MOLECULAR ANALYSIS OF THE EGGSHELL AND CUTICLE SURFACE OF THE POTATO CYST NEMATODE, GLOBODERA ROSTOCHIENSIS James Price A Thesis Submitted for the Degree of PhD at the University of St Andrews 2019 Full metadata for this item is available in St Andrews Research Repository at: http://research-repository.st-andrews.ac.uk/ Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/18248 This item is protected by original copyright Molecular analysis of the eggshell and cuticle surface of the potato cyst nematode, Globodera rostochiensis James Price This thesis is submitted in partial fulfilment for the degree of Doctor of Philosophy (PhD) at the University of St Andrews May 2019 i Declarations Candidate's declaration I, James Price, do hereby certify that this thesis, submitted for the degree of PhD, which is approximately 56,000 words in length, has been written by me, and that it is the record of work carried out by me, or principally by myself in collaboration with others as acknowledged, and that it has not been submitted in any previous application for any degree. I was admitted as a research student at the University of St Andrews in September 2015. I received funding from an organisation or institution and have acknowledged the funder(s) in the full text of my thesis. Date Signature of candidate Supervisor's declaration I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of PhD in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. Date Signature of supervisor Date Signature of supervisor ii Permission for publication In submitting this thesis to the University of St Andrews we understand that we are giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. We also understand, unless exempt by an award of an embargo as requested below, that the title and the abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker, that this thesis will be electronically accessible for personal or research use and that the library has the right to migrate this thesis into new electronic forms as required to ensure continued access to the thesis. I, James Price, confirm that my thesis does not contain any third-party material that requires copyright clearance. The following is an agreed request by candidate and supervisor regarding the publication of this thesis: Printed copy No embargo on print copy. Electronic copy No embargo on electronic copy. Date Signature of candidate Date Signature of supervisor Date Signature of supervisor iii Underpinning Research Data or Digital Outputs Candidate's declaration I, James Price, understand that by declaring that I have original research data or digital outputs, I should make every effort in meeting the University's and research funders' requirements on the deposit and sharing of research data or research digital outputs. Date Signature of candidate Permission for publication of underpinning research data or digital outputs We understand that for any original research data or digital outputs which are deposited, we are giving permission for them to be made available for use in accordance with the requirements of the University and research funders, for the time being in force. We also understand that the title and the description will be published, and that the underpinning research data or digital outputs will be electronically accessible for use in accordance with the license specified at the point of deposit, unless exempt by award of an embargo as requested below. The following is an agreed request by candidate and supervisor regarding the publication of underpinning research data or digital outputs: No embargo on underpinning research data or digital outputs. Date Signature of candidate Date Signature of supervisor Date Signature of supervisor iv K, K. R., E. M. & J. H. Acknowledgments I am not one for grand declarations and pouring my heart into words, however, although by this point my creativity lacks, my gratitude does not. I have thoroughly enjoyed my PhD but don’t think I could have if it was not for the supervision provided by both Prof. John Jones and Prof. Terry Smith. They were both always willing to listen to my concerns and questions and supported me whenever I was doubting my work. Both have taught me a lot that I hope to replicate one day and for all of this, I am eternally grateful. I cannot thank the Perry foundation enough for the support they provided. Where others didn’t think this work was worth funding, they stepped up and provided me with more than I hoped for. Not only did this allow me to carry out the work in this thesis but also enabled me to travel and present my results conferences in London, Durham, Braga (Portugal) and Ghent (Belgium). Time management for this work was often a struggle due to actively working between the James Hutton Institute and the University of St. Andrews. However, colleagues from both sites always made me feel at home wherever I was working. I would like to extend a special thanks to friends from the NemaLab at the James Hutton Institute; Kerry, Shona, Akis and Jamie. My gratitude also goes to colleagues, past and present from the TKS lab at the University of St Andrews. Throughout my research, I have met many other PhD students and friends whom I would like to thank for always saying ‘why not?’ when I suggested a long weekend on a distant Scottish island. Merci aussi à Adeline, pour avoir supporté mes excentricités, m’avoir épaulé, et pour toute l’aide que tu m’as apportée au cours des deux dernières années. Finally, I must thank my parents, Andrew and Patricia Price. I cannot put into words how grateful I am for their encouragement, their presence and their constant support. vi Abstract Plant-parasitic nematodes regulate their interactions with the external environment and their host. The surface of the nematodes, whether the cuticle or the eggshell, plays a key role in this process. Work undertaken in this project aimed to exploit new genome resources for potato cyst nematodes (PCN) to allow characterisation of these structures. The eggshell is a key survival structure for PCN. Hatching of juvenile potato cyst nematodes occurs in response to host derived hatching factors. Little is known about the molecular mechanisms that underpin hatching in response to these host diffusates and nothing is known about the molecular composition of the PCN eggshell. Methods that allow extraction of proteins and lipids from large numbers of isolated PCN eggshells were developed. These extraction techniques permitted identification of a range of proteins present in the eggshell, including chondroitin proteoglycans and a calcium dependent phospholipid-binding annexin. The annexin was focused on due to the apparent major role of calcium in PCN hatching. This annexin was localised to the eggshells of Globodera rostochiensis by immunofluorescence. To our knowledge, this makes the annexin the first eggshell protein to be identified and localised in any plant- parasitic nematode. The annexin was shown to bind to phospholipids and RNAi of the annexin showed that reducing expression of the eggshell annexin alters nematode hatching patterns in response to host hatching factors. The eggshell permeability barrier is essential for the development of a juvenile within the embryo. Eggshell lipid analysis has given insight into components of the permeability barrier. Identified lipid species highlight potential function within the nematode hatching cascade through calcium interaction or down stream signalling. Attempts to identify the presence of nematode specific glycolipids, ascarosides, within the PCN eggshell showed that ascarosides or ascarylose were not detectable within PCN eggs, cysts or juveniles. vii It has previously been shown that the infective juveniles are able to alter their surface composition to avoid damage from host defence mechanisms. Here, methods that allow labelling of proteins present on the cuticle surface of PCN were developed. Three proteins; Galectin-5, Cri-2 and a nematode specific hypothetical protein that are present on the surface of PCN second stage juveniles were further investigated. These proteins were tested for their potential to suppress host defences through a variety of protein- small molecule interactions. This analysis showed that surface coat associated galectins could offer protection to an invasive juvenile through sequestering cell wall breakdown products, silencing detection of the pathogen. viii Abbreviations ALF160 Ascaroside #18 (produced in-house) ANOVA Analysis of variance APN Animal-parasitic nematode ASCR Ascaroside avr Avirulence genes BLAST Basic Local Allignment Search Tool BSA Bovine Serum Albumin cDNA Complementary DNA CE Collision energy CpG Chondroitin proteoglycan cps Counts per second cv. Cultivar Da Daltons DAG Diacylglycerol DAMP Damage Associated Molecular Pattern DCM Dichloromethane DEPC Diethyl pyrocarbonate dH2O Distilled water DIG Digoxegenin DNA Deoxyribonucleic acid DNTB 5,5'-dithiobis-2-nitrobenzoic acid dpi Days post infection DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid ESI-MS-MS Electrospray tandem mass spectrometry Electrospray ionisation time of flight mass ESI-TOF spectrometry ix EV Empty