Filamentous Growth in Eremothecium Fungi

FACULTY OF SCIENCE

UNIVERSITY OF COPENHAGEN

PhD thesis

Therése Oskarsson

Filamentous growth in Eremothecium fungi

Molecular characterization of the Ashbya gossypii ARF3 module

Academic advisors: Steen Holmberg, Department of Biology, University of Copenhagen

Co - supervisor: Jürgen Wendland, Carlsberg laboratory Submitted: 13/03/2014

Institutnavn: Natur- og Biovidenskabelige Fakultet

Name of department: Department of Biology

Author: Therése Oskarsson

Titel og evt. undertitel: Filamentous growth in Eremothecium fungi

Title / Subtitle: Molecular characterization of the Ashbya gossypii ARF3 module

Academic advisor: Professor Steen Holmberg

Submitted: 2014-03-13

Grade:

”The world is a thing of utter inordinate complexity and richness and strangeness that is absolutely awesome.”

Douglas Adams, 1952-2001

Preface

This thesis encompasses the results of three years of PhD studies at Carlsberg laboratory and the Univerity of Copenhagen. My research was a part of the Ariadne project, funded by the Marie Curie Programme and was mainly carried out at Carlsberg laboratory, Copenhagen, Denmark, under the supervision of Prof. Jürgen Wendland and Prof. Steen Holmberg. Five weeks was also spent working with the development of Eremothecium pathogenicity assays in the group of Prof. Antonio di Pietro, University of Córdoba, Córdoba, Spain.

This thesis begins with a general introduction of fungal biology and the topics presented in this thesis and the metods used during my research. Results and discussion of the three topics which has been the focus my research are presented in three separate chapters, followed by a general summary and future topics that could be adressed given more time. nnnnnnnnnnnnnnnnnnnnnnn

Contents

PREFACE ...... I

CONTENTS ...... II

SUMMARY ...... IV

RESUMÉ ...... V

ACKNOWLEDGEMENTS ...... VI

ABBREVIATIONS ...... VIII

CHAPTER 1: INTRODUCTION ...... 10

1.1 Fungi as model organisms ...... 10 1.1.1 Ashbya gossypii ...... 10 1.1.2 Eremothecium cymbalariae ...... 13

1.2 Factors for polarized hyphal growth ...... 13 1.2.1 Dynamics of the apical Spitzenkörper ...... 13 1.2.2 Phosphoinositides ...... 15 1.2.3 The Actin cytoskeleton ...... 15 1.2.4 Clathrin mediated endocytosis ...... 17

1.3 The Ras superfamily of small GTPases ...... 21 1.3.1 The mechanism of GTP/GDP switching ...... 22 1.3.2 The Arf3 small GTPase and its regulators ...... 24

1.4 Focus and aim ...... 26

CHAPTER 2: METHODS ...... 28

2.1 Strains, media and growth conditions ...... 28 2.1.1 Plate assays ...... 29 2.1.2 Pathogenicity assays ...... 29

2.2 Isolation of genomic DNA ...... 30

2.3 PCR and cloning ...... 31 2.3.1 Generation of deletion cassettes ...... 31 2.3.2 Vector generation by homologous recombination ...... 32 2.3.3 Cloning and functional expression of truncated AgGts1 ...... 32 2.3.4 Construction of E. cymbalariae centromere plasmids ...... 33

2.4 Transformation ...... 33

2.5 Cytological staining and microscopy ...... 34 ii

Contents

CHAPTER 3: FUNCTIONAL ANALYSIS OF THE A. GOSSYPII ARF3-YEL1-GTS1 MODULE ...... 35

3.1 Results ...... 35 3.1.1 Generation of A. gossypii arf3 and yel1 mutants ...... 35 3.1.2 Growth phenotypes and mycelial morphology ...... 37 3.1.3 Actin and chitin localization ...... 39 3.1.4 Endosome visualization using FM4-64 ...... 41 3.1.5 Truncation of AgGts1 ...... 44 3.1.6 AgGts1-GFP localization ...... 48

3.2 Discussion ...... 50 3.2.1 The A. gossypii Arf3-Yel1-Arf3 module ...... 50 3.2.2 AgGTS1-GFP localizes at sites of growth and septation ...... 52 3.2.3 Unregulated AgArf3 causes impaired endocytosis and morphological abnormalities ...... 52 3.2.4 AgARF3-dependent and independent apical actin localization ...... 54

CHAPTER 4: EREMOTHECIUM PATHOGENICITY ASSAYS ...... 56

4.1 Results ...... 56 4.1.1 Cellophane penetration assay ...... 56 4.1.2 Fruit pathogenicity assay ...... 57 4.2.3 Galleria mellonella killing assay ...... 58

4.2 Discussion ...... 59 4.2.1 Characteristics of filamentous growth in Eremothecium fungi ...... 59 4.2.2 Eremothecium spores causes G. mellonella immune reaction ...... 60

CHAPTER 5: ESTABLISHING MOLECULAR TOOLS IN E. CYMBALARIAE ...... 61

5.1 Results ...... 61 5.1.1 Establishing E. cymbalariae PCR based gene targeting ...... 61 5.1.2 Characterization of Ectec1 and ecym5230 ...... 64 5.1.3 Assessment of CEN and ARS function in E. cymbalariae ...... 66

5.2 Discussion ...... 68 5.2.1 Establishing PCR-based gene targeting in E. cymbalariae ...... 68 5.2.2 Wt-like phenotypes of E. cymbalariae TEC1 and ECYM5230 ...... 69 5.2.3 A. gossypii centromeres are stable in E. cymbalariae...... 70

CHAPTER 6: SUMMARY AND FUTURE PROSPECTS ...... 72

CHAPTER 7: REFERENCES ...... 73

APPENDIX I: STRAINS ...... 84

APPENDIX II: PRIMERS ...... 85

APPENDIX III: PLASMIDS ...... 87

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Summary

The fungal kingdom encompasses a diverse group of organisms some of which have a great impact on human lives, either as domesticated benefactors or as human and crop pathogens. Using the filamentous fungus Ashbya gossypii and its close relative Eremothecium cymbalariae as model organisms, this thesis deals with some of the aspects of hyphal growth, which is an important virulence factor for pathogenic fungi infecting both humans and plants.

Hyphal establishment through continuous polar growth is a complex process, requiring the careful coordination of a large subset of proteins involved in polarity establishment and maintenance, cytoskeleton dynamics and intracellular transport. The first part of this thesis addresses the A. gossypii Arf3 small GTPase and its GEF- and GAP regulators; Yel1 and Gts1, which has been implicated in polar growth in a wide range of organisms. We could demonstrate that manipulations of the regulatory AgGts1 rendered A. gossypii strains with severe actin localization, endocytic and morphological phenotypes, presumably due to unregulated AgArf3 activity. As a homolog of the extensively more studied mammalian ARF6, we hypothesize that the continuous AgArf3 activity causes an abnormal accumulation of PI(4,5)P2 in the plasma membrane, which in turn can be linked to the slow endocytic uptake of FM4-64, the sub-apical actin localization and subsequently the atypical mycelial morphology observed in Aggts1 mutants. Furthermore, we demonstrated that in addition to the AgArf3 GAP-regulatory activity of AgGts1, the protein could have additional actin organizing properties.

In the second and third part, this thesis addresses the use of A. gossypii and its relative E. cymbalariae as model organisms for filamentous growth. A series of assays analyzed the capability of Eremothecium genus fungi to invade and colonize both plant- and insect hosts. We found that neither A. gossypii nor E. cymbalariae are able to penetrate any host tissue, and although A. gossypii is classified as a plant pathogen it is strictly dependent on its insect vectors for infection. In addition, we optimized a series of molecular tools for E. cymbalariae to enable a faster and more efficient approach for genetic comparisons between Eremothecium genus fungi.

Resumé

Svampe er en stor gruppe af organismer med stor mangfoldighed. Nogle arter af svampe har påvirket mäniskors liv, enten som velgørere eller ved at forårsage sygdom på personer eller afgrøder. Denne afhandling avvänder trådsvampen Ashbya gossypii og dens nære slægtning Eremothecium cymbalariae som modelorganismer at analysere nogle aspekter involveret i trådet vækst, en væsentlig faktor der bidrager til evnen af svampe til at forårsage sygdom hos både personer eller planter.

Hyfer i trådsvampe dannes ved kontinuerlig polar vækst, en kompleks proces, der kræver omhyggelig koordinering af et stort antal proteiner, der bidrager til etablering og vedligeholdelse af polaritet. Den første del af denne afhandling omhandler ARF3 i A. gossypii og dens GFF og GAP regulatorer, YEL1 og GTS1. Disse gener koder for proteiner der tidligere er identificeret i polar vækst i forskellige organismer. Gennem manipulation af den regulatoriske AgGTS1 kan vi påvise at ureguleret AgArf3 aktivitet forårsager store defekter i lokalisering af actin cytoskelet, cellemorfologi og evnen til endocytose. Vores hypotese er, at hvis AgArf3 er en homolog af pattedyrsproteiner Arf6, kan de have lignende funktioner i cellen. Baseret på funktion hos Arf6, forårsager ureguleret AgArf3 aktivitet en unormal akkumulering af plasma membranbundet

PI(4,5)P2. Denne ændring i plasmamembraet kan igen knyttes til de fænotyper observerede i Aggts1; langsom endocytose, subapikal lokalisering af aktin og unormal mycelmorfologi. Vi kan også vise på ekstra funtioner af AgGts1 udover at regulere AgArf3 hvis proteinet påvirker organiseringen af actin uafhængig af AgArf3.

Den anden og tredje del er afsat til brugen af A. gossypii og E. cymbalariae som modelorganismer for trådformede vækst. I en række forsøg blev analyseret svampe evne til at invadere og kolonisere forskellige vævstyper fra både plante-og dyreriget. Vi fandt, at ingen af de testede svampe selvstændigt kan trænge ind i vævet i en værtsorganisme, og skønt A. gossypii er klassificeret som et plantpatogen er afhængighed af et insekt vektor at inficere sin vært. For yderligere at lette brugen af E. cymbalariae som en model organisme, har vi udviklet en serie af molekylære værktøjer til hurtigere og mere effektive sammenligninger mellem svampe fra slægten Eremothecium.

Acknowledgements

I would like to express my deepest and sincerest thanks to all the people who have supported me and in any way contributed to the completion of this thesis.

First of all, I would like to thank Professor Jürgen Wendland for giving me the opportunity to join his lab at Carlsberg laboratory as a PhD student. It has been three interesting and challenging years that I will never forget.

Thanks to Professor Steen Holmberg for being my academic advisor and for the support during the completion of this thesis.

Klaus Lengeler, thank you for all the comments, discussions and support during the completion of this manuscript. Also, thank you for always taking the time to answer any questions that has arisen during these three years, no matter how time-consuming or trivial they might have been.

Andrea Walther, thank you for all the support during endless microscopy sessions for always being available for comments and discussion during the progression of my project, and for always being interested in new results and developments.

Thank you Lisa for sharing projects, office space and travelling time, working with you has always been inspirational. And thank you for always being a good friend when I have needed one.

In addition to the above mentioned persons, I would like to mention the other members of the Carlsberg yeast group: Ana, Claudia, Davide, Jevgenia and Natalia. Thank you for creating the culturally colorful, friendly environment in which we all shared a piece of life for a while. I will miss you.n

To all the PIs, post docs and researchers involved in the Ariande project, thank you for organizing such an interesting and exciting project and for all the effort put in to courses, discussions and excursions. Special thanks to Professor Antonio di Pietro who invited me into his lab in Cordóba, and to all the members of his lab who made me feel welcome during my stay there.

Acknowledgements

To my fellow PhD students: Clara, Elisabetta “Betta”, Elzbieta “Ellie”, Filomena, Katja, Lisa, Mennat, Miriam, Pankaj, Sonia and Vikram. We started out as associates in a common project, but after three years of sharing research, fortunes, troubles and humor you all have become great friends and I wish you all the best!

In addition to the gratitude I owe all of those directly involved i this project, there are some persons whom deserve my depest thanks on a more personal level: Anna, Josefin and Linda, thank you for being the amazing friends that you are and for helping me to stay focused during these challenging years.

Most importantly, I would like to thank my wonderful family, my father Jan-Erik, mother Margareta, sisters Emelie and Frida and my sweet little nephew Thor. Whithout your love and support this thesis would not have been possible.

Therése Oskarsson

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Abbreviations

a a amino acid ADP adenosine diphosphate AFM Ashbya full media Ag Ashbya gossypii ARF ADP ribosylation factors (protein family) ARF3 ADP ribosylation factor (gene) ARS autonomously replicating sequence bp base pairs CDE centromeric DNA element CEN centromere CFW calcofluor white CSM complete supplement mixture (media) DIC differential interference contrast Ec Eremothecium cymbalariae EDTA ethylenediaminetetraacetic acid ER endoplasmic reticulum GAP GTPase activating protein GDP guanosine diphosphate GEF guanine nucleotide exchange factor GFP green fluorescent protein GLN glutamine-rich (protein domain) GTP guanosine triphosphate GTS1 glycine threonine serine repeat 1 (gene) kb kilo (x1000) base-pairs NPF nucleation-promoting factor ORF open reading frame PBS phosphate buffered saline PCR polymerase chain reaction PDB potato dextrose broth (media)

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Abbreviations

PEG poly-ethylene glycol pFA plasmid for Functional Analysis PH pleckstrin homology PI phosphoinositide PI(4,5)P2 phosphatidylinositol-4,5-bisphosphate PRO proline-rich (protein domain) RAS (protein superfamily) Sc Saccharomyces cerevisiae SDS sodium doedylsulphate STE NaCl-Tris-EDTA STM saccharose-TRIS-MgCl2 TE Tris-EDTA (buffer) TEC1 transposon enhancement control 1 (gene) TES 2-((1,3-dihydroxy-2-(hydroxymethyl)propanyl)amino)ethanesulfonic acid Tris tris(hydroxymethyl)aminomethane UBA ubiquitinin associated (protein domain) YEL1 yeast EFA6-like (gene) WASP Wilscott-Aldrich syndrome protein wt wild-type X-Gal 5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside YPD yeast peptone dextrose (media) YT yeast extract and tryptone (media)

Chapter 1: Introduction

1.1 Fungi as model organisms The fungal kingdom encompasses a large and diverse group of organisms, spanning from single cell yeasts to multicellular, filamentous molds and mushrooms. Approximately 800000 fungal species are described today, some of which influence human lives in several ways (Hedges, 2002). Some fungi are beneficial for us; e.g. the yeast Saccharomyces cerevisiae, which was one of the first organisms domesticated by humans. Today, S. cerevisiae is perpetually used in baking and alcohol fermentation, and with the development of the fields of molecular biology and biotechnology new functions for the yeast has emerged, both as a model organism and as a tool for processing and developing biological compounds (Botstein and Fink, 2011).

Other fungi are pathogenic and affect us either directly by causing disease in primarily immunocompromised individuals (Pfaller and Diekema, 2007), or indirectly by infecting cereal and crops, significantly reducing the crop yield worldwide each year (Dean et al., 2012). Due to the impact on human lives by pathogenic fungi, a significant research effort today is concentrated pathogenic fungi, including this thesis. Mapping and characterizing conserved virulence factors might not only result in increased understanding of the fungal infection process, but could also render new targets for antifungal drug development (Botstein and Fink, 2011, Hedges, 2002, Mustacchi et al., 2006).

1.1.1 Ashbya gossypii The ascomycete Ashbya gossypii, also known as Eremothecium gossypii, is a haploid, strictly filamentous fungus of the Eremothecium genus discovered by Ashby and Novell in 1926. Using insects of the Pyrrhocoridae family, i.e. “cotton-stainers”, as vectors, A. gossypii acts as a plant pathogen, causing stigmatomycosis in cotton and dry rot in citrus fruit (Ashby and Novell, 1926, Dietrich et al., 2013). While the fungus had a significant agricultural impact at the time of discovery, the development of modern day insecticides has more or less eliminated fungal infection by insect vectors. Today, A. gossypii is primarily used in biotechnology due to its Riboflavin overproducing properties (Kato and Park, 2012, Wendland and Walther, 2005a).

Chapter 1: Introduction

The A. gossypii life-cycle (Figure 1) is initiated by the germination of the needle-shaped ascospore, which after germination forms multinucleated hyphae with compartments separated by chitin-rich septa. Germination starts with isotropic growth, coupled with an increase of nuclei through meiosis. After switching to polarized growth, juvenile mycelia is formed which is characterized by lateral branching. As the hyphae mature, they switch to dichotomous hyphal branching and increases growth speed from 6-10 µm/h to 200 µm/h (Wendland and Walther, 2005b, Knechtle et al., 2003). No sexual cycle has been identified in A. gossypii (Wasserstrom et al., 2013), but spores are formed in sporangia of older hyphae, a process which is linked to the overproduction of Riboflavin, also known as vitamin B2 (Kato and Park, 2012, Stahmann et al., 2001).

Figure 1. The A. gossypii life cycle. The A. gossypii life cycle is initiated with a short period of isotropic growth (A), followed by the appearance of a primary germ tube along the gem bubble equatorial line. After the establishment of a second germ tube (B), the juvenile mycelia is characterized by lateral branching (C) and the growing hypha is divided into multinucleated compartments by chitin-rich septa. In the mature mycelia, growth speed increases 20-fold, and the hyphae switches from lateral- to dichotomous tip branching (D-E). Sporulation occurs in older hyphal compartments, a process linked to the overproduction of Riboflavin (F).

Chapter 1: Introduction

As a member of the Saccharomyces complex clade 12 (Figure 2), A. gossypii is a close relative of S. cerevisiae (Kurtzman and Robnett, 2003). However, while the S. cerevisiae ancestor went through a whole genome duplication event, the A. gossypii linage evolved a compact genome. The completion of the A. gossypii genome sequencing in 2004 revealed a small 9.2 million bp genome, with roughly 5000 genes located on seven chromosomes. In addition, the genome shows only few duplications and lacks transposons and sub-telomeric repeats altogether. Despite the differences in both phenotype and genome evolution between A. gossypii and S. cerevisiae, they remain close relatives with a genetic functional homology of 95 %, and a high degree of synteny (Dietrich et al., 2004, Prillinger et al., 1998). The close relationship to S. cerevisiae, together with the development of efficient tools for molecular manipulations (Steiner et al., 1995, Wright and Phillipsen, 1991, Wendland et al., 2000, Wendland and Walther, 2005a), A. gossypii has emerged as an interesting model organism for studying evolution and filamentous growth in fungi.

Figure 2. Fungi of the Saccharomyces complex. Clade 1 and Clade 12, the Saccharomyces and Eremothecium clades, are highlighted in the phylogenetic tree. Modified from from Kurtzman and Robnett, 2003.

Chapter 1: Introduction

1.1.2 Eremothecium cymbalariae Eremothecium cymbalariae, isolated by Borzi in 1888, is a filamentous ascomycete of the same genus as A. gossypii. The E. cymbalariae genome was published in 2011, and reveals large similarities to the A. gossypii genome. The 9.7 million bp encodes 4712 genes, of which 97 % have an A. gossypii homolog and 95 % have a homolog in S. cerevisiae (Wendland and Walther, 2011, Kurtzman and Robnett, 2003).

E. cymbalariae grows as multinucleated hyphae with dichotomous hyphal tip branching, however lacking the chitin-rich septa found in A. gossypii. As A. gossypii, no sexual cycle has been identified in E. cymbalariae, but needle-shaped ascospores are produced in aerial sporangia: However, it produces fewer spores than its relative, and also lacks the characteristic riboflavin overproduction displayed in A. gossypii (Wendland and Walther, 2011).

1.2 Factors for polarized hyphal growth Polar growth can be seen in a varying extent in all eukaryotic organisms. Unicellular organisms perform short growth periods, e.g during cell division, while multicellular organisms use continous polar growth for the development of specialized cell structures like roots, neurons and the fungal hyphae. As the mechanism for polarity establishment is highly conserved, and hyphal formation by continuous polar growth is a key feature for many pathogenic fungi, studying the molecular processes that govern hyphal development might offer new targets for the development of antifungal drugs. Discussed below are some of the most important factors for polarized growth, and their impact on the development of the fungal hyphae.

1.2.1 Dynamics of the apical Spitzenkörper Elongated cell structures like the fungal hyphae require a long-term maintenance of polarity, requiring careful coordination of continuous delivery of membrane and cell wall material, up- regulation of the polarisome complex and remodeling of the cytoskeleton (Figure 3). In the fungal hyphae, a specialized structure called the Spitzenkörper organizes the addition of new plasma membrane- and cell wall compounds by coordinating actin polarization and vesicle trafficking at the hyphal apex (Grove and Bracker, 1970, Girbardt, 1957, Sudbery, 2011). Regulated by small GTPases of the Ras superfamily (Park and Bi, 2007), secretory vesicles are 13

Chapter 1: Introduction

transported along the cytoskeletal microtubules from the Golgi apparatus towards the hyphal apex (Howard and Aist, 1980, Fischer et al., 2008). At the Spitzenkörper, the vesicles switch from microtubule tracks to actin filaments leading directly to the plasma membrane. The rapid and dynamic polymerization of actin at the Spitzenkörper tip is critical for vesicle delivery and maintenance of hyphal polarity and growth. Thus, an essential feature for the Spitzenkörper is the polarisome protein complex, which mediates actin polymerization and forms a link between the plasma membrane and the growing actin filament (Fischer et al., 2008, Uphadyay and Shaw, 2008).

Just below the hyphal apex and the Spitzenkörper is an area characterized by a high amount of endocytic vesicle formation, detectable by an accumulation of actin patches (Taheri-Talesh et al., 2008). Just as exocytosis is essential for delivering new material to the hyphal apex, the endocytic machinery is thought to recycle and redistribute polarity-associated proteins from areas no longer at the hyphal apex (Atkinson et al., 2002, Shaw et al., 2011).

Figure 3. Cytoskeleton- and organelle organization of the growing hyphae. Hyphal growth is mediated by a specialized structure, the Spitzenkörper, which organizes actin cable polymerization by the polarisome protein complex as well as transport of secretory vesicles to the plasma membrane. Polarity-associated membrane bound proteins are recycled by endocytosis, and endocytic vesicles are formed at an area close to the Spitzenkörper.

Chapter 1: Introduction

1.2.2 Phosphoinositides Although a minor constituent of the plasma membrane, phosphoinositides (PIs) are a group of phosphorylated lipids which has been found to have a wide range of cellular functions, including acting as secondary messengers in signal transduction cascades, membrane anchors for various proteins, organelle organization and vesicular trafficking. The regulatory role of PIs such as the plasma membrane-associated PI(4,5)P2, is assigned to the irregular distribution throughout the membranes. While PIs are synthesized from a at the associated membrane by specific inositide kinases, and the kinase activity is in turn regulated by proteins of the Ras superfamily of small GTPases (Mayinger, 2012, Oude Weernink et al., 2007, Balla, 2005, Brown et al., 2001)

As PI(4,5)P2 is located at the plasma membrane it is the PI most associated with polar growth. In

S. cerevisiae PI(4,5)P2, generated by the kinase Mss4, has been shown to mediate both the assembly of clathrin coated endocytic vesicles and interactions between the plasma membrane and the actin cytoskeleton (Desrivières et al., 1998, Gaidarov and Keen, 1999, Sun et al., 2007)

1.2.3 The Actin cytoskeleton The microtubule and actin cytoskeletons are essential for many diverse features of an eukaryotic cell, including vesicular transport and polar growth. Although the function of the microtubule cytoskeletion is mostly assinged to nuclear- and organelle migration, it is also involved in polar growth as the microtubule cytoskeleton functions as tracks for vesicle transport, linking the ER and Golgi apparatus to sites of growth (Barnes et al., 1990, Lichius et al., 2011). In contrast, the dynamic actin cytoskeleton is associated with actin cables and patches at the growing hyphal tip, structures linked to short-distance vesicle transport and endocytosis. In addition, actin rings is associate with the formation of septa throughout the hyphae (Evangelista et al., 2001, Wendland and Walther, 2005b).

The assembly of the actin cytoskeleton is mediated by a large array of proteins, many of which are regulated by membrane-bound PIs (Hilpelä et al., 2004). The most commonly involved in actin nucleation are proteins of the formin family and the Arp2/3 complex (Figure 4). In S. cerevisiae, formins mediate polymerization of unbranched actin cables used in vesicle transport (Aghamohammadzadeh and Ayscough, 2010). As key components of the polarisome protein

Chapter 1: Introduction

complex, formins act by binding actin monomers and incorporation them to the growing filament (Evangelista et al., 2001, Rida and Surana, 2005, Pollard, 2007). This formin-dependent actin polymerization is regulated by small GTPases of the Ras superfamily, which activate formin activity by blocking an auto-inhibitory binding between to formins (Li and Higgs, 2003, Wang et al., 2009, Evangelista et al., 1997). In S. cerevisiae, the bestt studied formin regulator is the conserved small GTPase Cdc42. In its active, GTP-bound state, Cdc42 associates with the plasma membrane and recruits Spitzenkörper polarisome components, including the formin Bni1. Thus, in addition to activation of the formin activity, Cdc42 acts as a linker between the polarisome and the plasma membrane which enables direct targeting of the actin filament to the plasma membrane (Evangelista et al., 1997, Chen et al., 2012).

Figure 4. Actin nucleation by formins and the Arp2/3 complex. (A) In the hyphal apex, polarisome associated formins mediate nucleation of unbranched actin filaments. (B) A branched actin network is assembled by the Arp2/3 complex assisted by associated NPFs such as the WASP-homolog Las17. Once activated, Arp2/3 anchors actin filaments to the preexisting actin network.

Cortical actin patches consists of clusters of branched actin filaments, primarily associated with endocytosis (Pollard, 2007, Aghamohammadzadeh and Ayscough, 2010). The clustering of actin filaments depend on the Arp2/3 complex, a conserved, seven subunit protein complex which anchors new actin branches to a preexisting actin network. As the Arp2/3 complex has a low intrinsic actin nucleation ability, actin assembly is activated by nucleation promoting factors, NPFs (Machesky et al., 1994, Winter et al., 1999). Several NPFs have been identified in yeast so

Chapter 1: Introduction

far, including the strong WASP-like homolog Las17 and the weaker Abp1 and Pan1 (Lee et al., 2000, Walther and Wendland, 2004b, Boettner et al., 2012, Machesky and Insall, 1998). Like formins, NPFs are regulated by small GTPases of the Ras superfamily (Goley and Welch, 2006, Bompard and Caron, 2004).

1.2.4 Clathrin mediated endocytosis In eukaryotic organisms, vesicle trassport is not only a key festure for filamentous growth but also essential for the biological functions of the cell. Intracellular transport is necessary for transporting newly synthesized proteins between the ER and the Golgi apparatus for various post- translational modifications before further distribution to the correct subcellular localization. Exocytosis enables correct positioning of membrane bound proteins as well as the excretion of soluble proteins into the extracellular fluid, necessary for cell-cell signaling. In turn, endocytosis enables internalization of proteins and lipids from the cell surface, and is used to recycle plasma membrane-bound proteins and to regulate their expression and localization (Figure 5) (Takai et al., 2001, Kaksonen et al., 2003).

Figure 5. Intracellular protein traffic. Newly synthesized proteins are transported between the endoplasmic reticulum, Golgi apparatus and lysosomes for post-translational modifications and further to the correct subcellular localization. Endocytosis is used to recycle and control the localization of protein components of the plasma membrane.

Chapter 1: Introduction

Several different kinds of endocytosis have been identified, but the most studied and best characterized is clathrin-mediated endocytosis which exists in all eukaryotic organisms. Still, the clathrin-mediated endocytic machinery is very complex, requiring the timing and coordination of a large number of proteins required for cargo sorting, membrane invagination and vesicle scission (Figure 6) (McMahon and Boucrot, 2011, Kaksonen et al., 2003). Most of the key genes encoding S. cerevisiae endocytic proteins have homologues in A. gossypii, (Table 1) and for this reason the endocytic machinery is thought to function in a similar manner for both species.

Figure 6. Organization of the components of clathrin mediated endocytosis. Early endocytic factors Syp1 and Ede1, and clathrin subunits assemble at the site of endocytosis. While Syp1 causes formation of a membrane curvature, Ede1 recruits other proteins of the endocytic complex. Coat proteins like the Sla1- End3-Pan1 complex mediate the assembly of the clathrin coat, and form a link between the forming vesicle and the actin cytoskeleton through the NFP Pan1. Assembly of the actin filament network by the Arf2/3 complex mediated further membrane invagination and the formin vesicle is released from the plasma membrane by accumulation of amphiphysins. Once the scission event has released the vesicle, the clathrin coat is disassembled, an event regulated by several proteins including small GTPases of the Ras superfamily

In S. cerevisiae, nucleation is initiated by the early arriving BAR-protein Syp1 and the EH- domain protein Ede1, which binds to the phosphatidylinositol-containing plasma membrane and cause a membrane curvature (Heath and Insall, 2008, Antonescu et al., 2011) and enables interaction with other proteins in the endocytic pathway (Confalonieria and Di Fiorea, 2002, McMahon and Boucrot, 2011). Also arriving at this time point is the clathrin heavy- and light chain. The subunits interact to form clathrin penta- and hexameres which serve as building blocks 18

Chapter 1: Introduction

for the clathrin coat (Figure 7) (Ungewickell and Branton, 1981, Crowther and Pearse, 1981, Higgins and McMahon, 2002).

Table 1. S. cerevisiae clathrin-mediated endocytic proteins and their A. gossypii homologues. The S. cerevisiae actin-clathrin linker Sla2 does not have a homologue in A. gossypii. S. cerevisiae proteins A. gossypii homologues Function(s) in endocytosis Early nucleation Ede1 ABR149W Protein-protein interactions Syp1 AEL147W Membrane curvature Chc1 AER359W Clathrin heavy chain Clc1 AGR309C Clathrin light chain Mid- to late nucleation and coat assembly Sla2 - Actin-Clathrin linker Sla1 AGR170C Actin assembly, Sla-End3-Pan1 complex End3 AER416C Actin assembly, Sla-End3-Pan1 complex Pan1 ADR018C Actin assembly, Sla-End3-Pan1 complex Yap1801 AEL209W Clathrin adaptor, Pan1 interaction Yap1802 AEL209W Clathrin adaptor, Pan1 interaction Ent1 ACL157C Clathrin adaptor, Actin assembly Ent2 ACL157C Clathrin adaptor, Actin assembly Las17 AGR285W Nucleation-promoting factor Myo3 AEL306C Nucleation-promoting factor Myo5 AEL306C Nucleation-promoting factor Abp1 AGL237C Nucleation-promoting factor Arp2 ADR316W Actin filamentation Arp3 AFR419C Actin filamentation Scission Rvs161 AER193W Actin filamentation Rvs167 AFR140C Actin filamentation Vps1 ABL001W GTPase, Actin organization Dnm1 AAL174C GTPase Slj2 (Inp52) AFL228W Polyphosphatidylinositol phosphatase Uncoating Ark1 ADL217W Kinase Prk1 ADL217W Kinase Arf3 ACL078W GTPase Gts1 ACL055W ArfGAP Lsb5 AFR709C Actin filamentation, Pan1 disassembly

Chapter 1: Introduction

Figure 7. Structure and assembly of the clathrin coat. Clathrin trisklerions (A) are composed of three heavy chains and three light chains. The trisklerions polymerize to form penta- and hexamere units (B) which build the clathrin coat in the forming vesicle (C).

In mammals, the AP2 complex plays a central role in clathrin coat formation and cargo selection. In contrast, although the AP2 complex is part of yeast endocytosis, it is not essential (Schmid, 1997, Yeung et al., 1999). Instead, continuous clathrin recruitment to the forming vesicle is dependent on the clathrin adaptors Yap1801/2 and Ent1/2 (Wendland et al., 1999a, Stahelin et al., 2003). As the coat protein Sla2 arrives at the nucleation site, it functions as a linker between the plasma membrane, the clathrin coat and the NPF Pan1 of the Sla1-End3-Pan1 complex (McCann and Craig, 1997, Tang et al., 2000). Further membrane invagination at the site of nucleation is driven by accumulation of actin filaments assembled by the Arp2/3 complex, which is activated by several NPFs present at the endocytic site (Boettner et al., 2012, Aghamohammadzadeh and Ayscough, 2010).

Scission of the newly formed vesicle from the plasma membrane is achieved by the amphiphysins Rvs161 and Rvs167 (Youn et al., 2010), together with Vps1 and Dmn1, related to mammalian dynamin (Smaczynska-de Rooij et al., 2010, Boettner et al., 2012, Gammie et al., 1995, McMahon and Boucrot, 2011). Once the scission event releases the vesicle from the cell membrane, the clathrin coat is disassembled. Several of the coat proteins, including the Pan1- Sal1-End3 complex and the Ent1/2 clathrin adaptors, are regulated by phosphorylation. Thus, several proteins implicated in endocytic vesicle uncoating are kinases, including Ark1 and Prk1 or phosphatases, like synaptojanins and small GTPases (Toret et al., 2008, Costa and Ayscough, 2005). Actin filaments protruding from the newly formed vesicle forms a link to the actin cytoskeleton, which is used as a track for transportation throughout the cell (Toshima et al., 2006, Qualmann and Kessels, 2002)

Chapter 1: Introduction

1.3 The Ras superfamily of small GTPases In the 1980s, the first small GTPases of the Ras superfamily, Ha-Ras and Ki-Ras, were identified as human oncogenes (Takai et al., 2001). Since then, a large number of small GTPases, varying between 20-40 kDa, has been identified in various eukaryotic species spanning mammals, insects, plants and fungi. The characterization of the small GTPases implements them in diverse cellular processes such as vesicular traffic, cell signaling, cytoskeleton organization and gene expression. Since their discovery, the over 150 members of the Ras superfamily have been divided into four sub-branches depending on sequence and biological function: Rho, Rab, Ras and Arf (Table 2) (Takai et al., 2001, Wennerberg et al., 2005).

Table 2. Proteins of the Ras superfamily No. genes Ras sub-family Protein function(s) Yeast Mammalia Arf Intracellular traffic 7 16 Rab Intracellular traffic 11 42 Nucleocytoplasmic transport Ran 2 1 Cytoskeleton organization Ras Gene expression 4 19 Gene expression Rho 6 19 Cytoskeleton organization

Small GTPases function as regulators of cellular processes by switching between an active and inactive state in a GTP-dependent manner. A common feature for all small GTPases is the GTP/GDP binding consensus sequence, the G-domain, which renders a high affinity for binding GTP and GDP (Bourne et al., 1991). The GTPase cycling between the active GTP-bound state and the inactive GDP-bound state is a naturally slow process, but the cycling is enhanced by two groups of regulatory proteins: Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). The inactive small GTPase is activated by disassociation of the bound GDP, catalyzed by the regulatory GEF (Rensland et al., 1991, Shapiro et al., 1993). The subsequent binding of GTP causes a conformational change of the protein, allowing interaction between the small GTPase and the downstream effector. In turn, the small GTPase is inactivated by a rate-limiting hydrolysis of GTP. This reaction is regulated by GAPs, which increase the intrinsic GTPase, promoting the transition into the inactive, GDP bound form (Figure 8) (Trahey and McCormick, 1987, Takai et al., 2001).

Chapter 1: Introduction

Figure 8. Illustration of small GTPase switching between active and inactive states. An upstream signal triggers GEF aided GDP-GTP exchange, causing a conformational change which activates the GTPase. In turn, the small GTPase is inactivated by GAP-assisted hydrolysis of GTP to GDP and Pi.

1.3.1 The mechanism of GTP/GDP switching The 166 residue, 20 kDa G-domain of guanine nucleotide binding proteins like the small GTPases of the Ras superfamily, is highly conserved and the mechanism of GTP/GDP binding is similar for all proteins. The G-domain structure consists of five α-helices organized around a six stranded β-sheet. The guanine nucleotide binding properties arise from five conserved sequence motifs (Figure 9) (Bourne et al., 1991). While the N/TKXD and GXXXXGKS/T motifs bind any nucleotide phosphate (Saraste et al., 1990), the aspartic acid side chain of the DXXG motif and the alanine of the S/CAK/L/T motif render the guanine nucleotide specificity (Zhong et al., 1995). Two areas in the G-domain are particularly prone to conformational changes when interacting with GTP/GDP. The conformation of these areas, named Switch I and Switch II, are very similar in all guanine nucleotide binding proteins when bound to GTP, whereas the structure varies to some extent in the GDP bound state (Corbett and Alber, 2001, Vetter and Wittinghofer, 2001). In the activated G-domain, the GTP is fixated by hydrogen bonds between GTP-oxygen and NH-groups in threonine and glycine residues in Switch I and II, respectively (Vetter and Wittinghofer, 2001).

Chapter 1: Introduction

Figure 9. Conserved sequence elements for the guanine nucleotide binding properties of the G-domain. Sequence elements I and IV enable a general affinity for nucleotide phosphates, while elements III and V renders guanine nucleotide specificity.

The slow GDP release from the G-domain is catalyzed by GEFs (Figure 10A). Different families of GEFs may be structurally unrelated, but they all catalyze the release of GTP in a similar manner (Cherfils and Chardin, 1999). By forcing a structural change of the G-domain through Switch I and II interaction, the bonds between the G-domain P-loop, the GTP and the magnesium ion is inhibited and the GDP is released (Kawashima et al., 1996, Vetter and Wittinghofer, 2001, Cherfils and Chardin, 1999). A new guanine nucleotide, together with a Mg2+ ion, rapidly replaces the GDP once the GEF has dissociated from the G-domain. (Zurita et al., 2010).

The process of GTP-to-GDP conversion varies between protein families. While G-domains of the G-protein family appear to be catalytically active due to an intrinsic arginine residue (Mittal et al., 1996), G-domain hydrolysis of GTP in the RAS superfamily is an approximately hundred- fold slower process (Rensland et al., 1991, Shapiro et al., 1993, Bourne et al., 1991). In order to rapidly cancel G-domain protein signaling, the speed of hydrolysis of GTP to GDP is enhanced up to 105-fold by GAP regulators (Gideon et al., 1992). The GAP-dependent switch from the active, GTP–bound state to the inactive GDP-bound state is a coordinated event which requires an Mg2+ ion to interact with the oxygen atoms of two water molecules, two phosphoryl groups of the bound GTP, an α-helix serine residue and a Switch I threonine residue (Figure 10C) (Zurita et al., 2010, Vetter and Wittinghofer, 2001). As the GAP regulator interact with Switch II, a Switch II glycine residue is repositioned and a GAP arginine finger is placed at the GTPase active site. The positively charged arginine triggers the displacement of one water molecule from the active site, creating an electrostatic field which catalyzes the hydrolysis of GTP to GDP and Pi (Scheffzek et al., 1996, Mittal et al., 1996, te Heesen et al., 2007).

Chapter 1: Introduction

Figure 10. Mechanism of G-domain GTP/GDP switching. (A) Small GTPases are activated by its corresponding GEF. The GEF forces a conformational change in the G-domain Switch regions and P- loop, which breaks the binding of the GDP and magnesium ion. (B) Free cytosolic GTP associates with the small GTPase through interactions between the GTP, Switch I and II, the G-domain P-loop and one magnesium ion. (C) Hydrolysis of bound GTP to GDP is mediated by the regulatory GAP protein, which inserts a catalytically active arginine finger in the G-domain active site. The charged arginine triggers a magnesium-dependent hydrolysis reaction resulting in GDP and Pi. Modified from Vetter and Wittinghofer, 2001.

1.3.2 The Arf3 small GTPase and its regulators ADP-ribosylation factors, Arfs, are a group of proteins within the Ras superfamily of small GTPases. Many Arfs are key regulators during different stages of intracellular membrane trafficking and they have been implicated in regulation of both clathrin and COPI-coated vesicle formation and trafficking between the ER, the Golgi apparatus and the plasma membrane (Spang et al., 2010, D’Souza-Schorey and Chavrier, 2006, East and Kahn, 2011). One of the best studied Arfs is the mammalian Arf6, which has been demonstrated to regulate endocytic vesicle formation and actin cytoskeleton organization (Toret et al., 2008). The regulatory properties of Arf6 are thought to be partly mediated by activation of enzymes responsible for increased membrane content of PI(4,5)P2 (Honda et al., 1999, D’Souza-Schorey and Chavrier, 2006). In turn, PI(4,5)P2 is known to effect clathrin-mediated endocytosis, capping of actin filaments and several actin binding proteins (Wenk and De Camilli, 2004, Hilpelä et al., 2004, Antonescu et al., 2011). Other possible effectors of Arf6 are the clathrin adaptor AP2, kinases implemented in vesicle scission and the Rac1 GTPase (D’Souza-Schorey and Chavrier, 2006).

S. cerevisiae Arf3, homologous to mammalian Arf6 is not as extensively studied as its mammalian counterpart, and the precise role of Arf3 is unclear (Lee et al., 1994). Initial characterization has identified Arf3 as a non-essential protein in polarity establishment rather

Chapter 1: Introduction

than endocytosis (Huang et al., 2003). However, later studies have shown that Arf3 might be involved in endocytosis by increasing the plasma membrane content of PI(4,5)P2 (Smaczynska- de Rooij et al., 2008) and through recruitment of the coat protein Lsb5 (Costa and Ayscough, 2005, Toret et al., 2008). There is also indications that Arf3, like mammalian Arf6, might have a role in actin assembly (Lambert et al., 2007). A characteristic feature for the S. cerevisiae Arf3, is an N-terminal glycine myristoylation (Huang et al., 2003), which enables Arf3-membrane interaction. In the GDP-bound Arf3, the N-terminal helix is folded into the G-domain, while conformational changes during GDP/GTP switching causes a delocalization of the myristoylated helix enabling helix-membrane interaction (Itzen and Goody, 2011, Antonny et al., 1997).

Like all small GTPases, Arf3 cycles between an active, GTP-bound and an inactive, GDP- bound state, aided by specific GEF and GAP regulators. Yel1 has been identified as the sole Arf3-GEF (Figure 11), and displays a significant C-terminal homology to Efa6, a GEF of mammalian Arf6. (Gillingham and Munro, 2007a, Franco et al., 1999). The Sec7-domain of Ye1l is a common feature for all ArfGEFs, and catalyzes nucleotide exchange through interactions with the Arf Switch I and Switch II regions. The structural change of the Arf G-domain enables the GEF Sec7-domain glutamic finger to compete electrostatically with the bound GDP, thereby efficiently ejecting it from the G-domain altogether (Casanova, 2007, Renault et al., 2003). In addition to the Sec7 domain, Yel1 contains a C-terminal PI(4,5)P2–interacting PH domain, which is thought to mediate Yel1 localization at the plasma membrane (Casamayor and Snyder, 2002, Gillingham and Munro, 2007a).

Figure 11. Domain organization of S. cerevisiae Arf3, Yel1 and Gts1.

Chapter 1: Introduction

Arf hydrolysis of GTP to GDP is catalyzed by specific GAPs, defined by the presence of the ArfGAP domain: a four cysteine zinc-finger motif, terminating with a catalytic arginine residue

(CX2CX16CX2CX4R) (Cukierman et al., 1995). There is a general idea that ArfGAPs may not only function as regulators of Arf activity, but also downstream effectors of Arf (Spang et al., 2010). Indeed, Gts1, the only GAP identified for S. cerevisiae Arf3, was initially characterized as a clock gene, and has since then been implicated in several cellular processes including oscillation of energy metabolism, clathrin mediated endocytosis and flocculation (Yaguchi et al., 2007, Bossier et al., 1997, Xu and Tsurugi, 2007). Most GAPs have additional domains, and the domain organization between different GAPs can vary significantly. The S. cerevisiae Gts1 has two domains downstream of the catalytic ArfGAP; the central ubiquitinin-associated, UBA, domain and the C-terminal glutamine-rich, GLN, domain. The functions of the UBA and GLN domains in Gts1 has not previously been described, although UBA-motifs are usually implemented in either binding of ubiquitinin directly, or binding of ubiquitinated proteins (Hofmann and Bucher, 1996).

1.4 Focus and aim This thesis is part of a project within the Marie Curie Initial Training Network, Ariadne, aimed at identifying and characterizing conserved fungal virulence factors which might pose targets for the development of new antifungal drugs. While research in the consortium spans human- and plant pathogens, this thesis is mainly focused on the use of Ashbya gossypii as a model organism for filamentous fungi due to the molecular tools readily available for manipulation of the A. gossypii genome.

Hyphal growth is a key virulence factor for many pathogenic fungi, and several steps of hyphal formation and growth are regulated by members of the conserved Ras superfamily of small GTPases. For this purpose, this thesis aims to characterize and analyze the interactions of the A. gossypii homolog of the S. cerevisiae small GTPase ARF3, and its regulators, YEL1 and GTS1. Previous studies in S. cerevisiae have implemented Arf3 in establishment of polarity, actin assembly and endocytosis, all important factors for filamentous growth. However, the precise function and mode of action of Arf3 is not clear, and the use of a filamentous fungus might give additional clues to how Arf3 regulates polarity and hyphal growth. 26

Chapter 1: Introduction

To enable a more correct comparison between A. gossypii and the fungal pathogens targeted by the Ariadne project, another aim of this thesis has been the development of pathogenicity assays to establish if, and how Eremothecium fungi might invade host tissue. To enable further investigation of the Eremothecium genus, the final part of this thesis addresses the development of molecular tools for E. cymbalariae, a close relative to A. gossypii.nnnnnnnnnnnnnnnnnnnnnnn

Chapter 2: Methods

2.1 Strains, media and growth conditions All strains, primers and plasmids used and generated in these experiments are listed in Appendix I, Appendix II and Appendix III, respectively.

A. gossypii leu2 was used as background strain for gene deletion, and served as wt control during phenotyping of mutant strains. All A. gossypii strains were grown at 30 °C in AFM media, consisting of 2 % glucose, 1 % yeast extract, 1 % casein peptone and when necessary 2 % agar for solidification. For antibiotic selection, 200 µg/ml G418/Geneticin and/or 50 µg/ml ClonNat was used. A. gossypii was sporulated in CSM minimal medium supplemented with 0.1 % myo- inositol consisting of 2 % glucose, 0.17 % yeast nitrogen base w/o amino acids and w/o ammonium sulphate, 0.069 % CSM with 0.1 % asparagine, and when necessary 2 % agar for solidification and antibiotic selection as described above. After 5-7 days, the mycelia were degraded by incubation at 37 °C in 1x TE buffer (10 mM Tris, 1 mM EDTA) with 1 mg/ml Zymolyase, and the spores were harvested by centrifugation and resuspension in 0.03 % Triton X-100 spore buffer.

DBVPG7215 was used for optimization of gene deletion, plasmid stability- and pathogenicity assays in Eremothecium cymbalariae. All E. cymbalariae strains were cultivated in the same media and under the same conditions as A. gossypii. E. cymbalariae was sporulated on AFM agar plates. After 5-7 days at 30 °C, the spores were harvested by mechanical disruption of the sporangia. 10 ml 0.03 % Triton X100 spore buffer was added to the over-grown agar plate and the sporangia were disrupted using a sterile spatula, releasing the spores into the spore buffer. The spore buffer and the spores were collected and the spores were harvested by centrifugation before immediate use.

Saccharomyces cerevisiae BY4741 was used for pAG marker exchange and GFP integration by homologous recombination. In all cases, S. cerevisiae was grown on YPD media, consisting of 2 % glucose, 1 % yeast extract, 2 % casein peptone and when necessary 2 % agar for solidification. For antibiotic selection, 200 µg/ml G418/Geneticin and/or 50 µg/ml ClonNat was used.

Chapter 2: Methods

Fusarium oxysporum f. sp lycopersici strain 4287 (FGSC 9935) was used as a positive control in Eremothecium genus pathogenicity assays. F. oxysporum microconidia was obtained from cultures grown in PDB as described elsewhere (di Pietro and Roncero, 1998). The spores were obtained by sterile filtration and used immediately after isolation.

E. coli DH5α was used for cloning and propagation of plasmids used in this study. E. coli was grown in 2YT media containing 1.6 % tryptone peptone, 1% yeast extract, 0.5 % NaCl and when necessary 2 % agar for solidification. For antibiotic selection, 100 µg/ml Ampicillin or 50 µg/ml Kanamycin was used. Propagated plasmids were isolated using the PureYieldTM Plasmid Midiprep System (Promega) (Ish-Horowicz and Burke, 1981).

2.1.1 Plate assays Phenotypic analysis of colony growth and temperature stress was performed by spotting fresh mycelia samples at the marked center of AFM agar plates with appropriate selection. Plates were incubated at temperatures ranging from 20 °C to 37 °C for 7 days, after which the colony diameter was measured. For each assay, were combined and summarized as average growth for each genotype and temperature. For each sample growth condition, the standard deviation was calculated according to the formula: - 2/(n-1).

2.1.2 Pathogenicity assays The ability of Eremothecium fungi to penetrate physical barriers and colonize various tissues was assayed using artificial media as well as fruit- and animal tissue. The ability to penetrate a physical barrier was assayed during optimal conditions in artificial AFM- or CSM agar media. Circular cellophane sheets, approximately the size of a petri dish, were prepared by autoclaving in distilled water. Under sterile conditions, a single cellophane sheet was transferred to solidified agar plates, after which 5 µl spore suspension was inoculated at the center of the cellophane sheets and inoculated plates were incubated at 30 °C for 2-6 days. After removal of the cellophane sheet, the plate was incubated for an additional 2-3 at 30 °C in order to evaluate mycelial regrowth.

Chapter 2: Methods

Fruit pathogenicity assays were performed on fresh, undamaged apples and oranges. The fruit were sterilized in 70% ethanol for 5 min, and under sterile conditions the fruit was cut into 0.5 mm slices and placed in petri dishes. For the apple assay, the exposed fruit tissue was inoculated with 5µl spore suspensions from A. gossypii, E. cymbalariae and F. oxysporum. For the orange assay, 5 µl spore suspension from the three species was inoculated separately in the exocarp, mesocarp and endocarp to assess penetration of different parts of the orange tissue. The samples were incubated at 30 °C and 100 % humidity. After 7 days, the colony growth on each fruit slice was assessed. Tissue from the downward facing surface of the fruit slice was examined by microscopy for the presence of mycelia.

The larvae of the greater wax moth, Galleria mellonella, were used for assaying the ability of Eremothecium fungi to colonize animal tissue. A. gossypii, E. cymbalariae and F. oxysporum spores were injected into Galleria mellonella larvae according to a protocol described elsewhere (Navarro-Velasco et al., 2011). Before injection the spores were suspended in PBS to a density which could pass the syringe. Heat-killed F. oxysporum microcornidia and A. gossypii spores, generated by 1 h incubation at 65 °C, were used as negative control. The G. mellonella larvae were incubated at 37 °C and scored daily as healthy (no melanization), sick (moderate melanization) or dead (intense melanization and non-responsive to physical stimuli).

2.2 Isolation of genomic DNA Genomic DNA was obtained by alkaline extraction and isopropanol precipitation (Birnboim and Doly, 1979). The cell wall was digested by incubation at 37 °C in STE buffer (1 M Sorbitol, 50 mM Tris, 100 mM EDTA) with 1 mg/ml Zymolyase and 50 µg/ml RNAse A. Proteins were denatured by addition of SDS to a final concentration of 1 % followed by incubation at 65 °C for 30 min. Potassium acetate to a final concentration of 0.64 M, followed by 30 min incubation on ice. Cell debris was removed from the supernatant by centrifugation, and isopropanol was used to precipitate the DNA. For recovery of plasmids from S. cerevisiae and Eremothecium fungi, the genomic DNA was subsequently transformed into E. coli DH5α by electroporation.

For isolation of larger amounts of A. gossypii and E. cymbalariae DNA, vacuum filtered mycelia were frozen using liquid nitrogen and pulverized using a mortar. The pulverized mycelia was

Chapter 2: Methods

dissolved in TES buffer (100 mM Tris pH 8, 10 mM EDTA, 1 % SDS) and incubated at 60 °C for 1 h. After centrifugation, 1 volume of 5 M ammonium acetate was added to the supernatant, followed by incubation on ice for another 1 h. After removal of cell debris by centrifugation, the DNA was obtained by isopropanol precipitation, dissolved in TE buffer (10 mM TRIS pH 8, 1 mM EDTA) with 50 µg/ml RNAse A and incubated at 37 °C for 1 h (Birnboim and Doly, 1979). Highly purified DNA was obtained by including a phenol extraction step prior to isopropanol precipitation (Kirby, 1956).

2.3 PCR and cloning

2.3.1 Generation of deletion cassettes For PCR-based gene targeting, primers were designed to amplify a dominant resistance marker, adding target ORF specific flanks enabelling genomic integration of the cassette through homologous recombination. To enable correct integration of the resistance markers, 40- and 100 bp homology regions were used for A. gopssypii and E. cymbalariae respectively. The deletion cassettes were amplified by standard PCR conditions (Sambrook and Russel, 2001): 95 °C initial denaturation for 2 min, followed by 35 cycles of 1 min denaturation at 95 °C, 1 min annealing at 52 °C, 2 min elongation at 72 °C and 5 min final elongation at 72°C.

Gene disruption cassettes were generated in a two-step process (Noble and Johnson, 2005). The target ORF was amplified from genomic DNA using PCR conditions as described above. For EcTEC1, an amplicon-native XbaI restriction site and a XhoI restriction site added by the antisense primer, was used to clone the amplicon into the basic vector pBluescript SK+. The KANMX resistance marker (Steiner and Philippsen, 1994) was released from pFA-KANMX6 by EcoRV/PvuII restriction enzyme digest, and cloned to pSK-EcTEC1 through an EcoRV restriction site (Sambrook and Russel, 2001). Clonal insertion of the KANMX marker disrupted the EcTEC1 ORF into a 0.93 kb upstream fragment and a 1.36 kb downstream fragment. Before transformation of E. cymbalariae, the TEC1::KANMX disruption construct was released by XbaI and XhoI restriction enzyme digest.

In homokaryotic mutants, correct integration of the marker was verified by PCR at both up- and downstream flanks of the resistancee marker. In addition, complete loss of the target ORF was

Chapter 2: Methods

verified by PCR amplification of an internal, ORF-specific fragment. This fragment could be amplified in wt and heterokaryotic strains, but not in homokaryotic strains.

2.3.2 Vector generation by homologous recombination S. cerevisiae homologous recombination was used to generate a C-teminal tagged AgGTS1-GFP construct and for pAG URA3- to NATMX marker exchange. Integration cassettes were amplified by PCR, using primers adding flanking 40 bp homology regions for plasmid integration. Using pFA-MoGFP-NAT1 and pFA-AgNATMX4 as templates, the cassettes were ampliofied standard PCR conditions: 95 °C initial denaturation for 5 min, followed by 35 cycles of 1 min denaturation at 95°C, 1 min annealing at 52-55 °C, 2 min elongation at 72 °C and 5 min final elongation at 72 °C (Sambrook and Russel, 2001). For the AgGTS1-GFP construct, the GFP-NAT1 cassette was integrated into pAG19275(GTS1) and NATMX marker exchange was performed on pAG19275(GTS1) and pAG17522(ARF3). Correct integration of the GFP-NAT cassette was verified by sequencing performed by LCG Genomics, Germany.

2.3.3 Cloning and functional expression of truncated AgGts1 A. gossypii Gts1 domains were identified by comparing the amino acid sequence (Gattiker et al., 2007) to conserved domain motifs using PROSITE (Sigrist et al., 2012). Predicted domains from the PROSITE analysis served as template for two AgGTS1 truncations, named after the domains in the truncated protein: AgGts1(ArfGAP), containing the N-terminal ArfGTPase activating protein (ArfGAP) domain, and AgGts1(UPG), containing the C-terminal Ubiquitin associated- (UBA), Proline-rich (PRO) and Glutamine-rich (GLU) domains.

The AgGTS1(ArfGAP) and AgGTS1(UPG) sequences were amplified from A. gossypii genomic DNA by standard PCR conditions (Sambrook and Russel, 2001): 95 °C initial denaturation for 2 min, followed by 35 cycles of 30 sec denaturation at 95 °C, 30 sec annealing at 52 °C, 2 min elongation at 72 °C and 5 min final elongation at 72 °C. Primers were designed to add 5’XhoI and 3’XbaI sites to the amplicons. While the AgGTS1(ArfGAP) sequence contained the native AgGTS1 start codon, an ATG start codon was added directly in front of the AgGTS1(UPG) sequence. The AgGTS1 truncations were cloned to pRS418-AgTEFp-LacZ through XbaI/XhoI

Chapter 2: Methods

restriction sites, enabling the use of the AgTEF promoter for expression regulation (Sambrook and Russel, 2001). Correct insert was verified by sequencing of the resultant plasmids. All sequencing reactions were performed by LCG Genomics, Germany.

2.3.4 Construction of E. cymbalariae centromere plasmids pFA-KANMX6 was used as vector backbone for construction of a low copy-number plasmids with E. cymbalariae CEN/ARS. Centromeres from chromosome I and V, EcCEN1 and EcCEN5, were amplified by standard condition PCR: 94 °C initial denaturation for 2 min, 35 cycles of 1 min denaturation at 93 °C, 1 min annealing at 52 °C, 2 min elongation at 72 °C and a final 5 min elongation at 72 °C. Using SpeI and SacI restriction sites added to the amplicons by the forward and reverse primers, the centromere sequences were cloned into pFA-KANMX6 (Sambrook and Russel, 2001). The resultant plasmid was sequenced by LCG Genomics, Germany.

2.4 Transformation Transformation of A. gossypii and E. cymbalariae was performed by electroporation according to the PCR-based gene targeting protocol (Wendland et al., 2000). After transformation the samples were incubated on AFM agar plates for 6 hours at 30 °C after which the plates were covered by 7 ml 0.5 % agarose with G418/Geneticin for a final antibiotic concentration of 200 mg/ml. Recombinant heterokaryotic colonies were isolated after 2-3 days incubation at 30 °C. IN order to isolate single homokaryotic mycelia, spores obtained from heterokaryotic mycelia were germinated overnight under selective conditions at room temperature. The next day, growing germlings were micomainpulated using a MSM Micromanipulator (Singer Instruments). Two separate isolates were obtained for each gene deletion.

Transformation of S. cerevisiae BY4741 was performed according to the LiAc/ss carrier DNA method (Gietz and Woods, 2002). The samples were plated on YPD agar plates with 50µg/ml ClonNat, and incubated at 30°C for 3-4 days.

Transformation of E. coli was achieved by electroporation (Dower et al., 1988). After 1 h incubation at 37 °C, the transformants were plated on 2YT agar plates with appropriate antibiotic

Chapter 2: Methods

selection. For blue-white screening of lacZ disruption by clonal insert (Vieira and Meesing, 1982), X-gal to a final concentration of 50 µg/ml was added to the growth medium. Transformants were selected after overnight incubation at 37 °C.

2.5 Cytological staining and microscopy A. gossypii and E. cymbalariae mycelial morphology was analyzed using confocal microscopy. For differential interface contrast (DIC) and GFP imaging, a small mycelial inoculum from an overnight liquid culture was grown in 10 ml AFM with appropriate selection at 30 °C for 4 h. Microscopy was performed using an Axio Imager M1 microscope (Zeiss, Germany) and mycelial images were taken using a MicroMax1024 CCD camera (Princeton Instruments, USA). Microphotographs were edited using Metamorph 7 software (Molecular Devices LLC, USA). Fluorescence microscopy was performed using the appropriate filter settings for GFP fluorescence (excitation 470±20 nm, emission 525 ±25 nm), DAPI (excitation 350 ±25 nm, emission 460 ±25 nm) and RED (excitation 545 ±15 nm, emission 620 ±30 nm).

A. gossypii septal sites were stained using CFW (Sigma Aldrich) (Pringle et al., 1989). The stain was added to A. gossypii liquid cultures to a final concentration of 40µg/ml. After incubation at room temperature for 10 min, the sample was observed with the microscope using the DAPI filter set. Actin was stained using Rhodamin-Phalloidin (Molecular Probes) on A. gossypii mycelia fixed in PBS with 3.7 % formaldehyde (Oberholzer et al., 2002). The samples were observed with microscope using the GFP filter set. To visualize endosomes and vacuoles in A. gossypii, 3- 4 h old mycelial cultures were harvested, diluted in 200 µl AFM with 2 µM FM4-64 (Molecular Probes) and incubated at room temperature for 15-120 min. Internalization of the FM4-64 dye was observed by microscopy using the RED filter set (Fischer-Parton et al., 2000). nnnnnnnnnnnnnnnnn

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

Chapter 3: Functional analysis of the A. gossypii Arf3- Yel1-Gts1 module

3.1 Results

3.1.1 Generation of A. gossypii arf3 and yel1 mutants S. cerevisiae GTS1 was initially characterized as a clock-gene, since deletion mutants have few phenotypes except an oscillation phenotype regarding timing of budding (Mitsui et al., 1994). As GTS1 has previously been deleted in A. gossypii we could compare S. cerevisiae and A. gossypii gts1 deletion strains and in contrast to S. cerevisiae, a strong growth phenotype was immediately obvious in A. gossypii gts1 strains. As Gts1 is an Arf3-GAP regulatory protein, we wanted to generate deletion strains of the AgArf3 small GTPase and the Arf3-GEF AgYel1, which would enable characterization of the whole A. gossypii Arf3 small GTPase module.

Agarf3 and Agyel1 deletion mutants were generated using PCR-based gene targeting, during which the target ORF was replaced with a G418/Geneticin resistance marker, GEN3 (Wendland et al., 2000). Primer pairs #6195-#6196 and #6201-#6202, corresponding to A. gossypii genes ACL078W (ARF3) and ABR218C (YEL1) were designed to amplify the GEN3 cassette (Figure 12) (Sambrook and Russel, 2001), and homocaryotic Agarf3 and Agyel1 mutants were verified by PCR (Figure 13).

Figure 12. A. gossypii gene deletion by PCR-based gene targeting. Deletion- cassettes containing a resistance marker and flanking homology region to the target ORF is amplified using S1-21 primers. The deletion cassettes integrate in the genome by homologous recombination, replacing the target ORF with the resistance marker. Correct integration of the deletion cassette is verified by PCR up- and downstream of resistance marker using G1-G2 and G3-G4 primers. Complete deletion of the target gene is verified by amplification of a target OFR specific sequence, using I1-I2 primers.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

Figure 13. Agarose gel electrophoresis of PCR verification fragments. AgARF3 (upper panel) and AgYEL1 (lower panel) deletion were verified by amplifying the GEN3 flanking regions using G1-G2 and G3-G4 primers (see methods). Loss of the target ORF was verified by amplification of an ORF-specific sequence using I1-I2 primers.

Figure 14. Agarose gel electrophoresis of pAG17522(ARF3) (upper panel) and pAG19275(GTS1) (lower panel) internal fragments. Complementation of A. gossypii arf3 and gts1 phenotypes was verified by PCR using ORF-specific internal primers.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

No phenotypes could be observed in Agyel1 but phenotypes observed in the Aggts1 and Agarf3 strains (see section 3.1.2) could be complemented by re-introduction of the deleted ORF using pAG plasmids pAG19275(GTS1) and pAG17522(ARF3). In addition to the AgGTS1 ORF, pAG19275 contains the partial sequences of AgMND1 and AgATG1. pAG17522 contains the full length AgRKI1 ORF as well as the partial sequences of AgRPS7A/B and ACL079C. Re- introduction of the ORFs was verified by PCR amplification of the ORF-specific internal sequence (Figure 14).

3.1.2 Growth phenotypes and mycelial morphology As the obvious growth retardation of the Aggts1 mutants was one of the reasons for our interest in the Arf3 small GTPase complex, the full scale of Aggts1 growth retardation was carefully documented. Along with the Agarf3 and Agyel1 mutants, Aggts1 growth were investigated by growth- and temperature assays, where a small mycelial inoculum was allowed to grow on AFM agar plates for seven days at 20, 30 and 37 °C. While the Agarf3 and Agyel1 mutants show wt- like growth during all tested conditions, the Aggts1 strains display severe growth retardation with successively decread growth at 20, 30 and 37 °C (Figure 15). The decreased growth phenotype of the Aggts1 mutant was completely rescued by introduction of the AgGTS1 ORF in pAG19275(GTS1) (see section 3.1.5, Figure 24).

Figure 15. Colony growth of A. gossypii arf3, yel1 and gts1. Colony growth after 7 days on AFM media, 20-37°C, represented as median growth and percentage of wt growth. Standard deviation error bars are calculated for values with 5 or more replicates.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

By measuring Aggts1 radial growth continuously over 7 days, it was concluded that the retarded growth phenotype of Aggts1 is most likely due to a generally slower growth and not because of a log-phase at any time during growth (Figure. 16) The slow growth rate of Aggts1 mutants has not been seen in S. cerevisiae, which maintain wt-like growth at 30 °C (Mitsui et al., 1994).

Figure 16. A. gossypii wt and gts1 colony growth on full media, 30 °C, 3-7 days after inoculation.

DIC imaging of the Aggts1 mutant mycelia show an abnormal hyphal morphology (Figure 17). While wt A. gossypii has smooth hyphae with dichotomous branching in mature mycelia, Aggts1 mutants are irregularly shaped with indications of sub-apical hyphal swelling. Lateral branching is observed in an irregular pattern and although dichotomous tip branching is most prevalent; tip branching into 3 or 4 new hyphae is readily observed. Aggts1 also exhibits a sporulation deficiency which were complemented by re-introduction of the AgGTS1 ORF. Standard condition sporulation in liquid media generated no detectable spores, and spores from solid media were extremely scarce and of an abnormal morphology (Figure 17).

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

FIgure 17. DIC imaging of wt and Aggts1 hyphal- and spore morphology. Arrows indicates Aggts1 sub- apical hyphal swelling and multiple hyphal tip branching. Scale bar for hyphal images is 25 µm and for spore images 10 µm.

3.1.3 Actin and chitin localization The actin cytoskeleton is tightly coupled to the maintenance of polar growth and endocytosis. In growing hyphae the actin filaments accumulate at the site of growth, the Spitzenkörper. Additionally, actin patches occur at a high frequency at an area just below the site of growth, an area associated with endocytic recycling of proteins associated with polar growth (Shaw et al., 2011, Sudbery, 2011). ScArf3 has previously been implicated to be in is involved in development of polarity (Huang et al., 2003) and the Agwal1 and Agsac6 mutants, which have similar growth retardation phenotypes as Aggts1, have sub-apical actin localization phenotypes (Walther and Wendland, 2004a, Jorde et al., 2011). Thus, we analyzed the actin localization in the deletion mutants of the A. gossypii Arf3 small GTPase complex by Rhodamin-Phalloidin actin staining. In wt A. gossypii as well as the Agarf3 and Agyel1 deletion strains, actin filaments and patches

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

cluster at the growing hyphal tip. Actin patches can also be seen evenly distributed throughout the hyphae. The Aggts1 actin cytoskeleton is not polarized at the hyphal tips, but like Agwal1 and Agsac6 (Walther and Wendland, 2004a, Jorde et al., 2011), at an area in the sub-apical hyphae. There is also a clear decrease of actin patches distributed in hyphae of the Aggts1 deletion mutnt (Figure 18).

Figure 18. Actin localization in A. gossypii mutant hyphae. Agyel1 and Agarf3 have wt-like actin localization at the hyphal tip. The dot-like structures in the hyphae represent actin patches, i.e. former sites of endocytosis. Aggts1 have a sub-apical actin localization phenotype (hyphal tip is marked by (*) and a decreased amount of actin patches in the hyphae. Scale bar is 10 μm

Deletion of AgWAL1 causes a septation defect in A. gossypii (Walther and Wendland, 2004a). Due to the accumulation of chitin in mature A. gossypii septa, any septation phenotypes can be easily visualized by chitin staining using the fluorescent dye CFW. Due to the similar growth-, sporulation and actin localization phenotypes between Agwal1 and Aggts1, we investigated the ability of Agarf3, Agyel1 and Aggts1 to form mature chitin-rich septa. While chitin accumulates at septal sites in all three mutants, there is a difference in the septal distribution throughout the hyphae between the A. gossypii wt and Aggts1. Compared to wt septation, Aggts1 show an

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

irregular pattern of septation, and furthermore, an increased chitin content in the cell wall at sites of sub-apical swelling (Figure 19).

Figure 19. Accumulation of chitin at septal sites in A. gossypii mutant hyphae. While Aggts1 has chitin-rich septa, the septal sites are irregularly dispersed throughout the hyphae. Increased flourescence of the hyphal cell wall visualizes the sub-apical hyphal swellings observed in Aggts1. Scale bar is 10 μm.

3.1.4 Endosome visualization using FM4-64 While mammalian Arf6 has a clear role in uncoating of clathrin-coated endocytic vesicles (Toret et al., 2008), the role or the S. cerevisiae homologue Arf3 and its GAP and GEF regulators are less clear. Although not essential, ScArf3 is implicated in endocytosis through a yet unknown mechanism (Huang et al., 2003). In order to study whether deletion of the AgArf3 small GTPase module has any effects on endocytosis, the deletion strains were stained with the lipophilic stain FM4-64, and endocytic uptake of the dye enabled tracing of both early endosomes and vacuoles at time points 15, 60 and 120 min after addition of the dye (Figure 20).

In A. gossypii wt, endosomes are rapidly visualized by FM4-64. Stained endosomes could also be detected in Agyel1, and to a lesser extent Agarf3 within 15 min after addition of the dye. Endosome progression from endocytosis and early endosome to vacuolar fusion occurs similar to wt in Agyel1. Although the FM4-64 uptake in Agarf3 is slower than wt, the progression of the

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

Figure 20. Endocytic uptake of the lipophilic dye FM4-64 in Agarf3, Agyel1 and Aggts1. Hyphae were grown in AFM and pictures were acquired using fluorescence microscopy at time points 15, 60 and 120 min after addition of the dye. Uptake of the dye is delayed in various degrees in the Agarf3 and Aggts1, furthermore Agarf3 appear to have an endosome-vacuolar fusion phenotype. Scale bar is 10 µm.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

endosome through the hyphae appears to be wt-like until the point of vacuolar fusion. 60 to 120 min after addition of FM4-64, the dye stains the vacuoles of the wt hyphae. However, Agarf3 appear to have a endosome-vacuolar fusion phenotype, the endosomes cluster together in the older hyphae, but does not appear to fuse in order to form vacuoles. The endosome fusion phenotype is rescued by re-introduction of the AgARF3 ORF in pAG17522(ARF3) (Figure 21).

Figure 21. Complementation of the Agarf3 endosome fusion phenotype by pAG17522(ARF3). The dye can be detected in the vacuoles of A. gossypii wt and pAG17522(ARF3) complementation strain 60 min after addition of the FM4-64 dye. In Agarf3, endosomes cluster without fusing to form a vacuole.

FM4-64 staining of Aggts1 shows a delay in internalization of the dye at the hyphal tip compared to the A. gossypii wt, and early endosomes are not detectable until 60 min after addition of the dye. A slower rate of dye internalization is also indicated by the rate of which the FM4-64 dye reached the older hyphae. Vacuoles of the Aggts1 mutant are visualized after 120 min, while the dye is detectable in the older wt hyphae already after 15 min, and is clearly visible after 60 min.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

3.1.5 Truncation of AgGts1 Due to the many, and severe phenotypes of Aggts1, most of which could not be observed in the corresponding S. cerevisiae deletion strains, we wanted to investigate whether AgGts1 might have any functions additional to the Arf3-GAP activity. Using PROSITE (Sigrist et al., 2012), four AgGts1 domains were identified and used for two truncated versions of AgGts1 (Figure 22). Primers #6338-#6339 and #6340-#6341 were designed to amplify the truncations, which were cloned to shuttle vector pRS418-AgTEFp-LacZ. Expression of the AgGTS1(ArfGAP) truncation in the Aggts1 background was verified by XbaI/XhoI restriction enzyme digest of the rescued plasmid. The AgGTS1(UPG) truncation was verified by PCR amplification of an internal fragment (Figure 23).

Figure 22. The domains of A. gossypii Gts1 as identified by PROSITE: the Arf GTPase activating protein (ArfGAP) domain, the ubiquitinin associated (UBA) domain, the proline-rich (PRO) domain and the glutamine-rich (GLN) domain. Two truncated versions of the Gts1 protein were created, ArfGAP (a a 1- 174) and UPG (a a 166-471).

As stated before, colonies of the Aggts1 mutants show a significantly slower growth than A. gossypii wt, with a correlation between growth retardation and increasing temperature. The growth retardation observed in Aggts1 is complemented by introduction of the full length AgGTS1 sequence. AgTEFp overexpression of the AgGTS1(ArfGAP) sequence complements the Aggts1 mutant phenotype up to 80 % at low- to moderate temperatures (20-30 °C), but only to 35 % at 37 °C. The AgTEFp regulated AgGTS1(UPG) truncation is insufficient for complementation of the Aggts1 growth retardation and only rescues 35% of the wt growth speed (Figure 24).

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

Figure 23. Verification of AgGTS1(ArfGAP) and AgGTS1(UPG) in the Aggts1 background. AgGTS1(UPG) is verified by PCR and AgGTS1(ArfGAP) is verified by XbaI/XhoI restriction enzyme digest. To the left are the general plasmid map used for transformation of Aggts1.

Figure 24. Colony growth of A. gossypii gts1 deletion and complementation strains. Aggts1 is transformed using plasmids containing the full length AgGTS1 ORF, and two AgGTS1 truncations: AgGTS1(ArfGAP) and AgGTS1(UBA). Colony growth is measured after 7 days on AFM media, 20-37°C, and represented as median growth and percentage of wt growth.Standard deviation error bars are calculated for values with 5 or more replicates.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

A clear phenotype of Aggts1 is the sub-apical actin localization similar to those of Agwal1 and Agsac6 (Walther and Wendland, 2004a, Jorde et al., 2011). Fluorescence microscopy of Rhodamin-Phalloidin stained Aggts1 complemented with the full length AgGTS1 ORF, AgGTS1(ArfGAP) and AgGTS1(UBA) indicate that the actin miss-localization phenotype is complemented by all three versions of the Gts1 protein (Figure 25).

Figure 25. Rhodamin-Phalloidin staining of AgGTS1 deletion, truncation and complementation strains. The hyphal tip of gts1 is marked by (*). Scale bar is 10 µm.

In S. cerevisiae, the Arf3GAP activity of Gts1 depends on the highly conserved, N-terminal ArfGAP domain (Cukierman et al., 1995). In order to investigate whether the ArfGAP domain is essential for the Arf3-GAP function of AgGts1, we analyzed the ability of the truncated AgGTS1(ArfGAP) to complement the Aggts1 slow endocytosis phenotype. Endosome staining using FM4-64 indicate that the slow endocytic rate of Aggts1 is restored to wt-like when complemented with a plasmid expressing the AgGTS1(ArfGAP) sequence (Figure 26). Wt-like endocytic rate was also restored by introduction of the complete AgGTS1 ORF. In contrast, the AgGTS1(UBA) truncation, which lacks the catalytic ArfGAP domain responsible for regulation of AgArf3 activity, is unable to restore the Aggts1 endocytic rate.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

Figure 26. Visualization of endocytosis by FM4-64. Hyphal tip endosomes are visible after 15 min in Aggts1 strains complemented by the full length AgGTS1 ORF and the truncated AgGTS1(ArfGAP). AgGTS1(UBA) does not complement the Aggts1 slow endocytosis phenotype. Scale bar is 10 µm.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

3.1.6 AgGts1-GFP localization Proteins of the Ras superfamily are generally conserved, and many of their GEF and GAP regulators show a moderate substrate unspecificity in vitro. Even so, in vivo, every small GTPase have specific regulators, thus, it is generally thought that GEF and GAP specificity depends on subcellular co-localization with the target substrate (Takai et al., 2001). In order to study the localization of AgGts1, we constructed an AgGts1-GFP fusion protein by integrating a MoGFP- NAT1 cassette downstream of the AgGTS1 locus in pAG19275(AgGTS1) through homologous recombination in S. cerevisiae.

The MoGFP-NAT1 cassette was amplified by primers #6180-#6181, designed to add 40 bp homology regions to pAG19275(AgGTS1). Integration of the GFP cassette into the pAG plasmid enables expression of the GTS1-GFP be regulated by the endogenous GTS1 promoter (Figure 27). The GFP-fusion construct was introduced in the Aggts1 background, and in addition to compementing the Aggts1 growth phenotype, the mutant was verified by restriction enzyme digest of the rescued plasmid (Figure 28). Florescence microscopy of A. gossypii hyphae expressing the AgGts1-GFP construct show pattern of GFP signaling along the cell membrane, with distinct increase in florescence at actin patches, hyphal tips and septal sites (Figure 29).

Figure 27. Generation of pAG19275(AgGTS1-GFP) by homologous recombination of MoGFP-NAT1 to pAG19275(AgGTS1), using short flanking regions homologous to the pAG plasmid. The final construct, pAG19275(AgGTS1-GFP), enables expression of the AgGTS1-GFP fusion protein under control of the endogenous AgGTS1 promoter.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

Figure 28. Verification of Aggts1 pAG19275(AgGTS1-GFP) by NcoI restriction enzyme digest of rescued plasmids.

Figure 29. AgGts1 localization in A. gossypii hyphae. Localization of C-terminal AgGts1-GFP in a growing hypha with a well-defined septal site. Photographs show DIC images of growing hyphae, the AgGts1-GFP signal (green), and DIC/GFP as well as bright field (Red)/GFP overlay. Bright field image is not shown. Scale bar is 10 µm.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

3.2 Discussion Conserved small GTPases of the Ras superfamily have been shown to be key regulators of many stages during hyphal growth, an important virulence factor of many pathogenic fungi. One of the more extensively studied small GTPases is the mammalian Arf6, which regulates different stages of vesicle trafficking and actin cytoskeleton organization at the plasma membrane, and is associated with polar cells such as neurons, phagocytic cells and tumor cells. S. cerevisiae Arf3 has previously been established as the homolog of the extensively studied mammalian Arf6, and is also involved in endocytosis and actin cytoskeleton rearrangements. However, unlike mammalian Arf6, a link between ScArf3 and polarity has not been established.

In this series of experiments we studied the A. gossypii homologs of the S. cerevisiae Arf3 module and the mammalian Arf6, in an effort to evaluate the impact of the Arf3 module on the endocytosis and hyphal polarity. As A. gossypii is a close relative to S. cerevisiae, the Arf3 module is assumed to function in an equivalent manner, while the strictly filamentous nature of A. gossypii enables characterization of the Arf3 module on polar cells.

3.2.1 The A. gossypii Arf3-Yel1-Arf3 module The S. cerevisiae Arf3, like its mammalian homolog, is thought to modulate the local plasma membrane content of PI(4,5)P2 through activation of phosphoinositide kinases, e.g. S. cerevisiae Mss4 (Smaczynska-de Rooij et al., 2008, Desrivières et al., 1998, Honda et al., 1999). As there is an accumulation of evidence suggesting that increasing plasma membrane concentrations of the lipid PI(4,5)P2 have a regulatory role in both endocytic vesicle generation and uncoating, changes in plasma membrane composition might render an explanation to the phenotypes observed in A. gossypii arf3 and gts1mutants.

Although the effect of A. gossypii Arf3 on PI(4,5)P2 pools were not investigated in this project due to time limitations, the equivalent S. cerevisiae model might explain the phenotypes observed in A. gossypii arf3. In S. cerevisiae, Arf3-mediated increase of the membrane PI(4,5)P2 content facilitates endocytosis as PI(4,5)P2 acts as an anchor for many endocytic proteins through their

PI(4,5)P2-binding domains (Smaczynska-de Rooij et al., 2008). Supporting the link between

Arf3, PI(4,5)P2 and endocytosis proposed for S. cerevisiae, we can demonstrate that while A. 50

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

gossypii arf3 deletion strains are viable, FM4-64 uptake at the hyphal tip occurs at a lower extent compared to A. gossypii wt hyphae (Figure 20-21), indicating a slower vesicle formation. In addition, the effect of Arf3 on PI(4,5)P2 concentrations can also be linked to the observed Agarf3 inability of endosome-vacuole fusion. Vesicle coat disassembly is thought to be crucial for membrane fusion, and endosome fusion phenotypes similar to Agarf3 has previously been observed in mutant strains deficient in uncoating (Pishvaee et al., 2000, Harris et al., 2000). While several mechanisms cooperate in the regulation of vesicle uncoating, one important factor is the dephosphorylation of PI(4,5)P2 to phosphoinositole by synaptojanin, which stimulating endosome coat disassembly (Sun et al., 2007, Toret et al., 2008, Chung et al., 1997). Assuming that Agarf3, like the Scarf3 mutants, lacks the increased PI(4,5)P2 concentration associated with vesicle formation, synaptojanin-mediated PI(4,5)P2 dephosphporylation is drastically reduced. While other mechanisms triggering coat disassembly might still exist, this could reduce the rate of uncoating and give rise to the observed endosome fusion phenotype (Toret et al., 2008, Brown et al., 2001).

While 15 GEFs has been identified for the mammalian Arf6, YEL1 encodes the only identified Arf3 GEF in both S. cerevisiae and A. gossypii. Through the catalytic Sec7 domain, Yel1 promotes Arf3 activation by accelerating GDP- to GTP-exchange (Casanova, 2007, Gillingham and Munro, 2007a, Gillingham and Munro, 2007b), and deletion of YEL1 severely delays Arf3 re-activation after GTP hydrolysis (Gillingham and Munro, 2007a). A complete abolishment of AgArf3 activity in Agyel1 deletion strains would render phenotypes similar to the ones observed in Agarf3, however, we could not identify any non-wt Agyel1 phenotypes suggesting an AgYel1- independent AgArf3 activity. This could be due to one or more yet unidentified AgArf3 GEFs which may possess overlapping functions with AgYel1, or it may be caused by the initial activity of newly synthesized AgArf3 proteins, which could associate with GTP and remain active until inactivated by AgGts1 GAP activity (Zurita et al., 2010). Previous studies of both mammalian and fungal Arf3 has shown that while inactive Arf3 is cytosolic, active Arf3 interacts with the plasma membrae through a myrisoylated N-terminal α-helix (D’Souza-Schorey and Chavrier, 2006). Thus, any AgArf3 activity in Agyel1 deletion strains might be determined by examining the localization of an AgArf3-GFP construct.

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

In contrast to Agyel1 and Agarf3 deletion strains, deletion of AgGTS1 renders an overactive AgArf3. As AgGts1 is the only known AgArf3 GAP based on homology to S. cerevisiae, deletion strains is severely crippled in GTP hydrolysis, which efficiently renders the AgArf3 small GTPase unable to “shut off” (Gideon et al., 1992, Cukierman et al., 1995, Yaguchi et al., 2007). Deletion of AgGTS1 causes a drastic phenotype in hyphal growth and morphology (Figure 15- 17), actin assembly (Figure 18) and endocytosis (Figure 20). However, due to the nature of AgGts1 as an AgArf3 GAP regulator, the severe phenotypic effects observed in Aggts1 deletion strains could be caused either directly by the loss of AgGTS1, or indirectly by the overactive AgArf3. In an effort to establish the causative factor for the Aggts1 phenotypes, the possible roles for AgGts1 are discussed further below.

3.2.2 AgGTS1-GFP localizes at sites of growth and septation GFP-tagged proteins are commonly used for studying cellular processes and in situ protein localization in various species (Shimomura et al., 1962). The localization pattern of the AgGTS1- GFP fusion protein (Figure 29) is consistent with the phenotypes observed in Aggts1 deletion strains. Localization at actin patches is normally associated with proteins of the endocytic complex and coat proteins, suggesting that AgGts1, like ScGts1 might be directly involved in the formation of the endocytic vesicle (Yaguchi et al., 2007). Co-localization with the Spitzenkörper is usually assigned to proteins involved in polarity establishment and polar growth, including organization of the actin cytoskeleton (Araujo-Palomares et al., 2009, Delgado-Alvarez et al., 2010). The link between AgGts1 and actin is further supported by the association of AgGts1 to sites of septation, an event that is tightly linked to the organization of the actin cytoskeleton in constricting actomyosin rings (Walther and Wendland, 2003, Chang et al., 1996).

3.2.3 Unregulated AgArf3 causes impaired endocytosis and morphological abnormalities As discussed above, the phenotypic effects observed in Aggts1 deletion strains could be caused by the inability of AgArf3 to transition from the active GTP-bound state to the inactive GDP- bound state. Similar morphological-, polarity- and endocytic phenotypes as observed in Aggts1 have been described for a constitutively active mammalian Arf6 in several independent studies

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

(Albertinazzi et al., 2003, Brown et al., 2001, D’Souza-Schorey and Chavrier, 2006, Paleotti et al., 2005), and a role for S. cerevisiae Arf3 in polarity and endocytosis has been suggested as well (Huang et al., 2003, Tsai et al., 2008, Smaczynska-de Rooij et al., 2008). The truncated AgGTS1(ArfGAP), rendering an artificial AgGts1 protein, lacking the proposed UBA, PRO and GLN domains were used to analyze whether the phenotypes observed in Aggts1 were caused by the unregulated AgArf3. Since the known function of the remaining ArfGAP domain is to act as a catalyst of GTP hydrolysis (Scheffzek et al., 1996), it enables proper AgArf3 regulation. The ability of AgGTS1(ArfGAP) to complement several of the Aggts1 phenotypes show the importance of the ArfGAP domain in the function of AgGts1, further indicating that the observed phenotypes could be due to inability of AgArf3 regulation, rather than a direct consequence of the Aggts1 deletion.

The morphological and endocytic effects of unregulated AgArf3 activity give clues to the protein mechanism of action. While many functions has been suggested for the mammalian and fungal homologs of AgArf3 (Costa et al., 2005, Huang et al., 2003, Lambert et al., 2007, Brown et al., 2001, Radhakrishna et al., 1999), substantial evidence for Arf3-mediated accumulation of plasma membrane PI(4,5)P2 has been shown in both mammals and other fungal species (Honda et al., 1999, Brown et al., 2001, Smaczynska-de Rooij et al., 2008). Mammalian Arf6 overexpression studies have shown that unregulated Arf6 activity causes the formation of vacuoles containing plasma membrane components, efficiently removing endocytic factors like PI(4,5)P2 from the plasma membrane (Aikawa and Martin, 2005, Brown et al., 2001, Aikawa and Martin, 2003). As

PI(4,5)P2 is an essential component for endocytic vesicle formation, the unregulated Arf6 activity causes a secondary inhibitory effect on endocytosis (Smaczynska-de Rooij et al., 2008, Brown et al., 2001), as observed in the delayed internalization of FM4-64 by Aggts1.

Inhibition of endocytosis could render a possible explanation to the abnormal hyphal morphology and growth rate observed in Aggts1. Hyphal growth is mediated at the apical Spitzenkörper structure, through the careful coordination of exo- and endocytosis (Grove and Bracker, 1970, Girbardt, 1957). While exocytosis functions to maintain hyphal polarity and growth by addition of membrane compounds at the hyphal apex, endocytosis regulates the distribution of the same compounds from the area below the Spitzenkörper. Defective endocytosis caused by the overactive AgArf3 could potentially redistribute polarizing proteins normally concentrated at the

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

apex to the sub-apical hyphae, which would give rise to the morphological abnormalities and reduced growth rate seen in Aggts1 (Taheri-Talesh et al., 2008, Atkinson et al., 2002, Shaw et al., 2011).

3.2.4 AgARF3-dependent and independent apical actin localization A clear phenotype of Aggts1 is the localization of the actin cytoskeleton not at the hyphal apex but at an area in the sub-apical hyphae (Figure 18). The finding that both the AgArf3-regulating AgGTS1(ArfGAP)- and the AgGTS1(UPG), which lacks the ArfGAP domain responsible for AgArf3 regulation, rescues the actin localization phenotype gives rise to several possible roles for AgGts1 in regulation of the actin cytoskeleton dynamics.

As discussed previously, the notion that PI(4,5)P2 regulates several aspects in the actin cytoskeleton dynamics in S. cerevisiae suggests a causative role for the unregulated AgArf3 in the

Aggts1 sub-apical actin localization. In mammals, high plasma membrane content of PI(4,5)P2 stimulates actin polymerization by activating WASP family NPFs and the Arp2/3 complex

(Rohatgi et al., 1999, Miki et al., 1996). PI(4,5)P2 further effects actin cytoskeleton dynamics by stimulating uncapping of actin filaments, enabling further actin polymerization (Kim et al., 2007), and inhibiting the activity of actin-depolymerizing proteins (Janmey et al., 1987).

Unregulated AgArf3 could mediate a constant PI(4,5)P2-dependent actin polarization at the area of increased endocytosis below the apical Spitzenkörper. An additional consequence of the increased sub-apical actin accumulation would then be the depletion of free actin monomers from the cytoplasm, efficiently rendering actin polymerization at the hyphal apex impossible.

However, the finding that the sub-apical actin localization is rescued by introduction of the AgGTS1(UPG) construct, lacking the AgArf3-regulatory ArfGAP domain, suggest that AgGts1 has an additional role in the dynamics of the actin cytoskeleton. The mechanism of which AgGts1 might aid in the actin assembly is largely unknown, although a link between Gts1, Arf3 and the endocytic coat protein Lsb5 has been identified in S. cerevisiae (Toret et al., 2008, Costa and Ayscough, 2005). To further suggest a role for AgGts1 in actin organization, the sub-apical actin localization in Aggts1 is similar to those observed in Agsac6 and Agwal1 deletion strains (Jorde et al., 2011, Walther and Wendland, 2004a). As SAC6 encodes the only actin-bundling fimbrin

Chapter 3: Functional analysis of the A.gossypii Arf3-Yel1-Gts1 module

identified in A. gossypii (Adams et al., 1989), and AgWAL1 encodes a NPF homologous to the mammalian WASP (Walther and Wendland, 2004a), they are both directly implicated in the organization of the actin cytoskeleton, and like Aggts1, they both have endocytic deficiencies and show signs of abnormal polar establishment (Jorde et al., 2011, Walther and Wendland, 2004a). Although the relationship between AgGts1, AgSac6 and AgWal1 is unclear, it does highlight the link between endocytosis, polarity establishment and the dynamics of the actin cytoskeleton and shows the complexity of continuous filamentous growth.

Chapter 4: Eremothecium pathogenicity assays

4.1 Results Although A. gossypii is known for causing stigmatomycosis in cotton and dry rot in citrus fruit, the exact mechanism of infection is not known (Wendland and Walther, 2005a). A. gossypii has already been used as a model organism for filamentous growth, and development of pathogenicity assays for the Eremothecium genus might increase the applicability of A. gossypii to other pathogenic fungi. Here we use pathogenicity assays developed for the pathogenic fungi F. oxysporum to analyze the pattern of pathogenicity in the Eremothecium genus, were A. gossypii and E. cymbalariae are considered representative species.

4.1.1 Cellophane penetration assay Physical barriers are the main protection against pathogens in both plant and animal tissue and the penetration of a physical barrier is often a key event during infection. To explore the ability of A. gossypii and E. cymbalariae to penetrate a physical barrier by sheer force, the cellophane penetration assay is set up to form a barrier between the fungus and the nutrient-rich media. While the positive control, F. oxysporum, penetrates the cellophane within 2 days of growth, and regardless of media condition, neither A. gossypii or E. cymbalariae hyphae penetrates the mycelia during the 6 days trial (Figure 30).

Figure 30. Cellophane penetration by A. gossypii, E. cymbalariae and F. oxysporum when grown on full media. Upper row: Photos of fungal colonies before removal of the cellophane. Bottom row: mycelial regrowth after removal of the cellophane sheet. F. oxysporum was incubated 4 days while A. gossypii and E. cymbalariae 6 days before removal of the cellophane sheet.

Chapter 4: Eremothecium pathogenicity assays

4.1.2 Fruit pathogenicity assay The fruit pathogenicity assay was constructed to evaluate the ability of A. gossypii to grow on the surface of-, and invade fruit tissue. After 7 days at 30 °C and 100 % humidity, the positive control F. oxysporum has invaded and caused rotting of the apple tissue as well as the orange exo-, meso- and endocarp (Figure 31). Microscopic analysis of the fruit tissue confirms that the mycelia have completely invaded the fruit tissue and mycelia can be identified in tissue samples opposite of the site of infection. Microscopic analysis shows that even though E. cymbalariae spores were present in both the apple and fruit assays, the spores were unable to germinate in the fruit tissue and growing mycelia could not be identified in any assay. While the A. gossypii wt and leu2 spores were unable to germinate in the apple assay, hyphal filaments could be found in the orange endocarp assay. However, microscopic analysis of the orange tissue suggests that the colonies are superficial at the sites of infection, and the mycelia do not penetrate the orange tissue.

Figure 31. Invasive growth of apple and orange tissue. A. gossypii wt, Agleu2 and F. oxysporum wt spore suspensions were inoculated at the exposed tissue surface. The pictures are taken after 7 days incubation at 30 °C, 100 % humidity.

Chapter 4: Eremothecium pathogenicity assays

4.2.3 Galleria mellonella killing assay G. mellonella larvae were chosen as an animal model for Eremothecium fungal infections, and sickness could easily be scored by daily control of larval melanization. As melanization of the normally pale yellow larvae is a clear indicator of activation of the innate immune system, melanized larvae were scored as sick or, if non-responsive to physical stimuli -dead (Burgwyn Fuchs et al., 2010). Injection of A. gossypii and E. cymbalariae spores into G. mellonella larvae induced a rapid melanization reaction of the hemocoel. Within 15 min, 40 % of the larvae injected with A. gossypii spores, and all 100 % of the larvae injected with E. cymbalariae spores showed a clear darkening of the hemocoel. The rapid, intense melanization response also occurs in all 100 % of the heat killed A. gossypii spores used as a negative control. In contrast, the control group with larvae injected with F. oxysporum microcornidia shows no signs of illness at this point.

Daily scoring of the fungal infection progress revealed that the F. oxysporum control kills 100 % of the larvae within 4 days of injection. Spores from A. gossypii and E. cymbalariae kill more than 50 % of the larvae in each assay over the course of 7 days (Figure 32). However, larvae injected with heat-killed A. gossypii spores have a 100 % mortality rate in just 2 days after injection. Furthermore, microscopical analysis of the larval hemocoel does not reveal any germinated pores of any Eremothecium fungi, which suggests that the G. mellonella larvae are not killed by an infection, but rather a physical effect of the spores.

Figure 32. Virulence of A. gossypii, E. cymbalariae and F. oxysporum on G. mellonella. Kaplan-Meier plots of G. mellonella survival after injection of spores to the larval hemocoel.

Chapter 4: Eremothecium pathogenicity assays

4.2 Discussion Fungal pathogens have great impact on human society, both through human infections and agricultural and economic loss. In order to fight fungal infections, and to find new targets for antifungal chemicals it is necessary to increase the understanding of the mechanisms of fungal growth and pathogenesis. Knowledge of A. gossypii mechanism of infection could greatly enhance the use of the fungus as a model organism for filamentous growth, thus, we aimed at developing assays for evaluating the ability of Eremothecium fungi to infect and cause disease in plant and animal tissue. While not taking into account any molecular mechanism for infection, we investigated whether Eremothecium fungi could penetrate a physical barrier, a key event during host infection, and proliferate and cause disease in fruit and animal tissue.

4.2.1 Characteristics of filamentous growth in Eremothecium fungi In order to infect host tissue, many pathogenic fungi have evolved mechanisms to penetrate host defense barriers. Either by degrading enzymes or sheer force, the fungal hyphae invades the host tissue where it can proliferate in a nutrient-rich environment (Dean et al., 2012). By exploring the capability of invasive growth in fungi of the Eremothecium genus, we hoped to gain insight in the mechanism of pathogenicity for A. gossypii.

Previous reports have shown that phytopathogenic fungi such as F. oxysporum are able to penetrate an artificial barrier such as a cellophane sheet and invade the underlying media, and deletion of genes encoding essential pathogenicity factors often reduces the ability of pathogenic fungi to invade cellophane covered agar (Pérez-Nadales and Di Pietro, 2011, Rispail and Di Pietro, 2009). In order to analyze the invasive growth of Eremothecium species, we systematically compared cellophane invasion of A. gossypii, E. cymbalariae and F. oxysporum. As shown previously, F. oxysporum was able to penetrate the cellophane and invade the underlying media within days of vegetative growth. In contrast, while the vegetative growth of A. gossypii and E. cymbalariae was wt-like, they were both unable to penetrate the cellophane.

A. gossypii has previously been reported as a cotton- and citrus fruit pathogen spread by insects of the Pyrrhocoridae family, although the mechanism of infection is not clear (Ashby and Novell, 1926, Dietrich et al., 2013). We can demonstrate that E. cymbalariae is non-pathogenic, and unable to germinate in neither apple- nor orange tissue. While not being able to germinate in

Chapter 4: Eremothecium pathogenicity assays

apple tissue, A. gossypii spores geminate in the nutrient rich orange endocarp, but do not invade the tissue. Additionally, despite the fact of being the cause of dry rot, and irregardless of growing in the endocarp, A. gossypii did not germinate- or grow on the surface of the citrus fruit. Due to the apparent inability of A. gossypii to invade host tissue, we hypothesize that the fungus is dependent on insects of the Pyrrhocoridae family not only as a vector for spreading, but also for the mechanical force necessary for gaining access to the sites of growth. The use of insect vectors for fungal plant infection has been noted previously (Medrano et al., 2009), and as Pyrrhocoridae insects are known pests feeding on both cotton seeds and various fruit, the A. gossypii spores might be delivered to the infection site through the insect feeding on the plant host (Kimura et al., 2008).

4.2.2 Eremothecium spores causes G. mellonella immune reaction Designing animal models for fungal pathogenicity there are ethical and logistical reasons for using invertebrates for initial infection models (Arvanitis et al., 2013). While several invertebrate animal models have been developed, we chose the larvae of the greater wax moth, G. mellonella for assaying Eremothecium animal infection. The relatively large size of G. mellonella larvae enabled injection of spores to the larval hemocoel without causing significant trauma to the larvae, although injection of Eremothecium spores cause a rapid melanization of the larvae. As the G. mellonella immune system is based on humoral phagocytic cells, which after ingestion of the pathogen forms melanin-rich aggregates in the hemocoel (Ratcliffe, 1985, Lionakis, 2011), the rapid melanization strongly suggest an intense immune reaction to the Eremothecium spores.

While the G. mellonella immune response to Eremothecium spores can be followed by scoring melanization and dead larvae (Burgwyn Fuchs et al., 2010, Navarro-Velasco et al., 2011), it is unlikely that the Eremothecium fungi are able to establish an infection. The observation that heat- killed A. gossypii spores also induces rapid melanization, and the fact that no germinated spores could be identified in the larval hemocoel, suggests that the larval melanization is caused by the humoral phagocytes reacting to the presence of spores and does not correspond to the progress of fungal infection. Thus, it is likely that under the tested conditions, A. gossypii and E. cymbalariae are non-virulent in the sense that they do not invade invertebrate host tissue.

Chapter 5: Establishing molecular tools in E. cymbalariae

5.1 Results

5.1.1 Establishing E. cymbalariae PCR based gene targeting In an effort to establish PCR-based gene targeting in E. cymbalariae, a close relative to A. gossypii, we sought to analyze whether 100 bp homology regions would suffice for correct resistance marker integration into the E. cymbalariae genome. As target genes, we chose ECYM7434 and ECYM5230. While ECYM5230 encodes a hypothetical protein with no identifiable homologs in A. gossypii or S. cerevisiae, the 2292 bp ECYM7435 ORF encoding a homolog to A. gossypii transcriptional regulator Tec1.

For PCR-based gene targeting, deletion cassettes with 100 bp homology regions were amplified using primers #6333-#6334 (EcTEC1) and #6329-#6330 (ECYM5230) and ~100ng of DNA were transformed into E. cymbalariae germlings in order to obtain two independent strains for each target gene. The number of transformant colonies were compared those generated using EcTEC1::KANMX disruption cassettes with a 10-fold increase in homologt region size (Table 3). No difference in correct KANMX integration could be observed when comparing gene deletion using 100 bp homology regions and ~1000 bp homology regions. Two independent homokaryotic strains were isolated for ecym5230, Ectec1 and EcTEC1::KANMX (Figure 34).

Table 3. Transformation of gene disruption constructs and gene deletion cassettes in E. cymbalariae. pRS417-AgTEFp-GFP was used as positive control. Transformants Sample Deletion method Homology region obtaied/analyzed/verified

ECYM5230 deletion cassette 100 bp 5/5/4

EcTEC1 deletion cassette 100 bp 9/5/5 TEC1::KanMX EcTEC1 0.93-1,36 kb 6/5/5 disruption cassette Positive control - - >100

Negative control - - 1

Chapter 5: Establishing molecular tools in E. cymbalariae

PCR amplification of the target ORF up- and downstream regions verified that the PCR-based deletion cassettes integrated into the genome and replaced the ORF with the KANMX marker. Since E. cymbalariae mycelia consist of multinucleated compartments, primary heterokaryotic transformants contain both wt- and mutant nuclei. Sporulation and selective germination of uninuclear spores enabled isolation of homokaryotic mutant mycelia, in which loss of the target ORF could be verified by PCR amplification of an internal, ORF-specific fragment. In TEC1::KANMX disruption mutants, the internal fragment is not missing, but integration of the KANMX marker should lead to an 1.,4 kb increase of the amplicon size (Figure 33).

Figure 33 Construction of a disruption cassette for gene deletion of TEC1 in E. cymbalariae. The amplified EcTEC1 ORF is cloned into the basic plasmid pBluescript SK+. ORF disruption is achieved by clonal insertion of the KANMX resistance marker, containing the E. coli KanR gene flanked by the A. gossypii TEF promoter and terminator sequences. The disrupted ORF creates two flanks, which were used for homologous recombination.

Chapter 5: Establishing molecular tools in E. cymbalariae

Figure 34. Agarose gel electrophoresis of PCR verification fragments. EcTEC1::KANMX gene disruption (upper panel), EcTEC1 (middle panel) and ECYM5230 (bottom panel) deletions were verified by amplifying the KANMX flanking regions using G1-G2 and G3-G4 primers. Loss of the target ORF was verified by amplification of an ORF-specific sequence using I1-I2 primers.

Chapter 5: Establishing molecular tools in E. cymbalariae

5.1.2 Characterization of Ectec1 and ecym5230 When optimizing gene deletion in E. cymbalariae, we chose to target two genes which pose special interest areas that might improve the use of E. cymbalariae as a model organism beside A. gossypii: the unknown, no-homolog gene ECYM5230 and EcTEC1, encoding a transcriptionregulator involved in filamentous and invasive growth as well as sporulation (Grünler et al., 2010).

The homokaryotic E. cymbalariae tec1 and ecym5230 deletion strains were used to investigate possible roles for the corresponding EcTec1 and Ecym5230 proteins. Growth assays of the E. cymbalariae mutants on full media at 30 °C, indicate wt-like phenotypes of both ecym5230 strains and Ectec1 deletion strains, independent of method for integration of the KANMX marker (Figure 35 and Figure 36). DIC imaging of hyphal morphology is consistent with the wt-like phenotypes of the ecym5230, Ectec1 and EcTEC1::KANMX mutants (Figure 37). Furthermore, the increased sporangia or spore formation observed in A. gossypii could not be observed microscopically in Ectec1 deletion mutants (data not shown).

Figure 35. Colony growth of E. cymbalariae ecym5230, Ectec1 and EcTEC1::KANMX mutants. Colony appearance after 7 and 14 days of growth on full media, 30 °C.

Chapter 5: Establishing molecular tools in E. cymbalariae

Figure 36. Colony growth of E. cymbalariae ecym5230, tec1 and TEC1::KANMX mutants. Median colony growth (mm) after 7-14 days on AFM media at 30 °C. Standard deviation error bars are calculated for strains with 5 or more replicates.

Figure 37. Differential interference contrast (DIC) imaging of E. cymbalariae wt, Ectec1, EcTEC1::KANMX and ecym5230. Scale bar is 50 µm.

Chapter 5: Establishing molecular tools in E. cymbalariae

5.1.3 Assessment of CEN and ARS function in E. cymbalariae The ARS sequence motif enables plasmid propagation in eukaryotes, and are essential for plasmid replication. In contrast, centromeres are not essential for eukaryotic vectors, but they do increase the mitotic and meiotic stability of the plasmid (Clarke and Carbon, 1980). While S. cerevisiae ARS, but not CENs, are stable in A. gossypii (Wright and Phillipsen, 1991), it is unknown whether S. cerevisiae and A. gossypii CEN/ARS elements are stable in E. cymbalariae. Alignments of the A. gossypii and E. cymbalariae centromeric DNA elements, CDEs, together with the S. cerevisiae CDE consensus sequences, show that the CDEs, specially the cysteine residues, are conserved between the species (Figure 38). Thus, we analyzed the stability of plasmids containing ARS/CEN sequences of S. cerevisiae and A. gossypii in E. cymbalariae, and compare those to the stability of the native ARS/CEN sequences.

Figure 38: Interspecies alignment of centromeric DNA elements CDEI, CDEII and CDEIII. Nucleotide sequences from A. gossypii and E. cymbalariae are compared to the corresponding consensus sequences of S. cerevisiae. The less conserved CDEII sequences are represented by sequence length and percentage of A-T content. In the S. cerevisiae consensus sequences, U represents purine (A or G), R represents A or T, and N represents any base (A, T, C or G).

Chapter 5: Establishing molecular tools in E. cymbalariae

The plasmid pFA-KANMX6 served as backbone for construction of two E. cymbalariae centromeric vectors rendering G418/Geneticin antibiotic resistance. E. cymbalariae genomic sequences containing ARS elements and CEN1 or CEN5 were amplified using primers #4800- #4801 and #5030-#4817 respectively, and cloned to pFA-KANMX6 through SpeI/SacI restriction sites added during PCR amplification (Figure 39).

Figure 39. E. cymbalariae CEN/ARS vectors. (A) Construction of E. cymbalariae CEN1 and CEN5 plasmids by cloning the centromeric sequence to pFA-KanMX6 through SpeI/SacI restriction sites. (B) The structures of CEN1 and CEN5, showing the number and relative positions of the autonomously replicating sequences (ARS), as well as the centromere DNA element (CDE) I, II and III.

E. cymbalariae germlings were transformed with the pFA-KANMX6-EcCEN1 and pFA- KANMX6-EcCEN5 as well as plasmids containing A. gossypii and S. cerevisiae CEN/ARS (Figure 40). Initial transformants were obtained for all plasmids during selective growth on 200 µg/ml G418/Geneticin. However, pRS-plasmids with ScCEN/ARS sequences appear unstable, and antibiotic resistance is lost within a few days of growth in liquid selective full media (Data not shown). Even so, ScCENs are more stable on solid media and the plasmids are maintained during selective growth (Figure 40 C-D). The vector with an AgCEN/ARS is mitotically stable with 72.2% plasmid retention after 10 days incubation without selective pressure. In comparison, a sole A. gossypii ARS renders 100% plasmid loss in non-selective conditions (Figure 40 A-B). Both EcCEN plasmids have an observed antibiotic retention of 100 % after 10 days of non- selective growth (Figure 40 E-F). However, in both cases introduction of EcCEN plasmids also

Chapter 5: Establishing molecular tools in E. cymbalariae

cause a slow growth phenotype which raise the question of genomic integration. Furthermore, we were unable to recover the plasmids by re-transformation of E. cymbalariae DNA into E. coli.

Origin Resistance Plasmid CEN ARS marker A pLC-Shuttle-A A. gossypii A. gossypii KanMX, NatMX B pHC-Shuttle-B - A. gossypii KanMX C pRS417-ScTEFp1-LacZ S. cerevisiae S. cerevisiae GEN3 D pRS415-KanMX S. cerevisiae S. cerevisiae KanMX E pFA-EcCEN1 E. cymbalariae E. cymbalariae KanMX F pFA-EcCEN5 E. cymbalariae E. cymbalariae KanMX

Figure 42. Loss of G418/Geneticin resistance in E. cymbalariae plasmid transformants. Upper row: regrowth on selective full media after 10 days of selective growth. Lower row: regrowth on selective full media after 10 days of non-selective growth. CEN and ARS sequences are obtained from A. gossypii (A- B), S. cerevisiae (C-D) and E. cymbalariae (E-F). Table: CEN and ARS sequences tested originate from A. gossypii, E. cymbalariae and S. cerevisiae

5.2 Discussion

5.2.1 Establishing PCR-based gene targeting in E. cymbalariae Homologous recombination has been established as a tool for engineering DNA mutations in many fungal species, and under optimal conditions it allows for a single mutation without any unspecific genomic alterations (Klinner and Schäfer, 2004). However, in many species homologous recombination is unspecific, and the transformation efficiency often requires time-

Chapter 5: Establishing molecular tools in E. cymbalariae

consuming screening for the correct mutation. In an effort to reduce non-specific integration, sequences for homologous recombination are usually several hundred bp, and construction of the cassettes is often a time-consuming multi-step process (Krawchuk and Wahls, 1999, Tatebayashi et al., 1994).

Gene deletion in the filamentous fungus A. gossypii is uncommonly efficient, and a mere 40 bp homology region is sufficient for a correct integration of a resistance marker cassette into a specific locus in the A. gossypii genome. The efficient homologous recombination of A. gossypii has enabled the development of PCR-based gene targeting, which uses a one-step PCR reaction for deletion cassette amplification, and is therefore time-saving compared to the traditional use of gene disruption cassettes (Wendland et al., 2000). In an effort to establish PCR-based gene targeting in E. cymbalariae, a close relative to A. gossypii (Wendland and Walther, 2011), we used deletion cassettes with 100 bp homology regions for transformation of E. cymbalariae mycelia. Our results show that although the initial transformation frequency of E. cymbalariae is relatively low, correct integration of the deletion cassette could be verified in the vast majority of the tested transformants (Table 3). Furthermore, there was no obvious difference in transformation frequency using PCR-based deletion cassettes with 100 bp homology regions and the disruption cassette with ~1000 bp homology regions. PCR verification of the correct integration confirms that the PCR-based gene targeting in E. cymbalariae is accurate, and correct integration of the marker occurs with high fidelity.

5.2.2 Wt-like phenotypes of E. cymbalariae TEC1 and ECYM5230 The growth phenotypes and hyphal morphology of Ectec1 and ecym5230 are both equivalent to E. cymbalariae wt. The lack of ECYM5230 orthologs were the main cause of our interest in the gene, and the reason for creating an ecym5230 deletion strain. However, while we can show that ECYM5230 is non-essential, the lack of a deletion phenotype severely limits the understanding of the gene function. Additional clues to the Ecym5230 protein function could be gained by further genetic manipulations, e.g. Ecym5230-GFP localization and expression studies, but due to time limits, neither was done in this study.

Chapter 5: Establishing molecular tools in E. cymbalariae

Unlike ECYM5230, EcTec1 have known orthologs in both S. cerevisiae and A. gossypii and in both species TEC1 is a transcription regulator implicated in mating, filamentous growth and adhesion. Be that as it may, the S. cerevisiae and A. gossypii deletion phenotypes differ in some aspects which caused our interest in the E. cymbalariae TEC1. While S. cerevisiae tec1 deletion strains are incapable of pseudohyphal- and invasive growth (Köhler et al., 2002), Agtec1 strains are more similar to the Ectec1 strains and maintain filamentous growth at a slightly reduced rate compared to A. gossypii wt. Furthermore, A. gossypii TEC1 deletion strains do not abolish invasive growth, and also displays a clear over-sporulation phenotype (Grünler et al., 2010, Grünler, 2010). Although no clear oversporulation phenotype was observed in E. cymbalariae tec1 deletion strains, both phenotypes have to be further evaluated in E. cymbalariae at a later occasion.

5.2.3 A. gossypii centromeres are stable in E. cymbalariae The development of interspecies shuttle vectors has greatly facilitated genetic engineering and DNA manipulations in several species (Clarke and Carbon, 1985). In order to establish a system of stable shuttle vectors for the filamentous fungus E. cymbalariae, we have compared the stability of replication origins and centromeres originating from S. cerevisiae, A. gossypii and E. cymbalariae during selective and non-selective growth.

Plasmid pHC-Shuttle-B, containing only an A. gossypii self-replicating sequence, AgARS, is rapidly lost during non-selective growth. There is a general consensus that the instability of an ARS-only plasmid is caused by an inability of the plasmid to segregate during mitotic division (Stinchcomb et al., 1979, Clarke and Carbon, 1985). Even though the hyphae of Eremothecium fungi are multinucleated, the nuclei in growing hyphae move towards the hyphal apex, and non- segregating plasmids will have a strong bias towards remaining in the nuclei of the older hyphae (Gibeaux et al., 2012). Thus, even though ARS-only plasmids might be present in older mycelia, there is little to no chance of migration of an ARS-only plasmid to the hyphal apex at the edge of a colony. Introduction of a functional centromere into the plasmid backbone enables segregation of the plasmid throughout the hyphae (Murray and Szostak, 1983). As G418/Geneticin resistance is maintained in hyphal samples transformed with an AgCEN-plasmid, pLC-Shuttle-A, we conclude that A. gossypii centromeric sequences mediate mitotic segregation in E. cymbalariae.

Chapter 5: Establishing molecular tools in E. cymbalariae

While G418/Geneticin resistance is maintained using native E. cymbalariae centromeres, transformed mycelia also show a slow grow phenotype. Failed efforts to recover the EcCEN plasmids by transformation of E. coli with E. cymbalariae genomic DNA further strengthens the hypothesis that using native E. cymbalariae centromeres enables genomic integration of the plasmid.

Plasmids containing S. cerevisiae CEN6 and ARSH4 sequences are not stable in E. cymbalariae. As the G418/Geneticin resistance is maintained during selective growth on solid media, the ScARS is functional, however the resistance marker loss in liquid cultures suggest that the ARS activity might be weak. In addition to analyzing the ScCEN/ARS activity, the use of two G418/Geneticin resistance marker modules, KANMX and GEN3, enabled us to assess the function of A. gossypii and S. cerevisiae TEF promoter and terminator activity in E. cymbalariae. Both modules contain the E. coli KanR gene: in the KANMX module, expression is regulated by the AgTEF promoter and terminator (Steiner and Philippsen, 1994), and in GEN3 it is regulated by the ScTEF promoter and terminator (Wendland et al., 2000). As G418/Geneticin resistant transformants are generated using either KANMX or GEN3 plasmids, expression regulation in E. cymbalariae can be achieved using either S. cerevisiae or A. gossypii regulatory sequences.

Chapter 6: Summary and future prospects

The conserved nature of small GTPases makes them interesting targets for comparative studies. This study focus on the A. gossypii ARF3 module due to the impact of excess of Arf3 activity on filamentous growth, an important factor for fungal pathogenicity. Comparing the A. gossypii ARF3 small GTPase with its yeast and mammalian homologs, it is likely that Arf3 acts by modulating the plasma membrane content of the lipid PI(4,5)P2 by directing the activity of phosphatidylinositide kinases. While the unregulated activity of Arf3 causes endocytic, actin localization- and morphological phenotypes which can all be linked to an excess of membrane

PI(4,5)P2 content, the direct effect of Arf3 on membrane composition and PI(4,5)P2 concentrations has not been addressed in this study. In addition to addressing the correlation between Arf3 and PI(4,5)P2, further linkage between the excess of Arf3 activity in the gts1 deletion strains could be demonstrated through either overexpression of the YEL1 GEF, or construction of a constitutively active ARF3.

In order to compare A. gossypii to other fungi, we established a set of pathogenicity assays to determine the limits of filamentous growth in species of the Eremothecium genus. In addition, this study also establishes molecular tools for E. cymbalariae, a close relative to A. gossypii. Due to the fact that the yeast S. cerevisiae only does short rounds of polar growth during budding, it is valuable to compare the impact of the modified Arf3 module in other filamentous fungi, such as E. cymbalariae. As we established that neither A. gossypii or E. cymbalariae are able to penetrate host tissue, and thus are limited in the development of an infection structure, it would be interesting to evaluate the Arf3 module in a pathogenic fungus. This could possibly address the importance of filamentous growth during fungal infections and would enlighten whether the Arf3 module poses potential targets for antifungal drugs. In addition, the conserved nature of small GTPases, further investigations of the A. gossypii ARF3 module could be valuable when compared to mammalian Arf6, a known oncogene. As Arf6 is regulated by several different GEFs and GAPs, research in the regulation of Arf6 activity is particularly difficult. The fact that A. gossypii ARF3 only has one GAP and one GEF, GTS1 and YEL1 respectively, facilitates deletion of regulatory proteins and enables further understanding of the Arf3-, and consequently the mammalian Arf6 function.

Chapter 7: References

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Appendix I: Strains

Strain Genotype Source Derivate of Ashbya gossypii ATCC10895 wt Ashby and Novell, 1926 - Agleu2 wt (leu2) Mohr and Philippsen ATCC10895 ALK17-1 gts1::GEN3, leu2 Lengeler, this study Agleu2 ALK17-2 gts1::GEN3, leu2 Lengeler, this study Agleu2 ATO010 arf3::GEN3, leu2 This study Agleu2 ATO012 arf3::GEN3, leu2 This study Agleu2 ATO002 yel1::GEN3, leu2 This study Agleu2 ATO004 yel1::GEN3, leu2 This study Agleu2 ATO005 gts1::GEN3/GTS1, leu2 This study ALK17-1 ATO006 gts1::GEN3/GTS1, leu2 This study ALK17-2 ATO007 gts1::GEN3/GTS1-GFP, leu2 This study ALK17-1 ATO013 gts1::GEN3/GTS1(ArfGAP), This study ALK17-1 leu2 ATO014 gts1::GEN3/GTS1(ArfGAP), This study ALK17-2 leu2 ATO017 gts1::GEN3/GTS1(UBA), leu2 This study ALK17-1 ATO018 gts1::GEN3/GTS1(UBA), leu2 This study ALK17-2 ATO015 arf3::GEN3/ARF3, leu2 This study ATO010 ATO016 arf3::GEN3/ARF3, leu2 This study ATO012 AWE37 wal1::KanMX6, leu2 Walther and Wendland Agleu2 2004 ASJ22 sac6::KanMX6, leu2 Jorde et al., 2011 Agleu2 Eremothecium cymbalariae DBVPG7215 wt Kurtzman - ETO12 tec1::GEN3 This study DBVPG7215 ETO13 tec1::GEN3 This study DBVPG7215 ETO14 TEC1::KanMX This study DBVPG7215 ETO15 TEC1::KanMX This study DBVPG7215 ETO16 ecym5230::GEN3 This study DBVPG7215 ETO17 ecym5230::GEN3 This study DBVPG7215 ETO18 pHC-NatMX-KanMX This study DBVPG7215 ETO19 pLC-KanMX-LacZ This study DBVPG7215 ETO20 pFA-EcCEN1 This study DBVPG7215 ETO21 pFA-EcCEN5 This study DBVPG7215 Saccharomyces cerevisiae BY4749 MATa, his3, leu2, met15, ura3 Euroscarf CEN.PK2 MATa/α, his3, leu2, trp1, ura3 Hoepfner et al, 2000 F234 Carlsberg brewery strain Carlsberg YTO001 pRS418-MoGFP-Nat1 This study BY4749 YTO002 pRS418-AgTEFp-GTS1-GFP This study BY4749 YTO003 pRS418-AgTEFp-GTS1-GFP This study CEN.PK2 YTO004 pRS418-AgTEFp-GTS1-GFP This study F234 Fusarium oxysporum f. sp. Lycopersici FGSC 9935 wt O'Donnell et al. 2004 FGSC 9935

Appendix II: Primers

Primer Primer name Primer sequence 5’-3’ No. 392 5’-GFP cataaccttcgggcatggcatc 511 AgTEF-prom aggatttgccactgaggttcttc 529 pFAup ttgtgcgttagaacgcggctac 530 pFAdo tgcaggttaacctggcttatc 1112 G2new gccagtttagtctgaccatc 1113 G3new tcgcagaccgataccagg 1202 N2new gcgtttccctgctcgcaggtc 1214 ScTEFp-up1 gggtaatttgtcgcggtctggg 1215 ScTEFt-down1 gcccatcagattgatgtcctcc 4321 G1-ACL055W cgatcacgtgacaatgcaac 4322 G4-ACL055W gtcctactactaggtggcg S1-ACL055W CAGCAAGAGGTAAAGCCAGAGAACGGTGGGCTTCGACGGACA 4323 GGCgaagcttcgtacgctgcaggtc S2-ACL055W TCATGGCTCTAACTGAGCCCCTGATGAGATGTGCTTGTGGCGT 4324 CActgatatcatcgatgaattcgag 4325 I1-ACL055W ctcgctgaatagcgccag 4326 I2-ACL055W cctgctgtaaagagccatg 4800 3'-Ecym_CEN1 CTTCTTactagtcgctaactctcgtgccattgg 4801 5'-Ecym_CEN1 AAACTAgagctcctgtccctgaccttgacg 4817 3'-Ecym_CEN5 CTTCTTactagtctattccataaactttctataacgg 4832 G1-EcymTEC1 cattgcagcagcagcagcaag 4833 G4-EcymTEC1 ggtaacggttcagtacagtg 4834 I1-EcymTEC1 cttcgggcacggatcattcgg 4835 I2-EcymTEC1 ggcgtactacgctcgctac 5030 CEN5-1 AAACTAgagctcccgtctgggcagttgatatgag 6062 TATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATACCA pAGlib_swap1 Cgaagcttcgtacgctgcaggtc 6063 AATTTGTGAGTTTAGTATACATGCATTTACTTATAATACAGTttttta pAGlib_swap2 ggggcagggcatgctcatg 6180 CCAACGGCCTCGCAGGGCGCTTATCCTGGCTACAACTACCAG AgGTS1-GFP-S1 ATgggtgctggcgcaggtgcttc 6181 GTTACGTTTGTTTCACGTGGTTTTTAATTACGTACTGATACAGA AgGTS1-GFP-S2 Atctgatatcatcgatgaattc 6195 AGACTACCGAAGTCTAGCGGCGTGGGCAAGATCACAGACGGC S1-ACL078W GCAgaagcttcgtacgctgcaggtc 6196 CACGGCCGCGCCGGCCCCGCTCTACTGTTACAAGCCCGCGCA S2-ACL078W GGCctgatatcatcgatgaattcgag 6197 G1-ACL078W ttggacgatctacggtgtacg 6198 G4-ACL078W accgacaacaacaacctgctc 6199 I1-ACL078W agttcaacatgtgggacgttgg 6200 I2-ACL078W agctggttgttcaggttgtcg 6201 S1-ABR218C GAGCTACTGTTTCCTGGGGAGACAGAGTAAGGCGCACGCCGA TATgaagcttcgtacgctgcaggtc 6202 S2-ABR218C CTTTGGTGGTACATAGGAATGATACATAATGGAAACTCAGGTT CGctgatatcatcgatgaattcgag 6203 G1-ABR218C cattctcgtggaagacacacc 6204 G4-ABR218C cggtcagcatgtgtaacatgc 6205 I1-ABR218C acgaacgacaacagccatagc 6206 I2-ABR218C caagttgtctggctgttgctc

Appendix II: Primers

Primer Primer name Primer sequence 5’-3’ No. 6327 G1-Ecym5230 ctgtctatatataccaagag 6328 G4-Ecym5230 ctgctctctgtgtaactgcg 6329 AGACACAACCAACACTCTATATATATATATACACTCTCACCCAT S1-Ecym5230 ATACGACGCCGCCGCACACTGCATCGACTCTGACATCTAAAAA CACAAATCAAACAgaagcttcgtacgctgcaggtc 6330 GGCCACCCGGCAACGCCACTACTACTATGTTCTAATATATGCT S2-Ecym5230 TCGTTCGCCATGTATTGTGTGTACGATACACCAACATGCAACA CCGGGGCTACTTATctgatatcatcgatgaattcgag 6331 I1-Ecym5230 gttgcgctgcattgcgctg 6332 I2-Ecym5230 cacccggcaacgccactac 6333 S1-EcTEC1 ATGATGAATGAAGAAGGAGCAGGGGCTAGGTTTGAGAATCTGT TTAATTCGCAGCTGTATTACCAGCAGCCAGGAGGAGGGGGGG GTGGGGATGGTCAGGgaagcttcgtacgctgcaggtc 6334 S2-EcTEC1 TTATCCTGACCGAGAGGCGTCATCAGAATATTCTTGAGCTTCA AGAGAAACAACGGTCTGCTGCTGTGGGAGCGGTATGCTCTGT TGGGGGGACACATGCctgatatcatcgatgaattcgag 6336 3'-EcTEC1-XbaI ctgtcatctagacatattccaacggtgttgtagg 6338 5'-GTS1fwd-XhoI ctgtcactcgaggatactaaagcaccatgcgc 6339 5'-GTS1rev-XbaI ctgtcatctagactagctattcagcgagctgtag 6340 3'-GTS1fwd-XhoI ctgtcactcgagatgtacagctcgctgaatagcg 6341 3'-GTS1rev-XbaI ctgtcatctagactacatctggtagttgtagc Lower case letters represent homology region to PCR target sequence. Upper case letters represent bases for homologous recombination. Italics/underlined represent restriction sites. Bold letters represent added codons.

Appendix III: Plasmids

Plasmid Plasmid Name Marker(s) Source No. 120 pSK+Bluescript Amp (Short et al., 1988) 121 pFA-KanMX6 Amp, Kan (Wach et al., 1994) 150 pRS415-KanMX Amp, Kan (Schade et al., 2003) 651 pRS417-AgTEFp-LacZ Amp, GEN3 (Dünkler and Wendland, 2007) 690 pRS417-AgTEFp-GFP Amp, GEN3 (Dünkler and Wendland, 2007) 706 pHC-Shuttle-AgTEFp-StlacZ Amp, Kan (Gastmann et al., 2007) 738 pLC-Shuttle-KanMX-NATMX3 Amp, Nat, Kan (Gastmann et al., 2007) 840 pFA-AgNATMX4 Amp, Nat Lab collection C169 pRS417-ScTEFp-LacZ Amp, GEN3 Lab collection C455 pRS418-AgTEFp-LacZ Amp, Nat Lab collection C470 pFA-MoGFP-Nat1 Amp, Nat Lab collection C682 pFA-EcCEN1 Amp, Kan This study C686 pFA-EcCEN5 Amp, Kan This study C781 pAG19275(GTS1)-NatMX Amp, Nat This study C783 pAG17522(ARF3)-URA3 Amp, URA3 (Wendland et al., 1999b) C787 pAG19275(GTS1-GFP) Amp, Nat This study C832 pRS418-AgTEFp-GTS1(ArfGAP) Amp, Nat This study C834 pSK+EcymTEC1 Amp This study C841 pSK+EcymTEC1::KanMX(fwd) Amp, Kan This study C842 pSK+EcymTEC1::KanMX(rev) Amp, Kan This study C871 pRS417-AgTEFp-(GTS1-GFP) Amp, Kan This study C936 pAG19275(GTS1)-URA3 Amp, URA3 (Wendland et al., 1999b) C938 pRS418-AgTEFp-GTS1(UBA) Amp, Nat This study C943 pAG17522(ARF3)-NatMX Amp, Nat This study