genome annotation, gene characterization, and the functional analysis of natural antisense ...

313 Pages·2013·5.88 MB·English

GENOME ANNOTATION, GENE CHARACTERIZATION, AND THE
FUNCTIONAL ANALYSIS OF NATURAL ANTISENSE TRANSCRIPTS IN THE
FUNGAL PLANT PATHOGEN Ustilago maydis

A dissertation submitted to the Committee of Graduate Studies
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
in the faculty of Arts and Science

TRENT UNIVERSITY
Peterborough, Ontario, Canada
© Copyright by Michael Edward Donaldson, 2014
Environmental and Life Sciences Graduate Program
January 2014
ABSTRACT

Genome annotation, gene characterization, and the functional analysis of natural
antisense transcripts in the fungal plant pathogen Ustilago maydis
Michael Edward Donaldson

Ustilago maydis (DC) Corda is the causal agent of ‘common smut of corn’. Completion of
the U. maydis lifecycle is dependent on development inside its host, Zea mays. Symptoms
of U. maydis infection include chlorosis and the formation of tumours on all aerial corn
tissues. Within the tumours, thick-walled diploid teliospores form; these are the
reproductive and dispersal agent for the fungus. U. maydis is the model to study
basidiomycete biotrophic plant-pathogen interactions. It holds this status in part because
of the completely sequenced 20.5 Mb genome; however, thorough genome annotation is
required to fully realize the value of this resource. The research presented here improved
U. maydis genome annotation through the analysis of cDNA library sequences and
comparative genomics. These analyses identified and characterized pathogenesis-related
genes, and identified putative meiosis genes. This enabled the use of U. maydis as a model
for investigating ‘host-induced’ meiosis. Further, the cDNA library analyses identified
non-coding RNAs (ncRNAs) and natural antisense transcripts (NATs). NATs are
endogenous RNA molecules with regions complementary to a protein-coding transcript.
Although NATs have been identified in a wide variety of mammals, plants, and fungi, very
few have been functionally characterized. Over 200 U. maydis NATs were annotated by
analyzing full-length cDNA sequences. NAT structural features were characterized.
Strand-specific RT-PCR was used to detect NATs in U. maydis and in a related smut
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fungus, U. hordei. The data supported a common role for NATs in smut teliospore
development, independent of the RNA interference pathway. Analysis of the expression
of one U. maydis NAT, as-um02151, in haploid cells, led to a model for NAT function in U.
maydis during teliospore dormancy. This model proposed NATs facilitate the
maintenance of stored mRNAs through the formation of double-stranded RNA. In testing
this model, it was determined that the deletion of two separate upstream regulatory regions,
one of which contained a ncRNA (ncRNA1), altered NAT levels and decreased
pathogenesis. These studies strengthened U. maydis as a model organism, and began the
functional investigation of NATs in U. maydis, which identified a new class of fungal
pathogenesis genes.

NOTE: The appendices and supplementary materials are presented in electronic form on a
compact disc, which is attached at the end of the thesis.

KEYWORDS: Ustilago maydis, cDNA library analysis, genome annotation, natural
antisense transcripts, mRNA stability, pathogenesis

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ACKNOWLEDGEMENTS

During an introductory lecture for the Master of Biotechnology program at the University
of Toronto, Dr. Paul Horgen proclaimed 'an M. Biotech student can do anything'. Since
2005, I have found myself striving to live up to that adage, but importantly; it was as an M.
Biotech candidate that I met Dr. Barry Saville. It was in a basement lecture hall,
affectionately called 'the dungeon' that Dr. Saville and I sketched out what would become
the foundation for my PhD thesis project. I am indebted to Dr. Saville for his financial
support, technical guidance, literary teachings, encouragement, and friendship. While at
UofT, several other researchers were responsible for putting me on the right path. It is
with heartfelt thanks that I acknowledge Eric Ho, Dr. Matt Cahill and Dr. Alexander Zahiri
for teaching me the molecular and bioinformatic techniques that are required to work with
Ustilago maydis. I also thank Dr. Yohannes Tadesse for his patience and for shaping how
I interact with younger researchers.
Since moving to Peterborough, many people have helped make my time at Trent
University a positive experience. I thank Linda Cardwell, Mary Lynn Scriver, Jane
Rennie, Erin Davidson, and Chris McWilliam for their administrative support. I am
grateful to members of the Saville laboratory, including: Erin Morrison, Kayla Marsh,
Kitty Cheung, Colleen Doyle, Amanda Seto, and Scott Lambie. Their thoughtful
suggestions and critical reviews of the manuscripts presented in this thesis are appreciated.
I am grateful for Laura Peers, Michael Kalisz, Jen Dart, and Christine Russell, whose
administrative and technical support reached beyond the boundaries of their job
descriptions as a member of Dr. Savilles lab. I thank Dr. Christopher Kyle and Dr. Leslie
Kerr for advice on life and experimental design. I am also appreciative for the feedback
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provided by Dr. Craig Brunetti and Dr. Janet Yee as members of my supervisory
committee. Additionally, with the financial support of Dr. Saville, I was encouraged to
attend numerous scientific conferences. I am indebted to the members of the fungal
research community, and specifically thank Dr. William Holloman, Dr. Michael Perlin, Dr.
Flora Banuett, and Dr. Guus Bakkeren for their time and thoughtful advice.
I appreciate my friends for their encouragement and for not giving up on our
friendships, even when I was immersed in my own work. I thank my family (the
Donaldsons) and the Warrener family, for their support and for showing interest in my
work, even when I couldn't convey its meaning in layman's terms. To my parents, Ted and
Lynda, and my in-laws, Jay and Ginny, I am especially grateful for their words of
encouragement and for their kindness. Lastly, I thank my wife, Carolyn, for her
unwavering support. I am a very lucky man.

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TABLE OF CONTENTS

PAGE

TITLE PAGE ........................................................................................................... i
ABSTRACT ............................................................................................................. ii
NOTE ....................................................................................................................... iii
KEYWORDS ........................................................................................................... iii
ACKNOWLEDGEMENTS ..................................................................................... iv
TABLE OF CONTENTS ......................................................................................... vi
LIST OF TABLES ................................................................................................... ix
LIST OF FIGURES .................................................................................................. xi
LIST OF ABBREVIATIONS .................................................................................. xiii
CHAPTER 1 General introduction

An overview of the fungal kingdom ............................................................. 1
Fungi as model organisms ............................................................................ 2
Ustilago maydis as a model for biotrophic plant pathogens ........................ 3
Fungal genome annotation ........................................................................... 6
Research objectives ...................................................................................... 8

CHAPTER 2 Identification and analysis of genes expressed in the Ustilago
maydis dikaryon: uncovering a novel class of pathogenesis
genes

Preface .......................................................................................................... 12
Abstract ........................................................................................................ 14
Introduction .................................................................................................. 16
Materials and Methods ................................................................................. 18
Results .......................................................................................................... 27
Discussion .................................................................................................... 34
Conclusions .................................................................................................. 43
Tables and Figures ........................................................................................ 45
Supplementary Material Captions ................................................................ 54
Acknowledgements ...................................................................................... 54
References .................................................................................................... 55

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CHAPTER 3 Investigating the Ustilago maydis/Zea mays pathosystem:
transcriptional responses and novel functional aspects of a
fungal calcineurin regulatory B subunit

Preface .......................................................................................................... 59
Abstract ........................................................................................................ 61
Introduction .................................................................................................. 63
Materials and Methods ................................................................................. 65
Results .......................................................................................................... 73
Discussion .................................................................................................... 79
Conclusions .................................................................................................. 91
Tables and Figures ........................................................................................ 92
Supplementary Material Captions ................................................................ 100
Acknowledgements ...................................................................................... 100
References .................................................................................................... 101

CHAPTER 4 Ustilago maydis transcript features identified through
full-length cDNA analysis

Preface ............................................................................................................ 107
Abstract ........................................................................................................ 109
Introduction .................................................................................................. 110
Materials and Methods ................................................................................. 112
Results .......................................................................................................... 118
Discussion .................................................................................................... 125
Conclusions .................................................................................................. 134
Tables and Figures ........................................................................................ 135
Supplementary Material Captions ................................................................ 145
Acknowledgements ...................................................................................... 145
References .................................................................................................... 146

CHAPTER 5 Bioinformatic identification of Ustilago maydis meiosis genes

Preface .......................................................................................................... 150
Abstract ........................................................................................................ 151
Introduction .................................................................................................. 152
Teliospore dormancy and germination ......................................................... 153
Meiosis in basidiomycetes ............................................................................ 154
Possible meiotic genes identified from U. maydis cDNA libraries .............. 159
Identification of U. maydis genes involved in meiosis using reciprocal
best hit blastp ................................................................................................ 161
Putative meiosis orthologs in U. maydis ...................................................... 164
Synaptonemal complex formation ................................................................ 165
Control of meiotic gene expression .............................................................. 166
Conclusions .................................................................................................. 170
Tables and Figures ........................................................................................ 171
Supplementary Material Captions ................................................................ 173
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Acknowledgements ...................................................................................... 173
References .................................................................................................... 174

CHAPTER 6 Natural antisense transcripts in fungi

Preface .......................................................................................................... 177
Abstract ........................................................................................................ 178
Introduction .................................................................................................. 179
Non-coding RNAs and natural antisense transcripts .................................... 180
Mechanisms for regulating gene expression ................................................ 182
Natural antisense transcript mediated gene control in fungi ........................ 185
Genome-wide identification of natural antisense transcripts in fungi .......... 194
Conclusions .................................................................................................. 201
Tables and Figures ........................................................................................ 203
Supplementary Material Captions ................................................................ 206
Acknowledgements ...................................................................................... 206
References .................................................................................................... 207

CHAPTER 7 Ustilago maydis natural antisense transcript expression alters
mRNA stability and pathogenesis

Preface .......................................................................................................... 212
Abstract ........................................................................................................ 214
Introduction .................................................................................................. 215
Results .......................................................................................................... 218
Discussion .................................................................................................... 230
Experimental Procedures .............................................................................. 243
Tables and Figures ........................................................................................ 254
Supplementary Material Captions ................................................................ 264
Acknowledgements ...................................................................................... 264
References .................................................................................................... 266

CHAPTER 8 General discussion

Introduction .................................................................................................. 272
U. maydis cDNA library analysis ................................................................. 272
U. maydis genome annotation ...................................................................... 275
Characterization and functional investigation of U. maydis natural
antisense transcripts ...................................................................................... 279
The ‘post-transcriptome sequencing’ era emerges ....................................... 283
Conclusions .................................................................................................. 286
References .................................................................................................... 292

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LIST OF TABLES

PAGE

Table 2.1 Primers used in this study ...................................................................... 45
Table 2.2 Fungi used in tblastn searches ............................................................... 47
Table 2.3 EST representation among cDNA libraries and functional
associations ............................................................................................ 48
Table 2.4 Classification of filament-specific DIK represented genes assessed
by tblastn and blastp analysis ................................................................ 49
Table 3.1 U. maydis strains used in this study ....................................................... 92
Table 3.2 Identification of U. maydis genes selectively expressed during
infection of corn .................................................................................... 93
Table 3.3 Representative Z. mays transcripts selectively expressed during
U. maydis infection ................................................................................ 94
Table 3.4 Characterization of calcineurin pathway mutants ................................. 95
Table 4.1 Assessment of current MUMDB gene models ...................................... 135
Table 4.2 Features of U. maydis FLcDNA sequences ........................................... 136
Table 4.3 EST libraries and UTR length ............................................................... 136
Table 4.4 UTR lengths associated with position of antisense complementarity ... 136
Table 4.5 Start of transcription dataset motif characteristics ................................ 137
Table 4.6 ISTD motif characteristics ..................................................................... 137
Table 4.7 Promoter dataset motif characteristics ................................................... 138
Table 4.8 Characteristics of known polyadenylation signal sequences identified
in U. maydis ............................................................................................ 139
Table 4.9 Poly(A) addition site motif characteristics ............................................ 140
Table 5.1 Reciprocal best hit blastp summary ....................................................... 171
Table 5.2 Core eukaryotic meiosis genes .............................................................. 171
Table 6.1 Experimentally characterized natural antisense transcripts in fungi ..... 203
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Table 6.2 Characteristics of fungal natural antisense transcripts .......................... 204
Table 7.1 Characteristics of sense-antisense transcript pairs ................................ 254
Table 7.2 Summary of RT-PCR screen confirming in silico analysis ................... 254
Table 7.3 Antisense expression summary ............................................................. 254
Table 7.4 Strains used in this study ....................................................................... 255
Table 8.1 Experiments to investigate the NAT-related phenotypes ...................... 287

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