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Edited by
Karl Esser

THE MYCOTA
A Comprehensive Treatise on Fungi
as Experimental Systems for Basic and Applied Research

Biochemistry
and Molecular Biology
III
Third Edition

Dirk Hoffmeister
Volume Editor
The Mycota
Edited 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
3rd edition ed. by J. Wendland
II Genetics and Biotechnology
1st and 2nd edition ed. by U. Kück
III Biochemistry and Molecular Biology
1st and 2nd edition ed. by R. Brambl and G. Marzluf
3rd edition ed. by D. Hoffmeister
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
3rd edition ed. by I.S. Druzhinina and C.P. Kubicek
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.A. Brakhage and P.F. Zipfel
VII Systematics and Evolution
1st edition ed. by D.J. McLaughlin, E.G. McLaughlin, and P.A. Lemke{
2nd edition ed. by D.J. McLaughlin and J.W. Spatafora
VIII Biology of the Fungal Cell
1st and 2nd edition ed. by R.J. Howard and N.A.R. Gow
IX Fungal Associations
1st and 2nd edition ed. by B. Hock
X Industrial Applications
1st edition ed. by H.D. Osiewacz
2nd edition ed. by M. Hofrichter
XI Agricultural Applications
1st and 2nd edition ed. by F. Kempken
XII Human Fungal Pathogens
1st edition ed. by J.E. Domer and G.S. Kobayashi
2nd edition ed. by O. Kurzai
XIII Fungal Genomics
1st edition ed. by A.J.P. Brown
2nd edition ed. by M. Nowrousian
XIV Evolution of Fungi and Fungal-Like Organisms
Ed. by S. Pöggeler and J. Wöstemeyer
XV Physiology and Genetics: Selected Basic and Applied Aspects
Ed. by T. Anke and D. Weber
The Mycota
A Comprehensive Treatise on Fungi as
Experimental Systems for Basic and Applied
Research

Edited by K. Esser

III
Volume Editor:
Biochemistry and Molecular
Biology
3rd Edition
D. Hoffmeister
Series Editor
Professor Dr. Dr. h.c. mult. Karl Esser
Allgemeine Botanik
Ruhr-Universität
44780 Bochum, Germany
Tel.: +49 (234)32-22211
Fax.: +49 (234)32-14211
e-mail: Karl.Esser@rub.de

Volume Editor

Professor Dr. Dirk Hoffmeister

Friedrich-Schiller-Universität
Pharmazeutische Mikrobiologie
Winzerlaer Straße 2
07745 Jena
Germany
Tel.: +49 (3641) 949851
Fax: +49 (3641) 949852
e-mail: dirk.hoffmeister@leibniz-hki.de

ISBN 978-3-319-27788-2 ISBN 978-3-319-27790-5 (eBook)

DOI 10.1007/978-3-319-27790-5

Library of Congress Control Number: 2016932310

Springer Cham Heidelberg New York Dordrecht London

# Springer International Publishing Switzerland 1996, 2004, 2016
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the
material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
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The use of general descriptive names, registered names, trademarks, service marks, 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.
The publisher, the authors and the editors are safe to assume that the advice and information in this
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herein or for any errors or omissions that may have been made.

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media

(www.springer.com)
Karl Esser
(born 1924) is retired Professor of General Botany
and Director of the Botanical Garden at the Ruhr-
Universität Bochum (Germany). His scientific work
focused on basic research in classical and molecular
genetics in relation to practical application. His stud-
ies were carried out mostly on fungi. Together with
his collaborators he was the first to detect plasmids in
higher fungi. This has led to the integration of fungal
genetics in biotechnology. His scientific work was
distinguished by many national and international
honors, especially three honorary doctoral degrees.

Dirk Hoffmeister
(born 1972) is a mycologist and Full Professor for
Pharmaceutical Microbiology at the Friedrich-Schil-
ler-University Jena (Germany). He is also affiliated
with the Leibniz Institute for Natural Product
Research and Infection Biology—Hans Knöll Insti-
tute. Particularly emphasizing the basidiomycetes,
research in his group is focused on the biochemical
and genetic basis underlying fungal natural product
biosyntheses.
ThiS is a FM Blank Page
Series Preface

Mycology, the study of fungi, originated as a sub discipline of botany and was a
descriptive discipline, largely neglected as an experimental science until the early
years of this century. A seminal paper by Blakeslee in 1904 provided evidence for
self-incompatibility, termed “heterothallism,” and stimulated interest in studies
related to the control of sexual reproduction in fungi by mating-type
specificities. Soon to follow was the demonstration that sexually reproducing
fungi exhibit Mendelian inheritance and that it was possible to conduct formal
genetic analysis with fungi. The names Burgeff, Kniep and 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 a period of interest in mutation induction and analysis of mutants
for biochemical traits. Such fundamental research, conducted largely with Neu-
rospora crassa, led to the one gene:one enzyme hypothesis and to a second Nobel
Prize for fungal research awarded to Beadle 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 soon able to
compete with bacterial systems in the molecular arena.
The experimental achievements in research on the genetics and molecular
biology of fungi have benefited more generally studies in the related fields of
fungal biochemistry, plant pathology, medical mycology, and systematics. Today,
there is much interest in the genetic manipulation of fungi for applied research.
This current interest in biotechnical genetics has been augmented by the develop-
ment of DNA-mediated transformation systems in fungi and by an understanding
of gene expression and regulation at the molecular level. Applied research initia-
tives involving fungi extend broadly to areas of interest 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 through-
out this series of volumes, the names adopted for major groups of fungi (repre-
sentative genera in parentheses) are as follows:
Pseudomycota
Division: Oomycota (Achlya, Phytophthora, Pythium)
Division: Hyphochytriomycota
viii Series Preface

Eumycota
Division: Chytridiomycota (Allomyces)
Division: Zygomycota (Mucor, Phycomyces, Blakeslea)
Division: Dikaryomycota
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 forms insofar as they ingest nutrients by phagocytosis, lack a cell
wall during the assimilative phase, and clearly show affinities with certain proto-
zoan taxa.
The series throughout will address three basic questions: what are the fungi,
what do they do, and what is their relevance to human affairs? Such a focused and
comprehensive treatment 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 give serious and sustained consideration to any reclassification of major
fungal groups, has delayed early publication. We wish, however, to provide a
preamble on the nature of fungi, to acquaint readers who are unfamiliar with
fungi with certain characteristics that are representative of these organisms and
which make them attractive subjects for experimentation.
The fungi represent a heterogeneous assemblage of eukaryotic microorgan-
isms. Fungal metabolism is characteristically heterotrophic or assimilative for
organic carbon and some nonelemental source of nitrogen. Fungal cells charac-
teristically imbibe or absorb, rather than ingest, nutrients and they have rigid cell
walls. The vast majority of fungi are haploid organisms reproducing either
sexually or asexually through spores. The spore forms and details on their
method of production have been used to delineate most fungal taxa. Although
there is a multitude of spore forms, fungal spores are basically only of two types:
(1) asexual spores are formed following mitosis (mitospores) and culminate
vegetative growth, and (2) sexual spores are formed following meiosis (meios-
pores) and are 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 direct
recovery of meiospores coupled with extended vegetative haploidy, that have
made fungi especially attractive as objects for experimental research.
 Series Preface ix

The ability of fungi, especially the saprobic fungi, to absorb and grow on rather
simple and defined substrates and to convert these substances, not only into
essential metabolites but into important secondary metabolites, is also noteworthy.
The metabolic capacities of fungi have attracted much interest in natural products
chemistry and in the production of antibiotics and other bioactive compounds.
Fungi, especially yeasts, are important in fermentation processes. Other fungi are
important in the production of enzymes, 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 and animal hosts. Fungi even grow on other fungi. They are found in
aquatic as well as soil environments, and their spores may pollute the air. Some
are edible; others are poisonous. Many are variously associated with plants as
copartners in the formation of lichens and mycorrhizae, as symbiotic endophytes
or as overt pathogens. Association with animal systems varies; examples include
the predaceous fungi that trap nematodes, the microfungi that grow in the
anaerobic environment of the rumen, the many insect associated fungi, and the
medically important pathogens afflicting humans. Yes, fungi are 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 such studies 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 leadership shown by the Volume Editors in selecting topics and assembling
the experts. We have all been a bit ambitious in producing these volumes on a
timely basis and therein lies the possibility of mistakes and oversights in this first
edition. We encourage the readership to draw our attention to any error, omis-
sion, or inconsistency in this series in order that improvements can be made in
any subsequent edition.
Finally, we wish to acknowledge the willingness of Springer-Verlag to host
this project, which is envisioned to require more than 5 years of effort and the
publication of at least nine volumes.

Bochum, Germany KARL ESSER

Auburn, AL, USA PAUL A. LEMKE
April 1994 Series Editors
.
Volume Preface

The years in between the previous and the current third edition of The Mycota III
witnessed a dramatic change in how we address scholarly questions in fungal
biology. Above all, the rapidly advancing technologies to generate massive
amounts of nucleic acid sequence data that enabled the exponential growth of
the number of sequenced genomes and the increased quality of transcriptomes
have provided insight into the function and evolution of fungi to an extent we
would probably not have even dreamed of 10 years ago. Consequently, results
reviewed in the newly written or updated chapters of this volume underscore the
copious availability of genomes, transcriptomes, or proteomes, which has become
the expected norm.
To truly advance fungal biology, we need to be careful in order not to get
sidetracked and drowned in an ocean of computer-produced data. “We are at the
start of what will be one of the most exciting periods of advance and discovery in
the history of our field.” These words, written by Professors Robert Brambl and
George A. Marzluf in the preface of the second edition of The Mycota III, should
encourage us, more than ever, to be thoughtful in asking the right questions and
to test hypotheses—beyond the in silico level—experimentally through wet-
bench work.
To produce the third edition of The Mycota III, the Editor was privileged to
work with diligent and enthusiastic experts as both recurring and first-time
chapter contributors. The Editor is both pleased and thankful of the five returning
authors/author teams and additionally excited to have many new authors. It was
the aim of the Editor to cover fungi as broadly and comprehensively as possible.
Accordingly, chapters highlighting the aspects of zygomycete and basidiomycete
biology appear to complement contributions that focus on precious model spe-
cies, such as Aspergilli. Also, primary metabolism, gene regulation, and signal
transduction were further emphasized in the new volume, e.g., with chapters on
major metabolic routes, RNA interference, regulation in plant pathogenic fungi,
but also on global regulation of Aspergillus natural product biosyntheses, the
latter to reflect the markedly increased interest in fungal secondary metabolites.
The Editor cordially thanks Dr. Andrea Schlitzberger of Springer Publishers
for excellent and competent guidance throughout the production process and,
last but not least, the Series Editor, Professor Emeritus Karl Esser, for precious
advice and encouragement that was always combined with a fine sense of humor.

Jena, Germany Dirk Hoffmeister

October 2015
.
Contents

Regulation of Gene Expression

1 Molecular Biology of Asexual Sporulation in Filamentous Fungi . . . 3

HEE-SOO PARK, JAE-HYUK YU

2 Insight into Fungal Secondary Metabolism from Ten Years of LaeA

Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
JIN WOO BOK, NANCY P. KELLER

3 RNAi Function and Diversity in Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

ÅKE OLSON

4 Fungal Molecular Response to Heavy Metal Stress . . . . . . . . . . . . . . . . . . 47

MATTHIAS GUBE

5 Control of Gene Expression in Phytopathogenic Ascomycetes During

Early Invasion of Plant Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
CANDACE E. ELLIOTT

Signal Transduction

6 Fungal MAP-Kinase-Mediated Regulatory Pathways . . . . . . . . . . . . . . . . 97

STEFAN IRNIGER, ÖZLEM SARIKAYA-BAYRAM, ÖZGÜR BAYRAM

7 Heterotrimeric G Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

REKHA DEKA, ARIT GHOSH, RANJAN TAMULI, KATHERINE A. BORKOVICH

Molecular Biology and Biochemistry of Fungal Carbohydrates

8 The Cell Wall Polysaccharides of Aspergillus fumigatus . . . . . . . . . . . . 147

MARK J. LEE, DONALD C. SHEPPARD

9 Chitin Synthesis and Fungal Cell Morphogenesis . . . . . . . . . . . . . . . . . . . 167

CESAR RONCERO, ALBERTO SANCHEZ-DIAZ, M.-HENAR VALDIVIESO

10 Trehalose Metabolism: Enzymatic Pathways and Physiological

Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
MARTA RUBIO-TEXEIRA, GRIET VAN ZEEBROECK, JOHAN M. THEVELEIN
xiv Contents

Molecular Aspects of Biochemical Pathways

11 Regulation of Fungal Nitrogen Metabolism . . . . . . . . . . . . . . . . . . . . . . . . 281

RICHARD B. TODD

12 Regulation of Sulfur Metabolism in Filamentous Fungi . . . . . . . . . . 305

JOHN V. PAIETTA

13 The Regulation of Carbon Metabolism in Filamentous Fungi . . . 321

JOAN M. KELLY

14 Special Aspects of Fungal Catabolic and Anabolic Pathways . . . . . 341

MATTHIAS BROCK, ELENA GEIB

15 Genetic and Metabolic Aspects of Primary and Secondary

Metabolism of the Zygomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
KERSTIN VOIGT, THOMAS WOLF, KATRIN OCHSENREITER, GÁBOR NAGY,
KERSTIN KAERGER, EKATERINA SHELEST, TAMÁS PAPP

Biosystematic Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

List of Contributors

ÖZGÜR BAYRAM
Department of Biology, Maynooth University, National University of Ireland
Maynooth, Maynooth Co. Kildare, Ireland

JIN WOO BOK

Department of Medical Microbiology and Immunology, University of Wisconsin-
Madison, Madison, WI, USA

KATHERINE A. BORKOVICH
Department of Plant Pathology and Microbiology, University of California,
Riverside, CA, USA

MATTHIAS BROCK
Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product
Research and Infection Biology e. V., -Hans Knöll Institute, Jena, Germany
Institute for Microbiology, Friedrich Schiller University Jena, Jena, Germany
Fungal Genetics and Biology Group, School of Life Sciences, University of
Nottingham, Nottingham, UK

REKHA DEKA
Department of Plant Pathology and Microbiology, University of California,
Riverside, CA, USA

CANDACE E. ELLIOTT
School of Biosciences, The University of Melbourne, Melbourne, VIC, Australia

ELENA GEIB
Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product
Research and Infection Biology e. V., -Hans Knöll Institute, Jena, Germany
Fungal Genetics and Biology Group, School of Life Sciences, University of
Nottingham, Nottingham, UK

ARIT GHOSH
Department of Plant Pathology and Microbiology, University of California,
Riverside, CA, USA
xvi List of Contributors

MATTHIAS GUBE
Soil Science of Temperate Ecosystems, Georg August University Göttingen,
Göttingen, Germany
Microbial Communication, Friedrich Schiller University Jena, Jena, Germany

STEFAN IRNIGER
Department of Molecular Microbiology and Genetics, Georg August University,
Göttingen, Germany

KERSTIN KAERGER
National Reference Center for Invasive Mycoses, Leibniz Institute for Natural
Product Research and Infection Biology – Hans-Knöll-Institute, Jena, Germany

NANCY P. KELLER
Department of Medical Microbiology and Immunology, University of Wisconsin-
Madison, Madison, WI, USA

JOAN M. KELLY
Department of Genetics and Evolution, The University of Adelaide, Adelaide,
Australia

MARK J. LEE
Department of Microbiology and Immunology, McGill University, Montréal, QC,
Canada

GÁBOR NAGY
Faculty of Science and Informatics, Department of Microbiology, University of
Szeged, Szeged, Hungary

KATRIN OCHSENREITER
Karlsruhe Institute of Technology (KIT), Institute of Process Engineering in Life
Sciences, Section II: Technical Biology, Karlsruhe, Germany

ÅKE OLSON
Department of Forest Mycology and Plant Pathology, Swedish University of
Agricultural Sciences, Uppsala, Sweden

JOHN V. PAIETTA
Department of Biochemistry and Molecular Biology, Wright State University,
Dayton, OH, USA

TAMÁS PAPP
Faculty of Science and Informatics, Department of Microbiology, University of
Szeged, Szeged, Hungary

HEE-SOO PARK
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI,
USA
 List of Contributors xvii

CESAR RONCERO
Departamento de Microbiologia y Genetica, IBFG, CSIC, Universidad de
Salamanca, Salamanca, Spain

MARTA RUBIO-TEXEIRA
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology,
KU Leuven, Leuven, Belgium
Department of Molecular Microbiology, VIB, Leuven-Heverlee, Flanders,
Belgium

ALBERTO SANCHEZ-DIAZ
Instituto de Biomedicina y Biotecnologia de Cantabria (IBBTEC), Universidad de
Cantabria, CSIC, Santander, Spain
Departamento de Biologia Molecular. Facultad de Medicina, Universidad de
Cantabria, Santander, Spain

ÖZLEM SARIKAYA-BAYRAM
Department of Biology, Maynooth University, National University of Ireland
Maynooth, Maynooth Co. Kildare, Ireland

EKATERINA SHELEST
Systems Biology/Bioinformatics, Leibniz Institute for Natural Product Research
and Infection Biology – Hans-Knöll-Institute, Jena, Germany

DONALD C. SHEPPARD
Department of Microbiology and Immunology, McGill University, Montréal, QC,
Canada
Department of Medicine, McGill University, Montréal, QC, Canada
Department of Microbiology & Immunology, McGill University, Montréal, Qc,
Canada

RANJAN TAMULI
Department of Biosciences and Bioengineering, Indian Institute of Technology
Guwahati, Guwahati, Assam, India

JOHAN M. THEVELEIN
Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology,
KU Leuven, Leuven, Belgium
Department of Molecular Microbiology, VIB, Leuven-Heverlee, Flanders,
Belgium

RICHARD B. TODD
Department of Plant Pathology, Kansas State University, Manhattan, KS, USA

M.-HENAR VALDIVIESO
Departamento de Microbiologia y Genetica, IBFG, CSIC, Universidad de
Salamanca, Salamanca, Spain
xviii List of Contributors

KERSTIN VOIGT
Jena Microbial Resource Collection, Leibniz Institute for Natural Product
Research and Infection Biology – Hans-Knöll-Institute, Jena, Germany
Department of Microbiology and Molecular Biology, Institute of Microbiology,
University of Jena, Jena, Germany

THOMAS WOLF
Systems Biology/Bioinformatics, Leibniz Institute for Natural Product Research
and Infection Biology – Hans-Knöll-Institute, Jena, Germany

JAE-HYUK YU
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI,
USA
Department of Genetics, University of Wisconsin-Madison, Madison, WI, USA

GRIET VAN ZEEBROECK

Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology,
KU Leuven, Leuven, Belgium
Department of Molecular Microbiology, VIB, Leuven-Heverlee, Flanders,
Belgium
Regulation of Gene Expression
1 Molecular Biology of Asexual Sporulation in Filamentous Fungi

HEE-SOO PARK1, JAE-HYUK YU1,2

CONTENTS ation, progression, and completion of asexual

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 sporulation are regulated by various positive
II. Asexual Sporulation in Aspergillus and negative genetic elements that direct the
nidulans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 expression of genes required for the proper
A. Morphology of Asexual Structure . . . . . . . . . . . 3 assembly of the conidiophore and the forma-
B. Regulators of Asexual Development . . . . . . . . . 4
1. Central Regulators of Conidiation . . . . . . . . 4 tion and maturation of conidiospores (Adams
2. Controllers of the Central Regulators . . . . . 6 et al. 1998; Park and Yu 2012). This chapter
3. Feedback Regulators of Conidiation . . . . . 10 presents a snapshot of the up-to-date molecular
III. Asexual Sporulation in Penicillum biology of conidiation in the three model fungi
marneffei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Aspergillus nidulans, Penicillium marneffei,
A. Morphology of Asexual Structure . . . . . . . . . . 10
B. Regulators of Asexual Development . . . . . . . . 12 and Fusarium graminearum.
IV. Asexual Sporulation in Fusarium
graminearum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
A. Morphology of Asexual Structure . . . . . . . . . . 12
B. Regulators of Asexual Development . . . . . . . . 13 II. Asexual Sporulation in Aspergillus
V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 nidulans

A. Morphology of Asexual Structure

I. Introduction A conidiophore is a specialized asexual struc-
ture, which is the most remarkable characteris-
Filamentous fungi are of great importance to tic of a specific fungal species (Adams et al.
humankind as pathogens, environmental recy- 1998; Yu 2010). The formation of conidio-
clers, industrial producers, and agricultural phores in A. nidulans is highly sophisticated
aids. Fungi produce spores as the primary and can be divided into several differential
means of reproduction, propagation, and infec- stages (Fig. 1.1) (Mims et al. 1988; Timberlake
tivity (Ebbole 2010). Fungi use asexual sporula- 1990). First, conidiation starts from the foot
tion as a main reproductive mode (Adams et al. cells, thick-walled cells, which elongate into
1998; Ni et al. 2010). Asexual nonmotile spores, the air to produce aerial stalks. After the exten-
called conidia (conidiospores), are formed on sion of stalks ceases, the tip of stalks then starts
the specialized developmental structure called to swell and form apical vesicles, which contain
conidiophore. The entire processes of the initi- multiple nuclei. Through a budding-like pro-
cess, 60 metulae are formed on the surface of
1
the vesicle and then each metula produces
Department of Bacteriology, University of Wisconsin-Madi-
2 buds that develop into uninucleate sterig-
son, Madison, WI 53706, USA; e-mail: jyu1@wisc.edu
2
Department of Genetics, University of Wisconsin-Madison,
mata, termed phialides. Phialides undergo
1550 Linden Drive, Madison, WI 53706, USA; e-mail: repeated asymmetric mitotic divisions to gen-
jyu1@wisc.edu erate chains of conidia (about 120 conidia per

Biochemistry and Molecular Biology, 3rd Edition

The Mycota III
D. Hoffmeister (Ed.)
© Springer International Publishing Switzerland 2016
4 H.-S. Park and J.-H. Yu
Central regulatory genes
mature conidia
immature conidia
phialides
metulae
StuA
vesicle
stalk
foot cell
Fig. 1.1 Conidiophore and the central regulators of conidiation in A. nidulans. Simplified illustration of the
conidiophore (left) and a genetic model of the central regulatory pathway of conidiation (right) are shown
phialide) (Adams et al. 1998; Mims et al. 1988).
Conidia are haploid cells and capable of form-
ing a new colony under appropriate conditions.
Soon after conidia are produced, they undergo
a maturation process, involving the modifica-
tion of the conidial wall and trehalose biogene-
sis for the establishment of conidial dormancy
(Ni et al. 2010; Sewall et al. 1990b; Timberlake
1990). The whole process of conidiation is
genetically programmed, and various regula-
tors are responsible for the progression of
each stage (Adams et al. 1998).
B. Regulators of Asexual Development
1. Central Regulators of Conidiation
a) BrlA
An essential initiation step of conidiation is the
activation of brlA encoding a C2H2 zinc-finger
transcription factor (TF) (Adams et al. 1988,
1990). The conidiophores of brlA null mutants
fail to form a vesicle and other asexual structures
and continue to grow conidiophore stalks (thus
termed “bristle”) (Clutterbuck 1969). Contrarily,
the overexpression of brlA causes termination of
hyphal growth coupled with the formation of
spores directly from the hyphal tips (Adams
et al. 1988), suggesting that brlA is sufficient to
induce the transition from polar hyphal growth
to asexual sporulation.
As a key TF, BrlA is necessary for activating
the expression of development-related genes
during the early phase of conidiation (Adams
VelB
WetA Feedback regulators
VosA
AbaA
MedA Developmental modifiers
BrlA
Inducers
et al. 1988; Boylan et al. 1987; Clutterbuck 1990;
Mirabito et al. 1989; Stringer et al. 1991; Timber-
lake 1991). The brlA mutants fail to accumulate
transcripts of certain development-specific
genes, including abaA, wetA, rodA, and yA
(Birse and Clutterbuck 1991; Boylan et al. 1987;
Mayorga and Timberlake 1990; Stringer et al.
1991). BrlA contains two C2H2 zinc-finger motifs
(Adams et al. 1988), which are important for
DNA binding and BrlA TF activity (Adams
et al. 1990). Mutation in either one of the two
motifs results in a complete loss of BrlA activity
(Adams et al. 1990). This central developmental
TF interacts with the BrlA response elements
(BREs; 50 -(C/A)(G/A)AGGG(G/A)-30 ) and acti-
vates mRNA expression of multiple develop-
mental genes (Chang and Timberlake 1993).
Chang and Timberlake demonstrated that brlA
expression in Saccharomyces cerevisiae caused
activation of the brlA-dependent gene, and mul-
tiple BREs were present in the promoter regions
of several developmentally regulated genes in A.
nidulans (Chang and Timberlake 1993).
Expression of brlA is subject to complex regulation.
The brlA gene consists of two overlapping transcripts,
designated brlAa and brlAb, which are regulated at
different levels and are individually required for proper
development (Han and Adams 2001; Han et al. 1993;
Prade and Timberlake 1993). The brlAb transcript
encodes two open reading frames (ORFs), brlAb ORF,
and a short upstream ORF (brlAb mORF) (Han et al.
1993). Expression of brlAa is only regulated at the
transcriptional level and requires both BrlA and AbaA
(Han et al. 1993). Expression of brlAb is regulated at
both the transcriptional and translational levels. For
 Molecular Biology of Asexual Sporulation in Filamentous Fungi
transcriptional control, there are multiple cis-acting
regulatory sequences involved in the activation or
repression of brlAb mRNA (Han and Adams 2001). In
vegetative cells, translation of the brlAb mORF represses
BrlA translation and thereby prevents premature devel-
opment (Han and Adams 2001; Han et al. 1993).
b) AbaA
AbaA is a presumed TF that is induced by BrlA
during the middle phases of asexual develop-
ment after differentiation of metulae and is
required for proper formation and function of
phialides (Boylan et al. 1987; Sewall et al.
1990a). The abaA null mutants bear an
abacus-like structure with swellings at intervals
in place of chains of conidia, suggesting its
possible role in cytokinesis (Clutterbuck 1969;
Sewall et al. 1990a). Non-sporulating conidio-
phores of abaA mutants are differentiated from
sterigmata but do not form conidiogenous
phialides, suggesting that abaA is required for
proper function of phialides as sporogenous
cells. The overexpression of abaA in vegetative
cells results in growth termination and cellular
vacuolization but no spore formation (Adams
and Timberlake 1990; Mirabito et al. 1989).
AbaA is a member of TEF-1 (Transcrip-
tional Enhancer Factor-1) family that contains
an ATTS (AbaA, TEC1p, TEF-1 sequence)/TEA
DNA-binding domain with a potential leucine
zipper for dimerization (Andrianopoulos and
Timberlake 1991, 1994; Mirabito et al. 1989).
Results from a gel shift assay showed that
AbaA interacts with the consensus sequence
50 -CATTCY-30 (AbaA response element
(ARE), where Y is a T or C) (Andrianopoulos
and Timberlake 1994). One or multiple AREs
are present in the promoter regions of several
developmental genes such as yA, rodA, wA, and
wetA, the upstream gene brlA and abaA itself
(Andrianopoulos and Timberlake 1994) (Ara-
mayo and Timberlake 1993; Mayorga and Tim-
berlake 1990; Mirabito et al. 1989; Stringer et al.
1991). Further studies demonstrated that AbaA
regulates the chitin synthase gene chsC (Ichino-
miya et al. 2005; Park et al. 2003), the velvet
genes vosA and velB (Ni and Yu 2007; Park
et al. 2012b), and a component of the axial
bud site marker Axl2 (Si et al. 2012) during
conidiation.
5
Genetic interaction between abaA and brlA is compli-
cated (Aguirre 1993; Aguirre et al. 1990). The overex-
pression of abaA in hyphae induces expression of brlA,
whereas the abaA null mutant also activates brlA
expression (Aguirre 1993), indicating that AbaA acts
as both an activator and a repressor of brlA. Further
studies proposed that AbaA exerts its repressive role in
brlA expression via activating VosA, a key feedback
negative regulator of brlA (Han and Adams 2001; Ni
and Yu 2007).
c) WetA
The wetA gene is activated by sequential
expression of brlA and abaA during asexual
sporulation, and WetA is essential for the mod-
ification of the conidial wall resulting in the
impermeable and resilient conidia (Marshall
and Timberlake 1991; Sewall et al. 1990b). The
phenotype of wetA null mutants is described as
“wet-white” because these mutants produce
colorless and autolytic conidia (Clutterbuck
1969; Sewall et al. 1990b). Conidia of the wetA
null mutant lack both the condensation of the
C2 wall layer and the formation of C3 and C4
layers (Sewall et al. 1990b). The wetA gene is
sufficient for the activation of the conidium-
specific gene, including wA, and the wetA null
mutant fails to accumulate mRNAs of
conidium-specific genes, indicating that WetA
may act as a (transcriptional) regulator of
conidium-specific genes (Marshall and Timber-
lake 1991; Sewall et al. 1990b). The overexpres-
sion of wetA in hyphae inhibits hyphal growth
and causes excessive branching without caus-
ing the activation of brlA and abaA expression
or the induction of precocious conidiation
(Marshall and Timberlake 1991). Overall,
WetA is required for spore maturation and is
proposed to be an activator of a set of
conidium-specific genes.
d) StuA and MedA
StuA (stunted) and MedA (medusa) function as
developmental modifiers that are necessary for
the precise organization of conidiophores
(Adams et al. 1998). The StuA is a sequence-
specific TF, which contains the APSES domain
(Dutton et al. 1997). Mutational inactivation of
stuA causes production of abnormal conidio-
phores with the lack of metulae and phialides.
6 H.-S. Park and J.-H. Yu
However, some stuA mutants can produce viable
conidia directly from vesicle (Clutterbuck 1969).
The stuA gene encodes two transcription units,
stuAa and stuAb, and transcription of both
mRNAs dramatically increases during the estab-
lishment of developmental competence (Miller
et al. 1991, 1992). StuA is required for proper
activation of brlA and repression of abaA (Dut-
ton et al. 1997). Apposite stuA expression is
required for the proper conidiophore morphol-
ogy, but StuA may not affect the temporal devel-
opment of cell types (Wu and Miller 1997).
Mutational inactivation of medA results in aber-
rant conidiophores with multiple layers of ster-
igmata before conidia formation (Clutterbuck
1969). While StuA is essential for spatial expres-
sion of brlA, MedA is required for proper tem-
poral expression of brlAa and brlAb. In
addition, MedA acts as a coactivator required
for abaA expression (Busby et al. 1996).
2. Controllers of the Central Regulators
a) FluG-Mediated Signaling Pathway
Developmental transition from vegetative
growth is highly complex and involves various
positive elements in response to internal and
external cues (Fig. 1.2) (Adams et al. 1998).
Two decades ago, Wieser et al. carried out
genetic analyses of recessive mutations and
identified six upstream developmental activa-
tors, including fluG, flbA, flbB, flbC, flbD, and
flbE (Wieser et al. 1994). The deletion of any one
of these genes leads to the fluffy phenotypes
(cotton-like colonies) coupled with decreased
brlA expression (Adams et al. 1998; Wieser
et al. 1994). A series of follow-up studies has
revealed the molecular mechanisms and genetic
relationships of these genes and proposed a
model for the roles of the upstream developmen-
tal activators in conidiation (Adams et al. 1998;
Etxebeste et al. 2010; Park and Yu 2012; Yu
2010).
FluG contains a glutamine synthetase I like
domain and is a key activator of asexual devel-
opment in A. nidulans (Lee and Adams 1994).
The fluG deletion mutant cannot produce nor-
mal conidiophores under standard culture con-
ditions, whereas the overexpression of fluG
causes conidiophore formation and brlA acti-
vation in liquid submerged culture (D’Souza
et al. 2001; Lee and Adams 1996). Lee and
Adams proposed that FluG is required for the
synthesis of an extracellular sporulation induc-
ing factor (ESIF, also known as the FluG factor),
which signals the activation of conidiophore
development (Lee and Adams 1994). Later
studies by Rodriguez-Urra and colleagues
have revealed that the FluG factor is a
diorcinol-dehydroaustinol adduct, which can
rescue the conidiation defects caused by the
deletion of fluG (Rodriguez-Urra et al. 2012).
The FluG-initiated developmental signaling
then leads to two independent regulatory pro-
cesses: (1) inhibition of hyphal growth via acti-
vation of FlbA and (2) activation of
developmental-specific regulatory cascades
(FlbB–FlbE) (Adams et al. 1998). Further
genetic studies have revealed that both FluG-
mediated processes occur via the removal of
repression of conidiation imposed by SfgA
(Seo et al. 2003, 2006). SfgA is an upstream
repressor of conidiation with a Gal4-type
Zn(II)2Cys6 binuclear DNA-binding domain.
Double mutant analyses indicate that SfgA
functions downstream of FluG but upstream
of FlbA–FlbD and BrlA (Seo et al. 2006).
FluG-initiated developmental-specific regu-
latory cascades (FlbB–FlbE) consist of at least
two separated pathways, FlbC and FlbE/FlbB/
FlbD, for activation of brlA (Etxebeste et al.
2010; Park and Yu 2012). FlbC is a C2H2 zinc-
finger protein that directly binds to the
promoter region of brlA and activates brlA
expression (Kwon et al. 2010a). Kwon et al.
also demonstrated that FlbC also binds to the
cis-element of abaA and vosA and activates
their expression (Kwon et al. 2010a). FlbE con-
tains two uncharacterized conserved domains
and co-localizes with FlbB, a basic leucine zip-
per (b-zip) TF, at the hyphal tip (Etxebeste et al.
2009; Garzia et al. 2009; Kwon et al. 2010b).
FlbE and FlbB together induce flbD expression
interdependently (Garzia et al. 2009; Kwon
et al. 2010b). Then, FlbB cooperates with
 Molecular Biology of Asexual Sporulation in Filamentous Fungi 7

Developmental FluG-mediated G protein

balancers signaling pathway signaling pathway

FluG ESIF

GPCR? GPCR
GTP GDP RicA
NsdC PpoA FadA GpgA FadA GpgA GpgA
GanB GDP GTP
GpgA
GanB
PpoC SfgA SfaD Pi SfaD SfaD SfaD
PpoB

GTP
NsdD

GDP

GDP
GTP
Pi
FlbA
OsaA RgsA

FlbC FlbB FlbE PkaA

NapA ZipA RgdA

FlbD
SltA AreA MtfA Transcription
factors

Asexual development RlmA

VosA
MpkB
P VeA

LreA VeA VelB VelC VosA

FphA LreA VeA
LreB MpkB
LreB FphA VelB VelB VosA
MkkB SteC
SteD

Light dependent Velvet family MAP Kinase

signaling pathway proteins signaling pathway

Fig. 1.2 A comprehensive model for the regulation of asexual sporulation in A. nidulans. This model includes
almost all elements that influence conidiation

FlbD (a cMyb-type transcription factor) for
brlA activation (Garzia et al. 2010). Overall,
FlbB~E play independent or interdependent
roles and are necessary for full activation of brlA.
In filamentous fungi, FlbA is the first studied regulator
of G-protein signaling (RGS) protein (Lee and Adams
1994; Yu et al. 1996). RGS proteins are a group of
proteins containing a conserved ~130 amino acid RGS
box that interacts with an activated GTP-Ga subunit
and increases its intrinsic GTPase activity, thereby
facilitating the hydrolysis of GTP-Ga (active form) to
GDP-Ga (inactive form) (Chidiac and Roy 2003;
McCudden et al. 2005). FlbA is the cognate RGS of the
FadA Ga protein, and together they govern the
upstream regulation of vegetative growth, develop-
ment, and biosynthesis of secondary metabolites
(Hicks et al. 1997; Tag et al. 2000; Yu and Keller 2005;
Yu et al. 1996). As an RGS, FlbA is presumed to enhance
the intrinsic GTPase activity of FadA (Yu et al. 1996,
1999). Loss of flbA function results in a similar fluffy
autolytic phenotype without sporulation caused by
dominant mutations in FadA (Hicks et al. 1997; Lee
and Adams 1994; Yu et al. 1996).
b) Heterotrimeric G Protein Signaling Pathways
In A. nidulans, two main G heterotrimeric G
protein signaling pathways involving FadA and
GanB Ga subunits play a predominant role in
controlling growth, development, stress
response, and secondary metabolism (Yu
2006). For more information on G protein sig-
naling pathways, please refer to Chap. 7. FadA
(fluffy autolytic dominant A) was identified and
characterized by studying dominant activating
mutants (G42R, R178L, G183S, R178C, and
Q204L) that exhibit the fluffy autolytic pheno-
type (Wieser et al. 1994; Yu et al. 1996, 1999).
Dominant interfering mutation (G203R) in
FadA caused hyperactive sporulation and
reduced hyphal growth (Hicks et al. 1997; Yu
8 H.-S. Park and J.-H. Yu
et al. 1996). This FadA-mediated signaling
needs to be attenuated by FlbA (RGS) for
proper asexual and sexual development to
occur (Hicks et al. 1997; Wieser et al. 1997).
Similar to FadA, GanB (G protein alpha subunit
in A. nidulans) negatively controls asexual
development (Chang et al. 2004). While the
ganB null or dominant interfering (G207R)
mutants produce conidiophores in liquid
submerged culture, constitutively active GanB
mutations (Q208L and R182L) cause severe
defects in asexual development (Chang et al.
2004).
GanB-mediated signaling is in part activated by the
putative GDP/GTP exchange factor, RicA (Kwon et al.
2012), and is negatively controlled by RgsA (Han et al.
2004a). These two Ga subunits work with the SfaA(Gb):
GpgA(Gg) dimer (Lafon et al. 2005; Rosen et al. 1999;
Seo et al. 2005) and inhibit asexual development in part
via the cyclic AMP (cAMP)-dependent protein kinase
PkaA (Shimizu and Keller 2001). Recently, Kong and
colleagues characterized the Gb-like protein B CpcB
that is required for proper conidiation and brlA expres-
sion in A. nidulans (Kong et al. 2013). Overall, these
results indicate that individual G protein components
are associated with asexual development and may play
differential roles in conidiation (Yu 2006).
c) MAP Kinase Signaling Pathways
Mitogen-activated protein kinase (MAPK)
pathways respond to various environmental
cues and amplify the signals, leading to appro-
priate cellular responses in fungi (Gustin et al.
1998). A. nidulans contains four MAPK genes:
mpkA, mpkB, mpkC, and hogA (Galagan et al.
2005). Among these, MpkB, a homologue of
Fus3p in S. cerevisiae, is associated with con-
idiation, sexual development, and secondary
metabolism (Atoui et al. 2008; Bayram et al.
2012; Jun et al. 2011; Kang et al. 2013; Paoletti
et al. 2007). The MAPK kinase kinase SteC, a
component of the MAP kinase module Ste11–
Ste7–Fus3 complex, is also required for proper
formation of conidiophore (Wei et al. 2003).
The deletion of mpkB causes increased brlA
expression and the formation of conidiophores
in liquid submerged cultures (Kang et al. 2013).
Moreover, the DmpkB mutant produces abnor-
mal conidiophores and exhibited a decreased
number of conidia (Bayram et al. 2012; Jun et al.
2011; Kang et al. 2013). Interestingly, the dele-
tion of mpkB results in decreased VeA phos-
phorylation and formation of the VeA–VelB
heterodimer, which acts as a key and essential
activator of sexual development (Bayram et al.
2012). MpkB also interacts with VosA in the
nucleus. These results suggest that MpkB is
also involved in the regulation of the velvet
proteins’ activities, thereby drastically affecting
fungal development (Bayram et al. 2012). In-
depth information on MAP kinase pathways is
found in Chap. 6.
d) The Velvet Family Proteins
The velvet family proteins, mainly VeA, VelB,
VelC, and VosA, play a central and global role
in coordinating fungal growth, development,
virulence, and metabolism in ascomycetes and
basidiomycetes (Bayram and Braus 2012; Ni
and Yu 2007). These proteins define a new
fungi-specific TF class that contains the velvet
domain with DNA-binding activity (Ahmed
et al. 2013; Ni and Yu 2007). Interestingly, the
velvet regulators form diverse complexes that
play differential roles in coordinating various
biological processes in fungi (Bayram et al.
2008; Park et al. 2012b, 2014; Sarikaya Bayram
et al. 2010). VeA is the founding member of the
velvet regulators playing a key role in the light-
dependent developmental control. The veA1
mutation abolishes the light affected develop-
mental changes and causes severely restricted
sexual development with increased conidiation
(Kafer 1965; Mooney and Yager 1990). Further
studies demonstrated that the nuclear localiza-
tion and complex formation of VeA are impor-
tant for the regulation of light-controlled
development (Bayram et al. 2008; Stinnett
et al. 2007). In the dark, VeA predominantly
localizes in the nucleus and forms the VeA–
VelB heterodimer, which controls sexual devel-
opment or the VelB–VeA–LaeA heterotrimeric
complex that regulates secondary metabolism
(Bayram et al. 2008). VeA also interacts with the
light complex components, which balance asex-
ual and sexual development (Purschwitz et al.
2008, 2009). Recently, Bayram et al. demon-
strated that phosphorylation of VeA is regu-
lated by the MAP kinase MpkB (AnFus3)
(Bayram et al. 2012). Similar to VeA, VelB is
 Molecular Biology of Asexual Sporulation in Filamentous Fungi
required for sexual development and produc-
tion of certain secondary metabolites (Bayram
et al. 2008). In addition, VelB functions as a
positive regulator of conidiation (Park et al.
2012b). The velB deletion mutant exhibits the
decreased conidiospore number, whereas the
overexpression of velB results in enhanced
conidial production (Park et al. 2012b).
Another multifunctional regulator VosA func-
tions as a key repressor of conidiophore forma-
tion and an essential activator of trehalose
biogenesis in conidia (Ni and Yu 2007). VosA
directly binds to the promoter of brlA and
represses brlA expression in vegetative cells
(Ahmed et al. 2013; Ni and Yu 2007). VosA
physically interacts with VelB, VosA, or VelC,
and each complex is presumed to control dif-
ferent sets of genes thereby regulating various
aspects of fungal development (Park et al.
2012b, 2014; Sarikaya Bayram et al. 2010).
VelC is important for balancing asexual and
sexual development, and acts as an activator
of sexual development, likely by binding to
VosA during the early stages of sexual develop-
ment thereby leading to the increased forma-
tion of VelB–VeA that is essential for the
initiation of sexual fruiting (Park et al. 2014).
As a consequence, VelC indirectly inhibits
asexual development in A. nidulans.
e) Light and Signals
Light is a key environmental cue affecting
diverse cellular processes, including fungal
development and secondary metabolism in A.
nidulans (Bayram et al. 2010; Mooney and
Yager 1990). For example, light induces coni-
diation but represses sexual development and
ST production (Bayram et al. 2010). In fungi,
several photoreceptors play a crucial role in
light-response processes (Bayram et al. 2010).
The red-light sensor FphA is a fungal phyto-
chrome that stimulates asexual development
(Blumenstein et al. 2005). The deletion of fphA
causes reduced mRNA accumulation of the
brlA and fluffy genes and decreased conidial
production (Blumenstein et al. 2005; Ruger-
Herreros et al. 2011). LreA and LreB are main
components of the blue light-sensing system,
and they form complexes with FphA (Pursch-
witz et al. 2008). The deletion of lreA or lreB
9
causes slightly increased conidiospore produc-
tion in light and dark conditions, suggesting
that LreA and LreB repress asexual develop-
ment (Purschwitz et al. 2008). These sensors
form the photoreceptor complex LreA/LreB/
FphA/VeA, which activates the accumulation
of the brlA and fluffy genes (Ruger-Herreros
et al. 2011).
f) Developmental Balancers
Balance between asexual and sexual develop-
ment is regulated by various factors. First, cer-
tain sexual developmental activators
negatively control brlA expression and asexual
development. For instance, NsdC and NsdD
were initially identified as sexual activators
with DNA-binding domains (Han et al. 2001;
Kim et al. 2009; Lee et al. 2014). The deletion of
one of them results in the absence of sexual
fruiting bodies, whereas the overexpression of
these genes causes enhanced formation of Hűlle
cells (Han et al. 2001; Kim et al. 2009; Lee et al.
2014). Further studies have revealed that NsdC
and NsdD repress brlA expression and conidi-
ophore formation (Kim et al. 2009; Lee et al.
2014). Genetic analyses indicate that NsdD
functions downstream of FlbE/B/D/C and
upstream of brlA. Second, Psi factors (preco-
cious sexual inducers), derived from oleic, lino-
leic, and linolenic acids, control the balance
between asexual and sexual development
(Bayram and Braus 2012; Dyer and O’Gorman
2012). In A. nidulans, the three oxylipin bio-
synthetic genes ppoA, ppoB, and ppoC (psi fac-
tor producing oxygenase) are present (Brodhun
and Feussner 2011; Tsitsigiannis and Keller
2007; Tsitsigiannis et al. 2004, 2005). The dele-
tion of ppoA or ppoB causes increased conidial
production, suggesting that PpoA and PpoB
negatively affect asexual development (Tsitsi-
giannis et al. 2004, 2005). However, the deletion
of ppoC leads to decreased asexual sporulation,
suggesting that PpoC positively influences con-
idiation, and has antagonistic activity to PpoA
and PpoB (Tsitsigiannis et al. 2004, 2005).
Third, OsaA, an ortholog of Wor1 in Candida
albicans, has been identified by a gain-of-func-
tion genetic screen and is an orchestrator of
sexual and asexual development (Ni and Yu
10 H.-S. Park and J.-H. Yu
2007). The deletion of osaA causes enhanced
sexual fruiting with reduced conidiation, sug-
gesting that OsaA acts as a repressor of sexual
development and indirectly affects asexual
development in a positive way.
g) Other Transcription Factors
A diverse range of other TFs have been shown
to influence asexual development in A. nidu-
lans (Fig. 1.2). RgdA (retarded growth and
development) is a putative APSES TF, which is
required for proper conidial production and
brlA expression. The rgdA deletion mutant
exhibits a reduced number of conidia, and con-
idiophores of this mutant show irregular
shaped phialides. In addition, the deletion of
rgdA results in decreased mRNA accumulation
of brlA and abaA, suggesting that RgdA may act
as an upstream regulator of central regulatory
genes (Lee et al. 2013). MtfA with a C2H2 zinc-
finger domain is termed as a master TF regulat-
ing secondary metabolism and differentiation
(Ramamoorthy et al. 2013). The absence of
mtfA results in a drastic reduction of conidio-
phore formation, conidial production, and
expression of brlA. The zinc-finger TF SltA
mainly plays a role in cation homeostasis
(Shantappa et al. 2013). Conidia production
and brlA expression were reduced in the sltA
deletion mutant, suggesting that sltA is
required for normal conidiation. RlmA con-
taining a MADS-box domain is a major
MpkA-dependent TF, which regulates cell wall
integrity signaling (Fujioka et al. 2007; Kovacs
et al. 2013). The deletion of rlmA results in an
increased number of conidiospores and brlA
accumulation in surface cultures. In addition,
the rlmA deletion mutant produces conidio-
phore in liquid submerged culture, where WT
strains do not. Kovács et al. proposed that sfgA,
flbB, and flbE, upstream regulators of brlA, are
putative target genes of RlmA, indicating that
RlmA indirectly regulates brlA expression
(Kovacs et al. 2013). Two basic leucine zipper
(bZIP) transcription factors, NapA and ZipA,
affect asexual and sexual development. The
overexpression of napA or zipA causes
increased conidial production but decreased
sexual spore production (Yin et al. 2013).
AreB is a putative GATA zinc-finger TF con-
taining a leucine zipper motif at N-terminal
region and acts as a negative regulator of nitro-
gen catabolism (Conlon et al. 2001). The areB
deletion mutant exhibits reduced growth and
conidiation, suggesting that AreB is crucial for
normal growth and conidiation (Wong et al.
2009).
3. Feedback Regulators of Conidiation
Along with the formation of conidiospores,
expression of the key developmental activator
brlA is repressed, and many other spore-
specific genes are induced (Adams et al. 1998).
As mentioned, AbaA is required for the repres-
sion of brlA through the control of brlAb (Han
and Adams 2001). However, AbaA indirectly
represses brlA expression, because the pro-
moter of brlA does not contain AbaA response
element (Han and Adams 2001). Recent genetic
analyses demonstrated that the VosA–VelB
complex is involved in AbaA-mediated repres-
sion of brlA (Ni and Yu 2007; Park et al. 2012b).
During the conidiophore development, the
VosA and VelB proteins are completely
degraded. Then, in phialides, AbaA directly
binds to the promoter regions of vosA and
velB and induces their expression, thereby a
large amount of the VosA and VelB proteins
are newly synthesized in the developing cells
(Park et al. 2012b). VosA, then, interacts with
VelB, and the VosA–VelB heterocomplex binds
to the brlA promoter and represses brlA expres-
sion in conidia (Ahmed et al. 2013). Moreover,
the VosA–VelB complex regulates spore-
specific genes, suggesting that the VosA–VelB
dimeric complex acts as a key functional unit
regulating spore maturation (trehalose biogen-
esis and spore wall synthesis), viability, and
attenuating conidial germination in conidios-
pores (Ahmed et al. 2013; Park et al. 2012b).
III. Asexual Sporulation in Penicillum
marneffei
A. Morphology of Asexual Structure
Penicillium marneffei is an emerging fungal
pathogen and a dimorphic fungus, which
 Molecular Biology of Asexual Sporulation in Filamentous Fungi 11

A. The stages of conidiation in P. marneffei

Conidium

Hyphal growth Foot cell

B C
GPCR ? GPCR ?

γ GasC γ
GasA

β β

RasA
AbaA
StuA
PkaA DrkA SlnA CflB
PakB
MyoB
TupA

BrlA

StuA
AbaA

Fig. 1.3 Stages and regulation of conidiation in conidiation in P. marneffei. (c) Genes involved in the
P. marneffei. (a) The stages of conidiation in P. mar- conidiophore morphogenesis
neffei. (b) Simplified model for the genetic regulation of

grows in the yeast form at 37  C and the fila-
mentous form at 25  C. In the filamentous
phase, P. marneffei undergoes the complex
asexual development and forms conidiophores
(Andrianopoulos 2002; Pasricha et al. 2013). In
response to inducing environmental signals,
hyphal growth is arrested and the fungus starts
to form asexual structures. Multinucleate stalk
cells, formed from foot cells, are subsequently
septated and form the subapical branches
called rama. Two sterigmata, metulae and phia-
lides, are formed from the apical cells. Finally,
phialides produce conidia, resulting in the for-
mation of brush-like structures called penicillin
(Fig. 1.3a) (Andrianopoulos 2002; Roncal and
Ugalde 2003; Vanittanakom et al. 2006).
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arrive, so that by morning the place was covered with tents and
wagons, and swarming with people, horses, mules and cattle.
7th.—At an early hour we were again moving, in order to reach the
Elkhorn Ferry before any of the trains should take the precedence,
whereby we would have been detained. We had first to cross the
Papilion on a bridge, and as it was very narrow, and the road leading
to it very steep, we were obliged to unharness the mules from the
wagon, lest they might, by pushing one another, precipitate the
wagon and themselves into the stream and mud below. The wagon
was then pulled across the bridge by hand. Ascending the opposite
hill, we were again on the high prairie. Before us, twelve or fifteen
miles distant, could be discovered the timber of the Elkhorn, which
we expected to cross before noon;—to our right we could yet
perceive the timber of the Missouri, and the Old Council Bluffs,
where formerly there was a fort for the protection of the traders;—
and to our left the timber of the Platte.—
Arrived at the Elkhorn, we saw a considerable number of
Pawnees, who all appeared glad at our coming to visit them. Here
we learnt that a slight affray had taken place the day before,
between some Pawnees and a train of emigrants. The readers of the
Miscellany have perhaps read of such cases in the newspapers,
headed: “Depredation of the Pawnees (or some other Indians) upon
a train of emigrants,” and the like, where the blame is generally
attached to the Indians. I think it is due to the Indians to state here
that the fault does not always lie with them, but very often with the
whites. The road of the emigrants lies through the country belonging
to the Indians;—their hunting-grounds are traversed by the long lines
of white-covered wagons, and the buffaloes, the principal
subsistence of the Indians, are thereby chased away to more distant
and more secluded pastures, perhaps to regions where it would be
unsafe for them to hunt, on account of other tribes inimical to them;
and they have thereby been reduced to poverty and want. For all
these privations they have been promised presents, as a
compensation, from our Government, but thus far they have received
nothing. So when the emigrants are passing through their country,
they frequently apply to them for tobacco, or some other small
presents. It seems that in the above instance, while the Pawnees
were hovering around the train, they were refused a present, and
one of the men, with the ox-whip, struck an Indian, who came near,
which, of course, roused the feelings of the Indians. These
accordingly drove off a beef or two, which were then despatched.—
The Elkhorn at this place is a deep and rapid stream, about 20
yards wide. Two French halfbreeds are living here, in order to keep a
ferry for the emigrants.—Having passed over the Elkhorn, our road
lay across a bottom prairie extending between the Elkhorn and Platte
rivers. We now turned off from the wagon road, taking the village for
our landmark; without any track across the prairie, and soon arrived
on the north bank of the Platte.—The grass being somewhat more
advanced in the bottom than on the high bluff on the opposite side,
where the village is situated, the Pawnees had turned their ponies to
graze in the bottom, watched by the women and children.—Many
women were also engaged in digging for roots with their hoes,
provisions being at present very scarce among them. We were soon
surrounded by a crowd of young men, women and children, who by
their smiling countenances seemed to bid us welcome.—It is difficult
to describe our feelings on the present occasion. We were now in
sight of the village, where the people lived, whom we had come to
visit, in order to give them the opportunity of accepting or rejecting
the offer of having Missionaries to live among them, to lead them to
the Savior of sinners. A wide stream, over a mile across, separated
us from the object of our journey, and could not be crossed without
getting assistance from the very people, for whose benefit we had
come. From the conversation of Mr. Allis and Mr. Sharpee with the
Indians there seemed to be some difficulty in getting help, originating
in a jealousy existing between the Chief in our company and the
principal Chief of the village. Meantime it commenced raining, and a
strong, chilly wind was blowing, which forced us to wrap ourselves in
our blankets. In this perplexity nearly an hour was spent. At length a
messenger was despatched across the river to inform the principal
chief of the village, Siskatuppe, of our arrival, with the request that
he would send us some men to assist us to cross. After another
hour’s patient waiting we were cheered by seeing the chief with
about twenty men coming to our help.—The ford of the river is only
about four feet at the deepest places, but what makes this river
peculiarly perilous in crossing is the shifting quicksand at the bottom,
so that, while crossing, it is necessary to keep constantly moving;—
for the moment a person stops he begins to sink on account of the
uncertain foothold.—Our baggage was placed upon the backs of
Pawnees, who immediately started off with their load. The mules
having been unharnessed, and the harness placed in the wagon, a
long rope was tied from the end of the tongue of the wagon to each
single-tree. The Pawnees then took hold of the rope, while some
pushed behind at the wagon, and thus proceeded into the river. Mr.
Sharpee kindly offered br. Smith his horse, while he and br. Oehler
each took a mule, and Mr. Allis borrowed a pony of the Pawnees, the
mounted men taking up the rear. A full half hour was consumed in
crossing, and passing over two islands on our route. At length we
reached the opposite bank, where we were greeted by numbers of
Pawnees, who were awaiting our arrival. A difficulty which now
presented itself before us was to get the wagon up the steep bluff,
the sides of which had become slippery by the falling rain. To obviate
this difficulty it was necessary to go nearly a mile down the river,
through their cornfields, in the bottom, to a place where it was not
quite so steep as elsewhere.—At length, being arrived at the village,
we found, in spite of the rain, crowds standing around the chief’s
lodge, to receive and to welcome the missionaries.—
Having secured everything loose about the wagon that might be
liable to be stolen, and entrusted our baggage, harness and animals
to the care of the chief, we entered the lodge of our host. We were
not a little surprised, when we came in, to find that it was a spacious
apartment, a description of which will be given at another time. We
had hardly reached the place when a young gentleman in the
employment of the Government arrived in company with the United
States interpreter at Fort Kearney, a black man, who speaks the
Pawnee fluently. They had travelled the whole distance, without an
escort, alone;—Fort Kearney being about 150 miles further up the
Platte river. This young man was sent out to the different tribes of
Prairie Indians, the Pawnees among the rest, in order to invite them
to attend a General Council of Tribes at Fort Laramie, to be held
about the beginning of September.
 While Mr. Allis and ourselves, and the above-named persons from
Fort Kearney lodged with Siskatuppe, the principal chief of the
village, Mr. Sharpee put up at the lodge of the chief Gatarritatkutz,
who had travelled with us, and with whom he is accustomed to tarry
whenever he comes to the village to trade.
After we had spent an hour in drying our clothes, smoking, and
conversing with some of the chiefs and braves, a messenger arrived
from Mr. Sharpee, inviting us to be present at a feast of coffee and
crackers, which he had prepared for the chiefs, during which he
intended to make inquiries respecting his stolen horse. When we
arrived Mr. Sharpee informed us that, as the principal men were now
assembled, it would be a convenient opportunity for us to hold a
council with them. We accordingly commenced by informing them of
our object. The jealousy between the chiefs, however, became
apparent, and after some consultation among themselves
Siskatuppe intimated that some of the chiefs were not present, and
as the business before us was of great importance to the whole band
they ought also to be summoned. It was, therefore, resolved
immediately to adjourn to his lodge, in order to receive our
communications. Accordingly all the chiefs and braves having been
assembled, we were informed that they were now ready to hear us.
Br. Smith then in a speech, which was interpreted by Mr. Allis,
informed them of the object of our visit. Hereupon Siskatuppe made
an address, welcoming us in the kindest manner and hoping that we
might send missionaries among them. Br. Oehler then, in an
address, explained to them more particularly the object which
missionaries have in view, and that it was especially our present aim
among them to find out whether they were desirous not only of
having their outward condition bettered, but of having the
missionaries among them to teach them about a Savior, who came
into this world to save us from our sins, to preach whom was our first
and principal design. Several other chiefs and braves then made
speeches, expressive of their satisfaction at our coming to see them,
and welcoming us to live with them, promising us their protection; so
that we should in no wise be hindered by any of their people; and
that we might rest assured that our cattle and all other property
which we might bring along with us should not be destroyed or
molested by any one. Moreover, they acknowledged that they
needed instruction, and that they would all be willing to listen to us.
We were very much pleased with the apparent earnestness with
which these remarks were made, and have no doubt that they made
these promises in good faith. Before us were thus assembled the
nobility of the village,—the chiefs and braves, besides numbers of
the common people, at least 500 in all, sitting in solemn council.
What a spectacle for the humble missionaries of the cross! Here
were the representatives of a village, numbering at least 2,500 souls,
deliberating upon the acceptance or rejection of missionaries,—
holding a council, unconsciously, whether the time in the providence
of the Lord had at length arrived, when they should again be
instructed in the knowledge of the “Unknown God,” whom they, and
their fathers and forefathers have worshipped, though in great
ignorance and superstition. O, how cheering to our hearts, when we
were not merely coldly permitted to make our abode with them, so
that they might derive from us some temporal good, in supplying
their wants when hungry and destitute; but when we were hailed and
welcomed among them as the “Medicine men of the Great Spirit,” to
have whom among them, would better their outward condition, and
perhaps (as we ardently hope and pray, through the blessing of our
Lord,) make them a happy and christian nation. Of what vast
importance may have been this solemn hour for these people?—the
future, we humbly trust, will develop many happy results from the
decision of this council; but Eternity alone may reveal, that the
happiness or misery of many a soul, bought by the precious blood of
Jesus, was connected with the results of this occasion!
Our business with them being over, the above-named young man,
in the employ of the government, also made known to them that their
Great Father at Washington had invited them to a grand council of
the different nations, to be held at Fort Laramie for the purpose of
defining the territories of the different prairie tribes, who were also to
receive presents there from him, as a compensation for the losses
which they have in later years sustained, on account of the scarcity
of the buffaloes, occasioned by the great emigration to the far West,
etc.,—at all which they expressed their great satisfaction. After the
crowd had somewhat dispersed, a dish of soup, made of hominy and
beans, was placed before us, with two spoons, made of buffalo
horns, in the dish. Having fasted since sunrise, we could not
complain of our appetites, and the dish, though not attractive in its
appearance, was soon emptied of its contents. We then prepared
some coffee, and having supped, gave our chief and his family a
feast of the remaining coffee, with some crackers and slices of ham.
We had hardly finished, (much time was not consumed in washing
our dishes,) when a messenger arrived, inviting us to a feast, which
one of the chiefs had prepared for us. When we arrived, and had
seated ourselves on mats around the fire, (for there are no chairs in
a Pawnee lodge,) a dish of soup, made of corn, was again placed
before us. As our appetites had previously been satiated, we could
merely partake of a few spoonsful, to please our kind host. We had
hardly entered into a conversation when an invitation came from
another chief;—and so we were led from lodge to lodge, till we had
partaken of about a dozen feasts. At last we returned to the lodge of
Siskatuppe, and, having wrapped ourselves in our blankets, and laid
ourselves down on mats on the ground, were soon in the land of
visions.
May 8th.—Early in the morning we were awakened by the shrill
voices of the Pawnee women, who were engaged in cleaning up the
lodge, and collecting their hoes, previous to their going out to the
fields to prepare the ground for planting corn. Our breakfast being
over, and having had our animals brought from the pasture, we,
together with the gentleman from Fort Kearney and his interpreter,
started for the upper village, distant about 25 miles, accompanied by
our old friend Gatarritatkutz and another Pawnee. Our road was
again for several miles across the high prairie. We then descended
into the bottom prairie of the Platte, travelling about five miles in sight
of the river, when we made a halt near the stream in order to prepare
dinner. The situation here pleased us very much, as very suitable for
a mission station, should the Pawnees be permanently located
where they at present reside. The timber on the islands in the Platte
is very easily obtained here. The prairie bottom is from two to three
miles wide, gently ascending to the bluffs, and extends about ten
miles along the Platte, before the bluffs again approach the river. A
mile or two from where we took our lunch a beautiful spring of never
failing water gushes forth from the bluff. After dinner, having
permitted our animals to graze a while, we proceeded on our
journey, travelling partly on the high prairie, after ascending the bluff,
and partly in the bottom. At a certain place, as we were travelling
along, we noticed, that our Pawnee friends rode aside to a spot,
where their attention seemed to be rivetted upon something on the
ground. Inquiring what it was, they informed us, that at that place
about nine or ten months ago, a Sioux chief had been killed by the
Pawnees. It seems, he had made a hostile incursion upon the
Pawnees, with a company of his people, and having found some
squaws engaged at work in their fields, he had killed them. The
Pawnees, irritated at this unprovoked attack, immediately made up a
party, who hotly pursued their enemies, and, the horse of the Sioux
chief being wearied, and not able to keep up with the rest, he called
to his men: “Stop not for me, but save yourselves; I shall die
fighting.” His pursuers soon came up with him and killed him, fighting
bravely. The spot where he had been killed still presented the marks
of Indian barbarity; stones, arrows, and small pieces of the skull,
which had been cleft by their tomahawks, lying around,—the bones
having been carried away with the carcass by the wolves of the
prairie. We turned away in disgust from a place, which had been the
scene of such a barbarous atrocity, praying only the more fervently
to the Lord, that the passions of these poor people might be softened
down by the all-subduing influence of His blessed gospel, through
the happy effects of which alone, we have been made to differ.—
As we were approaching the upper village we observed sentinels
standing on the highest bluffs, posted there, it seems, to watch the
approach of any strangers. The first that we observed was at a
distance of five miles from the village. In the bottom prairie, numbers
of ponies, the property of the village, were grazing, watched by
women and children. As we approached the village, young men and
boys joined our caravan, and when at last we arrived there a dense
crowd of children surrounded us, eager to see the visitors of their
village, so that it was necessary for a chief to come to our aid, who
opened a way through the immense throng for our wagon to proceed
on to the lodge, where we were to put up. We were here, as well as
at the other village, struck with the large proportion of children, a
circumstance not generally observable among Indians. Mr. Allis
informed us that visiting them a few months previous (the smallpox
having appeared among some of the Indian tribes) he had
vaccinated about 1,500 under 14 years of age (in a population of
hardly 6,000); the last time that the whole tribe had been vaccinated,
having been 14 years ago.
The village stands on a rising ground, about three miles from the
river, and consequently the same distance from the nearest timber.
In a valley near by flows a beautiful stream, from which the people of
the village are supplied with water.
The lodge, where we were to remain, was the medicine lodge of
the village, and just as we entered it we found a company of about a
hundred men engaged in dancing a medicine dance, in order to
propitiate the Great Spirit, to grant them prosperity in the
approaching buffalo-hunting season, and protection from their
enemies. Their naked bodies were painted in the most grotesque
manner, their hair and weapons plumed with eagles’ feathers, and
thus armed with bows and arrows, spears, and shields, they were
dancing to the beat of the drum, intermingled with songs. Their yells
rent the air, while the very earth seemed to shake under their feet.
After we had sat in the lodge a few minutes, a dense crowd of two or
three hundred children filling up the space at the entrance, whose
curiosity was probably more attracted by us than by the dancers, a
chief came forth from the dancing party, with a whip in his hand, at
sight of whom the children made for the door, but as it took some
time before the crowd could get out by the narrow opening he
commenced plying his whip most unmercifully on the naked backs of
the poor children till the entrance was cleared. We were then
informed that on account of our arrival, out of deference to us, they
would now dance outside; if, however, we wished to look at them
while dancing, we were welcome to come out and see them.—After
a little while we went out and looked at them for some time, while
they were engaged in these religious exercises. Our hearts melted,
and our eyes filled with tears at the thought of the benighted state of
their minds, living without Christ, and without hope. We were not
long engaged in these mournful reflections, when a messenger
arrived inviting us to a feast. We followed the messenger, who led us
to a lodge, which we entered. Our host, who had prepared a feast for
us, was no other than the chief of the Grand Pawnee Band, and
principal chief of the whole Pawnee nation. He received us in a very
warm and affectionate manner, embraced us, and welcomed us
among his people. His name is Asseruregarrigu;—he seems to be
extremely old, on the verge of the grave, yet, in spite of his great
age, is still very much respected by his nation. After being invited to
sit down on mats, a dish of green corn soup was placed before us,
which was very palatable. The old man complained, that the corn
had not been sufficiently boiled, as he had ordered it to be put over
when he heard of our arrival, and had been anxious to be the first to
welcome us by a feast.—
Our repast being finished, we were yet invited to several feasts
prepared by the chiefs of the village, whereupon we returned to the
medicine lodge. In the evening a council of the chiefs and braves
was called, in order to inform them of the business upon which we
had come to visit them. We were received in the most cordial
manner, embraced by several of the chiefs, and after informing them
of our object the principal chief of the Pawnee tribe, aforementioned,
made a reply to the following effect: “It appears to me this evening as
though I had been dead a long time and had suddenly to-day risen
from the dead,—so glad am I to hear the news that teachers are
willing to come among us, in order to live with us and instruct us. We
shall welcome you among us, and the chiefs will see to it, that your
property and cattle shall be protected;—I hope you will come soon to
live with us. I am now a very old man,—I must soon go hence;
therefore come soon that I may behold the missionaries living among
my people before I die.” Speeches to a similar effect were then made
by Leezikutz, chief of the Republican band, Terrericawaw, chief of
the Topages (pronounced Tuppay) band, and two or three other
inferior chiefs, which were all interpreted; and finally a chief named
Lalogehanesharn (or Fatty, as he is called by the whites, from his
corpulence, something very unusual among wild Indians) closed by
making a long appeal to the chiefs, delivered in a very loud and
sonorous voice, exhorting them to keep the promises which they had
just made. “Don’t cheat,” said he; “don’t act deceitfully. You have now
promised these men that if they come to live among us you will take
them under your protection, and will always restrain your people
from molesting their property. Remember this, and now since you
have made these promises, see to it that you also keep them.” The
council then dispersed, not, however, before several chiefs had
again embraced us, whereupon we laid ourselves down in our
blankets upon the mats in the lodge for repose.
May 9th.—After breakfast we were invited to the principal chief’s
lodge, to attend a council which had been called for the purpose of
listening to the invitation sent to them by the President of the United
States through the above-mentioned agent, to attend a general
council at Fort Laramie. The Pawnees declared themselves satisfied
with the offers of government, and several speeches were then
made expressive of the prospect that ere long the condition of their
people would be bettered, especially since they might now indulge
the hope of soon having teachers among them to give them
instruction. During the council a severe thunderstorm was passing
over, and while the chief, Fatty, was speaking, after a loud peal of
thunder: “See,” said he, “the Great Spirit is pleased with us this
morning and expresses his satisfaction by speaking loudly to us!!”—
At the close we were yet invited to partake of a feast with them. A
large dish of corn-soup was brought in and set before the chiefs;—
the medicine man then came forward, and, taking a spoonful of the
soup, went to the fire, and making a small hole in the ashes he
poured it in. After putting the spoon back again into the dish he
returned to the sacrifice at the fire, which was blessed thrice by
holding both hands over it; then, turning round to the assembly, and
fronting the chiefs, looking up to heaven, he stretched out his hands
thrice in silent benediction, and then returned to his seat. The dish
was then passed round, each person partaking of a mouthful or two
of the soup.
The council being over, and the thunderstorm having somewhat
subsided, towards noon we started on our return, the object of our
visit to the Pawnee villages being now fully accomplished. We had
now only to retrace our steps, as that was the nearest way for us to
travel. We prepared our coffee and lunch at the same beautiful spot,
where we had halted yesterday. In getting a fire, however, to boil our
coffee, we had considerable trouble, the matches in our pockets
being damp, and the grass and wood being all wet from the rain,
which was yet falling. But at last our Pawnee friends succeeded in
finding some dry rotten wood, which, by means of paper and powder,
we succeeded in igniting. In the evening we arrived at the Lower
Village, where we staid over night at Siskatuppe’s lodge.
May 10th.—Towards morning a very heavy thundergust passed
over the village, and the water came pouring into the lodge, from the
small opening above (which is made to let out the smoke), and the
shrill voices of the women, who seemed to be scolding one another
while engaged in cleaning up the water, disturbed us considerably in
our slumbers. In the morning the Pawnees informed us that the
Platte was rising; we, therefore, hastened to get ready for travelling,
and crossed the river without much difficulty, in the same manner as
at the first time. Arrived at the opposite bank, we made a present of
some tobacco to our Pawnee friends, for assisting us in crossing the
river. A large company of Pawnees followed us, who intended going
to the Omahaw village in order to trade for corn. When we arrived at
the ferry of the Elkhorn, a heavy gust was threatening to overtake us,
and we had hardly crossed, secured our baggage, and got into the
hut of the ferrymen, when a furious hailstorm passed over us. The
rain having detained us so long, that it was impossible to reach
another camping ground by daylight, we pitched our tent here for the
night.
May 11th.—During the night, another gust passed over us, but our
tent kept us dry and comfortable. One of the ferrymen, who had
arrived during the night from Council Bluffs, informed us that the
bridge across the Papilion, over which we had passed, had been
washed away by the high water occasioned by the heavy rains, and
that he had been obliged to swim the stream, which had swollen to
the size of a river. We, therefore concluded to take another route,
which would lead along a high ridge between the Great and Little
Papilion, and strike the former opposite the Omahaw village, at the
confluence of the two streams, where we hoped to get assistance,
should we find any difficulty in crossing.
 When we arrived at the place, we found both streams very much
swollen by the heavy rains. Collecting some wood together, we
made a fire, and prepared our dinner, while the party of Pawnees,
who had followed us, were busied in crossing the Little Papilion, in
order to get to the Omahaw village. After we had finished our meal,
and had come to the crossing of the Great Papilion, preparations
were made for getting our wagon, baggage, and ourselves on the
other side and here we found Mr. Sharpee, who has travelled several
times to the Rocky Mountains, and was accustomed to meet with
such exigencies as the present, to be of invaluable service to us.
Under his direction the tent-cloth was spread out on the ground,
upon which was placed the wagon-body. The corners of the cloth
were then laid over the body, and around the whole a rope was
tightly tied to keep the cloth firmly adhering to it. Thus a boat was
soon constructed, in which the forewheels and tongue were put, and
then launched in the stream, with Mr. Sharpee and Mr. Allis on
board. A rope had previously been attached, the end of which an
Indian took in his mouth, and swam across, the boat being drawn
after him. The contents being quickly taken out on the other side, it
was towed back again by the Indian with Mr. Sharpee still in it. The
second load consisted of the hindwheels with Mr. Sharpee and br.
Oehler. Meanwhile another Indian had formed a boat of a buffalo-
skin, stretched out by sticks placed crosswise, in which the baggage
was all safely transported to the other side. The animals were driven
into the stream, and forced to swim across. Finally, the boat was
brought over the third time, and br. Smith and a lame Pawnee man
were taken to the other side. When the boat had made this its last
trip, it had not leaked more than about an inch of water. During the
whole time, while we were crossing, the rain was pouring down in
torrents upon us. Having now safely gained the other shore with all
our effects, and put everything in travelling order, we proceeded
about three miles farther, when we came to a slough, which had also
been filled up by the rains. It was impossible to ford it, at the place
where the road led across. After reconnoitering a little, we found a
place where the water was fordable, but with an almost
perpendicular bank of about ten feet on the other side. The mules
having been unharnessed, Mr. Sharpee and br. Oehler took them
across, although they almost stuck fast in the mud. Mr. Allis and br.
Smith then pushed the wagon into the slough, thus forming a bridge
for them to cross over. All hands were then employed in pulling the
wagon out of the mud up the bank, till the end of the tongue reached
the top, to which the doubletree was then tied. The mules being
reharnessed, and all the other available muscular power being
applied in pushing up the wagon, we finally succeeded in getting it
up on the bank. We had now yet two miles to travel, in order to reach
the Presbyterian mission station at Bellevue, and were truly thankful
that there were no more streams or sloughs to cross. About
sundown we arrived at our station, and were heartily welcomed by
Mr. McKinney and the Mission family, though our outward
appearance was not very prepossessing, our clothing being
bespattered with mud, from the various adventures of the day.—
On the 14th of May the steamboat El Paso came up the Missouri
to Council Bluffs, and on the morning of the 15th we took passage in
her down the river. On the evening of the 16th the boat arrived at
Weston, and in the afternoon of the 17th left there for Kansas, where
we arrived at dark. After breakfast on the following morning, the
18th, we proceeded to Westfield, eight miles, on foot, where we
arrived just as the congregation were leaving the church after the
service, which had been held by the national assistant, Frederic
Samuel—truly thankful to the Lord to find all well at home, after an
absence of nearly four weeks.
 DESCRIPTION
OF THE MANNERS AND CUSTOMS OF THE PAWNEE INDIANS
(By Br. D. Z. Smith.)

Bands or Clans.—All the Indian tribes are subdivided into bands

or clans. The Pawnees have among them four distinct bands, viz.,
the Loups (pronounced Loos), the Republicans, the Topages
(Tuppays), and the Grand Pawnees. Each band has at its head a
Chief, among whom, however, the Chief of the Grand Pawnees,
Asseruregarrigu, is considered the Principal Chief of the whole tribe.
Villages, Localities, former Missionaries, &c.—The Pawnees
live in two villages at present, both on the South side of the Platte or
Nebraska river. The lower village lies about 50 miles from Bellevue,
and about 10 or 15 miles above the mouth of the Elkhorn river, the
first important tributary of the Platte from the North; and the upper
village is situated about 20 or 25 miles above the lower, nearly
opposite the mouth of the Loup Fork of the Platte, the next branch of
any consequence above the Elkhorn. Both villages are situated on
eminences, so that the approach of an enemy can easily be
observed, and a sharp lookout is constantly kept in order to guard
against any sudden surprise from a hostile force. Sentinels are
constantly posted on all the surrounding heights, who can
immediately by signs, known among the Indians, transmit
intelligence in case of impending danger. During the night, sentinels
are constantly perched upon the tops of the lodges, to guard against
any unexpected nocturnal attack. When we were on our return, and
remained over night at the Loup village, a report, probably a false
alarm, had reached there during the day, that a large war-party of
Sioux had lately been seen near the head waters of the Elkhorn
river. During the night sentinels were as usual posted upon the
lodges, a little more noisy, however, than generally, yelling to one
another and singing, nearly the whole night. Upon our inquiring why
the sentinels made so much noise, we were informed that it was to
let the Sioux know, should they be near, that they might not expect to
find them off their guard.
Thus it will be observed that a certain kind of military organization
is constantly kept up at each village, rendered necessary by their
wars with their enemies. This unpleasant state of things can only be
removed through the benign influence of the Gospel, when “they
shall beat their swords into plough-shares, and their spears into
pruning-hooks: nation shall not lift up a sword against nation, neither
shall they learn war any more. But they shall sit every man under his
vine and fig-tree; and none .” (Mic. 4:3, 4.)
As hinted in a note in a previous communication, the Pawnee
villages are not located on their own land. Formerly the land on both
sides of the Platte river belonged to them. But about the year 1835
they concluded a treaty with the United States, in which they ceded
to the latter all their territory on the South side of the river. Their
villages were then situated about 150 miles further up the Platte on
the North side. The missionaries of the A. B. C. F. M. were then
residing with them, their mission stations being situated in the vicinity
of these villages. When the Pawnees were at their homes, which is
only four or five months of the year (the remaining months being
consumed on their summer and fall hunts), the missionaries were
engaged in teaching such of their children as could be induced to
attend school, and in instructing the people in the truths of religion,
as well as they could according to their limited knowledge of the
language. About six years ago, the powerful Sioux made repeated
incursions into the Pawnee country, and the strength of the latter
being very much weakened by their frequent wars with their
enemies, they were obliged to succumb, and at length forced to
leave their former location on the North side of the Platte, and to
remove to the South side, nearer to the white settlements on the
Missouri river. The missionaries, too, found it unsafe to remain, and it
was thought advisable to suspend their missionary operations
among them. Mr. and Mrs. Allis are remnants of this noble band of
missionaries, and, still cherishing the desire, that these benighted
but very interesting people might be enlightened by the gospel, have,
while their fellow-laborers have long ago left for other fields of
usefulness, still lingered behind—and toiled on, amidst many self-
denials, privations and discouragements, in doing whatever lay in
their power for the good of these Indians. Lately the government has
established Fort Kearney, not far distant from their former villages,
which, it is hoped, will be a security against future incursions, and
prove a safeguard, when our mission will once be established
among them.
Language.—The Pawnee language is said to be altogether
different from the languages of the surrounding prairie tribes. The
Otoes, the Ioways, the Osages, the Kanzas or Caws, the Omahaws,
the Puncaws, etc., seem all to speak dialects of the same language,
the Dakota; while the Pawnee language seems, by its great
difference, to point back to the earliest periods of the residence of
the Indians in this country, when a disruption may have taken place,
that formed them into a distinct nation.
Population, Lodges, etc.—The lower village consists of about 80
lodges, with a population of nearly 2,500; and in the upper village
there are between 140 and 150 lodges, the population amounting
perhaps to 3,500. On approaching a village, the lodges have the
appearance of so many small hillocks, of a conical form, huddled up
together in the closest possible manner, with only narrow passages
between for walking, and the rest of the space filled up by pens,
formed of stakes, for confining their ponies during the night, to guard
them from being suddenly taken off by a warlike party of another
tribe.
The Pawnee lodges are of a circular form, large and spacious in
the interior; many of them being 50 feet in diameter. Three and
sometimes four circular rows of forked trunks of trees are placed
upright, at appropriate distances from each other. The row nearest
the centre consists of only four such upright timbers, about 15 or 20
feet high, while the crotches in the more exterior rows are shorter
and more numerous, in proportion as the circumference is greater.
These forks or crotches support thick crossbeams, upon which a
frame of long poles is laid, extending from the ground at the outer
circumference of the lodge to the top, leaving only an opening at the
apex, of about four feet in diameter, to answer the double purpose of
letting in the light, and letting out the smoke. Upon the frame work of
poles, willow osiers are laid, and the whole is then covered with
prairie-sod from 12 to 18 inches thick. Immediately below the
opening above, a hole is sunk in the centre for the fire-place,
common to all the residents of the lodge. From five to ten families,
generally related together, have a common occupancy of one lodge,
governed by a head man, who may be styled the lodge chief. Around
the circumference of the lodge are recesses for sleeping, partitioned
off for each family, resembling the berths on board of steamboats,
many of which are screened in front and on the sides by willow-twigs
laid above each other, tied fast to a frame, which, partly with the
reddish bark on, and partly white from having the bark taken off,
exhibit considerable taste in those who wrought them. When thus
ornamented with twigs, a small opening about two by three feet is
left in front to admit the occupants. The bottom of the berth is raised
about two feet above the ground by means of large hewn sills,
overlaid by thick willow twigs, which are then covered with buffalo-
robes. The entrance of the lodge is always on the East side,
protected by a passage, closed above and on the sides, of about 15
feet in length—the door being a large buffalo robe, or several sewed
together, hanging before the inner entrance. On the side opposite to
the door of each lodge, a recess is reserved, for depositing the skull
of a buffalo, surmounted by shields, quivers of arrows, spears, bows,
skins, feathers, etc. used for hunting, for medicinal or religious
purposes, as the case may require. In the intermediate space
between the recesses and the fireplace, mats, about three by five
feet, made of rushes, are laid, at convenient distances, for seats;
while an extra supply of them, rolled up, and set aside at different
places in the lodge, are reserved for extraordinary occasions, as for
instance councils or feasts.
Subsistence.—What the seal is to the Greenlander the buffalo is
to the Pawnees and other Prairie Indians; and many of their songs,
in which reference is made to the buffalo, show that this animal is
held in high estimation among them. It will no doubt be a difficult
undertaking to wean them from an occupation, which is at the same
time a gratifying and exciting sport to them, and which imparts to an
individual who distinguishes himself in its pursuit, a character of high
standing among his tribe. The great scarcity of the buffalo, however,
of late years, since the immense emigration to the far west, has
pinched them with want and hunger, and it is evident that they
cannot subsist much longer by depending mainly upon this animal as
heretofore. This circumstance, though calling forth our warmest
sympathies in behalf of their wretched condition, may perhaps be the
means, in the hands of an Allwise Providence, of shaking that
tenacity and attachment so strongly developed in the Indian
character, for long cherished customs, and making them more
pliable in adopting the arts of civilization. Their attention must soon
be directed to agriculture and the raising of stock, or they must
starve.
While on the hunt, in the buffalo country, the Pawnees generally
have a good supply of fresh meat to subsist on. That portion of their
meat which is intended to be jerked, in order to take home with them,
they cut into narrow slips, and dry it over the fire, without any salt,
where, by the action of the smoke and sun, it soon dries. Several
slips are then plaited together, when it is put away into a skin, and
reserved for future use. The reason why no salt is added is probably,
first, because it is a scarce article, and secondly, because anything
that is salted and dried, imbibes moisture in damp weather, and
sooner becomes rancid than meat dried without salt.
Besides the buffalo, as their main subsistence, they raise some
corn, beans, and pumpkins. As they use no other agricultural
implements than hoes, their fields are not very extensive. For
suitable spots of cultivation they generally select the mouth of a
ravine, or any spot where, by the washing of the rains, the ground
has become loose and mellow, and consequently the sod may more
easily be extirpated than in the prairies generally, where the ground
can only be prepared by turning the thick sod by means of a large
prairie plough, and three or four yoke of oxen.
 Their fields are not enclosed by fences or any other kind of
protection. Having no other stock but horses, which are constantly
herded and watched during the day time, and shut up in enclosures
in the village during the night, fences are unnecessary. While absent
from their homes, their fields are subject to depredations from prairie
wolves and deer, which however no ordinary fence could restrain.
Because these poor people have no other agricultural implements
than hoes, the spots which they are obliged to select for fields are
often at great distances from each other, and frequently from five to
eight miles from their village. When abroad, to prepare and plant
them in the spring, and gather the corn in the fall, they are often
exposed to attacks of their enemies. While the husband is watching
from some neighboring eminence to guard against the stealthy
approach of any hostile force, the wive is engaged in the labor of the
field. With fear and trembling their field-labor is thus performed, and
many a one, while so employed, is suddenly fallen upon and killed
by a marauding party of enemies.
When we visited them, they particularly requested us “to beg their
Great Father, the President, that he should be so kind and again
break up some ground for them, as he had done in former years,
that they might be able to plant more corn.” We have brought in the
petition of these destitute people before the proper Department at
Washington, and it is greatly to be desired that our benevolent
Government may do something for them in their wretched state.
As has already been remarked, they were in an almost starving
condition when we visited them. The hunt had proved unsuccessful,
so that in the lower village they had no meat whatever, and had to
depend upon the little corn that they had raised the preceding year.
In the upper village they were also in a miserable condition, for,
though they had yet a little dried buffalo-meat, the Sioux had during
their absence on the hunt, destroyed their village (their present
lodges had been rebuilt since their return) and carried off a portion of
their corn, burnt another portion, and thus very little remained
concealed in the “caches,” which are made in their lodges.
Hospitality.—The Indian has always been renowned for his
hospitality. This trait of character is noticeable among all the tribes.