Analysis of sterol metabolism in the pathogenic oomycetes Saprolegnia parasitica and Phytophthora infestans

Paul Dahlin

Analysis of sterol metabolism in the pathogenic oomycetes Saprolegnia parasitica and Phytophthora infestans

Paul Dahlin

Abstract

The primary objective of this thesis was to investigate the sterol metabolism of two pathogenic oomycetes, specifically the processes of sterol synthesis and sterol acquisition in the fish pathogen Saprolegnia parasitica (Saprolegniales) and the plant pathogen Phytophthora infestans (Peronosporales). Furthermore, the effects of steroidal glycoalkaloids from Solanaceous plants, on P. infestans, were examined. The improved understanding of these processes should help to identify approaches for the identification of new oomycete inhibitors targeting sterol metabolism in agriculture and aquaculture farming systems, and to guide plant-breeding strategies to defend solanaceous plants against oomycetes. For these reasons, the molecular basis of the metabolic pathways of sterol synthesis and/or sterol acquisition was investigated. Sterols are derived from isoprenoids and indispensable in various biological processes. Our biochemical investigation of an oxidosqualene cyclase revealed that sterol synthesis in S. parasitica begins with the formation of lanosterol (Paper I), and a reconstruction of the complete sterol synthesis pathway to the final compound, fucosterol, in S. parasitica was performed using bioinformatics (Paper II). Complementary to this work, the extent to which P. infestans, which is incapable of de novo sterol synthesis, is able to modify exogenous- ly provided sterols was investigated by determining the growth impact of various sterol supplements in the growth media (Paper II). Building on the sterol investigations, the solanaceous sterol derivatives from the glycoalkaloid family were analysed. These compounds contain both a steroidal and a carbohydrate (glycan) moiety. Data obtained by feeding vari- ous deuterium-labeled sterols to potato shoots, supported the theory that steroidal glycoalkaloids in Solanum tuberosum are produced from cholesterol (Paper III). Since these steroidal glycoalkaloids are thought to play a role in plant defense, their physiological effects on P. infestans were investigated (Paper IV). Unexpectedly we found that non-glycosylated steroidal alkaloids had a greater inhibitory effect than steroidal glycoalka- loids. Steroidal glycoalkaloids derived from other Solanaceous species ex- hibited different physiological effects on the growth of P. infestans. This research was conducted on two oomycete species belonging to the Saprolegniales and Peronosporales orders, hence the results presented are likely to be representative of each of these two oomycete orders.

Sammanfattning

Det primära syftet med denna avhandling var att undersöka sterolmetabolismen hos två patogena oomyceter. Specefikt undersöktes processen för sterolsyntes och förvärv av steroler hos fiskpatogenen Saprolegnia parasitica (Saprolegniales) och växtpatogenen Phytophthora infestans (Peronosporales). Dessutom studerades effekterna av steroida glykoalkaloider från värdväxter tillhörande Solanaceae på P. infestans. Den förbättrade förståelsen av dessa processer bör bidra till att finna metoder för identifiering av nya inhibitatorer inriktade på sterolmetabolism i oomyceter, samt att ta fram strategier för växtförädling för att försvara växter mot oomyceter. Därför har den molekylära grunden för de metaboliska vägarna för sterol- syntes och / eller sterol förvärv undersökts. Steroler härrör från isoprenoider och är oumbärliga i olika biologiska processer. Vår biokemiska undersökning av en oxidoskvalencyklas visade att sterolsyntes i S. parasitica börjar med bildandet av lanosterol (Publikation I). En rekonstruktion av hela sterolsyntesvägen i S. parasitica till den slutliga föreningen, fucosterol, utfördes med hjälp av bioinformatik (Publikation II). Som komplement till detta arbete undersöktes i vilken utsträckning P. infestans som är oförmögen att syntetisera steroler de novo kan modifiera exogent tillhandahållna steroler. Detta gjordes genom att bestämma effekten på tillväxt av olika steroltillskott i tillväxtmediet (Publikation II). Med utgångspunkt i sterolstudierna analyserades sterolderivat från glycoalkaloidfamiljen från Solanaceae. Dessa föreningar innehåller både en steroid och en kolhydratenhet (glykan). Data som erhållits genom att mata potatisskott med olika deuterium-märkta steroler stödde teorin att steroida glykoalkaloider i Solanum tuberosum framställs från kolesterol (Publikation III). Eftersom dessa steroida glykoalkaloider tros spela en roll i växters försvar, undersöktes deras fysiologiska effekter på P. infestans (Publikation IV). Oväntat fann vi att icke-glykosylerade steroida alkaloider hade en större hämmande effekt än steroida glykoalkaloider. Steroida glykoalkaloider erhållna från andra Solanaceae arter uppvisade andra fysiologiska effekter på tillväxten av P. infestans. Denna forskning utfördes på två oomycetarter som tillhör ordningarna Saprolegniales och Peronosporales, därför kommer de resultat som presenteras sannolikt att vara representativa för dessa två order av oomyceter.

List of papers

The following papers, referred to by their Roman numerals, are the basis of this thesis.

I. Dahlin P, Srivastava V, Bulone V, McKee LS. 2016. The oxidosqualene cyclase from the oomycete Saprolegnia parasitica synthesizes lanosterol as a single product. Frontiers in Microbiology, doi: 10.3389/fmicb.2016.01802.

II. Dahlin P, Srivastava V, Ekengren S, McKee LS, Bulone V. Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans. “Accepted in PLoS ONE pending minor revision”.

III. Petersson EV, Nahar N, Dahlin P, Broberg A, Tröger R, Dutta PC, Jonsson L, Sitbon F. 2013. Conversion of exogenous cholesterol into glycoalkaloids in potato shoots, using two methods for sterol solubilisation. PLoS ONE 8(12): e82955. doi:10.1371/journal. pone.0082955

IV. Dahlin P†, Müller M†, Ekengren S, McKee LS, Bulone V. The impact of steroidal glycoalkaloids on the physiology of Phytophthora infestans, the causative agent of the potato late blight. “Accepted in Molecular Plant-Microbe Interactions pending minor revision”.

†These authors contributed equally to the work.

Author’s contribution to the papers:

Paper I: Participated in the experimental design, performed most experi- ments, analysed the main part of the data and wrote the first draft of the manuscript.

Paper II: Participated in the experimental design, conducted the experi- ments, analysed the data and wrote the first draft of the manuscript.

Paper III: Participated in the experimental design, the experiments, and the analysis of the data and contributed to the writing.

Paper IV: Participated in the experimental design, co supervised Master student Marion Müller, participated in analysing the data and contributed to the writing of the manuscript.

Contents

General introduction ...... 13 Oomycetes ...... 13 Phytophthora infestans (Mont.) de Bary ...... 16 Saprolegnia parasitica ...... 19 Sterols and glycoalkaloids ...... 22 Triterpene biosynthesis ...... 22 Sterols ...... 24 Glycoalkaloids from Solanaceous species ...... 27 Sterol and steroidal glycoalkaloid interactions with oomycetes ...... 31 Sterols ...... 31 Steroidal glycoalkaloids (SGAs) ...... 32

Aims of this thesis ...... 34 Comments on methodologies used ...... 35 Isolation, separation and analysis of sterols ...... 35 Isolation, separation and quantification of steroidal glycoalkaloids ...... 37 Results and discussion ...... 39 The oxidosqualene cyclase from the oomycete Saprolegnia parasitica synthesizes lanosterol as a single product (Paper I) ...... 39 Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans (Paper II) ...... 40 Conversion of exogenous cholesterol into glycoalkaloids in potato shoots, using two methods for sterol solubilisation (Paper III) ...... 42 The impact of steroidal glycoalkaloids on the physiology of Phytophthora infestans, the causative agent of the potato late blight (Paper IV) ...... 43 Conclusion ...... 44 Research perspectives ...... 45

Acknowledgements ...... 47

References ...... 49

Abbreviations

AACT Acetoacetyl-CoA thiolase C Cysteine CA Cycloartenol CAS Cycloartenol synthase DMAPP Dimethylallyl diphosphate DPMD 5-Diphosphomevalonate decarboxylase ELISA Enzyme-linked immunosorbent assay FPP Farnesyl diphosphate GC Gas chromatography GPP Geranyl diphosphate H Histidine HMG 3-Hydroxy-3-methylglutaryl HMG-CoA 3-Hydroxy-3-methylglutaryl CoA HMGR HMG-CoA reductase HMGS HMG-CoA synthase HPAEC-PAD High-Performance Anion-Exchange Chromatography Cou- pled to Pulsed Amperometric Detection HPLC High-performance liquid chromatography I Isoleucine IPP Isopentyl diphosphate LA Lanosterol LAS Lanosterol synthase MBD Methyl-β-cyclodextrin MEP Methylerythritol phosphate MS Mass spectrometry MVA Mevalonate MVAP Mevalonate-5-phosphate MVAPP Mevalonate-5-diphosphate MVK Mevalonate kinase (m/z) Mass-to-charge N Asparagine OS 2,3-Oxidosqualene OSC 2,3-Oxidosqualene cyclase PMVK 5-Phosphomevalonate kinase Q Glutamine SA Steroidal alkaloids SGA Steroidal glycoalkaloids

SpLASA Protein product of S. parasitica LAS gene SPRG_11783 SQE Squalene epoxidase SQS Squalene synthase T Threonine TLC Thin-layer chromatography V Valine Y Tyrosine

General introduction

The oomycetes or Oomycota, also referred to as “water molds”, represent a phylogenetic lineage of fungus-like organisms. This class of multicellular organisms includes a number of plant and animal pathogens such as the fish parasite Saprolegnia parasitica (order Saprolegniales) or the causative agent of the potato late blight, Phytophthora infestans (order Peronosporales). The plant pathogen P. infestans is commonly used as a model organism for oomycetes. However, members of the Saprolegnia genus are exclusively pathogens of water-borne animals, making comparisons with P. infestans not always relevant. S. parasitica was included in this study as a representative of oomycetous animal pathogens and for comparison with the crop pathogen P. infestans. Both pathogens share multiple biochemical features essential for growth, survival and reproduction. One striking difference between the orders they belong to is that Peronosporales species are incapable of de novo sterol syn- thesis, and require these essential isoprenoid-derived compounds to be acquired from their hosts. In contrast, Saprolegniales species are not dependent on external provision of sterols. A wide range of functions are attributed to sterols and sterol derivatives in eukaryotic organisms. In this introduction, the reader is first introduced to the oomycetes, with an emphasis on S. parasitica and P. infestans. Subse- quently, sterols and steroidal alkaloids are introduced, and their molecular, structural and functional properties as well as their impact on oomycete growth are discussed.

Oomycetes The oomycetes represent a class of eukaryotes encompassing several plant and animal pathogens accounting for severe economic losses in agriculture and aquaculture (van West, 2006; Beakes et al., 2012; Kamoun et al., 2015). Fossil records date the emergence of the oomycetes to about 300-315 million years ago (Krings et al., 2011). Due to their mode of nutrition and the fact that they typically show mycelial growth, these filamentous microorganisms were originally grouped within the class of fungi. However, numerous dis- tinct characteristics such as their ability to produce reproductive oospores, for which they are named, the occurrence of diploid nuclei in the vegetative

13 mycelium and of bi-flagellated zoospores, and mitochondria with tubular cristae distinguish the oomycetes from true fungi (Table 1). Additionally, the cell wall composition varies between these two classes of organisms: oomycete cell walls consist primarily of (1→3), (1→6) β-D-glucans and cellulose, while the fungal cell wall consists mainly of chitin, with some (1→3), (1→6) β-D-glucans and α-D-mannans (Table 1). Further, there are metabolic differences: oomycete lysine biosynthesis occurs via the dia- minopimelic acid pathway rather than via the alpha-aminoadipate pathway utilised by true fungi (Judelson & Blanco, 2005; Rossman & Palm, 2006; Hardham, 2007; Beakes et al., 2012). A further distinguishing characteristic for oomycetes is the presence of fucosterol as the end sterol (in cases where sterols are synthesised de novo), as opposed to ergosterol in fungi (Madoui et al., 2009; Gaulin et al., 2010).

Table 1. Key differences between oomycetes and true fungi.

Characteristic Oomycetes Fungi Sexual reproduc- Oospores form when oospheres are Sexual reproduction results in tion fertilised by nuclei from antheridia zygospores, ascospores or (Heterogametangia) basidiospores Vegetative nucle- Diploid Haploid or dikaryotic ar state Major cell wall (1→3),(1→6) β-D-glucans, cellulose Chitin, (1→3),(1→6) β-D- components (minute amounts of chitin in some glucans, α-D-mannans and species) cellulose in minor amounts Flagella type on Biflagellated: one posteriorly If flagellated: usually posteri- zoospores whiplash, one anteriorly directed or, whiplash of one type fibrous, ciliated Mitochondria Tubular cristae Flattened cristae Specific synthe- Fucosterol (sterol synthesising species Ergosterol sised sterol only) Lysine synthesis Diaminopimelic acid pathway Alpha-aminoadipate pathway Pigmentation Usually unpigmented Hyphae or spores are com- monly pigmented

Adapted from Rossman & Palm (2006) with additional information from Judelson & Blanco (2005); Hardham (2007); Madoui et al. (2009); Gaulin et al. (2010); Beakes et al. (2012).

The oomycetes are most closely related to diatoms and brown algae, which belong to the Stramenopiles (Heterokonta) superphylum (Cavalier-Smith & Chao 2006; Lamour et al., 2007; Beakes & Sekimoto, 2009; Beakes et al., 2012; Raffaele & Kamoun, 2012; Figure 1). Nonetheless, oomycetes are superficially still referred to in the literature as fungus-like organisms. Within the Stramenopiles, they are placed together with the Hyphochytridiomycetes and considered members of the pseudofungi (see Cavalier-Smith & Chao 2006; Beakes et al., 2012; and references therein).

14

tissue, forming a germ tube that infects plant cells, or indirectly germinate by releasing zoospores. The biflagellate motile zoospores lose their flagellae before forming cytospores, which subsequently germinate and form germ tubes that infect plant cells (Judelson & Blanco, 2005; Walker & van West, 2007; Fry, 2008). The sexual reproductive cycle occurs only once annually, when two mating types, referred to as type A1 and type A2, interact.

Figure 3. Life cycle of Phytophthora infestans divided into the sexual and asexual cycles (adapted from Kessel & Förch, 2006).

Until the 1970s, the mating types A1 and A2 were reported together only in Mexico, restricting the dominant global population of lineages US-1 and HERB-1 (both mating type A1) to asexual reproduction (Niederhauser, 1991; Spielman et al., 1991; Fry et al., 1993; Yoshida et al., 2013). After a replacement of these lineages by a more virulent P. infestans strain possessing both mating types A1 and A2 which reached Europe in the early 1980s, P. infestans outbreaks have been much more difficult to control (Spielman et al., 1991; Fry, 2008).

The host plant Solanum tuberosum The potato plant, S. tuberosum, is a tuber-forming plant indigenous to the Andes region, and in terms of production volume is now the most important

17 non-cereal crop in the world (FAO, http://www.fao.org, 2016). This perennial dicotyledonous plant is a starch tuberous crop that is a staple of the diet of more than a billion people. Potato additionally plays important roles in industrial applications where starch and starch derivatives are used (Vreugdenhil et al., 2007; CIP, http://cipotato.org, 2016). Generally, potatoes yield considerably more calories and protein compared to wheat, maize or rice per cultivated hectare per day (Rubatzky & Yamaguchi, 1997). Due to increasing global demand, it is presumed that global crop production needs to double by 2050 to feed a growing human population (Ray et al., 2013). As consequence of the increasing demands to supply the world with food, sus- tainable potato production has to be maximized. Pest management is a vital element of potato agriculture. Besides the oomycete P. infestans, potato plants are attacked by numerous pathogens, including fungi of the Rhizoctonia and Sclerotinia genera, Erysiphe cichoracearum, the causative agent of the powdery mildew, Spongospora subterranean causing powdery scab, or the agent of the disease blackleg, Pectobacterium carotovorum (EPPO/OEPP, 1994; Hallmann et al., 2009). In addition, viruses such as potato leafroll virus and animals such as nematodes, the Colorado potato beetle and some aphids, also have a detrimental impact on potato yield (EPPO/OEPP, 1994; Vreugdenhil et al., 2007; Hallmann et al., 2009). A potential defense mechanism against some of the mentioned pathogens is attributed to the potato derived steroidal glycoalkaloids α-solanine and α-chaconine (Milner et al., 2011). These steroidal glycoalkaloids (SGA) will be described later in the introduction.

Control strategies against Phytophthora infestans Good plant protection practices and integrated pest management control of late blight (IPM-LB) are specified by the European and Mediterranean Plant Protection Organization (EPPO) guidelines where a combination of preven- tive measures, cultural practices, genetic plant sources and the application of specific fungicides are described (EPPO/OEPP, 1994). Generally, all potato farming systems, whether conventional or organic, im- plement pest control strategies. This includes the selection of P. infestans-resistant cultivars of S. tuberosum, and crop rotation over the years in order to reduce the population of crop-specific pathogens. High hilling of the soil to protect tubers from P. infestans spores and controlled irrigation to minimize the duration of infection favor leaf wetness, which can additionally reduce P. infestans infection of potato tubers and plants (EPPO/OEPP, 1994; Hallmann et al., 2009; Rietman et al., 2012). Complementary agricultural farming practices are appropriate fertilization for the plant state, weed management to eradicate potential P. infestans hosts, the destruction or roguing of diseased plants to eliminate P. infestans inoculum sources, and optimized tuber depth and soil moisture to eliminate conditions that favor P. infestans (Colon et al., 1992; EPPO/OEPP, 1994;

18 Porter et al., 2005). Despite these preventive measures, untreated P. infestans infection of farmed potatoes exerts a devastating effect on crop yield up to entire crop losses (EPPO/OEPP, 1994; Hallmann et al., 2009). For this reason, conventional farming involves the application of fungicides to save infected potato cultures (EPPO/OEPP, 1994). During P. infestans favorable weather conditions and high availability of inoculum, fungicides can be applied before possible outbreaks as an effective preventive step (EPPO/OEPP, 1994; Hallmann et al., 2009). Limited application of fungicides to low-susceptibility S. tuberosum culti- vars as an additional protective measure showed great results at preventing infection in the past (Kirk et al., 2005; Fry, 2008; Mayton et al., 2008). However, with the appearance of more aggressive P. infestans strains, and increasing reports of resistance to important chemicals such as metalaxyl and mefenoxam, control of late blight is becoming more difficult (Nærstad, 2000; Grünwald et al., 2006; Fry, 2008). The development of new fungicides and selective chemical application strategies are of great importance to con- trol or reduce P. infestans outbreaks.

Saprolegnia parasitica The endemic fish pathogenic oomycete S. parasitica is found globally in freshwater lakes and streams, as well as in controlled environments like fish farms and aquaria. The oomycete can infect and destroy wild and cultivated fish by causing the disease saprolegniasis (Figure 4).

Figure 4. Brown trout (Salmo trutta) infected by Saprolegnia parasitica, showing severe skin lesions (Photo: Reiner Kühnis and Michael Kugler, 2015).

S. parasitica is usually considered as a secondary pathogen infecting the host under favoring conditions, such as skin necrosis, lesions and weak immunity (van West, 2006). However, some virulent S. parasitica strains can cause primary infection in salmon (van West, 2006). Grey-white lesions with fila- mentous mycelial elements appearing as cotton wool-like clusters are the characteristics for saprolegniasis, which are often found on incubating fish eggs, or brood fish skin or fins (van West, 2006; Phillips et al., 2008). The cellular necrosis, dermal and epidermal damage on fish, caused by

19 S. parasitica can lead to death if not treated (van West, 2006). Generally, the losses attributed to S. parasitica infections represent several millions of dollars per annum (van West, 2006).

Life cycle of Saprolegnia parasitica The diploid life cycle of S. parasitica has several unique and distinct developmental stages. Like P. infestans, S. parasitica can reproduce both sexually and asexually (Figure 5; van West, 2006). During asexual reproduc- tion, sporangia are formed in the rounded ends of vegetative hyphae, and eventually release zoospores. These single nucleated biflagellated cells encyst. From the resulting primary cyst, new free-swimming secondary zoospores develop which subsequently form a secondary cyst with so called “boat hook” structures. These structures have been suggested to enhance attachment to the host. Attached cysts germinate and penetrate via a germ tube in the underlying tissue of the host. If no suitable host is present, the secondary cyst can form an encysted secondary zoospore in a developmental process termed polyplanetism (Beakes, 1983; van West, 2006; Phillips et al., 2008). It is presumed that this process enhances the likelihood for S. parasitica to find and infect a host (Phillips et al., 2008).

Figure 5. Life cycle of Saprolegnia parasitica divided into the sexual and asexual cycles (picture adapted from van West, 2006).

20 Control strategies of Saprolegnia parasitica Since the international ban of the effective anti-S. parasitica drug malachite green in 2002, due to its carcinogenic and toxicological properties, fish farms have been facing an emerging and serious management problem (van West, 2006). To combat these problems, preventive measures are generally used and represent a cost-efficient farming approach to maintain fish health (Meyer, 1991; Lamour & Kamoun, 2009). Preventive strategies primarily involve the management of farms to ensure low-stress environments for the animals, since stressed fish show impaired immune responses and are thus more susceptible to infection (Lamour & Kamoun, 2009). Consequently, environmental control and the isolation of infected fish upon identification of infection are key management strategies (Meyer, 1991, Pillay & Kutty, 2005). Nonetheless, saprolegniasis is still very common in fish farms (van West, 2006). Therefore, new approaches to disease control and prevention are highly sought after since the banning of malachite green. Formalin can be used to treat fish and fish eggs (Gieseker et al., 2006; Lamour & Kamoun, 2009; Leal et al., 2016) and is widely used to control saprolegniasis. Formalin is however known to be toxic to fish at high concentrations (Gieseker et al., 2006; Khodabandeh & Abtahi, 2006; Leal et al., 2016), and is likely to be banned in the future (Lamour & Kamoun, 2009). Salt water treatment also has a concentration-dependent inhibitory effect on Saprolegnia sp. (Ali, 2005; Khodabandeh & Abtahi, 2006), but this is not a feasible approach to treat freshwater fish (Lamour & Kamoun, 2009). Some common antifungal and antimicrobial compounds, such as amphotericin B, chitosan and Bronopol (2-bromo-2-nitropropane-1,3-diol) are effective against Saprolegnia sp. infections (Bly et al., 1996; Muzzarelli et al., 2001; Branson, 2002). It is assumed that these compounds target the oomycete cell membrane, reducing its stability and integrity, ultimately leading to cell death. Unfortunately, further research is required before these compounds can be applied on a large scale in aquaculture (Lamour & Kamoun, 2009). Likewise, the antifungal drug clotrimazole, which targets the S. parasitica sterol demethylase enzyme (CYP51) required for sterol synthesis, must be more thoroughly investigated before use in aquaculture, although prelimi- nary work suggests that the drug has the same inhibitory potential as mala- chite green (Warrilow et al., 2014). Indeed, targeting sterol synthesis as a mean of inhibiting S. parasitica growth is a promising approach, and will be discussed in more detail below. Besides drug development against S. parasitica, an alternative approach is the development of fish vaccines, as currently utilized to prevent certain bacterial and viral infections (Gudding et al., 1999; Earle & Hintz, 2014). Further molecular studies on interactions between S. parasitica and its hosts are needed to develop a successful vaccine against this pathogen (Earle & Hintz, 2014).

21 Sterols and glycoalkaloids Sterols are triterpenoids that are essential for a broad range of vital cellular processes. Triterpenes are hydrocarbons involved in various processes and molecular interactions, and present a great structural variety and diverse functions (Bach & Rohmer, 2012). They are found in light-harvesting organs in photosynthetic organisms, have roles in stabilizing lipid bilayers, are in- volved in hormone formation and modifications (e.g., abscisic acid, gibberellic acid and brassinosteroids in plants, steroid hormones in animals), and are used as building blocks for the synthesis of more complex biomole- cules such as carotenoids, saponins or alkaloids (Clouse, 2001; Bach & Rohmer, 2012; Mouritsen & Bagatolli, 2015).

Triterpene biosynthesis These organic compounds are derived from the elementary isoprenoid skele- ton, and are synthesised from the five-carbon precursor isopentenyl diphosphate (IPP) and its dimethylallyl diphosphate (DMAPP) isomer (Bach & Rohmer, 2012; Figure 6). In most organisms, IPP, the universal isoprenoid precursor, and DMAPP are synthesised via the cytosolic mevalonate (MVA) pathway (Figure 6). During the MVA pathway, three acetyl-CoA molecules are condensed by the en- zymes acetoacetyl-CoA thiolase (AACT) and HMGCoA synthase (HMGS) to form 3-hydroxy-3-methylglutaryl CoA (HMG-CoA). HMG-CoA reductase (HMGR) further converts HMG-CoA to mevalonate (MVA), from which the name of the pathway derives. After phosphorylation and decarboxylation by the enzymes mevalonate kinase (MVK), 5-phospho- mevalonate kinase (PMVK), and 5-diphosphomevalonate decarboxylase (DPMD), MVA is successively converted to IPP via mevalonate-5- phosphate (MVAP) and mevalonate-5-diphosphate (MVAPP; Figure 6). Plants and some photosynthetic organisms can additionally synthesise IPP and DMAPP via the plastidic methylerythritol phosphate (MEP) pathway (Vranova et al., 2013). For most bacteria, cyanobacteria, green algae and some red algae, the MEP pathway is the only pathway of isoprenoid biosyn- thesis, and these organisms lack MVA-pathway related enzymes (Masse et al., 2004; Lohr et al., 2012; Vranova et al., 2013 and references therein). Regardless of their biosynthetic pathway, the isoprene units derived from IPP and DMAPP are comprised of five carbons and give rise to various molecules. The monoterpene geranyl diphosphate (GPP, C10) consists of two isoprene units, while sesquiterpenes [such as farnesyl diphosphate (FPP, C15)] are built by three isoprene units, and the fusion of six isoprene units leads to triterpenes such as squalene (C30; Bach & Rohmer, 2012; Figure 6).

22 Figure 6. Triterpene biosynthesis via the MVA pathway leading to the sterol precursor 2,3 oxidosqualene. Molecule abbreviations: ACCOA, acetyl-CoA; AACCOA, aceto-acetyl-CoA; HMGCOA, 3-hydroxy-3-methylglutaryl-coenzyme A; MVA, mevalonate; MVAP, mevalonate phosphate; MVAPP, mevalonate diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; PSDP, presqualene diphosphate. Enzymatic steps are indicated with an asterisk above the enzyme abbreviation: AACT, acetoacetyl-CoA thiolase; HMGS, 3-hydroxy-3-methylglutaryl coenzyme A synthase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; MVK, mevalonate kinase; PMVK, phosphomevalonate kinase; DPMD, diphosphomevalonate decarboxylase; IDI, isopentenyl diphosphate isomerase; SQS, squalene synthase; SQE, squalene epoxidase; OSC, oxidosqualene cyclase.

23 As the prefix ‘tri’ indicates, three monoterpenes are used to assemble this chemical family with the molecular formula C30H64. In nature, a variety of triterpenes exist with linear structures such as squalene, or cyclic structures as in the sterols. Cyclic triterpenes are formed by the oxidation of the linear squalene at the first carbon-carbon double bond to form 2,3-oxidosqualene [catalysed by the enzyme squalene epoxidase (Sakakibara et al., 1995; Laden et al., 2000)]. The linear 2,3-oxidosqualene is then cyclised by oxido- squalene cyclases of different specificities, giving rise to various tetracyclic triterpenes such as sterols, or other pentacyclic triterpenes (Abe et al., 1993; Abe, 2007; Bach & Rohmer, 2012).

Sterols Sterols have been the focus of attention in many research fields, since the discovery of cholesterol, extracted from gallstones, over 250 years ago (Brown & Goldstein, 1986; Nes, 2011; and references therein). Cholesterol in particular is well studied and often described as “the most highly decorated small molecule in biology”. This is based on 13 Nobel prizes awarded to scientists working on the structure and physiology of this sterol (Brown & Goldstein, 1986). The importance of sterols for multicellular organisms ranges from physical to regulatory functions. Sterols stabilise the phospholipid bilayer of the plasma membrane to regulate membrane fluidity and permeability. Furthermore, steroidal hormones are required for cellular communication or in developmental processes. In some plants, sterols are the precursors of secondary metabolites such as steroidal glycoalkaloids or phytoecdysteroid related to plant defense properties (Dinan, 2001; Benveniste, 2004; Ginzberg et al., 2009; Milner et al., 2011; Mouritsen & Bagatolli, 2015). This highly diverse group of amphipathic molecules shares an overall feature in their native structure composed of a hydrocarbon steroid ring (C17) system consisting of four rings, named A, B, C and D (Figure 7).

Figure 7. The nomenclature and carbon numbering of steroids based on the IUPAC- IUB recommendation (Moss, 1989).

24 The basic 1,2-cyclopentanoperhydrophenanthrene ring skeleton, domain A, is substituted with a polar 3β-hydroxyl group at position C3, and an aliphatic side chain substitution of 8-10 carbon atoms is found at position C17 of the D ring. The stereochemistry of the side chains, critical for intermolecular interactions, can be modified during sterol synthesis. Additional modifica- tions of the ring structures such as double bonds in different numbers and positions are regularly found and affect the conformation and function of the sterol. In eukaryotic cells, a single or a very few major end sterols are typically found. For example, the most abundant sterol in animals is cholesterol. Ergosterol is the predominant sterol in fungi, while plants contain a series of 24-ethyl sterols, such as sitosterol, stigmasterol and campesterol. Nonetheless, cholesterol is present in fungi, plants and other organisms, in- cluding oomycetes (Madoui et al., 2009; Weete et al., 2010; Behmer et al., 2011; Warrilow et al., 2014).

Sterol biosynthesis Although sterol synthesis has been intensively studied in vertebrates, fungi, plants, and numerous sterols have been characterised in other eukaryotes, our knowledge of the sterol biosynthesis pathway is still considered incom- plete (Desmond & Gribaldo 2009; Fabris et al., 2014; Warrilow et al., 2014). The complexity of sterol synthesis is exemplified by the finding of only a few conserved enzymatic steps across kingdoms (Summons et al., 2006; Figure 8). For example, the cyclization of 2,3-oxidosqualene into lanosterol or cycloartenol by the oxidosqualene cyclase (OSC; E.C. 5.4.99.7) is known to be the most complex reaction in sterol synthesis and the OSC amino acid sequence is well conserved (Summons et al., 2006; Desmond & Gribaldo, 2009). OSC enzymes show variable specificity, producing different reaction products, and they are divided into two sterol synthesising groups. Lanosterol synthase enzymes (LAS; E.C. 5.4.99.7) cyclise 2,3- oxidosqualene to lanosterol, while cycloartenol synthase enzymes (CAS: E.C. 5.4.99.8) produce cycloartenol. The cyclization of oxidosqualene to either lanosterol or cycloartenol is the last common step among sterol syn- thesising organisms (Summons et al., 2006; Abe, 2007; Figure 8). Reaction products of the OSC enzyme differ because they arise from different mechanisms. A deprotonation of the C9 position of the protosteryl cation results in the formation of a double bond between C8 and C9, resulting in the cyclised lanosterol (Figure 8). Alternatively, a deprotonation at C19 of the protosteryl cation closes the cyclopropyl ring to form cycloartenol (Abe et al., 1993; Ohyama et al., 2009; Figure 8).

25

Figure 8. Sterol biosynthesis via lanosterol or cycloartenol to diverse end sterols among eukaryotic organisms. Sterols are synthesised from the precursor 2,3-oxidosqualene via the lanosterol or cycloartenol pathway. Variations between end sterols can be observed in the sterol nuclei or the substitution of the side chain.

Whether an OSC enzyme produces lanosterol or cycloartenol depends on minor differences in the amino acid pattern of the enzymes (Meyer et al., 2002; Sawai et al., 2006; Summons et al., 2006). It has been demonstrated that the introduction of simple point mutations at key positions can convert a CAS into a LAS, and vice versa (Meyer et al., 2002; Sawai et al., 2006). Specifically, the amino acids at positions 381, 449 and 453 (amino acid numbered for the human LAS enzyme) determine the final products of the specific LAS or CAS enzymes (Meyer et al., 2002; Kolesnikova et al., 2006; Sawai et al., 2006; Summons et al,. 2006). At these positions, the amino acids seem to be invariably Y, H and I in CAS enzymes (Figure 9). LAS enzymes are more variable, with either a T or Y in position 381, followed by C, Q, N or H in position 449, and finally a V at position 453 (Kolesnikova et al., 2006; Summons et al., 2006; Figure 9). Both lanosterol and cycloartenol then undergo further modifications to form the different end sterols presenting different substitutions and configurations fulfilling diverse functions (Grieneisen, 1994; Gunaherath & Gunatilaka; 2006, Milner et al., 2011; Nes, 2011; Figure 8).

26

tomato plants, and comprises the glycosidic units (β-D-galactose, (1→4) β-D-glucose, 1→2) β-D-glucose and (1→3) β-D-xylose (Friedman et al., 1997b).

Distribution of SGAs The levels of the potato SGAs α-solanine and α-chaconine in different plant tissues can differ strongly between plant cultivars, growth phase and condi- tions. In potato flowers, up to 5000 mg kg-1 fresh weight of SGAs have been reported, while in the pith of potato tuber no SGAs have been detected (Milner et al., 2011). For food safety reasons, because of their toxicity (discussed below), the SGA levels in commercially available potato tubers are limited to 200 mg kg-1 fresh weight (Korpan et al., 2004). The increase of potato SGAs in tubers/plants in response to abiotic and bio- tic factors has been studied extensively. Light exposure, heat and wounding of potato tubers can lead to a significant increase of SGAs (Milner et al., 2011; Petersson et al., 2013b).

Mode of action and physiological effects of SA and SGA Because of the toxicity of SGAs and SAs for numerous pests, pathogens and humans (Milner et al., 2011), their physiological effects have been investi- gated in vivo and in vitro (Roddick, 1979; 1989; Roddick & Rijnenberg, 1986; Keukens et al., 1992; 1995; 1996; Blankemeyer et al., 1995; 1997; 1998; Roddick et al., 2001).

Steroidal glycoalkaloid membrane disruption The ability of SGAs to disrupt membranes has been demonstrated for α-chaconine using in vivo assays. Synthetic liposomes consisting of a phos- pholipid/sterol membrane layer in which a peroxidase was encapsulated were exposed to α-solanine and α-chaconine. SGA concentrations in the range 75-150 μM resulted in concentration-dependent liposome leakage (Roddick & Rijnenberg, 1986; Roddick et al., 2001). Interestingly, no mem- brane disruption occurred when sterol-free phospholipid membranes were used (Roddick & Rijnenberg, 1986). Furthermore, SGA-dependent membrane disruption was affected by the pH and generally showed less activity with decreasing pH (Roddick & Rijnenberg, 1986, Fewell & Roddick, 1993; Milner et al., 2011). Fungi treated with the tomato SGA α-tomatine, revealed similar impaired growth at higher pHs, suggesting that the SGA are un-protonated, due to the increasing pH, and are therefore more active in a charged form (Sandrock & VanEtten, 1998; Friedman, 2002; Simons et al., 2006). Some SGAs showed synergistic effects when applied in combination, e.g., α-solanine and α-chaconine (Roddick et al., 2001). The effects of SGAs on complex membranes have been investigated using erythrocytes. The results reflecting hemolytic activity were similar to those

29 from the experiments with liposomes (Roddick et al., 2001). A depolariza- tion of the plasma membrane with a direct effect on membrane permeability and transport of ions was described also for Xenopus laevis embryos or frog skins exposed to SGAs (Blankemeyer et al., 1995; 1997; 1998). In particular, the carbohydrate side-chains of the SGAs seemed to have important effects on the depolarization of the membrane (Blankemeyer et al., 1997; 1998). Different mechanisms have been proposed for this phenomenon. In brief, SGAs are able to form complexes with membrane-located β-hydroxy sterols (Keukens et al., 1995; Stine et al., 2006). Increasing SGA accumulation sub- sequently rearranges the membrane by the formation of insoluble complexes that form membrane pores (Armah et al., 1999). Sterol-SGA complex accu- mulation leads to the build-up of an inner monolayer, which results in vesiculation of the membrane (Keukens et al., 1995). Moreover, when SGAs integrate in the membrane they migrate toward sterol-rich domains, thereby destabilizing lipid rafts, which presumably leads to mem- brane curvature (Keukens et al., 1995; Stine et al., 2006).

Steroidal glycoalkaloids as acetylcholinesterase inhibitors Acetylcholinesterase catalyses the breakdown of acetylcholine, a neuro- transmitter responsible for synaptic signal transmission (Tougu, 2001). The detection of an acetylcholinesterase inhibitory activity in potato water ex- tracts and other extracts from Solanaceous plants have already been reported in the 1950s (Orgell et al., 1958; Milner et al., 2011 and references therein). Direct studies of SGA inhibitory effects are summarised in a review by Milner et al. (2011). These studies show that the inhibitory effects on plasma or serum acetylcholinesterase (EC 3.1.1.7) or on butyrylcholin-esterase (EC 3.1.1.8) differed depending on the enzyme’s origin (fetal bovine serum vs. human serum) and on the SGA used (Milner et al., 2011). For example, α-tomatine showed a greater effect on human serum acetylcholinesterase than on acetylcholinesterase extracted from fetal bovine serum (Fletcher et al., 2004). Remarkably, the Colorado potato beetle acetylcholinesterase dis- played up to 150-fold lower sensitivity towards the potato SGA α-chaconine, compared to the enzymes of housefly, cottonwood leaf beetle, mosquito or the German cockroach (Wierenga & Hollingworth, 1992). Despite the importance of the glycosidic unit for acetylcholinesterase inhibi- tion, the inhibition coefficient also depended on the structure of the agly- cone. This could be demonstrated when a comparison of α-solanine and α-solasonine, or of α-chaconine and α-solamargine was performed. These SGAs possess the same glycosidic units, but differ in their aglycone structures (Milner et al., 2011).

30 Sterol and steroidal glycoalkaloid interactions with oomycetes

Sterols Despite the diversity and importance of sterols for eukaryotic organisms, no sterol biosynthesis pathway has been reported for several arthropods, nema- todes, oomycetes and some other organisms (Dutky et al., 1967; Grieneisen, 1994; Chitwood, 1999; Behmer & Nes, 2003; Gaulin et al., 2010). The oomycetes in particular are an important group for the study of sterol synthe- sis and acquisition, since they differ in their sterol needs within the group (Elliott et al., 1964; Gottlieb et al., 1978; Gaulin et al., 2010). For example, members of the order Saprolegniales have the ability to grow on sterol-free media, while oomycetes from the order Peronosporales must acquire sterols from their hosts (Gottlieb et al., 1978; Nes, 1987; Gaulin et al., 2010; Madoui et al., 2009). Genomic and transcriptomic data from S. parasitica and A. euteiches, which belong to the Saprolegniales, reveal the occurrence of most enzymes of a complete sterol biosynthetic pathway (Madoui et al., 2009; Jiang et al., 2013; Warrilow et al., 2014). The sterols synthesised by S. parasitica are cholesterol, desmosterol, 24-methylenecholesterol, fucosterol and lanosterol (Warrilow et al., 2014). A. euteiches additionally synthesises lathosterol and 7-dehydrocholesterol, but not desmosterol (Madoui et al., 2009). Notably fucosterol, a sterol first documented in brown algae, is commonly found in sterol-synthesising oomycetes (Elliott, 1977; Warner et al., 1982). Whether the cyclization product of 2,3-oxidosqualene is lanosterol or cycloartenol was strongly debated in the oomycete community during the 1980s, and until recently, the question is not answered unambiguously (Warner & Domnas 1981; Warner et al., 1982; Berg & Patterson, 1986; Nes et al., 1986; Briolay et al., 2009; Madoui et al., 2009; Warrilow et al., 2014). The absence of sterol synthesis in Peronosporales was experimentally pre- dicted and bioinformatically confirmed to lie at the oxidation of squalene to 2,3-oxidosqualene (Gottlieb et al., 1978; Wood & Gottlieb, 1978; Fabris et al., 2014). Feeding studies with radioactively labeled squalene did not show any conversion to 2,3-oxidosqualene in Phytophthora cinnamomi and bioinformatics analysis could not identify a squalene epoxidase in P. infestans (Gottlieb et al., 1978; Wood & Gottlieb, 1978; Fabris et al., 2014). However, some Peronosporales species which cannot synthesise their own sterols (Phytophthora sp. and Hyaloperonospora arabidopsidis), do encode a putative Δ7 sterol reductase for possible sterol modification (Gottlieb et al., 1978; Madoui et al., 2009; Gaulin et al., 2010). In spite of the lack of a squalene epoxidase, sterol modification was reported for Peronosporales fed with lanosterol or cycloartenol (Warner et al., 1982).

31 The ability to modify sterols acquired from other organisms has also been reported for sterol-dependent insects and nematodes (Chitwood, 1999; Behmer & Nes, 2003). Interesting preliminary data revealed an oxidative burst as a plant response as well as the upregulation of defense genes in the plant Medicago truncatula exposed to ergosterol (a sterol produced by fungi), but not to cholesterol or fucosterol (Gaulin et al., 2010). P. infestans has also been shown to inhibit the expression of the squalene synthase and induce the sesquiterpenoid cyclase in inoculated potato tubers. Both are key enzymes for sesquiterpenoid phytoalexins and sterol biosynthe- sis (Yoshioka et al. 1999). Moreover, screening for additional non-host resistance in Arabidopsis thaliana, P. infestans susceptible pen2 mutants [penetration-resistance genes (pen2)] were submitted to additional rounds of mutagenesis. The screen based upon P. infestans inoculation resulted in the identification of phospholipid: sterol acyltransferase (PSAT1) mutants with enriched meso- phyll cell death and increased callose deposition (Kopischke et al., 2013). The mutation in the PSAT1 gene resulted in impaired formation of sterol ester synthesis and increased sterol glycoside accumulation in the mutated plants. This suggests that sterol conjugates play an important role as defense response to P. infestans and other filamentous pathogens (Kopischke et al., 2013).

Steroidal glycoalkaloids (SGAs) Even though the inhibition of growth by SGAs has been described for nu- merous plant pathogens in vitro (Nes et al., 1982; Fewell & Roddick., 1997; Sandrock & VanEtten, 1998; Ito et al., 2007; Seipke & Loria, 2008; Munafo & Gianfagna, 2011), the limited field trials available on the effect of SGA- dependent resistance are contradictory and inconclusive (Sarquís et al., 2000; Andreu et al., 2001; Milner et al., 2011). The same lack of evidence exists for the effects of potato SGAs on P. infestans. Many results are unclear and studies performed on plants and tubers showed no direct impact or only weak effects of SGAs on the growth of P. infestans (Deahl et al., 1973; Sarquís et al., 2000; Andreu et al., 2001; Andrivon et al., 2003; Carlson-Nilsson et al., 2011). Some studies showed a correlation between low α-solanine contents of S. tuberosum and Solanum vernei lines with P. infestans spore production (Andrivon et al., 2003). A significant correlation between α-chaconine content and plant defense responses (lesion size) was shown in one study (Carlson-Nilsson et al., 2011). But no consistent effects on P. infestans growth could be assigned to α-chaconine or α-solanine (Andrivon et al., 2003; Carlson-Nilsson et al., 2011). Former investigations could not find any correlation between foliar or tuber SGA contents of S. tuberosum and resistance to P. infestans (Kuc,

32 1984; Sarquís et al., 2000; Andreu et al., 2001). This might be due to numerous reasons. Investigations on SGA effects showed that their toxicity often impacts specialised pathogens much less than non-host or generalist pathogens (Fewell & Roddick, 1993; 1997; Milner et al., 2011). Additionally, one publication suggests that P. infestans has the ability to deglycosylate α-solanine into solanidine to some extent (Holland & Taylor, 1979). This mechanism might detoxify SGAs, similar to what is known for some other pathogens of Solanaceae, such as the fungus Fusarium oxysporum f.sp. lycopersici, Septoria lycopersici or Cladosporium fulvum (Osbourn et al., 1995; Sandrock et al. 1995; Lairini et al., 1996; Milner et al., 2011). F. oxysporum detoxifies α-tomatine by the action of a tomatidinase enzyme (FoTom1), which releases the intact α-tomatine lycotetraose group from the aglycone tomatidine (Lairini et al., 1996). This enzyme is a glycoside hydrolase, and cleaves the glycosidic linkage between the sugar and the steroidal alkaloid moieties of α-tomatine. This SGA detoxification by tomatidinase is also believed to play a role in pathogenicity since the expression of the tomatinase FoTom1 is induced by α-tomatine (Roldán- Arjona et al., 1999). The cleaved aglycone tomatidine and the tetra- saccharide lycotetraose, additionally increase the pathogenicity of F. oxysporum by suppressing induced plant defense responses (Ito et al., 2004). A similar process of cleavage of the entire sugar moiety can be assumed for P. infestans since no partly deglycosylated intermediates were reported for the transformation from α-solanine to solanidine (Holland & Taylor, 1979). This led to the hypothesis that SGA detoxification might play a role in P. infestans pathogenicity, or as previously proposed, SGAs might serve as a sterol source for P. infestans (Andrivon et al,. 2003) and are deglycosylated for this purpose (Holland & Taylor, 1979).

33 Aims of this thesis

The aims of the investigations described in this thesis were to analyse sterol synthesis/modification and sterol acquisition in the oomycetes S. parasitica and P. infestans, to identify the origins of the SGAs in potato, and to understand their impact on P. infestans growth.

The following investigations were made:

1.) Identification and characterisation of the 2,3-oxidosqualene cyclase enzyme, which produces the key sterol intermediate of the sterol synthesising oomycete S. parasitica. 2.) Use of bioinformatics to analyse the sterol biosynthesis pathway of S. parasitica and to investigate the possibilities of P. infestans to alter sterols obtained from its host plants. 3.) Determine the origin of the SGA precursors in potato by using shoots fed with deuterium-labeled sterols. 4.) Analyse the direct impact of Solanaceous SGA and SA on growth of P. infestans under in vitro conditions. S. parasitica and P. infestans, each representing one of the main oomycete orders, are well suited for detailed investigations because they can be cul- tured in synthetic media, have a short life cycle in the laboratory, and most important, their genomes are fully sequenced and available at the BROAD Institute and NCBI databases (Paquin et al., 1997; Haas et al., 2009; Jiang et al., 2013).

34 Comments on methodologies used

The research depicted in this thesis encompassed various methods described in depth in the four manuscripts presented. Here, the relevant techniques used to analyse sterols and steroidal glycoalkaloids will be summarised. Based on the polarity of these compounds (Dinan et al., 2001), this section is separated in two parts, sterols and steroidal glycoalkaloids (SGAs).

Isolation, separation and analysis of sterols Sterols belong to the diverse class of lipids and are characterised by their solubility in different organic solvents, which is important for their extrac- tion, structural analysis and quantification (Dowhan et al., 2008). The sterols must be extracted and isolated prior to analysis to separate them from other molecules with similar properties (Azadmard-Damirchi & Dutta, 2006; Lagarda et al., 2006). For the extraction and isolation steps, liquid-liquid extraction methods such as the Bligh and Dyer or the Folch methods are typically used to obtain total lipid fractions (Iverson et al., 2001). Consecutively, different organic sol- vents were used in combination with an aqueous phase in order to separate polar from non-polar compounds from milled or homogenized samples (Abidi, 2001; Dinan et al., 2001; Iverson et al., 2001). In this thesis, the Bligh and Dyer method was used to extract sterols from leaves and stems of potato plants and from oomycete mycelium. In all cases, the biological material was frozen and homogenized into powder under liquid nitrogen (Bligh & Dyer, 1959; Manuscripts II, III and IV). The separation of sterols from the total lipid fraction can be achieved by various methods, such as thin layer chromatography (TLC) and solid-phase extraction (SPE) (Abidi, 2001; Azadmard-Damirchi & Dutta, 2006). As an example, Figure 11 shows the separation of lipids in total extracts of S. parasitica and P. infestans by 1D-TLC and 2D-TLC revealed by copper sulfate (CuSO4). Showing the diversity of lipids present and an efficient separation of sterols from the other groups of lipids. These sterols can be recovered by scraping off the TLC plate for further analysis. After separation by TLC, solid-phase extraction (SPE) was applied (Azadmard-Damirchi & Dutta, 2006). SPE is a quick method that yields a higher recovery of sterols per sample fresh weight than the TLC method, which is important for further analyses. The final sterol detection, identification and quantification are commonly performed using methods such as TLC, gas (GC) or liquid (LC) chromatography coupled to mass spectrometry (MS), or a flame ionization detector (FID) (Dais, 2010; Tan et al., 2013). In this project, GC-MS was chosen for sterol analysis.

35 rod pair is connected and displays the same electrical radio frequency. Depending on the m/z ratio of the ion, it will collide with the rods or be se- lected and reach the detector. The ionized molecules are detected by recording the charge induced to the current produced. Based on the generated signals, the different sterols were identified and quantified using an internal sterol standard (Aitzetmüller et al., 1998; Laakso, 2005; Manuscripts I-IV).

Isolation, separation and quantification of steroidal glycoalkaloids The extraction and separation of steroidal glycoalkaloids was performed using a method based on aqueous solvents, i.e., a weak acid to which methanol or tetrahydrofuran was added (Edwards & Cobb, 1996; Oleszek, 2002 and references therein). After separation of the SGAs from the cell debris by centrifugation, the clear solvent containing the SGAs was loaded to an activated SPE column for further purification. For the analysis of SGAs, several methods can be used. TLC, LC such as high-performance liquid chromatography (HPLC), GC, enzyme-linked immunosorbent assay (ELISA) and colorimetric methods have been applied to SGA analysis (Friedman et al., 1997a; Milner et al., 2011). However, the most common method is HPLC coupled to a UV-detector (wavelengths of 200-210 nm) or with pulsed amperometric detection (PAD) (Milner et al., 2011). The structural determination of the SGAs can be performed by MS or nuclear magnetic resonance spectroscopy (NMR) (Milner et al., 2011), respectively. In contrast with the GC separation of sterols for MS analysis, no derivatization or hydrolysis steps were needed for the analysis of SGAs. Therefore, the analysis of LC-MS separated and purified SGAs did not re- quire additional preparatory steps. In this study, TLC, LC-UV or LC-tandem MS were used for analysis.

TLC analysis of SGAs TLC separation is a powerful method to resolve SGA mixtures. Fractionated SGAs can be purified for further analysis. In this study, silica plates were used as solid phase and chloroform, methanol and aqueous ammonium hydroxide as liquid phase for the separation of the SGAs. Generally, a great variety of solvents can be used for SGA separation, such as chloroform- methanol-water, butanol-acetic acid-water, or chloroform-methanol-aqueous ammonium hydroxide (Friedman et al., 1997a; Simonovska & Vovk, 2000; Dinan et al., 2001). Different reagents can be applied to reveal SGA spots on TLC as described in Dinan et al. (2001). Here, a CuSO4 solution was used, followed by heating for the visualization of SGAs.

37 SGA analysis by liquid chromatography coupled to UV detection The detection of SGAs by UV has limitations because of their narrow absorption range between 200-215 nm (Friedman et al., 1997a; Milner et al., 2011). In this wavelength range, many small compounds can be detected, so a good separation is required. This puts further limitations on the choice of solvent, which has to be transparent to UV radiations for detection (e.g. water, methanol, ethanol, acetonitrile are suitable). The use of a gradient should be considered (Friedman et al., 1997a; Petersson et al., 2013a). However, based on the improvement of SGA extraction and cleanup proce- dures, the sensitivity of UV detection can be satisfactory, as shown in our study in Manuscript III. The SGAs obtained after SPE were separated on a C18 column with a mobile phase consisting of phosphate buffer and acetoni- trile; the UV detector was set to 202 nm.

SGA analysis by liquid chromatography-tandem mass spectrometry The advantage of tandem MS analysis is the ability to elucidate the structure of not only the aglycone, but also of the attached glycoside (Milner et al., 2011). In the current study, a triple quadrupole tandem MS analyser was used which consists of two quadrupole mass analysers that are connected in series. SGAs were ionized before selection in the first m/z selector (named Q1). The selected ions then reach the quadrupole collision cell (named Q2) where they are fragmented, producing characteristic daughter ions, which can be further selected in the quadrupole mass filter Q3 before detection. Thus, tandem MS is a scanning instrument that allows the elucidation of the structures of the SGAs, which represents a major advantage compared to single MS analysis. The high sensitivity and the short time required for each analysis makes LC- MS/MS the state-of-the-art method for detection and structural determina- tion of SGAs (Milner et al., 2011; Hossain et al., 2015). This way, changes in molecular weight can be detected as described in Manuscript III.

38 Results and discussion

The oxidosqualene cyclase from the oomycete Saprolegnia parasitica synthesizes lanosterol as a single product (Paper I) To identify the key intermediates in the sterol biosynthetic pathway of S. parasitica, bioinformatic, phylogenetic and biochemical approaches were combined. A search for the enzymes cyclising 2,3-oxidosqualene into lanosterol or cycloartenol in the S. parasitica genome led to the identifica- tion of two predicted oxidosqualene cyclase sequences. Gene SPRG_11783 encoded an 805-amino-acid protein containing two predicted catalytic domains, a squalene-hopene cyclase N-terminal domain (PF13249) and a squalene-hopene cyclase C-terminal domain (PF13243). The second selected gene SPRG_17895 represented a truncated duplicate of gene SPRG_11783. SPRG_17895 starts at amino acid 334 of full-length SPRG_11783, so it only contains the squalene-hopene cyclase C-terminal domain (PF13243). With three amino acid differences, the 472-amino-acid long SPRG_17895 protein shares over 99% similarity with the amino acid sequence of SPRG_11783. When lined up with the human lanosterol synthase sequence, the amino acid positions 381, 449 and 453 are distinctly conserved among lanosterol synthases and cycloartenol synthases. These important amino acid regions are represented by the positions 450, 518 and 522 in SPRG_11783 and 117, 185 and 199 in SPRG_17895, respectively. Both S. parasitica proteins dis- played an unusual YNV pattern in these positions. This pattern had earlier been observed in plants; the corresponding enzymes of A. thaliana and Lotus japonicus had been characterised as lanosterol synthases. Therefore, SPRG_11783 and SPRG_17895 are respectively referred to as SpLASA (SPRG_11783) and SpLASB (SPRG_17895) in the remainder of this text. Phylogenetic analysis of OSC enzymes grouped SpLASA and SpLASB within the Heterokonta superphylum. Four additional oomycete OSCs grouped closely within the selected S. parasitica protein sequences reflecting the presence of the same YNV pattern. Interestingly, the other Heterokonta OSCs displayed a YHI pattern linked to the conversion of 2,3-oxidosqualene into cycloartenol; i.e., they represented CAS enzymes. For functional characterisation of the genes encoding SpLASA and SpLASB, the coding sequences were amplified from cDNA by PCR and cloned into the expression vector pET-DEST42. A recombinant protein could be puri- fied using its His6 tag only for the E. coli clone harbouring the gene SpLASA. The reaction assay where SpLASA was incubated with the substrate 2,3- oxidosqualene yielded lanosterol as a unique product (Manuscript I). These experiments confirmed an enzyme concentration-dependent conversion of 2,3-oxidosqualene into lanosterol, pointing towards a lanosterol synthesis pathway in S. parasitica.

39 Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans (Paper II) To advance our understanding of sterol acquisition in oomycetes, the sterol- autotrophic S. parasitica and the sterol-heterotrophic P. infestans were com- pared. P. infestans is dependent on sterol supply for growth and reproduc- tion, but based on bioinformatic analysis does possess enzymes predicted to modify sterols. Based on the sterol profile of S. parasitica and a group of 13 predicted genes encoding enzymes involved in sterol biosynthesis, the complete sterol bio- synthetic pathway of S. parasitica was constructed, starting with the previously characterised S. parasitica lanosterol synthase enzyme (Figure 12; Manuscript I). Starting with lanosterol, sterol biosynthesis is predicted to follow a common path to zymosterol where it potentially diverges into a cholesterol/ desmosterol pathway and a fucosterol pathway. The cholesterol/desmosterol pathway likely diverges at the following sterol (5α-cholesta-7,24-dien-3β-ol) into a cholesterol and desmosterol branch. This is based on the two genes SPRG_05001 (Δ24 sterol methyltransferase) and SPRG_04988 (Δ24 sterol reductase). Metabolites might be exchanged between the three different branches. The expression of the genes encoding enzymes involved in sterol biosynthe- sis in S. parasitica mycelium was confirmed by quantitative reverse transcription-polynucleotide chain reaction (RT-qPCR) analysis. Interestingly, changes of the growth medium (use of Machlis, Yeast Mold or Peptone media) resulted in variations in the sterol profile but, had no obvious effects on gene expression levels. These changes in the sterol profile of S. parasitica had not been reported previously and should be the subject of further investigations. The ability of P. infestans to modify sterols acquired from its host was investigated. Previous sterol feeding studies on Peronosporales species had demonstrated that minor proportions of sterols were converted into alternative forms (Nes & Patterson, 1981; Warner et al., 1982; Nes & Stafford, 1983). Therefore, a sterol feeding study was per- formed using a group of sterols from plants, fungi and animals. Bioinformatic mining of the P. infestans genome resulted in the identifica- tion of two putative sterol synthesis-related genes encoding a putative Δ5 sterol desaturase (PITG_21426) and a putative Δ7 sterol reductase (PITG_13128). The putative Δ7 sterol reductase had been described previously for Phytophtora sp. by Madoui et al. (2009). The enzymes encoded by these two genes, the putative Δ5 sterol desaturase (PITG_21426) supposed to act on lathosterol, and the putative Δ7 sterol reductase (PITG_13128) supposed to act on ergosterol, had not been considered to play a role in P. infestans sterol modification before.

40

A targeted feeding study was applied for the corresponding enzymes evalu- ating the sterol profile after growth on two selected sterols, lathosterol and ergosterol. Sterol profiles and transcription of the two genes were analysed and compared with cultures grown on non-substrate sterols (β-sitosterol, cholesterol, lanosterol and zymosterol). The results did not confirm P. infestans sterol modification in vivo, nor did they show significant changes in the transcript abundance of the two selected genes. Hence, the function of the genes encoding a putative Δ5 sterol desatu- rase and a putative Δ7 sterol reductase, and the ability of P. infestans to modify sterols remain unclear.

Conversion of exogenous cholesterol into glycoalkaloids in potato shoots, using two methods for sterol solubilisation (Paper III) The Solanaceous steroidal glycoalkaloids are secondary metabolites derived from sterols. In potato, the glycosylation of the main sterol alkaloid requires three enzymes. Early research suggested cholesterol as an important inter- mediate for SGA synthesis, which would not be surprising since cholesterol represents a substantial part of the total sterols in Solanaceous plants such as potato. Therefore, the role of cholesterol in the synthesis of SGAs was examined. Etiolated potato sprouts were fed with deuterium-labelled cholesterol, 27- hydroxycholesterol and sitosterol solubilized in methyl-β-cyclodextrin (MBD), or solubilized by addition of the detergent Tween-80. D5- or D6- cholesterols were labelled in their parent ring structures A and B. D7-cholesterol was labelled in the sterol side chain, and so were the sterols D6-27-hydroxycholesterol and D7-sitosterol. Potato sprouts fed with 200 μg of labelled sterols were harvested after 3 or 5 weeks for SGA analysis. The results differed dramatically based on the sol- vent used. Solubilisation by MBD led to a greater conversion of precursors into SGAs than solubilisation using Tween-80. Additionally, the in vivo conversion of the side chain-labelled D7-cholesterol revealed that two hydrogen atoms were released during the SA ring closure. Side chain- labelled D7-sitosterol or D6-27-hydroxycholesterol were not recovered as SGAs in measurable quantity. The finding that only cholesterol was converted to the SGAs α-solanine and α-chaconine, supports and broadens the results of earlier investigations on SGA biosynthesis in potato.

42 The impact of steroidal glycoalkaloids on the physiology of Phytophthora infestans, the causative agent of the potato late blight (Paper IV) P. infestans which causes lesions on foliage and rotting of potato tubers is responsible for billions of dollars in potato loss globally. Solanaceous plants such as potato produce a selection of SGAs which are assumed to have plant protective properties (Friedman, 2002; 2004; Milner et al., 2011). The proposed mechanisms are attributed to their disruption of membranes (Milner et al., 2011). On the pathogen side, it has long been known that some organisms, e.g., Fusarium sp. (Roldán-Arjona et al., 1999; Pareja- Jaime et al., 2008) can deglycosylate and thereby detoxify SGAs. Apparently, P. infestans has a limited capacity to deglycosylate α-solanine into solanidine (Holland & Taylor, 1979). Therefore, we studied whether and how P. infestans can overcome SGA toxicity. P. infestans growth was analysed on media supplemented with SGAs com- pared to control media. The results indicated that the two common potato SGAs α-chaconine and α-solanine had no effect on the physiology of the mycelia. Contrarily, the non-glycosylated SGA precursor solanidine exhibited a greater physiological impact, inhibiting mycelial growth. While all tested potato-derived alkaloids were taken up by the mycelium, no degly- cosylation of SGAs by P. infestans was found. Solanidine trace analysis by GC-MS confirmed that neither α-chaconine nor α-solanine was converted to solanidine by releasing the trisaccharide unit (Manuscript IV). Since the genome of P. infestans contained several genes encoding potential SGA- active glycoside hydrolases, the effects of extracellular hydrolases on SGAs were investigated. Extracted P. infestans proteins secreted during growth on SGA enriched liquid media were tested for release of α-chaconine and α-solanine, L-rhamnose, D-glucose and D-galactose by HPAEC-PAD (High- Performance Anion-Exchange Chromatography Coupled to Pulsed Amperometric Detection). Additionally, secreted proteins were tested for their ability to release the three saccharide moieties linked to the model substrates aryl glycosides used as substrates. No release of L-rhamnose, D-galactose or D-glucose was detected (Manuscript IV). Additional studies on the inhibitory effects of SGAs and SAs derived from tomato and aubergine revealed small but important differences in their agly- cones and their glycosidic units. The growth results for non-potato derived SGAs and SAs suggest adaptation of P. infestans to some potato-derived SGAs. Altogether, this study could not confirm that P. infestans could deglycosylate α-solanine into solanidine as reported previously (Holland & Taylor, 1979). Yet, evidence for host-specific adaptation of P. infestans to potato SGAs other than by direct detoxification as shown for other organisms, could be found (Pareja-Jaime et al., 2008; Okmen et al., 2013).

43 Given that deglycosylation of the potato SGA α-chaconine or α-solanine to solanidine would make them toxic to P. infestans mycelium, the lack of a mechanism for hydrolysis is not surprising.

Conclusion In this thesis, the first isolation and characterisation of an oomycete 2,3- oxidosqualene cyclase (OSC) gene from the fish pathogen S. parasitica is described. The catalytic domain of the predicted OSC has a rare sequence motif common to oomycetes of the saprolegnian clade and some lanosterol synthases of plants. Cloning, over-expression and functional characterisation of the S. parasitica OSC confirmed that it had lanosterol synthase activity. These results open new opportunities for detailed structural and functional characterisation of 2,3-oxidosqualene cyclases (LAS) as novel targets for inhibitors of sterol biosynthesis in S. parasitica. Moreover, the reconstruction of the complete sterol synthesis pathway of S. parasitica allowed the identification and characterisation of LAS, which can be used as potential drug targets. The reconstruction of the sterol synthesis pathway for S. parasitica is also useful to understand the sterol composition of this organism and of the order Saprolegniales in general. Variations in sterol profiles dependent on the composition of the growth media led to the conclusion that sterol synthesis and modification are environmentally controlled in S. parasitica. The importance of sterols for oomycetes can also be exploited to control the sterol-heterotrophic P. infestans. In this context, the potential of P. infestans for the modification of sterols was examined, with the conclusion that under the growth conditions used such potential does not exist. Thus, the sterol uptake mechanism as such is the most promising target of control strategies. By investigating the steroidal origin of steroidal glycoalkaloids in potato shoots, we showed that cholesterol could be taken up and converted to SGAs. This conversion was not found for sitosterol and 27-hydroxy- cholesterol, leading to the conclusion that cholesterol is a specific SGA pre- cursor in potatoes. A side result was that methyl-β-cyclodextrin, a cyclic oligosaccharide, was ideally suited for solubilizing cholesterol for delivery into plants. This finding can be used further for the exogenous application of sterols and sterol derivatives, for example in the context of metabolic studies on potato plants or the investigation of sterol uptake by pests or pathogens. Despite the toxic effect of SGAs on many organisms, from microbes to humans, P. infestans displayed an unidentified resistance mechanism towards potato SGAs. In comparison, P. infestans was more susceptible towards the aubergine SGA solamargine and the tomato SGA α-tomatine.

44 However, the SAs of potato and aubergine showed a strong inhibitory effect on the growth of P. infestans mycelium compared to the tomato SA tomatidine. Ultimately, in planta experiments have to be performed to con- firm the effects of SGAs and SAs on the growth of P. infestans. However, the fact that SGAs are toxic to humans limits the potential for targeted breeding.

Research perspectives • In order to understand sterol synthesis and end sterol accumulation in oomycetes, RNA sequencing, proteomic and metabolomics analyses should be applied to reveal changes from the cellular transcriptome up to the final end sterol. A time-course proteomic study on different growth media could be useful to identify genes possibly responsible for end sterol changes and understanding variations in sterol composition in different growth condi- tions. • Targeted gene knock down of the characterised S. parasitica lanosterol synthase (gene SPRG_11783) might reveal the function of the uncharacterised gene SPRG_17895, potentially encoding a truncated lanosterol synthase. Additionally, the knock down of SPRG_11783 will reveal how essential SPRG_11783 is for the organism and for lanosterol synthesis in S. parasitica. The CRISPR/Cas9 approach can be used for tar- geted gene knock down as it was newly demonstrated to target an effector gene in Phytophthora sojae (Fang & Tyler, 2016). • S. parasitica genes SPRG_04988 (Δ24 sterol reductase) and SPRG_05001(Δ24 sterol methyltransferase) potentially responsible for directing the end sterol towards cholesterol, desmosterol and/or fucosterol accumulation, should be functionally characterised in vitro by cloning and overexpression, and in vivo by knock out to address their proposed function in end sterol changes. • Additional S. parasitica sterol synthesis inhibitory studies with previously characterised sterol synthesis inhibitors should be performed to determine their potential use as inhibitors of S. parasitica. • Regarding the non-sterol synthesizing oomycete P. infestans: sterol carrier proteins called elicitins could be investigated as potential drug targets to unable P. infestans sterol uptake. Selected elicitins could be cloned, overexpressed and purified for sterol and sterol analog-drug binding studies. • MBD-solubilized autofluorescent sterols (e.g. dehydroergosterol) fed to plants could be used to study in planta trafficking and uptake of sterols by, e.g., P. infestans or aphids. • To clarify the mechanisms of the predicted Δ5 sterol desaturase (PITG_21426) and Δ7 sterol reductase (PITG_13128), the genes could be cloned, overexpressed and functionally characterised.

45 • To understand the increased physiological effects of SGA synthesized from tomato or aubergine on P. infestans strain obtained from potato, the physio- logical effect on P. infestans strains obtained from tomato and aubergine plants could be studied. • Characterisation of the potential glycoside hydrolases selected during the work presented in Manuscript III.

46 Acknowledgements

I take this opportunity to express my deep sense of gratitude to the people who have made this thesis possible. I am grateful for all the support, invaluable constructive criticism and friendly advice throughout the years.

Firstly, I would like to thank my supervisors: Dr. Vincent Bulone who made the thesis possible; Dr. Lauren McKee for the continuous support in the lab, with the writing of the Manuscripts and her valuable comments on my thesis; Dr. Sophia Ekengren for introducing me to a new work environment and all her support during lab work and the writing process of my thesis; Dr. Katharina Pawlowski for always being there when needed, her ideas, exceptional input and immense knowledge, the support during laboratory questions and the writing process of my thesis. Furthermore, I want to thank Dr. Lisbeth Jonsson, Dr. Folke Sitbon and Dr. Lisa Beste for introducing me to, and broaden my knowledge on sterols and steroidal alkaloids.

Besides my advisors: I would like to thank Dr. Andrea Ruthes for her insightful comments, help and support on my thesis and her endless encou- ragement; Erik Peterson for his great support at SLU Uppsala; Hugo Melida and Vaibhav Srivastava for their kind support at KTH. A particular thank goes to Marion Mueller, I really enjoyed working with you and wish you all the best for the future.

Christian Brown, Stefan Klinter, Felicia Leijon, Erik Malm, Ashley Tan, Elzbieta Rzeszutek, Nuria Butchosa, Zhili Pang, Osei Ampomah, Cortwa Hooijmaijers, Antonio Martinez, Sujit Kootala, Francisco Vilaplana, Annie Inman, Sara Diaz, Kun Yao, Qijun Meng, Ela Nilson, Nina Bauer and the entire Glycoscience Division. Peter, Maya, Stephan, Henrik, Andrea and Lisa, thanks for the nice interaction and lab support when needed. I would also like to thank Felicia Leijon for the translation of the Abstract into Swe- dish.

A special thanks to all my former lab members at SU and SLU. In particular Aleksandra, Lina, Sandra, Denis, Sara M., Marco, Van, Irina, Andrea C., Diana, Afaf and Selchuck. A particular thanks to Denis for all your support, great scientific discussions and the great time spent together with you. Thank you Dr. Ulla Rasmussen, without you I would have not finished my PhD!

47 Special thanks to Monica Winder and Peter Hambäck for all their support during my PhD.

I also would like to thank Dr. Johannes Hallmann and Dr. Mark Martindale for encouraging my research career and for supporting me in my growth as a scientist.

I would like to thank all people that contributed to my publications whom I have not met in person, but who are linked to my thesis by their scientific input.

I would like to thank my opponent and thesis committee members for taking their time, for their presence at my defense and for all the scientific input.

And I would like to thank the Department of Ecology, Environment and Plant Sciences at Stockholm University to accept me as a PhD-student and for providing the financial support.

Ein besonderer Dank gilt meinen Eltern und Geschwistern, die mich auf meinem Weg durch das Studium begleitet und mich mit vielen lieben Worte moralisch unterstützt und gestützt haben.

To all who, in one way or another, were important for the accomplishment of this thesis and for my scientific development, Thank you!

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