 40 : Necrotrophic Fungi: Live and Let Die
Authors: Marty Dickman, Jan van Kan, Christopher Lawrence
Necrotrophic fungi are pathogens that obtain nutrients from dead cells. In this chapter three major fungal necrotrophs, Sclerotinia sclerotiorum, Botrytis cinerea, and Alternaria brassicicola, are compared and contrasted. All three fungi discussed in the chapter have recently completed genome sequences. Effective pathogenesis by S. sclerotiorum requires the secretion of oxalic acid (OA). The role of OA in fungal pathogenicity was originally demonstrated using a genetic approach. In all eukaryotes examined, reactive oxygen species (ROS) are produced during normal cellular metabolism. It is now evident that low, nonlethal concentrations of ROS can function beneficially as regulatory molecules in cell-signaling pathways. Programmed cell death (PCD) is an intentional cellular suicide that is genetically based. The result of PCD is the orderly removal of unwanted, unneeded, used, or pathological cells and under normal homeostatic conditions, is of benefit to the organism. Diseases caused by B. cinerea occur in important crop plants in all temperate climate zones, both during plant cultivation and on harvested commodities, often during storage. The SNF1 kinase plays a central role in carbon catabolite repression in Saccharomyces cerevisiae. Importantly, the addition of tryptone to spores of both Δabste12 and Δabnik1 during plant inoculation resulted in a complete restoration of pathogenicity. These results might suggest the presence of a previously undescribed nutrient- or polypeptide-sensing pathway downstream of Amk1/AbSte12 signaling pathways and a putative AbNIK1 osmoregulation pathway.
Chapter 41 : Epichloë Endophytes: Models of an Ecological Strategy
Authors: Carolyn Young, Heather H. Wilkinson
This chapter considers our current understanding of the epichloae, for which the recently sequenced Epichloë festucae is considered a model. The epichloae are in the family Clavicipitaceae within the order Hypocreales. While it was not the first recognition of an epichloë endophyte, the discovery of an introduced tall fescue population by University of Kentucky agronomists in the early 1930s led to the development of the (in)famously popular cultivar Kentucky-31. A flourish of studies comparing endophyte-infected (E+) and endophyte-free (E-) conspecific grasses in the 1980s and 1990s established a list of host fitness enhancements attributable to the endophyte: herbivore resistance, disease resistance, competitive ability, drought tolerance, heat tolerance, and tolerance to nutrient deficits. Researchers conducted a study to determine and compare the impacts of E+ and E- tall fescue on a natural grass community. The implications of this study reveal the concern that when introduced symbiota with pronounced fitness enhancements become established in nature, they run the risk of dominating communities and driving out some native plant species. While much of the understanding of ergot alkaloid biosynthesis has come from the characterization of the genes in the Claviceps spp., disruption and characterization of key pathway genes such as dmaW, lpsA, and lpsB have also been performed for epichloë endophytes. Most of the Epichloë and Neotyphodium endophytes screened to date are able to synthesize the alkaloid peramine.
Chapter 42 : Mycoparasitism
Authors: Ada Viterbo, Benjamin A. Horwitz
Mycoparasitism is considered a major contributor to fungus-fungus antagonism. The necrotrophs, primarily Trichoderma species, have a wider host range and less-specific mode of action, and perhaps for this reason more field and greenhouse trials have made use of these. Trichoderma species are focused in this chapter, because they have been the focus of the most work at the molecular and cellular levels. The interaction of Trichoderma with soilborne pathogenic fungi is an excellent example of necrotrophic mycoparasitism. Drastic reduction of intracellular cyclic AMP (cAMP) by knockout of adenylate cyclase leads to slow growth and loss of mycoparasitism in T. virens. In T. atroviride, mutants in the ortholog of the same mitogen-activated protein kinase (MAPK) gene, tmk1, showed increased coiling but reduced mycoparasitism in confrontation assays. A potentiation in the gene expression enables Trichoderma-treated plants to be more resistant to subsequent pathogen infection. Chytrids parasitizing vesicular arbuscular mycorrhizae were found in the fossil record of the Rhynie Chert, dating to more than 400 million years ago. There are theories that mycorrhizae themselves might have evolved from biotrophic fungal parasites of plants. Major features such as detection of the host, signal transduction, altered transcriptional patterns, and secretion of enzymes are likely to be shared, and this has provided working hypotheses to guide studies of mycoparasitism. Molecular mechanisms and genes manipulated to optimize biocontrol will be different for each type of interaction and for each niche within the complex web of fungus-fungus and fungus-root interactions.
XI ANIMAL PATHOGENS
Chapter 43 : Aspergillus fumigatus
Authors: Judith C. Rhodes, David S. Askew
Aspergillus fumigatus causes a spectrum of clinical entities, ranging from allergic rhinitis to invasive disease. Following the inhalation of conidia, allergic rhinitis and allergic bronchopulmonary aspergillosis (ABPA) result from overly exuberant immunological responses. Techniques for global analysis of transcription are revolutionizing one's understanding of fungal physiology, including the complex regulatory networks that facilitate adaptation to the host environment. The first large-scale analysis of the A. fumigatus transcriptome was published in conjunction with the initial report of the Af293 genome sequence. In this study, microarrays were used to gain insight into genes that are differentially regulated in response to a temperature shift, with the goal of understanding aspects of thermotolerant growth that are relevant to mammalian infection. A more targeted proteomics strategy has been used to uncover novel cellulases in the A. fumigatus proteome. The procedure focuses on the identification of β-glucosidase activity in the secreted proteome using a 2D-in-gel β-glucosidase activity assay combined with tandem mass spectrometry (MS). Programmed cell death (PCD) is an important stress response among metazoans, and components of the pathway are considered to be excellent targets for anticancer drug development. Typical primary pathogen has unique virulence traits that evolved in association with a host organism. These virulence factors are generally dispensable for growth outside the host but provide some competitive advantage to the organism when it is in the host environment.
Chapter 44 : Cryptococcus neoformans: Budding Yeast and Dimorphic Filamentous Fungus
Authors: James W. Kronstad, Jennifer K. Lodge, Joseph Heitman
Cryptococcus neoformans and Cryptococcus gattii are closely related basidiomycetous fungi that commonly infect humans to predominantly cause meningoencephalitis. Both grow as budding yeasts in the environment and in the infected host yet undergo a dimorphic transition to a filamentous monokaryon or dikaryon during sexual reproduction. This chapter covers recent exciting advances in the field with special consideration of features of the virulence and life cycle relevant to studies of filamentous fungi and the emergence of microbial pathogens successfully infecting animals. The iron regulatory network and iron acquisition functions have been examined in some detail for C. neoformans. Initially, the response of the fungus to iron deprivation was examined by transcriptional profiling, and this study identified general patterns of gene expression as well as specific iron-responsive functions. The latter genes encoded iron acquisition functions and a predicted mannoprotein described as a cytokine-inducing glycoprotein (Cig1). The availability of the genome sequences and molecular techniques for C. neoformans strains provides an opportunity to define the transcriptome of the pathogen under a variety of growth conditions. An interesting recent study on extracellular proteome targeted proteins associated with extracellular vesicles. The contribution of the α allele to pathogenicity is background dependent, and virulence is a quantitative trait, in which mating-type locus (MAT) interacts with other unlinked genes to contribute to virulence.
Chapter 45 : Histoplasma capsulatum
Author: Chad A. Rappleye
Molecular phylogenetic analyses reveal that Histoplasma capsulatum comprises at least seven distinct phylogenetic groups associated with different geographical locations. Molecular differences between Histoplasma strains have been identified using restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA, ribosomal DNA, and the YPS3 locus. The yeast phase of Histoplasma is the morphological form found in infected tissues and is the phase devoted to pathogenesis. When subjected to a temperature of 37⁰C, germinating Histoplasma conidia or hyphal cells undergo a morphological conversion to produce yeast phase cells. Genetic evidence demonstrating the role of these potential iron acquisition mechanisms during intramacrophage growth of Histoplasma yeast has only been demonstrated for siderophores (SID1). Consistent with its role in pathogenesis, α-glucan is synthesized solely by yeast phase cells; AGS1 is only expressed by Histoplasma in the yeast phase. The molecular details underlying the mechanisms promoting Histoplasma virulence are now beginning to be revealed with the application of molecular genetics. Investigation of geographically overlapping yet nonrecombining species at the genome level should also provide an important perspective on Histoplasma evolution. Genome comparisons with other species may also enhance our understanding of the issue of dimorphism. The virulence factors identified for Histoplasma have all relied upon the development of reverse genetics methodology to functionally demonstrate the role of suspected genes in virulence. The ability of forward genetics to identify novel or unsuspected genes involved in the biology of Histoplasma is now possible through insertional mutagenesis techniques.
Chapter 46 : The Fungal Pathogen Candida albicans
Authors: Saranna Fanning, Aaron P. Mitchell
Research on Candida albicans is driven by the major questions regarding any infectious microbe. This chapter first introduces the medical problem and then animal models of infection and genetic tools for Candida albicans manipulation. It then focuses on three topics-morphogenesis, adherence, and azole drug resistance-that have long held the attention of our research community, in order to give the reader a sense of key questions and prospects for future study. There is a growing selection of minihost models. Models such as Drosophila melanogaster, Caenorhabditis elegans, and Galleria mellonella cannot recapitulate all of the complexity of mammalian infection, but they lend themselves to highthroughput screens that would be prohibitive for many reasons with mammalian hosts. The glycophosphatidylinositol (GPI) proteins of C. albicans are linked to the β-1,6 glucans, and for many, their expression can vary depending on morphology. A final thought is that natural selection has probably acted on C. albicans most strongly as a commensal. Its functional repertoire has likely evolved to avoid inflammation of host tissues and to support effective competition with its bacterial cohabitants. The true logic behind deployment of C. albicans virulence factors may be most apparent when they are viewed as commensalism factors.
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Cellular and Molecular Biology of Filamentous Fungi — Recommend this title to your library


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