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Analysis of sterol metabolism in the pathogenic oomycetes Saprolegnia parasitica and Phytophthora infestans
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.ORCID iD: 0000-0002-9945-8059
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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 exogenously 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 various 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 glycoalkaloids.  Steroidal glycoalkaloids derived from other Solanaceous species exhibited 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.

Place, publisher, year, edition, pages
Stockholm: Department of Ecology, Environment and Plant Sciences, Stockholm University , 2016. , p. 59
National Category
Biochemistry and Molecular Biology
Research subject
Plant Physiology
Identifiers
URN: urn:nbn:se:su:diva-136551ISBN: 978-91-7649-652-7 (print)ISBN: 978-91-7649-653-4 (print)OAI: oai:DiVA.org:su-136551DiVA, id: diva2:1055152
Public defence
2017-01-27, sal P216, Arrheniuslaboratorierna, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.

Available from: 2017-01-02 Created: 2016-12-11 Last updated: 2017-01-27Bibliographically approved
List of papers
1. The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product
Open this publication in new window or tab >>The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product
2016 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 1802Article in journal (Refereed) Published
Abstract [en]

The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

Keyword
lanosterol biosynthesis, oomycete, Saprolegnia parasitica, oxidosqualene cyclase, sterols
National Category
Biological Sciences
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-136547 (URN)10.3389/fmicb.2016.01802 (DOI)000388698600001 ()
Projects
No
Available from: 2016-12-11 Created: 2016-12-11 Last updated: 2017-11-29Bibliographically approved
2. Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans
Open this publication in new window or tab >>Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets.

National Category
Biological Sciences
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-136550 (URN)
Available from: 2016-12-11 Created: 2016-12-11 Last updated: 2016-12-12Bibliographically approved
3. Conversion of Exogenous Cholesterol into Glycoalkaloids in Potato Shoots, Using Two Methods for Sterol Solubilisation
Open this publication in new window or tab >>Conversion of Exogenous Cholesterol into Glycoalkaloids in Potato Shoots, Using Two Methods for Sterol Solubilisation
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 12, article id e82955Article in journal (Refereed) Published
Abstract [en]

Steroidal glycoalkaloids (SGA) are toxic secondary metabolites naturally occurring in the potato, as well as in certain other Solanaceous plant species, such as tomato, eggplant and pepper. To investigate the steroidal origin of SGA biosynthesis, cut potato shoots were fed cholesterol labelled with deuterium (D) in the sterol ring structure (D-5- or D-6-labelled), or side chain (D-7-labelled), and analysed after three or five weeks. The labelled cholesterol and presence of D-labelled SGA were analysed by GC-MS and LC-MS/MS, respectively. When feeding D-labelled cholesterol solubilised in Tween-80, labelled cholesterol in free form became present in both leaves and stems, although the major part was recovered as steryl esters. Minor amounts of D-labelled SGA (alpha-solanine and alpha-chaconine) were identified in cholesterol-treated shoots, but not in blank controls, or in shoots fed D-6-27-hydroxycholesterol. Solubilising the labelled cholesterol in methyl-beta-cyclodextrin instead of Tween-80 increased the levels of labelled SGA up to 100-fold, and about 1 mole% of the labelled cholesterol was recovered as labelled SGA in potato leaves. Both side chain and ring structure D labels were retained in SGA, showing that the entire cholesterol molecule is converted to SGA. However, feeding side chain D-7-labelled cholesterol resulted in D-5-labelled SGA, indicating that two hydrogen atoms were released during formation of the SGA nitrogen-containing ring system. Feeding with D-7-sitosterol did not produce any labelled SGA, indicating that cholesterol is a specific SGA precursor. In conclusion, we have demonstrated a superior performance of methyl-beta-cyclodextrin for delivery of cholesterol in plant tissue feeding experiments, and given firm evidence for cholesterol as a specific sterol precursor of SGA in potato.

National Category
Biological Sciences
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-100392 (URN)10.1371/journal.pone.0082955 (DOI)000328705200118 ()
Available from: 2014-02-04 Created: 2014-02-03 Last updated: 2017-12-06Bibliographically approved
4. The impact of steroidal glycoalkaloids on the physiology of Phytophthora infestans, the causative agent of potato late blight
Open this publication in new window or tab >>The impact of steroidal glycoalkaloids on the physiology of Phytophthora infestans, the causative agent of potato late blight
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Steroidal glycoalkaloids (SGAs) are plant secondary metabolites known to be toxic to animals and humans and that exert putative roles in defence against pests. The proposed mechanisms of SGA toxicity are sterol-mediated disruption of membranes and inhibition of cholinesterase activity in neurons. It has been suggested that phytopathogenic microorganisms can overcome SGA toxicity by enzymatic deglycosylation of SGAs. Here we have explored SGA-mediated toxicity towards the invasive oomycete Phytophthora infestans, the causative agent of the late blight disease in potato and tomato, as well as the potential for SGA deglycosylation by this species. Our growth studies indicate that solanidine, the non-glycosylated precursor of the potato SGAs α-chaconine and α-solanine, has a greater physiological impact than its glycosylated forms. All of these compounds were incorporated into the mycelium, but only solanidine could strongly inhibit the growth of P. infestans in liquid culture. Genes encoding several glycoside hydrolases with potential activity on SGAs were identified in the genome of P. infestans and were shown to be expressed. However, we found no indication that deglycosylation of SGAs takes place. We present additional evidence for apparent host-specific adaptation to potato SGAs and assess all results in terms of future pathogen management strategies.

National Category
Biological Sciences
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-136549 (URN)
Available from: 2016-12-11 Created: 2016-12-11 Last updated: 2016-12-12Bibliographically approved

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