Neurospora: Genomics and Molecular Biology | Book
"state-of-the-art review" (IMA Fungus)
"a pleasure to read ... belongs in every lab that works on fungi and every academic library" (CIMB)
Caister Academic Press
Durgadas P. Kasbekar and Kevin McCluskey
Centre for Cellular and Molecular Biology, Hyderabad, India and Fungal Genetics Stock Center, University of Missouri-Kansas City, USA (respectively)
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Building on over 70 years of genetics research, Neurospora continues to be the leading model for the study of the genomics and molecular biology of filamentous fungi. The ease of culture, amenability to genetic and molecular genetic analysis, and the close correlation between genetic and biochemical traits are some of its advantages. Research with Neurospora has provided insights unachievable from work with simpler systems and difficult to extract from more complicated ones, cementing its position as a leading model system. In recent years the application of modern high throughput analyses had led to a deluge of information on the genomics and molecular biology of Neurospora. This timely book aims to distil the most important findings to provide a snapshot of the current research landscape.
In this book, internationally recognised Neurospora experts critically review the most important research and demonstrate the breadth of applications to industrial biology, biofuels, agriculture, and human health. The opening chapter is an introduction to the organism. Following chapters cover topics such as: carotenoid biosynthesis, polysaccharide deconstruction, genome biology, genome recombination, gene regulation, signal transduction, self-recognition, development, circadian rhythms and mutation. The book closes with a fascinating look at the history and future trends for research on Neurospora gene and genome analysis.
This volume is essential for everyone working with Neurospora and other filamentous fungi. A recommended book for all biology, agriculture and medical libraries.
"wide-ranging work aims to distil the most important findings and provide snapshots of the current research landscape ... by bringing together leading researchers ... very much a state-of-the-art review of Neurospora genetics ... will undoubtedly also be of value to those working in different model genetic fungal systems" from IMA Fungus 3: 59-60
"This terrific new book was a pleasure to read; it belongs in every lab that works on fungi and every academic library." from Curr. Issues Mol. Biol. (2014) 16: 7-8.
Table of contents
1. Neurospora: The Organism, its Genes and its Genome
A J F Griffiths
The fungus Neurospora has been the subject of a large variety of types of research in genetics and other aspects of biology. This chapter provides a short overview of Neurospora genetics, both as an introduction for those not familiar with this fungus as a genetic tool, and as a general context for the diverse research described in the ensuing chapters. The overview consists of a description of the organism, how it is used in genetics, and a brief account of how the nature of its genome has been revealed. References have been kept to a minimum in this introduction, so for more details of the approaches described see Davis, 2000.
2. The Fungal Sense of Nonself
Myron L. Smith and Denis L. Lafontaine
Fungal incompatibility systems are intricate and complicated, but unifying patterns are emerging that provide a better understanding of the basis for the fungal "sense of nonself". This chapter explores recent advances in understanding fungal vegetative incompatibility (VI) within the broader context of fungal nonself recognition systems. The initial molecular characterization of a limited number of VI factors in N. crassa and P. anserina has provided a set of search images that can be used to find incompatibility genes in other species. The common features discussed include shared structural aspects and sequence motifs of VI complexes, and the downstream processes triggered by nonself recognition, including VI-associated Programmed Cell Death. Finally, we speculate on the role and origin fungal VI systems.
3. Control of Branching in Neurospora crassa
Michael K. Watters
The growth and vegetative morphology of filamentous fungi is characterized by two seemingly related activities: Tip growth - the highly polarized extension of hyphal tips, and branching - the process by which new hyphal tips emerge. Tip growth and branching are crucial elements in the colonization and utilization of the organic substrate, keys to the ecological role of this fungus. There appear to be at least two distinct forms of branch formation. One (lateral branching) which results in the new tips which emerge from the side of the tip region of an existing hyphal tube near, but not at, the apex of the tip, and the second (apical branching) in which a growing tip splits to form a pair of tips emerging from what was the apex of the hyphal tip. This review presents a brief description of the tip growth and branching process with a focus on genetic and environmental influences on the control of branching. Evidence that lateral and apical branches are controlled by separate mechanisms will be discussed. An additional focus will be on evidence for a proposed homeostatic system which regulates branch density, including a description of mutants which appear to disrupt the proposed system.
4. Glycosyl Hydrolases: Modular Structure, Physiological Roles, Gene Amplification and Evolution
The ascomycete fungus Neurospora crassa has a profusion of glycosyl hydrolase genes encoding a wide range of physiological roles. Domain structure of glycosyl hydrolases is considered in the context of domain shuffling within and between families. Mechanisms of action of glycosyl hydrolases are illustrated. The conservation of sequence and structure of the cellulose-binding domain is examined in detail, as is the sequence and structural basis of endo- and exo-cellulases. The individual N. crassa enzymes are classified by predicted function, family, clade (superfamily) and fold. Gene amplification is analysed in N. crassa and ascomycete relatives, and the most highly amplified families are examined for evidence of the evolutionary timing of gene amplification events.
5. Quantitative genetics in Neurospora
Since the 1940s, researchers have used Neurospora species in pioneering genetic analyses providing many discoveries in genetics and molecular biology. Several complex phenotypes continue to be the focus of Neurospora research, including spore germination, hyphal growth, aerial hyphae formation and asexual spore production, meiosis and sexual spore development, epigenetics and circadian rhythm. Surprisingly, unlike the many genes that have been discovered to regulate Mendelian trains in N. crassa, studies of complex traits in are in their infancy and few researchers have taken advantage of natural variation in phenotype. Quantitative trait locus (QTL) mapping and genome-wide association mapping (GWA) have proven to be highly effective methods for studying genetically complex traits. These methods link phenotypic data and genotypic data in an attempt to explain the genetic basis of variation in complex traits. Here I present a review of the principles that underlie these analyses, as well as some common methods for the collection and analysis of QTL and GWA data. We also present a review of quantitative genetics studies in Neurospora. Currently, quantitative studies in Neurospora center around two primary areas of research: circadian clock and the identification of genes involved in the reinforcement of mating barriers between Neurospora crassa and Neurospora intermedia. Recently the emergence of short-read high-throughput sequencing methods have allowed for the sampling of variation within populations of N. crassa at reasonable cost and effort. We further describe the use of these sequencing methods to construct a data set that allows for genome-wide association studies with a Louisiana population of N. crassa.
6. Genetic Recombination in Neurospora crassa
David E. A. Catcheside, Frederick J. Bowring and P. Jane Yeadon
Beadle and Tatum's selection of Neurospora to test the hypothesis that genes encode proteins was strongly influenced by the life cycle of this filamentous fungus, which is particularly amenable to genetic experiments. Pertinent features for the student of recombination include two mating types and a mitotic division following meiosis prior to spore formation, yielding an ascus in which the position of each meiotic product mirrors the order of the eight DNA strands as they entered meiosis. This latter feature allows ready detection of rare recombination events. In this chapter we review the contributions Neurospora has made to our understanding of recombination. These include the first unequivocal demonstration of gene conversion, fine structure maps of eukaryotic genes, evidence for polarity in gene conversion and identification of recombination hotspots (recombinators) and the genes that regulate them. More recent contributions come from analyses using knockouts of genes involved in recombination, a fluorescent recombination reporter system and whole octad sequencing to reveal genome-wide recombination.
7. Neurospora Duplications, and Genome Defense by RIP and Meiotic Silencing
Durgadas P. Kasbekar
Repeat-induced point mutation (RIP) and meiotic silencing by unpaired DNA serve to prevent the accumulation of transposable elements and other repetitive DNA in the Neurospora genome. RIP occurs in the premeiotic dikaryon that forms following fertilization in a sexual cross, and induces G:C to A:T mutations in duplicated DNA, thus destroying any ORFs present in it. Meiotic silencing uses RNAi to eliminate transcripts of genes that do not pair properly in meiosis with a homologue in the same chromosomal location and thus silences them and even their homologous genes at other chromosomal locations, regardless of whether or not the latter are paired. Chromosome segment duplications (Dps) also are substrates for RIP. But RIP is less severe in Dp-borne genes than in small gene-sized duplications, and the presence ofDps in a cross can lower the efficiency of RIP in small duplications. Dps thus behave as dominant suppressors of RIP. Short Dps (i.e., < 200 kbp) that individually are unable to suppress RIP can nonetheless do so in multiply heterozygous crosses if the total amount of duplicated DNA exceeds ~300 kbp. This suggests that RIP suppression by Dps might occur via titration of the RIP machinery. Dp-heterozygous crosses are barren, but their productivity can be increased by suppression of meiotic silencing. Barrenness limits the vertical transmission of Dps, therefore meiotic silencing augments genome defense by RIP, but it might also protect the genome independently of RIP. Meiotic silencing is demonstrable using tester strains that silence genes whose silencing can produce striking ascus-development phenotypes. Crosses of the testers with many wild-isolated strains revealed meiotic silencing to be relatively weak and restricted only to the early perithecia of the cross. We hypothesize that genes essential for meiotic silencing might become unpaired with time, possibly aided by sequence polymorphisms between the tester and wild genomes, thus becoming self-silenced, and thereby shorten the duration of silencing. Some wild strains contain genetic factors that when brought together produce a synthetic RIP suppressor phenotype. These factors might represent sub-threshold sized and cryptic Dps.
8. Mutagen Response and Repair
George Wells Beadle and Edward Lawrie Tatum chose Neurospora crassa as a model organism to uncover the role of the gene in biochemical synthesis pathways. Their studies led them to propose the famous, one gene-one enzyme hypothesis, by generating Neurospora mutants by the treatment with the mutagen (X-ray irradiation) to asexual spores (Beadle and Tatum, 1941; Beadle and Tatum, 1945). This artificial mutagenesis was originally reported by Hermann Joseph Muller, who discovered the generation of Drosophila mutants by irradiation with X-rays. Artificial mutagenesis has since become a great tool to facilitate progress in the study of gene function. Although mutagen treatment does damage to DNA, most of the induced lesions are repaired effectively by many modes of DNA repair by which cells maintain the integrity of genome information. But a subset of lesions that escape this repair function can become fixed as mutation. Most mutations cause serious issues, including alteration of cell, tissue, and organ function, disease, tumor, cancer, apoptosis, and so on. However, mutations are also the motive force of evolution. In this chapter, I summarize mutagen response and repair in Neurospora and describe the various DNA damaging agents, mutations and mutagenesis, and DNA repair systems. By the end of the 20th century, many strains, showing mutagen sensitivity, had been isolated as DNA repair defective mutants, i.e. uvs- (ultraviolet sensitive-), mus- (mutagen sensitive-), mei- (meiosis), and upr- (ultraviolet photoreactivation) mutants in Neurospora. Analyses of the phenotype of these mutants and comparison with repair processes of other organism led to the identification of several DNA repair process. Now, through the contribution of the Neurospora genome project, we are able to find additional DNA repair genes in Neurospora based on their homology with genes of other organisms. To be sure, many mutagen sensitive strains are caused by abnormalities of the genes included in DNA repair systems. Still, there are some mutants where the responsible genes do not belong to any known DNA repair system. Understanding their phenotypes such as chromosome instability, mitochondrial dysfunction, and senescence is important to advance fundamental knowledge of gene and genome biology.
9. Regulation of Gene Transcription by Light in Neurospora
Maria Olmedo, Carmen Ruger-Herreros and Luis M. Corrochano
Fungi use light cues to acquire environmental information like day length throughout the year. Neurospora adapts its development and behaviour to the changing conditions of the environment using light as a signal for the regulation of gene transcription. Neurospora perceives light through the blue light photoreceptor WHITE COLLAR-1 (WC-1). WC-1 dimerizes with WC-2 to form the White Collar Complex (WCC) that activates the transcription of target genes by binding to Light Regulated Elements (LRE) in their promoters. The light-dependent accumulation of certain proteins is responsible for light responses. Light activates genes involved in a variety of processes including development of asexual spores and sexual structures, biosynthesis of photo-protective pigments, and entrainment of the circadian clock. However, the activation of gene transcription by light is transient. After extended illumination, the light-induced transcription ceases and further incubation in the dark is required before transcription in response to light is again activated. This feature, photoadaptation, depends on the blue-light photoreceptor VIVID (VVD). This chapter will present the mechanisms of light perception and regulation of gene expression that lead to light responses in Neurospora.
10. Regulation and Physiological Function of MAP Kinase and cAMP-PKA Pathways
Masayuki Kamei, Shinpei Banno, Masakazu Takahashi, Akihiko Ichiishi, Fumiyasu Fukumori and Makoto Fujimura
Signal transduction pathways play important roles in growth, differentiation, and pathogenicity of filamentous fungi. Neurospora crassa uses two-component histidine kinases and G protein-coupled receptors to sense environmental changes, including osmotic and oxidative stress, chemical challenges, mating pheromone, and nutrient limitation. The environmental signals detected by the receptor/sensor proteins are transmitted to the mitogen-activated protein (MAP) kinase and cAMP-dependent protein kinase (PKA) pathways, both of which play important roles in cellular physiology, including osmoadaptation, mating response, maintenance of cell wall integrity, asexual conidiation, hyphal fusion, circadian response, and accumulation of secondary metabolites. In general, activation of these protein kinases leads to the modification of downstream transcription factors and, consequently, to changes in gene expression. In this chapter, we provide a brief overview of the sensing and signal transduction systems in N. crassa, yeasts, and other filamentous fungi, and we focus on the recent progress in our understanding of three different MAP kinase pathways as well as the cAMP-PKA pathway in N. crassa.
11. Heterotrimeric G Proteins
James D. Kim, Patrick Schacht, Amruta Garud, Gyungsoon Park and Katherine A. Borkovich
One of the major systems used by Neurospora crassa to sense and respond to changes in the environment is the heterotrimeric G protein signaling pathway. This system translates signals detected by G protein coupled receptors (GPCRs) or the cytosolic protein RIC8 to an associated intracellular heterotrimeric G protein (α, β and Gγ subunit) to regulate GDP/GTP exchange on the Gα protein. Gα-GTP and the Gβ γdimer have the potential to regulate downstream effectors. In N. crassa, all five characterized G protein subunits have some function in sexual and asexual growth and development, nutrient sensing or stress responses. Biochemical evidence indicates that the Gβ and Gγ subunits form a heterodimer, and that loss of either subunit leads to degradation of Gα proteins. GPCRs have been implicated in the pheromone response (PRE-1 and PRE-2), perithecial development (GPR-1) and carbon sensing (GPR-4). GTP binding assays using purified proteins demonstrate that RIC8 activates GDP/GTP exchange on two Gα proteins. cAMP is an important second messenger that regulates aspects of asexual and sexual development. Furthermore, metabolomics experiments using 1H-NMR support a role for one G Gα protein in nutrient sensing.
12. Calcium Signaling
Ranjan Tamuli, Ravi Kumar, Dhruv Aditya Srivastava and Rekha Deka
The filamentous fungus Neurospora crassa possesses a complex Ca2+- signaling system consisting of 48 Ca2+-signaling proteins. Ca2+ is stored in several intracellular stores such as vacuoles, plasma membrane vesicles, microsomes, and mitochondria; however, second messenger systems responsible for Ca2+-release from internal stores have not been identified in N. crassa or any other filamentous fungi. The cytosolic free Ca2+ ([Ca2+]c) can be measured in living N. crassa by using Ca2+-sensitive devices such as microelectrodes, fluorescent probes, or aequorin which is a photoprotein isolated from the jellyfish Aeqorea victoria. In N. crassa, the [Ca2+]c is ~100 nM that -is effectively regulated by the Ca2+ signaling machinery -as high concentrations of Ca2+ are toxic, and minute change of [Ca2+]c may trigger several cell processes. In N. crassa, Ca2+ signaling is known to be involved in regulating several processes such as Ca2+ stress tolerance, circadian clocks, growth, ion transport, sexual development, and UV survival. The Ca2+-signaling genes and proteins in N. crassa have several characteristic sequence features. Analysis of Ca2+-signaling in mutants and the availability of the genome sequence has provided deep insight into the functions for some Ca2+-signaling genes in N. crassa.
13. Carotenoid Biosynthesis in Neurospora
Javier Avalos and Luis M. Corrochano
Neurospora produces a mixture of carotenoid and apocarotenoid pigments, with the orange xanthophyll neurosporaxanthin as the major component. The five genes needed to produce this carboxylic apocarotenoid, in sequential order al-3, al-2, al-1, cao-2 and ylo-1, are known and the encoding enzymes have been biochemically investigated. Neurosporaxanthin biosynthesis is induced by light at the level of transcription through the specific action of the White Collar complex, a photoreceptor and transcription factor. Additionally, the pathway is developmentally induced during conidiogenesis and carotenoids are accumulated in the conidia in the dark. The function of the carotenoids has not been well established, but different observations point to protective roles against sun exposure and oxidative stress.
14. The Neurospora Circadian System: From Genes to Proteins and Back, in Less Than 24 Hours
Alejandro Montenegro-Montero and Luis F. Larrondo
Circadian clocks confer close to 24-hours rhythms to a large number of processes in most organisms across different evolutionary lineages. These endogenous cellular timekeepers regulate rhythms in gene expression, physiology and behavior and enable organisms to anticipate predictable environmental variations. Studies conducted in the ascomycete Neurospora crassa have been instrumental in unveiling the molecular and genetic basis of the emergent property of time-telling. The Neurospora circadian system integrates a series of cellular processes, including light perception, phosphorylation and dephosphorylation, nuclear trafficking, signal transduction pathways, chromatin remodeling and transcriptional regulation among others, that give rise to a robust pacemaker capable of coordinating rhythmic control of several aspects of Neurospora biology, the most obvious one being the daily appearance of asexual spores. This chapter will provide an overview of the major advances in the field, with an emphasis on the later discoveries propelled by the release of the Neurospora genome and the adoption of functional genomic strategies. In addition, the state of the art in the studies of the Neurospora circadian system will be discussed, along with the main challenges and opportunities ahead.
15. Neurospora Gene and Genome Analysis: Past Through Future
Aric Wiest, Scott E. Baker and Kevin McCluskey
As modern biological research has developed, so has the analysis of Neurospora biology. From beginnings as a simple genetic system to the present where high throughput analysis enables questions in every area of biological inquiry, research on Neurospora continues to set a high standard for all filamentous fungal experimental systems. Analysis of materials developed over fifty years at the Fungal Genetics Stock Center (FGSC) using a variety of techniques including genetic mapping, cosmid walking, and gene and whole genome sequencing reveals both new information and reinforces discoveries made over many years. Neurospora is and will continue to be the premier organism for studies of the biology of filamentous fungi.
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(EAN: 9781908230126 9781908230775 Subjects: [microbiology] [genomics] [mycology] )