A study of the interaction between the plant pathogenic fungus Botrytis cinerea and the filamentous ssRNA mycoviruses Botrytis virus X and Botrytis virus F Barbara Boine A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biological Sciences. The University of Auckland, April 2012. i Abstract The ecological significance of mycoviruses is becoming increasingly recognised, not just for their potential as biocontrol agents but also as driving forces in the evolution and diversification of fungi. Therefore, it is important to understand how mycoviruses and fungi interact on the molecular and biochemical level. To this end the interaction between Botrytis cinerea and the mycoviruses Botrytis virus F and Botrytis virus X was studied. Relative and absolute real time PCR protocols were developed for monitoring the titres of BVX and BVF during transfection studies to monitor changes in virus titre in relation to phenotypic and metabolic changes in the fungal host. Phenotypic changes included severe phenotypical alterations, which were associated with extreme up regulation of carbohydrate, amino acid and lipid metabolism, and induction of stress responses (vacuolisation/cell lysis, increased pigmentation). To study the location and distribution of BVX in infected Botrytis the BVX coat protein was recombinantly expressed in E. coli, BVX specific polyclonal antibodies produced, and protocols developed for the serological detection and visualisation of BVX. Immuno-fluorescence microscopy was used to studying the distribution of BVX within growing Botrytis cultures indicated that the virus is present in aggregates located attached to the cell membrane, the septum, in spores, and in hyphal tips. A combination of light and electron microscopy showed that BVX is often closely associated with cell walls, suggesting that the virus may be moving across the cell wall by altering cell wall composition. If this is shown to be the case then it provides an alternative method to transmission via hyphal anastomosis, which is currently considered to be the only method of horizontal transfer. ii To Matthias iii ‘It takes a lot of courage to release the familiar and seemingly secure, to embrace the new. But there is no real security in what is no longer meaningful. There is more security in the adventurous and exciting, for in movement there is life, and in change there is power.’ — Alan Cohen iv Acknowledgment First, I would like to thank my partner Matthias Kessler, who came with me to New Zealand not knowing what to expect and how this journey would turn out. His love, support, encouragements as well as constructive critics helped me in so many ways to advance personally as well as professionally. Mein Liebchen, the PhD would not have been possible without you. Secondly, I would like to express my gratitude for my supervisor Associate Professor Mike Pearson. The trust, freedom and guidance I was allowed to experience under Mike’s supervision inspired me, let me see things from a different point of view and helped me to find my own way. His reliable support, professionally as well as privately, made me feel appreciated and welcomed. I am especially thankful for the quick and thorough reviewing, commenting and correcting of my thesis. Thank you Mike, it was a pleasure and an honor to have been one of your PhD students. Similarly, I would like to thank Ross Beever, who was also a great inspiration for me and I feel honored and blessed to have known him. He was and is missed immensely! I am also very thankful for the help and advice from my advisors Andy Bailey and Professor Gary Foster, and also for the lab training I received from Risha Patel in Bristol. Furthermore, this PhD thesis was only possible due to the financial support of the Bio-Protection Research Centre (Lincoln, Canterbury, New Zealand). I highly appreciated that I was able to attend several overseas and New Zealand conferences as well as workshops. Moreover, I would like to thank my colleagues and collaborators Sarah Jane Cowell, Kar Mun Chooi, Scott Harper, Colin Tan, Kieren Arthur, Joanne Smith, Nicole Anfang, Mahmoud Khalifa, Elizabeth Woo, Peter Deines, Christian Kost, Susanne Schmitt, Kelly Roberts, Sonia Lilly, Saadiah Arshed, Graham Bailey, Siri Alana McKelvie, Raphael Aggio, Silas G. Vilas-Boas, Jane Martin, Karin Hoksbergen and Dang Nguyen for their help, support and constructive discussions. As several of my colleagues were also my friends, I feel especially fortunate to have spent movie, dinner, and game night with them together. Thank you for these wonderful times! Being a PhD student at the University of Auckland and in Mike Pearson’s lab also taught me how to give a presentation at international conferences, to work under pressure and to find your way through unfamiliar territory, which were many things in the beginning because English and New Zealand were still foreign to me. The plant pathology community of Auckland and New Zealand helped me greatly to overcome this and made me feel home and welcomed. Especially, I would like to say thank you to Colleen Higgins, Robin McDiarmid, Dan Cohen, Matt Templeton and plant pathology meeting group for their support and help during lab meetings and conferences, but also with practical questions, and professional advice. v However, one of the most difficult parts of this PhD was the expression of the BVX coat protein. Without the help of Richard L. Kingston this success would not have been possible. Therefore, I owe Richard a big thank you for his ideas and advices. Furthermore, I want to thank Robert Sutherland for the preparation of the ultra-thin cross sections through Botrytis hyphae and very helpful discussions about fluorescence microscopy techniques. A lot of practical help with the electron and fluorescence microscope I also received from Adrian Turner. I am very grateful for his endless patience and kindness. Furthermore, I would like to thank Ian McDonald, who was always there when pictures needed to be taken or printed. During the last 4 ½ years, I also highly appreciated and enjoyed the support of my friends and my family. Catia Delmiglio was always there for me to cheer me up, to stand by me, to party with me and to give me a home when needed. Her advice and help throughout the entire PhD was extraordinary! I am also very grateful for the friendship with Gerard Clover and Sarah Burges who helped my partner and me in so many ways and we will never forget the understanding and comforting we received. Furthermore, I want to thank my friends Ines and Robert Winz, who helped out with accommodation many times and who were always there for me. Also, I want to thank Christian Paul, who visit me in New Zealand, and brought a little bit of home to me. And after all those years of studying, I finally get to thank my dear friend Katrin Kuscher, who always believed in me, encouraged me and was there for me when it was important. In this context, I would also like to thank Gisela Naujoks, who showed me how much fun science can be and that it is worth perusing your dreams. At last, I want to say a very big thank you to my family, who was and is very patient with me and supported and encouraged me during my whole life. Mutti, I thank you so much for being there for me, for soothing me, for putting no pressure on me and for waiting for me. Judi, mein Schwesterherz, I thank you for being such a great sister. I could always rely on your love and on your care. Vati, I thank you for being there me for as long as you could. You will always be in my heart. And the rhyme you told me once still stands: “Es gibt nichts Gutes, auβer man tut es!”. vi Contents Abstract ………………………………………………………………………..….……...ii Acknowledgment ………………………………………………………………………..…..…..…v Contents ………………………………………………………………………...…….…vii Chapter index ………………………………………………………………………….…...…vii List of tables ……………………………………………………………………..….………xiii List of figures ……………………………………………..……………………...……..….…xv Abbreviations …………………………………………………………….….……………...xxvi Formulas ……………………………………………………………..……..….......…..xxxi Thesis chapters ……………………………………………………………………….….…....…1 Appendices ……………………………………………………………………..….…...…217 Bibliography ………………………………………………………………………..…..…..237 Chapter index 1. Introduction 1 1.1 Botrytis cinerea 1 1.1.1 General background information 1 1.1.2 Taxonomy and genetic characteristics of Botrytis cinerea 3 1.1.3 Somatic compatibility 4 1.1.4 Characteristics, pathogenesis, and disease cycle of Botrytis cinerea 5 1.1.5 Chemical and microbial control 7 1.1.6 Biocontrol agents for Botrytis cinerea and their modes of action 8 1.2 Mycoviruses 10 1.2.1 Introduction 10 1.2.2 Diversity of mycoviruses 11 vii 1.2.3 Host range and incidence 15 1.2.4 Origin and evolution of mycoviruses 16 1.2.5 Transmission and movement of mycoviruses 17 1.2.6 Effects of mycoviruses on host phenotype 19 1.2.7 Mycoviruses as biological control agents 21 1.2.8 Molecular techniques and the study of the virus-fungal interactions 22 1.3. Mycoviruses of Botrytis cinerea 24 1.3.1 Introduction 24 1.3.2 Botrytis virus F and Botrytis virus X 25 1.3.2.1 Genetic characteristics of BVF and BVX 27 1.4 Research aims 31 2. Material and methods 32 2.1. Fungal isolates and plant material 32 2.2 Culturing conditions of Botrytis isolates 33 2.3 Molecular protocols 34 2.3.1 DNA and RNA extraction 34 2.3.2 PCR reactions and primer design 36 2.3.2.1 Primer design 36 2.3.2.2 General gel electrophoresis procedure 37 2.3.2.3 Screening for presence of plasmid-DNA inserts 37 2.3.2.4 Detection of viral RNA by reverse transcription PCR and real-time PCR 38 2.3.3 Cloning of DNA fragments 40 2.3.3.1 Ligations and digests 40 2.3.4 Transformation of Escherichia coli DHα5 cells 41 2.4 Partial virus purification 42 viii 3.