Fungi: Experimental Methods In Biology, Second Edition

349 Pages·2011·13.868 MB·English

by  Ramesh Maheshwari

Fungi
Experimental Methods In Biology
Second Edition
MYCOLOGY SERIES
Editor
J. W. Bennett
Professor
Department of Plant Biology and Pathology
Rutgers University
New Brunswick, New Jersey
Founding Editor
Paul A. Lemke
1. Viruses and Plasmids in Fungi, edited by Paul A. Lemke
2. The Fungal Community: Its Organization and Role in the Ecosystem, edited by Donald T. Wicklow
and George C. Carroll
3. Fungi Pathogenic for Humans and Animals (in three parts),
edited by Dexter H. Howard
4. Fungal Differentiation: A Contemporary Synthesis, edited by John E. Smith
5. Secondary Metabolism and Differentiation in Fungi, edited by Joan W. Bennett and Alex Ciegler
6. Fungal Protoplasts, edited by John F. Peberdy and Lajos Ferenczy
7. Viruses of Fungi and Simple Eukaryotes, edited by Yigal Koltin
and Michael J. Leibowitz
8. Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi, edited by
Sally A. Leong and Randy M. Berka
9. The Fungal Community: Its Organization and Role in the Ecosystem,
Second Edition, edited by George C. Carroll and Donald T. Wicklow
10. Stress Tolerance of Fungi, edited by D. H. Jennings
11. Metal Ions in Fungi, edited by Günther Winkelmann and Dennis R. Winge
12. Anaerobic Fungi: Biology, Ecology, and Function, edited by Douglas O. Mountfort and
Colin G. Orpin
13. Fungal Genetics: Principles and Practice, edited by Cees J. Bos
14. Fungal Pathogenesis: Principles and Clinical Applications,
edited by Richard A. Calderone and Ronald L. Cihlar
15. Molecular Biology of Fungal Development, edited by Heinz D. Osiewacz
16. Pathogenic Fungi in Humans and Animals: Second Edition, edited by Dexter H. Howard
17. Fungi in Ecosystem Processes, John Dighton
18. Genomics of Plants and Fungi, edited by Rolf A. Prade and Hans J. Bohnert
19. Clavicipitalean Fungi: Evolutionary Biology, Chemistry, Biocontrol, and Cultural Impacts,
edited by James F. White, Jr., Charles W. Bacon, Nigel L. Hywel-Jones, and Joseph W. Spatafora
20. Handbook of Fungal Biotechnology, Second Edition, edited by Dilip K. Arora
21. Fungal Biotechnology in Agricultural, Food, and Environmental Applications,
edited by Dilip K. Arora
22. Handbook of Industrial Mycology, edited by Zhiqiang An
23. The Fungal Community: Its Organization and Role in the Ecosystem, Third Edition,
edited by John Dighton, James F. White, and Peter Oudemans
24. F ungi: Experimental Methods in Biology, Ramesh Maheshwari
25. Food Mycology: A Multifaceted Approach to Fungi and Food, edited by Jan Dijksterhuis and
Robert A. Samson
26. The Aspergilli: Genomics, Medical Aspects, Biotechnology, and Research Methods, edited by
Gustavo H. Goldman and Stephen A. Osmani
27. Defensive Mutualism in Microbial Symbiosis, edited by James F. White, Jr. and Mónica S. Torres
28. Fungi: Experimental Methods In Biology, Second Edition, Ramesh Maheshwari
Fungi
Experimental Methods In Biology
Second Edition
Ramesh Maheshwari
Indian Institute of Science
Bangalore, India
Front cover: The tube-like hyphae of the pink-orange mold Neurospora crassa are divided into multinuclear compartments by
cross walls. The fungus was transformed with a gene from a jellyfish that encodes a green-fluorescent protein. Nuclei in living
hyphae tagged by fusion of GFP to histone H1 gene were visualized by confocal microscopy. The nuclei become spindle-shaped
(inset) as they move from one compartment to another compartment of branched hyphae through centrally perforated cross
walls. This image was provided by Nick D. Read of the University of Edinburgh and is used with permission from Elsevier.
Back cover: The plant-parasitic fungus Puccinia graminis tritici has been a scourge of mankind since ancient times. It infects
stems and leaves of the wheat plant and forms rust-colored pustules. Each pustule contains countless numbers of wind-dissem-
inated urediospores shown at top right. The fungus survives in stubble in the form of thick-walled bi-celled teliospores. The
photographs were provided by Jacolyn A. Morrison of Cereal Rust Research Laboratory at the University of Minnesota.
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Dedication
David D. Perkins
(1919–2007)
Contents
Preface to the First Edition ..............................................................................................................xv
Preface to Second Edition ..............................................................................................................xvii
About the Author ............................................................................................................................xix
Part I
The Unique Features of Fungi
Chapter 1
The Hyphal Mode of Life ..................................................................................................................3
1.1 Features of Hyphae .................................................................................................................3
1.1.1 Apical Extension ........................................................................................................3
1.1.2 Spread and Longevity ................................................................................................4
1.1.3 Large Surface Area ....................................................................................................6
1.2 Hyphal Structure .....................................................................................................................8
1.2.1 Cell Wall ....................................................................................................................8
1.2.2 Glucan ........................................................................................................................9
1.2.3 Hydrophobin ............................................................................................................11
1.3 Internal Structure ..................................................................................................................12
1.3.1 Microtubules and Actin Filaments ..........................................................................12
1.3.2 Spitzenkörper and Polarisome .................................................................................16
1.3.3 Calcium ....................................................................................................................17
1.3.4 Vacuole .....................................................................................................................18
1.3.5 Septa and Woronin Body .........................................................................................18
1.4 Networking ............................................................................................................................18
1.4.1 Branching .................................................................................................................18
1.4.2 Hyphal Fusion ..........................................................................................................19
1.5 Main Functions .....................................................................................................................20
1.5.1 Nutrient Uptake ........................................................................................................20
1.5.2 Protein Secretion ......................................................................................................20
1.6 Morphogenesis ......................................................................................................................21
1.6.1 Hyphal Differentiation .............................................................................................22
1.6.2 Rhizomorph .............................................................................................................23
1.6.3 Mushroom Fruiting Body ........................................................................................23
1.7 Autophagy .............................................................................................................................24
1.8 Concluding Remarks .............................................................................................................24
References ........................................................................................................................................24
Chapter 2
The Multinuclear Condition .............................................................................................................29
2.1 Nuclear Number and Hyphal Growth Rate ...........................................................................29
2.2 Chromosome Numbers .........................................................................................................29
2.2.1 Classical Methods ....................................................................................................29
2.2.2 New Methods ...........................................................................................................30
2.3 Multiple Genomes in Individual Nuclei ................................................................................32
2.4 Nuclear Division Cycle .........................................................................................................33
2.4.1 Temperature-Sensitive Mutants ...............................................................................34
2.4.2 Stages in Nuclear Division Cycle .............................................................................34
vii
viii Contents
2.5 Asynchronous Nuclear Divisions ..........................................................................................35
2.6 Nuclear Migration .................................................................................................................36
2.7 Nuclear Positioning and Gene Regulation ............................................................................36
2.8 Heterokaryosis ......................................................................................................................38
2.8.1 Sheltering of Lethal Mutation ..................................................................................39
2.8.2 Change in Nuclear Ratio ..........................................................................................40
2.8.3 Nuclear Competence ................................................................................................42
2.9 Parasexual Recombination ....................................................................................................43
2.10 Are All Nuclei Active Simultaneously? ................................................................................43
2.11 Concluding Remarks .............................................................................................................44
References ........................................................................................................................................44
Chapter 3
Spores: Their Dormancy, Germination, and Uses ...........................................................................47
3.1 Models for Cellular Processes ...............................................................................................47
3.2 Pleomorphism .......................................................................................................................48
3.3 Dissemination .......................................................................................................................50
3.4 Longevity ..............................................................................................................................50
3.5 Structure ................................................................................................................................50
3.5.1 Topography ..............................................................................................................50
3.5.2 Wall Structure ..........................................................................................................52
3.6 Water Relations .....................................................................................................................53
3.7 Endogenous Substrates ..........................................................................................................53
3.7.1 Lipids .......................................................................................................................53
3.7.2 Polyols ......................................................................................................................54
3.7.3 Trehalose ..................................................................................................................55
3.8 Link between Sporulation and Secondary Metabolism ........................................................55
3.9 Self-Reconditioning of Substratum .......................................................................................55
3.10 Germination Triggers ............................................................................................................56
3.10.1 Adhesion ..................................................................................................................56
3.10.2 Heat ..........................................................................................................................57
3.10.3 Light .........................................................................................................................57
3.10.4 Chemicals ................................................................................................................57
3.11 Material for Probing Fungal Physiology ...............................................................................58
3.11.1 Prerequisites for Development .................................................................................58
3.11.2 Release of Self-Inhibitors and Efflux of Carbon Compounds .................................58
3.11.3 Cold Dormancy ........................................................................................................59
3.11.4 Genome Activation ..................................................................................................59
3.11.5 Dark Fixation of CO ...............................................................................................60
2
3.11.6 Respiratory Increase ................................................................................................60
3.11.7 Regeneration of Reducing Power .............................................................................62
3.11.8 Synthetic Pathways ..................................................................................................62
3.11.9 Sensing, Signaling, and Transcriptional Changes ...................................................63
3.12 Applications ..........................................................................................................................64
3.12.1 Biocatalysis ..............................................................................................................64
3.12.2 Biocontrol .................................................................................................................64
3.13 Concluding Remarks .............................................................................................................65
References ........................................................................................................................................65
Contents ix
Part II
Fungi in Biosphere and Human Health
Chapter 4
Fungi as Scavengers .........................................................................................................................71
4.1 Decay of Wood and Litter .....................................................................................................71
4.2 Clues from Microscopy .........................................................................................................72
4.3 White- and Brown-Rot Fungi ...............................................................................................73
4.4 Litter Decomposers ...............................................................................................................74
4.5 Degradation of Cell Wall Polymers ......................................................................................75
4.5.1 Lignin .......................................................................................................................75
4.5.2 Cellulose ..................................................................................................................84
4.5.3 Hemicellulose ..........................................................................................................86
4.6 Reactive Oxygen Species ......................................................................................................86
4.7 Unsolved Problems ................................................................................................................87
4.8 Clues from Genome Sequence ..............................................................................................87
4.9 Physiological Processes .........................................................................................................88
4.10 Concluding Remarks .............................................................................................................88
References ........................................................................................................................................89
Chapter 5
Fungi as Symbiotic Partners ............................................................................................................91
5.1 Mycorrhiza ............................................................................................................................91
5.1.1 Types of Mycorrhiza ................................................................................................91
5.1.2 Identification ............................................................................................................93
5.1.3 Development ............................................................................................................93
5.1.4 Carbohydrate Transfer .............................................................................................94
5.1.5 Phosphorus Transfer ................................................................................................95
5.1.6 Nitrogen Transfer .....................................................................................................95
5.1.7 Decomposition of Plant Residues ............................................................................96
5.1.8 Mycorrhiza as Conduits of Photosynthetically Fixed Carbon Compounds ............96
5.1.9 Cheating in Plant-Fungus Marriages .......................................................................99
5.1.10 Genome Sequence ..................................................................................................100
5.2 Endophytic Fungi ................................................................................................................100
5.3 Lichens ................................................................................................................................101
5.3.1 Mycobiont and Photobiont .....................................................................................102
5.3.2 In Situ Study ...........................................................................................................102
5.3.3 Synthesis ................................................................................................................102
5.3.4 Movement of Carbohydrate ...................................................................................103
5.4 Concluding Remarks ...........................................................................................................103
References ......................................................................................................................................103
Chapter 6
Fungi as Plant Pathogens ...............................................................................................................107
6.1 The Rust Fungi ....................................................................................................................107
6.1.1 Thigmotropism and Thigmomorphogenesis ..........................................................108
6.1.2 Haustorium .............................................................................................................110
x Contents
6.2 Host Resistance and Pathogen Avirulence ..........................................................................112
6.3 Dual Role for Haustorium ...................................................................................................115
6.4 Effector Molecules ..............................................................................................................115
6.5 Concluding Remarks ...........................................................................................................117
References ......................................................................................................................................118
Chapter 7
Fungi as Chemical Factories ..........................................................................................................121
7.1 Fungal Factories ..................................................................................................................121
7.1.1 Penicillin ................................................................................................................121
7.1.2 Cephalosporin ........................................................................................................126
7.1.3 Statins.....................................................................................................................126
7.1.4 Anticancer Drugs ...................................................................................................127
7.1.5 Defensins ................................................................................................................127
7.2 Attractions ...........................................................................................................................128
7.3 Yield Improvement ..............................................................................................................130
7.4 Heterologous Protein Production ........................................................................................131
7.4.1 Chymosin ...............................................................................................................131
7.4.2 Lipases ...................................................................................................................131
7.4.3 Lactoferrin .............................................................................................................132
7.4.4 Human Vaccine ......................................................................................................132
7.5 Methylotrophic Yeasts .........................................................................................................132
7.6 New Methods of Yield Improvement ..................................................................................133
7.6.1 Increase in Gene Copy Number .............................................................................133
7.6.2 Manipulation of Morphology .................................................................................133
7.6.3 Hyperbranching Mutants .......................................................................................134
7.6.4 Modification of Cell Wall ......................................................................................135
7.6.5 Other Molecular Manipulations .............................................................................135
7.6.6 New Expression Hosts ...........................................................................................135
7.7 Biofuel Ethanol ...................................................................................................................136
7.8 Agrochemicals ....................................................................................................................138
7.9 Concluding Remarks ...........................................................................................................138
References ......................................................................................................................................139
Part III
Gene Silencing
Chapter 8
Transformation and Discovery of Gene-Silencing Phenomena .....................................................145
8.1 Transformation Procedure ..................................................................................................145
8.2 Homologous vs. Ectopic Integration of Transgene .............................................................146
8.3 Purification of Transformant ...............................................................................................146
8.4 Gene-Silencing Phenomena ................................................................................................147
8.4.1 Silencing by Mutation ............................................................................................147
8.4.2 Meiotic Silencing by Unpaired DNA (MSUD) ......................................................149
8.4.3 Silencing by DNA Methylation (MIP) ...................................................................152
8.4.4 Quelling .................................................................................................................152
8.5 RNA Silencing ....................................................................................................................155