Aphid-Borne Viruses of Potato: Investigations into Virus/Host/Vector Interactions, Serological Detection Using Recombinant Antibodies and Control Strategies By Ahmad Al-Mrabeh A thesis submitted in partial fulfillment of the requirements of the University of Newcastle School of Biology For the degree of Doctor of Philosophy (PhD) September 2010 The project was carried out at the Scottish Crop Research Institute (SCRI) i ACKNOWLEDGEMENTS At the end of this research journey, first and foremost I would like to present extreme respect to Prof. L.Torrance, my supervisor, who hosted me in her laboratory at SCRI, and introduced me to the world of plant virology. Secondly, Dr. B. Fenton and Dr. A. Ziegler are very much appreciated for contributing in my supervision at different stages of this project. Thirdly, I would like to express my sincere respect and gratitude to Mr. G. Cowan who has never hesitated to provide any kind of help. I am also indebted to my supervisors at Newcastle (Prof. A. Gatehouse, and Dr. E. Hack) for their helpful advice during the process of my project, and for agreement that I will do my research at SCRI. In addition, I am thankful to Dr. K. Emami who supervised me at laboratory at the early stage of my PhD at Newcastle, Dr. J. Jones (SCRI) for helping in Q-Bot filter printing, Dr. K. MacKenzie (BioSS) for helping in statistical analysis, and Mr. P. Smith (IT, SCRI) for proof reading the thesis. General thanks to all member of staff and student colleagues at SCRI and Newcastle University who I have met, it is important to acknowledge that without their help and support, it was difficult to complete this work. I would like to extend my thanks to my supervisors at Syria namely: Dr. M. Halloum, Dr. I. Ismail and Dr. M. Ahmad, for their ceaseless support and encouragement before and during this study. I am deeply grateful to scientists from the plant virus community who contributed towards my work particularly Prof. H. Barker, Dr. S. Blanc and Dr. J.J. Lopez-Moya. Last but not least, I would like to thank my wife Abeer, my son Myso for their patience and understanding all this period. And special thanks to my parents and friends from Syria for their support and encouragement during the whole period of study particularly S. Omran at Newcastle University who has been always a source of inspiration and enthusiasm. Ahmad ii DECLARATIONS I, Ahmad Al-Mrabeh, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I can confirm that this has been indicated in the thesis. Ahmad Al-Mrabeh ************************************** This is to certify that this copy of the thesis was checked and approved by the examiners. Dr Anne Borland iii CONTENTS FRONT PAGE I ACKNOWLEDGEMENTS II DECLARATION III TABLE OF CCONTENTS IV ABSTRACT XII LIST OF FIGURES XIII LIST OF TABLES XVIII LIST OF ABBREVIATIONS XXI LIST OF VIRUSES AND ACRONYMS XXV 1. CHAPTER1: LITRATURE REVIEW 1 1.1. Background 1 1.2. Viruses which naturally infect potato 2 1.3. Brief description of the most important aphid-borne potato viruses 5 1.3.1. Potato leafroll virus (PLRV) 5 1.3.2. Potato virus Y (PVY) 6 1.3.3. Potato virus A (PVA) 8 1.3.4. Potato virus V (PVV) 8 1.3.5. Potato virus S (PVS) 8 1.3.6. Potato virus M (PVM) 9 iv 1.4. PVY characteristics 9 1.4.1 Emergence of PVY as the main potato virus 9 1.4.2. Host range of PVY 10 1.4.3. Genetic diversity and PVY strains 10 1.4.4. Indicator plants for differentiation and propagation of PVY 11 1.5. Transmission of potato viruses by aphids 12 1.5.1 Virus – vector relationship 12 1.5.2. Mechanism of non-persistent virus transmission 13 1.5.2.1. Background 13 1.5.2.2. Helper component (HC-Pro) 14 1.5.2.2.1. Discovery and proporties of HC-Pro 14 1.5.2.2.2. HC-Pro function in virus transmission 15 1.5.2.3. Coat protein (CP) 16 1.5.2.4. Hypotheses for molecular mechanisms of potyvirus transmission 18 1.6. Aphid species transmitting potato viruses 19 1.7. Controlling the spread of aphid transmitted viruses on potato crop 25 1.7.1. Seed classification schemes 25 1.7.1.1.British Seed classification schemes 25 1.7.1.2.Aphid monitoring and forecasting 26 1.7.1.3.Interpreting aphid monitoring data 28 1.7.1.3.1. Relative efficiency factor (REF) 28 1.7.1.3.2. How REF is assigned? 30 1.7.1.3.3. Bait plant system 30 1.7.1.3.4. Aphid thresholds 31 1.7.1.3.5. Post-harvest testing/ ELISA 32 1.7.2. Breeding for virus and vector resistance 32 1.7.3. Chemical strategies for controlling potato viruses 35 1.7.3.1. Mineral oils 35 1.7.3.1.1. Mode of action of mineral oil 35 1.7.3.1.2. Mixing oils with Pyrethroids 36 1.7.3.2. Synthetic insecticides 36 1.7.3.2.1. Old classes of insecticides 37 v 1.7.3.2.2. New classes of insecticides 37 1.7.4. Non-chemical control strategies 38 1.7.4.1. Isolation of seed potato fields 38 1.7.4.2. Removing weeds and groundkeepers 38 1.7.4.3. Haulm destruction 39 1.7.4.4. Border Crops 40 1.7.4.5. Host masking and reflective mulching 41 1.7.4.6. Using natural products 41 1.8. Project’s aims 42 2. CHAPTER 2: GENERAL MATERIALS AND METHODS 44 2.1. Virus and aphid cultures 44 2.2. Virus transmission by aphids 45 2.3.Virus detection 45 2.3.1. General buffers for virus detection 46 2.3.2. ELISA 47 2.3.3. RT-PCR conditions for potyvirus detection 49 2.3.3.1.cDNA synthesis 49 2.3.3.2.PCR conditions 49 2.3.4. Nested RT-PCR 49 2.3.4.1.RNA extraction from aphids 49 2.3.4.2.RNA extraction from plants 50 2.3.4.3.First round – RT-PCR 50 2.3.4.4.Second round PCR 50 2.3.5. Electron microscopy (EM) negative staining 51 2.3.6. SDS-PAGE 51 2.3.6.1.Resolving gel 51 2.3.6.2.Stacking gel 52 2.3.6.3.Sample loading 52 2.3.6.4.Coomassie Blue gel staining 52 2.3.7. Western blot analysis 52 vi 2.3.8. Dot blot assay 53 2.4.Molecular biology methods 53 2.4.1. Nucleic acid methods 53 2.4.1.1.General media, buffers, and reagents 53 2.4.1.2.Agarose gel electrophoresis of DNA 55 2.4.1.3.DNA extraction from agarose gel 55 2.4.1.4.DNA recovery from PCR product and after enzyme digestion 56 2.4.1.5.DNA and RNA quantification 56 2.4.1.6.DNA ligation 56 2.4.1.7.Chemical transformation of E. coli 57 2.4.1.8.Transformation by electroporation 57 2.4.1.9.Colony pick PCR 58 2.4.1.10. PCR primers 58 2.4.1.11. DNA sequencing, editing, and alignment 60 2.4.1.12. Site-Directed Mutagenesis 60 2.4.2. Proteomic methods 61 2.4.2.1.General buffers and recipes 61 2.4.2.2.Protein expression and purification buffers 62 2.4.2.3.PVY purification 63 2.4.2.4.Virus quantification 64 2.4.2.5.Recombinant protein expression and induction 64 2.4.2.6.Bradford protein assay 64 2.4.2.7.Protein dialysis 64 2.4.2.8.Ni-NTA resin protein purification system 65 2.5. Statistical analysis 65 3. CHAPTER 3: POTYVIRUS AND APHID PROTEINS INTERACTION 66 3.1. Introduction 66 3.2. Materials and Methods 68 3.2.1. Expressed colony screening 68 3.2.1.1. Q-Bot Filter printing 68 3.2.1.2.Filter processing and overlay assay conditions 72 vii 3.2.2. Cloning aphid proteins into the pQE-30 72 3.2.3. Aphid cuticle proteins expression and purification 72 3.2.3.1.Whole cell extract 73 3.2.3.2.Periplasmic extract (PE) 73 3.2.3.3. Cleared lysate under native conditions 73 3.2.3.4.Cleared lysate under denaturing conditions 74 3.2.3.5. Inclusion body (IB) method 74 3.2.3.6.Native CUPs extraction 74 3.2.4. HC-Pro protein expression and purification 75 3.2.4.1.HC-Pro expression in E. coli 75 3.2.4.2.HC-Pro expression in plant 76 3.2.4.2.1. PVY-HC-Pro in PVX system 76 3.2.4.2.2. TuMV HC-Pro 77 3.2.4.2.3. TEV HC-Pro 77 3.2.5. Overlay assay conditions for Western blot 78 3.3. Experimental Results 79 3.3.1. Clones interacting with the full-length HC-Pro expressed in E. coli 79 3.3.2. Clones interacting with the N-terminus of HC-Pro expressed in E. coli 79 3.3.3. Expression of cloned cDNA inserts into PQE 85 3.3.4. Interaction between aphid proteins and antisera to CUPs 88 3.3.5. Interaction between P72F protein and PVX expressed HC-Pro 89 3.3.6. Experiments to study binding to HC-Pro of other potyviruses 91 3.3.7.