The mycota VIII, biology of the fungal cell 2nd ed k esser, r howard (springer, 2007)

His research interest is in the growth, morphogen-esis and pathogenmorphogen-esis of the human fungal pathogen Candida albicans and he has specific interests in the molecular genetics of

The MycotaEdited by

K Esser

The Mycota

I Growth, Differentiation and Sexuality

1st edition ed by J.G.H Wessels and F Meinhardt

2nd edition ed by U Kües and R Fischer

II Genetics and Biotechnology

Ed by U Kück

III Biochemistry and Molecular Biology

Ed by R Brambl and G Marzluf

IV Environmental and Microbial Relationships

1st edition ed by D Wicklow and B Söderström

2nd edition ed by C.P Kubicek and I.S Druzhinina

V Plant Relationships

1st edition ed by G Carroll and P Tudzynski

2nd edition ed by H.B Deising

VI Human and Animal Relationships

1st edition ed by D.H Howard and J.D Miller

2nd edition ed by A Brakhage and P Zipfel

VII Systematics and Evolution

VIII Biology of the Fungal Cell

Ed by R.J Howard and N.A.R Gow

XII Human Fungal Pathogens

Ed by J.E Domer and G.S Kobayashi

XIII Fungal Genomics

Ed by A.J.P Brown

The Mycota

A Comprehensive Treatise

on Fungi as Experimental Systems

for Basic and Applied Research

Edited by K Esser

2nd Edition Volume Editors:

R.J Howard · N.A.R Gow

With 86 Figures, 7 in Color, and 9 Tables

123

Professor Dr Dr h.c mult Karl Esser

Professor Dr Richard J Howard

DuPont Crop Genetics

Experimental Station E353

Powder Mill Road

Library of Congress Control Number: 2007927884

ISBN 978-3-540-70615-1 Springer Berlin Heidelberg New York

ISBN 3-540-60186-4 1st ed Springer Berlin Heidelberg New York

This work is subject to copyright All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permissions for use must always be obtained from Springer-Verlag Violations are liable for prosecution under the German Copyright Law.

Springer is a part of Springer Science+Business Media

springer.com

© Springer-Verlag Berlin Heidelberg 2001, 2007

The use of general descriptive names, registered names, trademarks, etc in this publication does not imply, even in the absence of

a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

Editor: Dr Dieter Czeschlik, Heidelberg, Germany

Desk editor: Dr Andrea Schlitzberger, Heidelberg, Germany

Cover design: Erich Kirchner and WMXDesign GmbH, Heidelberg, Germany

Production and typesetting: LE-TEX Jelonek, Schmidt & Vöckler GbR, Leipzig, Germany

(born 1924) is retired Professor of General Botany and Director

of the Botanical Garden at the Ruhr-Universität Bochum many) His scientific work focused on basic research in classicaland molecular genetics in relation to practical application Hisstudies were carried out mostly on fungi Together with his col-laborators he was the first to detect plasmids in higher fungi.This has led to the integration of fungal genetics in biotech-nology His scientific work was distinguished by many nationaland international honors, especially three honorary doctoraldegrees

human pathogen Histoplasma capsulatum at the Barnes

Hospi-tal medical campus of Washington University in St Louis, MO(USA) In 1981 he accepted a research scientist position at theDuPont Experimental Station in Wilmington, DE (USA) where

he conducted detailed studies of the rice blast pathogen

Mag-naporthe grisea and the cell biology of appressorium structure

and function Appointed in 2003 as a Research Fellow for CropGenetics, his laboratory now serves as the core biological imag-ing center for DuPont’s research and development interests

Neil A.R Gow

(born 1957) graduated from Edinburgh University and was

a postgraduate at Aberdeen University He was a toral fellow in Denver before returning to a faculty position

postdoc-at Aberdeen where he now holds a personal chair in MolecularMycology He is a founding member of the Aberdeen FungalGroup, which constitutes one of the single largest academiccentres for medical mycology He is the immediate Past Presi-dent of the British Mycological Society and is a Vice President ofthe International Society for Human and Animal Mycology andholds fellowships of the Institute of Biology, the Royal Society

of Edinburgh and the American Academy of Microbiology He

is currently the editor-in-chief of the journal Fungal Genetics

and Biology His research interest is in the growth,

morphogen-esis and pathogenmorphogen-esis of the human fungal pathogen Candida

albicans and he has specific interests in the molecular genetics

of cell wall biosynthesis in fungi and the directional growthresponses of fungal cells as well as the virulence properties ofmedically important fungal species

Mycology, the study of fungi, originated as a subdiscipline of botany and was a tive discipline, largely neglected as an experimental science until the early years of thiscentury A seminal paper by Blakeslee in 1904 provided evidence for selfincompatibil-ity, termed “heterothallism”, and stimulated interest in studies related to the control

descrip-of sexual reproduction in fungi by mating-type specificities Soon to follow was thedemonstration that sexually reproducing fungi exhibit Mendelian inheritance and that

it was possible to conduct formal genetic analysis with fungi The names Burgeff, Kniepand Lindegren are all associated with this early period of fungal genetics research.These studies and the discovery of penicillin by Fleming, who shared a Nobel Prize

in 1945, provided further impetus for experimental research with fungi Thus began aperiod of interest in mutation induction and analysis of mutants for biochemical traits

Such fundamental research, conducted largely with Neurospora crassa, led to the one

gene: one enzyme hypothesis and to a second Nobel Prize for fungal research awarded toBeadle and Tatum in 1958 Fundamental research in biochemical genetics was extended

to other fungi, especially to Saccharomyces cerevisiae, and by the mid-1960s fungal

systems were much favored for studies in eukaryotic molecular biology and were soonable to compete with bacterial systems in the molecular arena

The experimental achievements in research on the genetics and molecular biology offungi have benefited more generally studies in the related fields of fungal biochemistry,plant pathology, medical mycology, and systematics Today, there is much interest in thegenetic manipulation of fungi for applied research This current interest in biotechnicalgenetics has been augmented by the development of DNA-mediated transformationsystems in fungi and by an understanding of gene expression and regulation at themolecular level Applied research initiatives involving fungi extend broadly to areas ofinterest not only to industry but to agricultural and environmental sciences as well

It is this burgeoning interest in fungi as experimental systems for applied as well as

basic research that has prompted publication of this series of books under the title The

Mycota This title knowingly relegates fungi into a separate realm, distinct from that of

either plants, animals, or protozoa For consistency throughout this Series of Volumesthe names adopted for major groups of fungi (representative genera in parentheses) are

Division: Chytridiomycota (Allomyces)

Division: Zygomycota (Mucor, Phycomyces, Blakeslea)

Subdivision: Ascomycotina

Class: Saccharomycetes (Saccharomyces, Schizosaccharomyces)

Class: Ascomycetes (Neurospora, Podospora, Aspergillus)

Subdivision: Basidiomycotina

Class: Heterobasidiomycetes (Ustilago, Tremella)

Class: Homobasidiomycetes (Schizophyllum, Coprinus)

We have made the decision to exclude from The Mycota the slime molds which, although

they have traditional and strong ties to mycology, truly represent nonfungal formsinsofar as they ingest nutrients by phagocytosis, lack a cell wall during the assimilativephase, and clearly show affinities with certain protozoan taxa

The Series throughout will address three basic questions: what are the fungi, what dothey do, and what is their relevance to human affairs? Such a focused and comprehensivetreatment of the fungi is long overdue in the opinion of the editors

A volume devoted to systematics would ordinarily have been the first to appear

in this Series However, the scope of such a volume, coupled with the need to giveserious and sustained consideration to any reclassification of major fungal groups, hasdelayed early publication We wish, however, to provide a preamble on the nature offungi, to acquaint readers who are unfamiliar with fungi with certain characteristicsthat are representative of these organisms and which make them attractive subjects forexperimentation

The fungi represent a heterogeneous assemblage of eukaryotic microorganisms.Fungal metabolism is characteristically heterotrophic or assimilative for organic carbonand some nonelemental source of nitrogen Fungal cells characteristically imbibe or ab-sorb, rather than ingest, nutrients and they have rigid cell walls The vast majority of fungiare haploid organisms reproducing either sexually or asexually through spores Thespore forms and details on their method of production have been used to delineate mostfungal taxa Although there is a multitude of spore forms, fungal spores are basically only

of two types: (i) asexual spores are formed following mitosis (mitospores) and culminatevegetative growth, and (ii) sexual spores are formed following meiosis (meiospores) andare borne in or upon specialized generative structures, the latter frequently clustered in

a fruit body The vegetative forms of fungi are either unicellular, yeasts are an example,

or hyphal; the latter may be branched to form an extensive mycelium

Regardless of these details, it is the accessibility of spores, especially the directrecovery of meiospores coupled with extended vegetative haploidy, that have madefungi especially attractive as objects for experimental research

The ability of fungi, especially the saprobic fungi, to absorb and grow on rathersimple and defined substrates and to convert these substances, not only into essentialmetabolites but into important secondary metabolites, is also noteworthy The metaboliccapacities of fungi have attracted much interest in natural products chemistry and inthe production of antibiotics and other bioactive compounds Fungi, especially yeasts,are important in fermentation processes Other fungi are important in the production ofenzymes, citric acid and other organic compounds as well as in the fermentation of foods.Fungi have invaded every conceivable ecological niche Saprobic forms abound,especially in the decay of organic debris Pathogenic forms exist with both plant andanimal hosts Fungi even grow on other fungi They are found in aquatic as well assoil environments, and their spores may pollute the air Some are edible; others arepoisonous Many are variously associated with plants as copartners in the formation oflichens and mycorrhizae, as symbiotic endophytes or as overt pathogens Associationwith animal systems varies; examples include the predaceous fungi that trap nematodes,the microfungi that grow in the anaerobic environment of the rumen, the many insec-tassociated fungi and the medically important pathogens afflicting humans Yes, fungiare ubiquitous and important

There are many fungi, conservative estimates are in the order of 100,000 species,and there are many ways to study them, from descriptive accounts of organisms found

in nature to laboratory experimentation at the cellular and molecular level All suchstudies expand our knowledge of fungi and of fungal processes and improve our ability

to utilize and to control fungi for the benefit of humankind

We have invited leading research specialists in the field of mycology to contribute

to this Series We are especially indebted and grateful for the initiative and leadershipshown by the Volume Editors in selecting topics and assembling the experts We have allbeen a bit ambitious in producing these Volumes on a timely basis and therein lies thepossibility of mistakes and oversights in this first edition We encourage the readership

to draw our attention to any error, omission or inconsistency in this Series in order thatimprovements can be made in any subsequent edition

Finally, we wish to acknowledge the willingness of Springer-Verlag to host thisproject, which is envisioned to require more than 5 years of effort and the publication

of at least nine Volumes

Bochum, Germany

Auburn, AL, USA

April 1994

Karl EsserPaul A Lemke

Series Editors

In early 1989, encouraged by Dieter Czeschlik, Springer-Verlag, Paul A Lemke and

I began to plan The Mycota The first volume was released in 1994, 12 volumes followed

in the subsequent years Unfortunately, after a long and serious illness, Paul A Lemkedied in November 1995 Thus, it was my responsibility to proceed with the continuation

of this series, which was supported by Joan W Bennett for Volumes X–XII

The series was evidently accepted by the scientific community, because severalvolumes are out of print Therefore, Springer-Verlag has decided to publish completelyrevised and updated new editions of Volumes I, II, III, IV, V, VI, and VIII I am glad thatmost of the volume editors and authors have agreed to join our project again I would like

to take this opportunity to thank Dieter Czeschlik, his colleague, Andrea Schlitzberger,and Springer-Verlag for their help in realizing this enterprise and for their excellentcooperation for many years

Bochum, Germany

February 2007

Karl Esser

A place for Fungi in our world has been well established In the years since the early

1990s the body of evidence accumulating and defining these organisms as a separateKingdom among life on earth has been (almost) universally accepted On a molecularbasis, there remain a few questions concerning the deep divides low in the branches ofthe evolutionary tree And as one considers the mid- and finer branches, there will notlikely be any shock waves big enough to rattle the tree or our thinking of the Fungi (e.g.Eumycota) as a distinct group of organisms – but there remains much to be done andlearned Certainly the work of phylogeneticists is not over, but especially, nor is that

of cell biologists – far from it! Indeed the technological and conceptual advances made

in fungal cell biology have been so rapid that a vast literature is being generated thatexplores how fungal cells grow and divide Since the previous edition of this volume inthis series was published these new methods in single-cell imaging, video microscopy,functional proteomics and gene expression have been widely applied to core questionsrelated to fungal growth and development The current edition incorporates the latestresearch using these new approaches and new perspectives that have been gained Italso adds new chapters in contemporary topics that have emerged in recent years to theareas that have been reviewed in the past as core areas of fungal cell biology

What makes the fungal cell unique among eukaryotes and what features are shared?This volume addresses some of the most prominent and fascinating facets of questions

as they pertain to the growth and development of both yeast and hyphal forms of fungi,beginning with subcellular components, then cell organization, polarity, growth, differ-entiation and beyond – to the cell biology of spores, biomechanics of invasive growth,plant pathogenesis, mycorrhizal symbiosis and colonial networks Throughout this vol-ume, structural, molecular and ecological aspects are integrated to form a contemporarylook at the biology of the fungal cell

Chapter 1 endeavors to generate a new perspective and appreciation for the unique

qualities of the endomembrane system in filamentous fungi, as opposed to other

eu-karyotes and sometimes also yeast cells, by drawing connections between structuraland molecular data In filamentous fungi the tubular vacuole system is one component

of the endomembrane system, as described in Chap 2, which plays a most importantrole in transport – of nutrients, proteins and membrane elements – at the subcellularand intercellular level The importance of these vacuolar tubules has generally not been

widely appreciated but they are now beginning to take their rightful place alongside

vesicles as major mediators of cellular traffic Their vital role in intercellular traffic of

late diverging fungi is dependent upon the perforate septation of hyphae that enablesmore complex multicellular differentiation Chapter 3 offers a review of the Woroninbody, an organelle unique to these “advanced” members of the Eumycota (i.e not foundamong non-septate filamentous fungi) that is also indispensable in the formation oflarge multicellular structures The ability of these fungi to establish tissues and largeorgans, and shared with animals and plants, is a consequence of Woronin body function

in gating cell-to-cell movement and loss of contents upon cell injury and is thus of greatevolutionary significance

A molecular and genomic component to the analysis of each of these cellular stituents has been essential in bringing our current understanding to new levels Simi-larly, these same tools provide a fresh insight into the most obvious manifestation of thefungal cell, the cell wall, and Chap 4 includes additional impetus for new research efforts

con-that go beyond the Saccharomyces model This same message is delivered in Chaps 5

and 6 Central to this volume, and to fungal cell biology, is the topic of growth as itrelates to the polarity of yeast and hyphal cells These two chapters review these aspectsfrom different perspectives and thus provide a more comprehensive synthesis of thesimilarities and likely differences that underlie the biology of the major growth formsexhibited by cells of fungi Chapter 7 continues these considerations with regard to the

“pleomorphic” pathogen Candida albicans, an extremely important organism in human

health as well as a model allowing the functional dissection of morphogenetic

signal-ing pathways The very recently discovered participation by C albicans, and another important human pathogen C glabrata, in mating processes led to additional findings

as reviewed in Chap 8 that point to a possible connection between the phenotypicswitching processes of morphogenesis and pathogenesis, and an obvious practicalityfor understanding the biology of these growth forms and cellular events

The biology of fungal cells that enable the pathogenesis of plants is reviewed inthe following three chapters, from the very first stages of contact to the mechanisms ofinvasive hyphal growth and the various cell structures elaborated for this purpose That

hyphae can penetrate solid substances is one of the defining characteristics of the fungi –

as pointed out in Chap 10, this is important not only during plant pathogenesis, but inevery interaction between fungal cells and their environment The essential ecological

role of certain fungi with the ability to form mycorrhizae, a sophisticated symbiotic

relationship between roots and these fungi that is one of the most prevalent associations

in all terrestrial ecosystems, is described in Chap 12 Though obviously important,

we still known very little about the biology of these plant–fungus interactions but, asyou will read, many tools of contemporary cell biology are being applied to help fillthese knowledge gaps The final chapter of the volume concerns the form and function

of interconnected, self-organized fungal networks typically occupying many squaremeters of space that are ubiquitous in nature but about which we also know quite little

As a whole this volume offers many small windows through which the reader canappreciate both the unique and shared biology of the fungal cell as well as how and whythese organisms represent remarkable and fascinating models for study

Wilmington, Delaware, USA

Aberdeen, Scotland, UK

February 2007

Richard J HowardNeil A R Gow

Volume Editors

Research in cell biology has exploded over the past decade, rendering impossible thetask of mortals to stay abreast of progress in the entire discipline Anyone interested inthe biology of the fungal cell has most certainly noticed this trend, even in this fringefield of the larger subject Indeed, to understand the biology of the fungal cell is tounderstand its interactions with the environment and with other cells, encompassing

a tremendously broad array of subdisciplines In fact, the Mycota represent one of thelast, largely unexplored gold mines of biological diversity From cellular morphogenesis

to colony formation and pathogenesis, this volume provides examples of the breadthand depth of fungal cell biology Of course, there are many topics that could not beaddressed in such limited space, but no matter Our primary aim has been to provide

a selected sampling of contemporary topics at the forefront of fungal cell biology tofacilitate the dissemination of information across and between the many enclaves ofresearchers who study fungal cell biology These include cell biologists, cytologists,developmental biologists, ecologists, geneticists, medical mycologists, microbiologists,molecular biologists, plant pathologists, and physiologists – many of whom would never

consider themselves mycologists, and what a pity We hope that the current volume will, in

some ways, serve to bridge the gaps and inequalities that exist between these mycologistsand to unite their efforts toward the advancement of our science

This volume is divided into two parts The first part considers a sampling of

behav-ioral topics – how, or in what manner and to what effect, do cells of fungi behave in

various environments; how does environment influence cell biology; how do the cellsaffect their surroundings, animate and inanimate? Topics include invasive growth, adefining characteristic of the Mycota; controls of cell polarity and shape, and morpho-logical changes that are essential for the virulence of many pathogenic fungi; and adetailed consideration of the ways in which groups of cells of the same species form anindividualistic coordinated organism

The second part of the volume looks at the fungal cell as a structural continuum –from proteins, e.g hydrophobins, that manage patterns of growth and development inspace, to extracellular matrices, molecular connections between extra- and intracellulardomains, including the cytoskeleton, to the molecular patterns of genomes that dictatethings we do not yet know exist All of these topics are perfused by recent advances inmolecular genetics and are written at a time when fungal genome databases are justbecoming established as a tool for the future We hope that this volume will not onlydemonstrate that fungal cell biology is useful in representing accessible systems forexploration of biological systems as a whole, but also in illuminating aspects of fungalbiology that are unique and fascinating in their own right We are challenged by anamazing universe of fungal cell biology waiting to be explored

Wilmington, Delaware, USA

Aberdeen, Scotland, UK

March 2001

Richard J HowardNeil A R Gow

Volume Editors

1 The Endomembrane System of the Fungal Cell

T.M Bourett, S.W James, R.J Howard 1

2 Motile Tubular Vacuole Systems

A.E Ashford, W.G Allaway 49

3 The Fungal Woronin Body

T Dhavale, G Jedd 87

4 A Molecular and Genomic View of the Fungal Cell Wall

F.M Klis, A.F.J Ram, P.W.J De Groot 97

5 The Cytoskeleton and Polarized Growth of Filamentous Fungi

R Fischer 121

6 Polarised Growth in Fungi

P Sudbery, H Court 137

7 Signal Transduction and Morphogenesis in Candida albicans

A.J.P Brown, S Argimón, N.A.R Gow 167

8 Mating in Candida albicans and Related Species

13 Network Organisation of Mycelial Fungi

M Fricker, L Boddy, D Bebber 309

Biosystematic Index 331Subject Index 333

Department of Microbiology, Columbia University,

Hammer Building, 701 West 168th Street, New York, NY 10032

A.E Ashford(e-mail: a.ashford@unsw.edu.au)

School of Biological Earth and Environmental Sciences,

The University of New South Wales, Sydney, NSW 2052, Australia

D Bebber

Department of Plant Sciences, University of Oxford,

South Parks Road, Oxford, OX1 3RB, UK

L Boddy

Cardiff School of Biosciences, Cardiff University,

Cardiff, CF10 3US, UK

T.M Bourett(e-mail: timothy.m.bourett@cgr.dupont.com)

DuPont Crop Genetics, Wilmington, DE 19880-0353, USA

A.J.P Brown(e-mail: al.brown@abdn.ac.uk)

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen,Foresterhill, Aberdeen, AB25 2ZD, UK

H Court

Department of Molecular Biology and Biotechnology, Sheffield University,

Western Bank, Sheffield S10 2TN, UK

T Dhavale(e-mail: ns_tjdha@tll.org.sg)

Temasek Life Sciences Laboratory, National University of Singapore,

1 Research Link, Singapore 117604

R Fischer(e-mail: reinhard.fischer@bio.uni-karlsruhe.de)

Max-Planck-Institute for terrestrial Microbiology,

Karl-von-Frisch-Str., D-35043 Marburg

and

University of Karlsruhe, Institute for Applied Biosciences, Dept of Applied Microbiology,Hertzstrasse 16, D-76187 Karlsruhe, Germany

M Fricker(e-mail: mark.fricker@plants.ox.ac.uk)

Department of Plant Sciences, University of Oxford,

South Parks Road, Oxford, OX1 3RB, UK

N.A.R Gow(e-mail: n.gow@abdn.ac.uk)

School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen,Foresterhill, Aberdeen, AB25 2ZD, UK

P.W.J De Groot(e-mail: p.w.j.degroot@uva.nl)

Swammerdam Institute for Life Sciences, University of Amsterdam,

1018 WV Amsterdam, The Netherlands

A.R Hardham(e-mail: adrienne.hardham@anu.edu.au)

Plant Cell Biology Group, Research School of Biological Sciences,

The Australian National University, Canberra, ACT 2601, Australia

H.C Hoch

Department of Plant Pathology, Cornell University,

New York State Agricultural Experiment Station, Geneva, NY 14456

R.J Howard(e-mail: richard.j.howard@cgr.dupont.com)

DuPont Crop Genetics, Wilmington, DE 19880-0353, USA

S.W James(e-mail: sjames@gettysburg.edu)

Biology Department, Gettysburg College, Gettysburg, PA 17325, USA

G Jedd(e-mail: gregory@tll.org.sg)

Temasek Life Sciences Laboratory, and Department of Biological Sciences,

National University of Singapore, 1 Research Link, Singapore

F.M Klis(e-mail: f.m.klis@uva.nl)

Swammerdam Institute for Life Sciences, University of Amsterdam,

1018 WV Amsterdam, The Netherlands

F Martin(e-mail: fmartin@nancy.inra.fr)

UMR INRA/UHP 1136 ‘Interactions Arbres/Micro-Organismes’, IFR110,

Centre INRA de Nancy, 54280 Champenoux, France

N.P Money(e-mail: moneynp@muohio.edu)

Department of Botany, Miami University, Oxford, OH 45056, USA

A.F.J Ram(e-mail: ram@rulbim.leidenuniv.nl)

Institute of Biology, Clusius Laboratory, Leiden University,

2333 AL Leiden, The Netherlands

B.D Shaw(e-mail: bdshaw@tamu.edu, hch1@cornell.edu)

Department of Plant Pathology and Microbiology, Program for the Biology

of Filamentous Fungi, 2132 TAMU, Texas A&M University, College Station

TX 77843, USA

D.R Soll(e-mail: david-soll@uiowa.edu)

Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242, USA

P Sudbery(e-mail: P.Sudbery@shef.ac.uk)

Department of Molecular Biology and Biotechnology, Sheffield University,

Western Bank, Sheffield S10 2TN, UK

T.M Bourett1, S.W James2, R.J Howard1

CONTENTS

I Introduction 1

II Tools for Study of the Endomembrane System 2

III Secretory Pathway 24

A Endoplasmic Reticulum 24

B Golgi Apparatus 27

C Exocytosis/Secretion 32

IV Endocytic Pathway: Plasma Membrane, Endocytosis, Endosomes, and Vacuoles 36

V Enigmatic Compartments 39

VI Conclusions 40

References 42

I Introduction

The eukaryotic endomembrane system can be

de-fined as all the organelles comprising both the

endocytic and secretory pathways, including the

endoplasmic reticulum (ER), Golgi apparatus,

en-dosomes, multivesicular bodies, lysosomes,

vac-uoles, plasma membrane, and transport

interme-diates such as vesicles and microvesicles These

membrane-enclosed compartments form a

com-plex intracellular system that can comprise a large

percentage of the total cellular volume To

under-stand the interrelationships between these

intra-cellular compartments it is helpful to consider how

each might have evolved One of the most

signif-icant advances in evolution from prokaryotes to

eukaryotes was the development of extensive

cel-lular compartmentalization (Stanier 1970),

facili-tated by the proliferation of internal membranes

(Blobel 1980) This elaboration of internal

mem-branes allowed for an organelle-based division of

labor for the biochemistry that was previously

re-stricted to the surface of prokaryotic cells (Becker

and Melkonian 1996) This in turn allowed for the

development of large cells with vastly reduced

sur-1 DuPont Crop Genetics, Wilmington, DE 19880-0353, USA

2 Biology Department, Gettysburg College, Gettysburg, PA 17325,

USA

face area:volume ratios – the average eukaryotic cell

is 102–103times greater in volume than prokary-otes (Dacks and Field 2004)

Intracellular compartments can be divided into three distinct topological groups: (1) the nucleus and cytosol, (2) mitochondria, and (3) organelles of the endomembrane system, based upon the predominant means of protein transport within each group (Blobel 1980): gated between the cytosol and nucleus via nuclear pores, transmembrane in the case of mitochondria, and mainly vesicle-mediated Organelles are membrane-bounded compartments that contain specific chemistry The protein constituents of each organelle define its structure and function Since most proteins are synthesized in the cytosol, mechanisms exist for delivery of these proteins to the proper organelle Therefore, an understanding

of protein transport is inexorably connected with understanding the endomembrane system In large part organelle homeostasis is controlled

by limiting the flow of molecules both into and out of each compartment Thus, to understand the workings of the eukaryotic cell it is funda-mental to understand the defining biochemical activities for each organelle, how molecules move between them, and how the compartments are created and maintained For the compartments that comprise the endomembrane system this is

a daunting task considering all the interorganellar communication that occurs concurrently with the flow of biomaterials through the system It is even more remarkable in fungal hyphae, perhaps the ultimate fast growing polarized eukaryotic cell Cells of septate fungi can be upwards of 200 times longer than wide (and coenocytic Zygomyetes much longer than this) with a hyphal apex that extends a distance of up to four times the hyphal diameter every minute (Collinge and Trinci 1974; López-Franco et al 1995)

While there is certainly much overlap in the strategies adopted by various eukaryotes, the

Biology of the Fungal Cell, 2nd Edition The Mycota VIII

R.J Howard and N.A.R Gow (Eds.)

© Springer-Verlag Berlin Heidelberg 2007

endomembrane system of filamentous fungi has

several singular structural features that set it

apart from that of other higher eukaryotes For

example, the Golgi apparatus in filamentous fungi

lacks stacks of membrane cisternae and does not

disperse during mitosis Also, there is a lack of

structural evidence to support the existence of

clathrin-coated vesicles, vectors that are

respon-sible for the bulk of endocytosis and trans-Golgi

network trafficking Some of these differences

appear to be shared between filamentous fungi

and their yeast relatives while others are not

(Ta-bles 1.1–1.6) Whether these structural differences

underlie significant functional differences remains

to be answered and could perhaps be exploited

in the design of control strategies against fungi,

many of which have a significant negative impact

upon humankind

As we consider the endomembrane system of

filamentous fungi, we unavoidably focus on the

hy-phal tip cell (Fig 1.1) where the most obvious

prod-uct of that system, polarized growth, is manifest

Additionally, in terms of morphology and

ultra-structure, the hyphal tip cell is undoubtedly the

most studied of all fungal cells The overall

dis-tribution of endomembrane compartments related

to the tip growth process is carefully orchestrated

and maintained; and perturbation of the hyphal

apex is evidenced by a rapid redistribution of

cellu-lar endomembrane components, particucellu-larly those

associated with the Spitzenkörper For further

re-lated discussion, the reader is referred to Chaps 5

and 6 in this volume, respectively by Fischer, and

by Sudbery and Court

The present chapter was written in part to spur

further inquiries in this area by bringing together

disjointed sources of information By

emphasiz-ing morphogenesis and structure we aim to draw

connections between microscope-based structural

knowledge and molecular data, and hope that this

undertaking will generate a new perspective and

appreciation for the unique qualities of the

en-domembrane system in filamentous fungi

II Tools for Study

of the Endomembrane System

The discovery and manipulation of fluorescent

reporter molecules has revolutionized cell biology

and been exploited to study fungi (Cormack

1998; Lorang et al 2001; Czymmek et al 2005)

Fluorescent protein tagging methods have aidedgreatly investigations of the endomembranesystem of other eukaryotes (Hanson and Köhler2001) These probes can be used to determine thesubcellular distribution of a given molecule aswell as assess its mobility and potential protein–protein interactions In addition, fluorescentprotein markers can be used to label specificcompartments, monitoring their size, shape, mo-bility and time-resolved changes that occur duringdevelopment or in response to environmentalstimuli For example, a yeast deletion library wasused in conjunction with a background strainwith a plasma membrane-targeted GFP to identifygenes required for precise delivery of this protein

to its proper destination (Proszynski et al 2005).These types of studies could do much to advanceour understanding

Recent advances in gene targeting and the velopment of fusion PCR for gene-tagging havecombined to make large-scale gene and genome

de-manipulation feasible in Neurospora crassa,

As-pergillus nidulans, and A fumigatus First,

dis-ruption of the non-homologous end joining DNArepair pathway (NHEJ), by deletion of the KU70

or KU80 genes, essentially eliminates the ically difficult problem of inefficient gene target-ing in these fungi (Ninomiya et al 2004; da SilvaFerreira et al 2006; Krappman et al 2006; Nayak

histor-et al 2006) For example, in A nidulans cells

lack-ing KU70 or KU80, ∼90% of transformants are

Table 1.1 (on page 3–4) Endoplasmic reticulum proteins

in fungi A nidulans (An) ER proteins were identified

by tBlastn of the An genome (http://www.broad.mit.edu/

annotation/genome/aspergillus_nidulans/) using S

cere-visiae (Sc) proteins Sc proteins were obtained from Gene

Ontology annotation for yeast endoplasmic reticulum (www.yeastgenome.org) Proteins were further defined

by forward and reverse tBlastn and blastp between

Sc and An genomes, tBlastn of An proteins against the An genome, and tBlastn and blastp of An and Sc proteins to all Fungal Genome Initiative (FGI) genomes (http://www.broad.mit.edu/annotation/fgi/)

Table 1.2 (on page 5–7) Golgi proteins A nidulans (An)

ER proteins were identified by tBlastn of the An genome (http://www.broad.mit.edu/annotation/genome/aspergillus

_nidulans/) using S cerevisiae (Sc) proteins Sc proteins

were obtained from Gene Ontology annotation for yeast golgi (www.yeastgenome.org) Proteins were further defined by forward and reverse tBlastn and blastp between

Sc and An genomes, tBlastn of An proteins against the An genome, and tBlastn and blastp of An and Sc proteins to all Fungal Genome Initiative (FGI) genomes (http://www.broad.mit.edu/annotation/fgi/)

we were able to delete the cdc7 gene within a

re-combinationally suppressed chromosomal region

on LGVI of A nidulans, in which the apparent

genetic-to-physical distance was at least 7-fold panded (≥54 kb per map unit) relative to the av-erage of∼8 kb per map unit Repeated attemptsusing conventional strains failed to delete the lo-cus, but in the∆nkuA (∆KU70) background∼15%

ex-of transformants (4 out ex-of 30) were deleted forthis gene (laboratory of S.W James, unpublisheddata) Second, fusion PCR can be used to rapidlyproduce constructs for gene deletion, tagging, orpromoter replacement Two-way fusion PCR, orsingle-joint PCR, may be used to fuse any twoDNA fragments, e.g., the coding region of a genewith an inducible promoter; and three-way fusionPCR can be used to make deletion constructs (Yang

et al 2004; Yu et al 2004) Where the genome hasbeen sequenced, fusion PCR obviates the need forDNA cloning; PCR-generated fusion constructs can

be directly transformed into NHEJ-deficient hoststrains to delete, tag, or replace portions of genes

In A nidulans, useful tagging cassettes are

avail-able 21 for tagging a gene’s C-terminus with GFP

or mRFP for in vivo cytological studies; and withthe 15-amino-acid S-peptide for affinity purifica-tion of in vivo protein complexes (Yang et al 2004).Publicly available cassettes, strains, libraries, and

a host of other resources may be obtained via theFungal Genetics Stock Center (University of Mis-souri, Kansas City; http://www.fgsc.net/) Together,these recent advances in gene targeting and genemanipulation make it possible to rapidly target ev-ery gene in a fungal genome (e.g., see Nayak et al

Table 1.3. (on page 8–11) COPII (ER → Golgi) and COPI (Golgi → ER) transport processes A nidulans

(An) proteins were identified by tBlastn of the An genome (http://www.broad.mit.edu/annotation/genome/ aspergillus_nidulans/) using Sc proteins Sc proteins were obtained from Gene Ontology annotation for yeast (www.yeastgenome.org) Proteins were further defined by forward and reverse tBlastn and blastp between Sc and An genomes, tBlastn of An proteins against the An genome, tBlastn and blastp of An and

Sc proteins to all Fungal Genome Initiative (FGI) genomes (http://www.broad.mit.edu/annotation/fgi/), and by phylogenetic trees using the neighbor- joining method excluding positions with gaps (http://www.ebi.ac.uk/clustalw/index.html)

2006) A knockout project is currently underway

in N crassa, with∼800 genes deleted so far (Colot

et al 2006) Similar projects, along with ray libraries and related technologies, are planned

microar-or under development fmicroar-or a variety of fungi (fmicroar-orcurrent status, see http://www.fgsc.net/)

Tagging the C-terminus of certain brane proteins, such as those requiring C-terminalprenylation, could be problematic and may, for ex-ample, necessitate the incorporation of a prenyla-tion motif following the tag Alternatively, tagging

endomem-of the N-terminus would require, in many cases,that the tag be incorporated after the N-terminalcleavage site for the signal peptide

The power of fluorescent reporters has beenenhanced through the development of and ad-vancements in imaging methodologies such as

laser scanning confocal and multiphoton croscopy (König 2000; Zipfel et al 2003; Czymmek

mi-2005), making the use of these approaches evenmore attractive However, an element of cautionmust also be employed Controlling the level ofexpression of a fusion protein can alter its local-ization within the cell In a worst-case non-lethalscenario the fusion is non-functional and targeted

to the wrong intracellular location Functionalcomplementation of a mutant phenotype by thefusion protein is an important determinant ofproper targeting, as is antibody-based ultra-structural confirmation that the fusion is going

to the proper compartment (Hawes et al 2001).Since it is rare to have the native promoter for

a gene in hand, expression must be controlled

by the judicious choice of a known promoterwhose expression level in a given cell type is

Table 1.4 (on page 12–16) SNAREs, RABs, RAB regulators,

COGs, YIPs, and other SNARE mediators A nidulans (An)

SNARE and SNARE-related proteins, COGs, and YIPs were

identified by tBlastn and blastp using S cerevisiae (Sc)

proteins Sc proteins were obtained from Gupta and Heath (2002), and from Gene Ontology annotation for yeast proteins (www.yeastgenome.org) An proteins were defined

by forward and reverse tBlastn between Sc and An genomes (http://www.broad.mit.edu/annotation/genome/aspergillus _nidulans/), tBlastn and blastp of An proteins against the An genome, and tBlastn and blastp of An and Sc proteins to all Fungal Genome Initiative (FGI) genomes

(http://www.broad.mit.edu/annotation/fgi/) A nidulans (An) RABs were identified by tBlastn using S cerevisiae (Sc)

Rabs Sc Rabs were obtained from Pereira-Leal and Seabra

(2001) ER Endoplasmic reticulum, GA Golgi apparatus, VA vacuole, HVS homotypic vacuole sorting, Endo Endosome,

TGN trans-Golgi network, FUN function unknown, PM

plasma membrane, PVC prevacuolar compartment

in plant epidermal cells can lead to a distribution

of the fusion protein throughout the entire Golgistack, whereas under lower levels of expressionthe enzyme is restricted to the trans-face (Hawesand Satiat-Jeunemaitre 2005) Overexpressionmay also be required for microscopic detection ifexpression of the endogenous promoter is too low,

as appears to be the case with the majority of gene

products in the Saccharomyces genome (Natter

et al 2005)

Issues and uncertainties associated with

over-expression may be less problematic in A nidulans, where the widely used alcA alcohol dehydrogenase

gene promoter permits flexible control over els of expression, and where it is often possible tofine tune the expression of a protein based simply

lev-on the carblev-on source of the growth medium In

Aspergillus, the alcA alcohol dehydrogenase gene

promoter is repressed strongly by glucose Low

Table 1.5. (on page 17–18) Clathrins, clathrin

media-tors, and dynamins in fungi A nidulans (An) proteins were identified by tBlastn and blastp using S cerevisiae

(Sc) proteins Sc proteins were obtained from Gene Ontology annotation for yeast (www.yeastgenome.org).

An proteins were defined by forward and reverse tBlastn and blastp between Sc and An genomes (http://www.broad.mit.edu/annotation/genome/aspergillus _nidulans/), tBlastn and blastp of An proteins against the An genome, tBlastn and blastp of An and Sc pro- teins to all Fungal Genome Initiative (FGI) genomes (http://www.broad.mit.edu/annotation/fgi/), and by phylo- genetic trees using the neighbor-joining method excluding positions with gaps In addition, fungal dynamins were analyzed by blastp of An proteins vs Hs genome, and by phylogenetic trees with Sc, An, and Hs proteins, using the neighbor-joining method excluding positions with gaps

Table 1.6 (on page 19–22) Endosomal, vacuolar and

exocy-totic processes A nidulans (An) ER proteins were identified

by tBlastn using S cerevisiae (Sc) proteins Sc proteins were

obtained from Gene Ontology annotation for yeast some/vacuole (www.yeastgenome.org), and from Bowers and Stevens (2005) An proteins were defined by forward and reverse tBlastn and blastp between Sc and An genomes (http://www.broad.mit.edu/annotation/genome/aspergillus _nidulans/), tBlastn of An proteins against the An genome, and tBlastn and blastp of An and Sc proteins to all Fungal Genome Initiative (FGI) genomes (http://

endo-www.broad.mit.edu/annotation/fgi/) MVB Multivesicular body, PM plasma membrane, PI-3-P phosphatidylinositol 3-phosphate, VA vacuole

alcA-driven expression on glycerol are low enough

that in some cases expression is not sufficient tofully complement a mutation Conversely, while re-

pression of an alcA-gene fusion by glucose is

nor-mally tight enough to prevent complementation of

a corresponding mutation, the alcA promoter does

leak slightly, with greater expression on glucose than on rich-glucose media (e.g., see James

minimal-et al 1999) In some cases, this differential leakinesscan provide an additional and useful means of fine-tuning gene expression, especially in cases wherethe endogenous gene expresses at a very low level.Fungi are eminently co-operative to bothmolecular and classic genetics In combinationwith live cell imaging and fluorescent protein-tagged transformants, temperature-sensitivemutants and transient expression of gene productsunder the control of inducible promoters allow forthe dissection of cell biological processes Theseapproaches are particularly useful where null

mutations are lethal In A nidulans and N crassa,

for example, essential genes are commonly deletedfrom sheltered backgrounds containing an extra,inducible wild-type allele of the target gene.Issues of dosage and expression by the induciblecopy, following deletion of the endogenous gene

locus, may be mitigated in N crassa by a recently

introduced method for constructing minimallysheltered knockout mutants (Metzenberg 2005)

In this chapter, we use comparative genomics

to delineate many components of the

endomem-brane system of A nidulans and other fungi relative

to the budding yeast Saccharomyces cerevisiae,

with additional comparisons to human (Tables 1.1–1.6) Absent from this analysis are members of thesignal recognition particle, and the complement

of glycosyltransferases and related enzymes for and O-linked protein glycosylation, and secreted

N-extracellular proteins S cerevisiae was chosen

as the primary tool for comparison because thegenome is very well annotated, and provides

a robust ontology for genes of the endomembranesystem (www.yeastgenome.org) S cerevisiae

also was selected for its relative simplicity, alongwith its phylogenetic relationship to filamentousfungi That said, some caution is warranted in theuse of this organism as a basis for comparison

to other fungi, and in addition care must be

Fig 1.1 The tip region of an

Aspergillus nidulans hypha Most organelles of the fun- gal endomembrane system can be observed in this near median longitudinal section.

The mitochondria (m) in this

preparation are marginally contrasted. er Endoplasmic

reticulum, f filasome, g Golgi cisternum, mvb multivesicular body, mt microtubule Freeze substitution preparation Bar

1.0µm

used when inferring cellular functions from

comparative genomic approaches alone Although

comparative genomics has been used to assess

subcellular distribution of various biomolecules

and to compare cellular molecular machinery

(Borkovich et al 2004), sequence analysis is often

less than conclusive (Hansen and Köhler 2001) and

needs to be followed by concrete experimentation

(Gupta and Heath 2002) For example, deletion

of an A niger ortholog of the essential SEC4P

gene in S cerevisiae required for proper vesicular

transport was found not to be lethal (Punt et al

2001) This functional difference could not havebeen gleaned by sequence comparison alone

It may be significant to note that comparativegenomics has indicated that many components ofthe fungal cytoskeleton are more closely related

to their respective animal molecules than to those

found in S cerevisiae (Xiang and Plamann 2003).

There is a tendency for non-mycologists especially

to lump filamentous fungi together with the yeast

S cerevisiae but this is ill advised and capricious.

Furthermore, S cerevisiae evolved by a

whole-genome duplication event, followed by gene loss,

gene expansion, and gene-pair divergence, from

an ancestor closely related to Kluyveromyces waltii

(Kellis et al 2004) Thus, it is no surprise that

many fungi possess simpler and less redundant

gene sets for many processes and pathways This

is true for A nidulans and many other fungi, as

we demonstrate in this chapter (Tables 1.1–1.6)

In contrast, we discovered several endomembrane

processes and gene families that have expanded

relative to yeast In some cases the expanded

mem-bers show strong similarities to genes and their

associated pathways in animals (e.g., Table 1.2,

novel ERD2-related protein; Table 1.3, novel fungal

ARF/ARL GTPases and ARF-GAP; Table 1.4, novel

fungal RAB GTPases, RAB-GAPs, and RAB-GEFs;

Table 1.5, novel fungal dynamin-related proteins

and BAR domain protein)

As a general rule, two protein sequences were

considered homologous at an expected value

(e-value) ≤ 0.0001 (i.e., 10−4) Choice of e-value

cutoff for a significant Blast hit varies between

10−4to 0.1 (e.g., see Claverie and Notredame 2003;

Pevsner 2003; Buehler and Rashidi 2005), with the

caveat that alignment between short homologs,

especially those consisting of less than 100 amino

acids, may generate e-values higher than the cutoff

Some endomembrane proteins reported herein

are relatively short, less 100 amino acids, with

correspondingly high e-values against putative

Aspergillus orthologs (e.g., ERI1 and VMA21 in

Table 1.1, PBI2 and YOS1 in Table 1.4) In several

cases, we have reported potential homologs with

e-values greater than 0.1

Another major tool used to study the

en-domembrane system has been the transmission

electron microscope, with fungi being among

the first organisms to be studied (Bracker 1967,

1974; Bracker and Grove 1971) In the early days

many new structures were described, many of

which were later shown to be artifact (Heath and

Greenwood 1970) The fine structure of most

compartments of the endomembrane system was

first described based on electron micrographs

The quality of the information has been

im-proved through the adoption of the cryo-based

methodology of ultrarapid freezing and freeze

substitution (Howard and Aist 1979; Howard 1981;

see also Chap 2 in this volume) Much of the

earliest “fungal” work was done on the öomycetes,

organisms that since have been recognized as

taxonomically separate from true fungi (Wainright

et al 1993); and ultrastructural differences such

as those related to the Golgi apparatus bear this

out (Bracker et al 1996) The electron microscopecontinues to provide invaluable information in theform of high-resolution images, and molecularinformation can be obtained using immuno-based

and other affinity probes such as lectins The lectin

concanavalin A (ConA) has been used as a generalmarker for the fungal endomembrane system forboth light and electron microscopy (Bourett et al.1993; Bourett and Howard 1994) ConA-labeledorganelles include the plasma membrane, Golgiequivalents (see Sect III.B.), transport vesiclesand a class of tubular vacuoles, but not the ER(Fig 1.2B) For discussion of additional probes,such as the fluorescent FM dyes, see Sect IV Transmission electron microscope analysescan also be enhanced through the creation ofthree-dimensional renderings While this three-dimensional information can be extracted fromserial thin-section (e.g., Bourett and Howard 1994;Winey et al 1995) or HVEM semi-thick section(e.g., Howard 1981) analysis, higher resolutionimages can be obtained using semi-thick sectionsand electron tomography methodologies (Ladin-sky et al 1999; O’Toole et al 1999; Müller et al.2000; Harris et al 2005; Donohoe et al 2006)

Biochemistry has also played a role in

increas-ing our understandincreas-ing of compartmentalizationwithin the endomembrane system, especially in

studies of Saccharomyces cerevisiae where good

morphological data is often elusive For example,

it has proven to be very useful to analyze secretedproteins as they pass through successive compart-ments of the endomembrane system If the secre-tory pathway is blocked by either treatment with

a pharmacological agent or the introduction of

a genetic lesion, then the normally secreted tein will accumulate at some location(s) within thecell Through biochemical analysis the compart-ment where the accumulation occurred can be de-termined since the subcellular site for each sequen-tial modification to the secreted protein has beenestablished (Schekman 1992)

pro-III Secretory Pathway

A Endoplasmic Reticulum

The ER has an essential role in both lipid andprotein pathways as the site of biosynthesis ofnearly all cellular membranes and transmembraneproteins regardless of the ultimate destination

of these molecules Luminal proteins destined