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Verticillium dahliae transcription factors Som1 and Vta3
control microsclerotia formation and sequential steps
of plant root penetration and colonisation to induce disease
Dissertation
for the award of the degree
"Doctor rerum naturalium"
of the Georg-August Universität Göttingen
within the doctoral program Biology
of the Georg-August University School of Science
submitted by
Thuc Tri Bui
from Thai Nguyen, Vietnam
Göttingen 2017
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Thesis Committee
Prof. Dr. Gerhard H. Braus
Department of Molecular Microbiology and Genetics
Institute of Microbiology and Genetics
Georg-August Universität Göttingen
Prof. Dr. Stefanie Pöggeler
Department of Genetics of Eukaryotic Microorganisms
Institute of Microbiology and Genetics
Georg-August Universität Göttingen
Members of the Examination Board
Reviewer I
Prof. Dr. Gerhard H. Braus
Department of Molecular Microbiology and Genetics
Institute of Microbiology and Genetics
Georg-August Universität Göttingen
Reviewer II
Prof. Dr. Stefanie Pöggeler
Department of Genetics of Eukaryotic Microorganisms
Institute of Microbiology and Genetics
Georg-August Universität Göttingen
Further members of the Examination Board
Prof. Dr. Ivo Feussner
Department of Biochemistry of the Plant
Albrecht-von-Haller-Institute of Plant Sciences
Prof. Dr. Kai Heimel
Department of Molecular Microbiology and Genetics
Institute of Microbiology and Genetics
PD Dr. Michael Hoppert
Department General Microbiology
Institute of Microbiology and Genetics
Prof. Dr. Rolf Daniel
Department of Genomic and Applied Microbiology
Institute of Microbiology and Genetics
Date of oral examination: 21.11.2017
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Affirmation
I hereby declare that this thesis was written independently and with no other
sources and aids than quoted
Göttingen, 3.10.2017
Thuc Tri Bui
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This work was accomplished in the group of Prof. Dr. Gerhard H. Braus, at the
Department of Molecular Microbiology and Genetics at the Institute of Microbiology
and Genetics, Georg-August Universität Göttingen.
Parts of my work will be published in:
Tri-Thuc Bui, Rebekka Harting, Susanna A. Braus-Stromeyer, Van-Tuan Tran,
Oliver Valerius, Rabea Schlüter, Claire E. Stanley, Alinne Ambrósio, Gerhard H.
Braus (2017). Verticillium dahliae transcription factors Som1 and Vta3 control
microsclerotia formation and sequential steps of plant root penetration and
colonisation to induce disease. Submitted for publication.
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Table of contents
Summary 1
Zusammenfassung 2
1. Introduction 3
1.1 Verticillium dahliae – a pathogen of wilt diseases 3
1.1.1 V. dahliae is a threatening plant pathogenic fungus 3
1.1.2 Verticillium morphology 5
1.1.3 Disease symptoms of V. dahliae on tomatoes 7
1.1.4 V. dahliae disease cycle 8
1.2 Adhesion is essential for fungal pathogens 9
1.2.1 Adhesion in yeasts 10
1.2.2 Adhesion and virulence in filamentous fungi 13
1.2.3 Adhesion and virulence in V. dahliae 16
1.2.4 Wing helix-turn-helix DNA binding proteins 19
1.3 Regulation of conidia and microsclerotia formation 20
1.3.1 Regulation of conidation 20
1.3.2 Regulation of microsclerotia formation 21
1.4 Aim of this work 22
2 Materials and Methods 24
2.1 Materials 24
2.1.1 Chemicals 24
2.1.2 Primers 24
2.1.3 Plasmids 29
2.1.4 Organisms 30
2.1.4.1 Bacterial strains and their cultivation 30
2.1.4.2 Fungal strains and their cultivation 30
2.2 Methods 33
2.2.1 Bioinformatic analysis 33
2.2.2 Gene deletion, complementation, and overexpression 33
2.2.2.1 Gene deletion 33
2.2.2.2 Gene complementation 35
2.2.2.3 Gene overexpression 36
2.2.3 Genetic manipulations 37
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2.2.3.1 E. coli transformation 37
2.2.3.2 A. tumefaciens transformation 38
2.2.3.3 S. cerevisiae transformation 38
2.2.3.4 V. dahliae transformation 39
2.2.4 Confirmation of transformation 39
2.2.4.1 DNA purification 39
2.2.4.2 PCR amplification 41
2.2.4.3 Southern hybridization 42
2.2.5 Phenotypical analyses 42
2.2.5.1 Microsclerotia counting 42
2.2.5.2 Conidia examination 42
2.2.5.3 Hyphal branching test 43
2.2.5.4 Localisation study 43
2.2.5.5 Oxidative stress test 43
2.2.5.6 Adhesion examination 44
2.2.6 Plant infection test 44
2.2.6.1 Tomato infection study 44
2.2.6.2 Arabidopsis root infection test 45
2.2.6.3 Scan electron microscopy 45
2.2.7 Protein methods 46
2.2.7.1 Protein isolation 46
2.2.7.2 Proteomic analysis 46
2.2.7.3 Western hybridization 47
2.2.7.4 GFP trap assay 47
2.2.8 Gene expression quantification 47
3 Results 49
3.1 The transcription factors SOM1 and VTA3 can reprogram
non-adhesive yeast strain 49
3.1.1 SOM1 and VTA3 genes encode proteins comprising a LisH or a wing
helix-turn-helix DNA binding domain 49
3.1.2 Som1 and Vta3 are nuclear proteins 51
3.1.3 Som1 and Vta3 can rescue adhesion of FLO8-defective S. cerevisiae
strains 52
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3.1.4 Low expression of SOM1 can activate flocculation genes 54
3.1.5 Activation of VTA3 can stimulate expression of flocculation genes 55
3.2 Transcription factors SOM1 and VTA3 are required for morphology
and virulence in V. dahliae 56
3.2.1 Deletion and complementation of SOM1 and VTA3 in V. dahliae 56
3.2.2 Som1 promotes adhesion in V. dahliae 58
3.2.2.1 Som1 is necessary for hyphal clumping and suppresses biomass
formation 58
3.2.2.2 Som1 is needed for adherence on abiotic surfaces 60
3.2.3 Som1 and Vta3 are required for pathogenicity 62
3.2.3.1 Som1 and Vta3 are involved in fungal pathogenicity 63
3.2.3.2 Fungal Som1 and Vta3 are required for sequential steps of plant root
penetration and colonisation 65
3.2.4 Som1 and Vta3 support conidia and microsclerotia formation 67
3.2.4.1 Som1 and Vta3 promote conidia formation 68
3.2.4.2 Som1 and Vta3 control microsclerotia formation 69
3.2.5 Som1 and Vta3 antagonise in oxidative stress response 71
3.2.6 Som1 and Vta3 are needed for hyphal growth of V. dahliae on agar
plates 72
3.2.7 Som1 is essential for hyphal development in V. dahliae 75
3.2.8 Som1 and VTA3 regulate the expression of VTA genes and related
adhesion, conidia and microsclerotia formation, and virulence genes 79
3.2.8.1 Som1 and Vta3 regulate the expression of VTA genes 79
3.2.8.2 Som1 control expression of genes involved in adhesion 80
3.2.8.3 Som1 and Vta3 control expression of genes involved in conidia and
microsclerotia formation, oxidative stress response and virulence 83
3.2.8.4 Som1 interacts with protein Ptab while Vta3 interacts with the
transcriptional co-repressor Ssn6 85
3.3 A. fumigatus SOMA can rescue the deletion of SOM1 in V. dahliae 86
4. Discussion 89
4.1 The transcription factors Som1 and Vta3 support adhesion
of S. cerevisiae 89
4.1.1 Som1 presumably binds to promoter regions of flocculation genes in
S. cerevisiae for activation 89
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4.1.2 Vta3 might activate adhesion through repressing the negatively acting
SFL1 in S. cerevisiae 91
4.2 The Transcription factors Som1 and Vta3 promote fungal
development and virulence 92
4.2.1 Som1 and Vta3 control transcription factors for adhesion 92
4.2.2 Som1 controls adhesion and penetration in V. dahliae 94
4.2.3 Som1 and Vta3 promote pathogenicity 95
4.2.4 Som1 and Vta3 are essential for conidia and microsclerotia formation 96
4.2.5 Som1 and Vta3 antagonise the oxidative stress response 98
4.2.6 Som1 and Vta3 are required for hyphal development 101
4.3 AfSom1 and VdSom1 fulfil similar functions in plant and human
pathogens 102
4.4 Outlook 104
References 106
Abbreviations 120
List of Figures 122
List of Tables 125
Acknowledgements 126
Curriculum vitae 128
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Summary
Summary
Verticillium dahliae belongs to the soil-borne ascomycete fungi. It causes wilt
diseases and early senescence in more than 200 plant species including
economically important crops. It can exist in the soil without a host for a decade by
forming microsclerotia. Root exudates induce germination of microsclerotia. V.
dahliae enters its hosts through root infection, colonises the root cortex and invades
the xylem vessels. The host infection of pathogenic fungi requires penetration and
colonisation processes. The penetration of the root surface needs adhesive proteins
at several stages during the host-parasite interaction. Adhesion proteins are not well
described in V. dahliae whereas they are well studied in Saccharomyces cerevisiae.
S. cerevisiae Flo8 is a transcription factor of adhesion, which regulates the
expression of flocculation genes such as FLO1 and FLO11. The defective FLO8
strain is unable to adhere to agar plates or to flocculate in liquid medium. V. dahliae
nuclear transcription factors Som1 and Vta3 can rescue adhesion in a FLO8-
deficient S. cerevisiae strain. Som1 and Vta3 induce the expression of FLO1 and
FLO11 genes encoding adhesins. The SOM1 and VTA3 genes were deleted and
their function in fungal induced plant pathogenesis was studied by genetic, cell
biological, proteomic and plant pathogenicity experiments. V. dahliae Som1 and
Vta3 are sequentially required for root penetration and colonisation of the plant host.
Som1 supports fungal adhesion and root penetration and is required earlier than
Vta3 in the colonisation of plant root surfaces and tomato plant infection. Som1
controls septa positioning, the size of vacuoles, and subsequently hyphal
development including aerial hyphae formation and normal hyphal branching. Som1
and Vta3 control conidia and microsclerotia formation and antagonise in oxidative
stress response. The molecular function of Som1 is conserved between the plant
pathogen V. dahliae and the opportunistic human pathogen Aspergillus fumigatus.
Som1 controls the expression of genes for adhesion and oxidative stress response.
Som1, as well as Vta3, regulate a genetic network for conidia and microsclerotia
formation and pathogenicity of V. dahliae.
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Zusammenfassung
Zusammenfassung
Verticillium dahliae gehört zu den bodenbürtigen Askomyceten. Dieser Pilz
verursacht Welke-Erkrankungen und verfrühtes Altern in mehr als 200
verschiedenen, auch ökonomisch wichtigen Pflanzen. Verticillium kann im Boden
ohne Wirtspflanze durch die Bildung von Mikrosklerotien bis zu 10 Jahre überleben.
Wurzelexsudate induzieren die Auskeimung der Mikrosklerotien. V. dahliae infiziert
seinen Wirt durch die Wurzel, besiedelt den Wurzelkortex und dringt dann in die
Xylemgefäße ein. Die Infektion des Wirts durch pathogene Pilze erfordert
Penetrations- und Kolonisierungsprozesse. Am Eindringen durch die
Wurzeloberfläche sind adhäsive Proteine an verschiedenen Stellen der Wirt-
Parasit-Interaktion beteiligt. Adhäsive Proteine sind in S. cerevisiae gut untersucht,
während nur wenig über sie in V. dahliae bekannt ist. Der Adhäsions-
Transkriptionsfaktor Flo8 aus Hefe reguliert die Expression der sogenannten
„Flocculation“-Gene wie zum Beispiel FLO1 und FLO11. Ein Stamm ohne FLO8 ist
nicht in der Lage an Agarmedium zu haften und in Flüssigmedium auszuflocken.
Die im Zellkern lokalisierten Transkriptionsfaktoren Som1 und Vta3 können die
Adhäsion in einem S. cerevisiae Stamm, welchem FLO8 fehlt, wiederherstellen.
Som1 und Vta3 induzieren die Expression von FLO1 und FLO11, welche Adhäsine
kodieren. Die SOM1 und VTA3 Gene wurden deletiert und ihre Funktion in der durch
Pilze verursachten Pflanzenpathogenese wurde durch genetische, zellbiologische,
Proteom- und Pflanzenpathogenitätsexperimente untersucht. V. dahliae Som1 und
Vta3 sind sequenziell für die Penetration und Kolonisation des Pflanzenwirts
erforderlich. Som1 unterstützt die pilzliche Adhäsion sowie das Eindringen in die
Wurzel. Somit wird es früher für die Besiedlung der Pflanzenwurzeloberfläche und
die Tomateninfektion benötigt als Vta3. Som1 kontrolliert darüber hinaus die
Positionierung von Septen und die Größe von Vakuolen und folglich auch die
Entwicklung von Hyphen inklusive der Bildung von Lufthyphen und normalen
Hyphenverzweigungen. Som1 und Vta3 beeinflussen die Bildung von Konidien und
Mikrosklerotien und wirken sich in der Antwort auf oxidativen Stress entgegen. Die
molekulare Funktion von Som1 ist zwischen dem Pflanzenpathogen V. dahliae und
dem opportunistischen Humanpathogen Aspergillus fumigatus konserviert. Som1
kontrolliert die Expression von Genen welche für Adhäsion und die Antwort auf
oxidativen Stress benötigt werden. Sowohl Som1 als auch Vta3 regulieren ein
genetisches Netzwerk für die Bildung von Konidien und Mikrosklerotien sowie die
Pathogenität von V. dahliae.
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Introduction
1. Introduction
1.1 Verticillium dahliae – a pathogen of wilt diseases
Verticillium species are soil-borne plant pathogenic fungi which cause high
losses of crops (Pegg & Brady, 2002; Berlanger & Powelson, 2005). The name
Verticillium was given because of the phialide arrangement in verticillate shape
around the conidiophores (Pegg & Brady, 2002; Berlanger & Powelson, 2005).
There are three different species of Verticillium including Verticillium albo-atrum,
Verticillium dahliae, and Verticillium longisporum (Pegg & Brady, 2002). However,
the first Verticillium strain was detected in 1879 (Reinke & Berthold, 1879) until
1913 V. dahliae which causes wilt on dahlia (Asteraceae family) was first described
(Isaac, 1947).
1.1.1 V. dahliae is a threatening plant pathogenic fungus
V. dahliae strains cause wilting diseases and early senescence in more than
200 plant species of economically important crops including tomato, potato, cotton,
cabbages, and strawberries (Pegg & Brady, 2002). This species is causing significant
loss in crop yield and are widely spread.
A V. dahliae strain can enter the root and develop resting structures not only
in more than 200 plant host species but also in non-host plants (Pegg & Brady, 2002;
Berlanger & Powelson, 2005). After entering the plant roots, this fungus produces
conidia which are transported in the transpiration stream to any part of the plant. In
which, conidia germinate and colonise the plant (Pegg & Brady, 2002; Berlanger &
Powelson, 2005). V. dahliae produces resting structures which are called
microsclerotia to survive in the soil without host plants. Microsclerotia are formed in
dry tissues when the plant dies. Thick cell walls with dense melanin deposits of
microsclerotia protect the fungus against extreme temperatures, enzymatic lysis,
and UV light. They still can recover from animal feces after staying two days in the
stomach (Pegg & Brady, 2002; Berlanger & Powelson, 2005). Therefore,
microsclerotia can survive without hosts for a decade in the soil (Pegg & Brady,
2002). Additionally, V. dahliae can enter non-host plants and produce
microsclerotia, which cause no symptoms or diseases (Pegg & Brady, 2002;
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Introduction
Berlanger & Powelson, 2005). Because of these reasons, it is not easy to treat
V. dahliae even when using crop rotations or fungicides (Pegg & Brady, 2002).
Therefore, V. dahliae causes a loss of billions of dollars in annual crops worldwide
(Pegg & Brady, 2002; Berlanger & Powelson, 2005).
Conidia and microsclerotia of V. dahliae are easily transported worldwide by
different ways. For instance, conidia are tiny cells which can be transported through
xylem vessel systems with the transpiration stream from the root vessels into the
shoot and thereby distributing the fungus to the whole plant. Therefore it is easy
transported with crop products (Pegg & Brady, 2002; Berlanger & Powelson, 2005;
Inderbitzin et al., 2011b). Furthermore, they are easily transported by air for 20 feet
or by water stream such as rivers and irrigation canals when the water was re-used.
V. dahliae may also be distributed by contaminated seeds, insects, vegetative
cutting, transplant, hand tools or farm machinery (Pegg & Brady, 2002; Berlanger &
Powelson, 2005). Microsclerotia can also be found in seeds of infected plants,
therefore, they are easily transported worldwide when crop products are exported
or imported (Pegg & Brady, 2002). Nowadays, this fungus can be found worldwide
in countries with cool or warm climate (Figure 1).
Figure 1. V. dahliae distribution. V. dahliae species were found worldwide from cool to
warm climate. They are more common in Europe, America, and Africa than in Asia and
Australia. Figure taken from http://www.plantwise.org/KnowledgeBank/Datasheet.
aspx?dsid=56275.
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Introduction
1.1.2 Verticillium morphology
Hyphae of V. dahliae are mostly haploid, but hyphal tips may be
multinucleate. They are hyaline, simple or branched, septated and multinucleated.
Hyphal septa are perforated, but nuclei have not been reported to traverse the pore
(Pegg & Brady, 2002; Berlanger & Powelson, 2005). The hyphal extension is directly
proportional to the availability of growth capabilities. Diffusible morphogenic factors
in V. dahliae inhibit hyphal elongation and conidiation and induce lateral branching
(Brandt, 1967). Conidia are single cells which are born on phialides (Figure 2a, b, c).
These phialides are arranged in whorls around conidiophores which are branched
aerial hyphae(Pegg & Brady, 2002; Berlanger & Powelson, 2005). Each phialide
carries a mass of conidia which are named conidiospore cluster in the following
(Figure 2c). Conidia are hyaline and ovoid to an elongated shape. They have thin cell
walls without melanin deposits (Figure 2d). Conidia are very small (3.5-5.5 µm) and
are transported easily with the transpiration stream in plants (Pegg & Brady, 2002).
Fungal materials such as microsclerotia can be therefore found in whole plants
including seeds.
The resting structures, which are formed by hyphal welting, are usually found
in the dead tissue of infected plants. They have thick cell walls with dense melanin
layers. Black microsclerotia are found in the dead plant as black dots (Figure 2e, f, g)
(Pegg & Brady, 2002; Berlanger & Powelson, 2005; Inderbitzin et al., 2011b). These
structures help V. dahliae to survive in the dead plants and the soil up to 15 years
or after going through the animal stomach (Pegg & Brady, 2002).
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Introduction
Figure 2. Conidia and microsclerotia of V. dahliae. (a) Whorl phialide. (b) Solitary
phialide. (c) Branched conidiophore. (d) Conidia. (e) Microsclerotia in planta. (f)
Microsclerotia on agar plates. (g) The structure of a single microsclerotium. The arrowhead
shows a conidiospore cluster. This figure is modified from Inderbitzin et al. (2011a).
6
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