Biosynthesis of biphenyl and dibenzofuran phytoalexins in Sorbus aucuparia cell cultures Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Mohammed Nabil Ahmed Khalil aus Kairo / Ägypten 1. Referent: Professor Dr. Ludger Beerhues 2. Referent: Privatdozent Dr. Wolfgang Brandt eingereicht am: 27.05.2013 mündliche Prüfung (Disputation) am: 14.08.2013 Druckjahr 2013 „Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes“ Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Chizzali C, Khalil MNA, Beurle T, Schuehly W, Richter K, Flachowsky H, Peil A, Hanke MV, Liu B, Beerhues L: Formation of biphenyl and dibenzofuran phytoalexins in the transition zones of fire blight-infected stems of Malus domestia cv. Holsteiner Cox and Pyrus communis cv. Conference . Phytochemistry 77: 179-185 (2012). Khalil MNA, Beuerle T, Müller A, Ernst L, Bhavanam VBR, Liu B, Beerhues L : Biosynthesis of the biphenyl phytoalexin aucuparin in Venturia inaequalis-treated Sorbus aucuparia cell cultures. Submitted (2013). Khalil MNA, Brandt W, Beuerle T, Liu B, Beerhues L: Charcterization of two cDNA encoding O-methyltransferases participating in biosynthesis of phytoalexins in Sorbus acuparia cell cultures. In preparation (2013). Tagungsbeiträge Khalil MNA, Beuerle T, Liu B, Beerhues L: Molecular analysis of biphenyl biosynthesis (Vortrag) Black Forest Retreat 2012 on Molecular Plant Science. Herzogenhorn, Freiburg, Sep 10th-13th. Khalil MNA, Beuerle T, Liu B, Beerhues L: Biosynthesis of biphenyl and dibenzofuran phytoalexins in Sorbus aucuparia cell suspension cultures (Vortrag) Tagung der Sektion ‘Pflanzliche Naturstoffe’ der Deutschen Botanischen Gesellschaft im Michaeliskloster Hildesheim, 30.09-02.10.2012 Acknowledgment Thanks to GOD, the source of all knowledge by whose abundant grace this work has come to fruition. He guided us to see his greatness in his creatures. I would like to express my deep appreciation and gratitude to my supervisor Prof. Dr. Ludger Beerhues, for giving me the opportunity to join his workgroup in context of the GERLS scholarship program, for the valuable inspiring scientific discussions and his affectionate, friendly way of guidance and supervision. His trust, support and careful listening and comprehension make it possible to surpass the difficulties of work. Thanks for respecting and discussing my ideas and opinions, even when they appear silly to me or they are not convincing to you. Working with you and in your workgroup taught me many lessons about science and life. I owe much of the success of this work and most of the knowledge I expanded during the scholarship to Dr. Till Beuerle. Thanks a lot for your help and guidance during the chemical synthesis of the substrates and references which were so essential for performing this research and the useful fruitful tips and recommendations throughout the work. Thanks for teaching me that before performing an experiment, it is so important to think about its outcome, asses the alternatives and at the end find a compromise between the resources and goals. For me, you are a great teacher who gives his students all his experience to save their time and efforts and then give them the freedom to research freely. Thanks for your continuous support and respect even when I have done mistakes and your brotherly advices and discussions. Thanks and appreciation to Dr. Benye Liu for his valuable helpful advices in the work, his welcoming approachable personality and kindness. Any time I could ask or discuss anything with him. Sincere appreciation and gratitude to PD Dr. Wolfgang Brandt for performing the modeling work with diligence and enthusiasm and meeting our many questions and requests with patience and comprehension. I am grateful to Prof. Dr. Ludger Ernst for measuring and interpreting the NMR data. I am grateful and thankful for Dr. Helge Scharnhop and Dr. Cornelia Chizzali for establishing the fundamentals of this work, my work was only a continuation for what you started. Your results and the problems you faced helped to save my time and efforts. I appreciate the kind help and the friendly support I get from my colleague Dr. Cornelia Chizzali. Great thanks and appreciation to Dr. Rainer Lindigkeit for his support during the work in the isotope laboratory. Because of his efforts and care, the work flows smoothly in the institute. I was lucky to work with a group of kind, helpful and patient colleagues. To enumerate Mrs. Claudine Theuring and Mrs. Kathrin Meier for their help with the handling of radioactive isotopes, Mrs. Carolin Rattunde for her guidance and help by the real time-qPCR and answering my questions, Mrs. Ines Rahaus and Mrs. Doris Glindemann for help with everyday working. I would like to express my sincere gratitude for Mrs. Ines Rahaus for her moral and affectionate support and continuous encouragement throughout the PhD work especially in the first year, when I faced a lot for disappointing results. Listening to your monthly radio program and the discussions ideas about arts, social and cultural issues were a rewarding experience and gave me new perspectives. Thanks for spreading optimism and fun in the stringent scientific life. Thanks Doris for the many presents you gave to me every Christmas and feast, for alerting me, in a friendly way, to my mistakes and asking about my family. I extend my appreciation to my colleagues, Islam El-Awaad , Dr. Andreas Müller, Dr. Iman Abdel-Rahman, Malte Büttner, Frauke Gumz for their indispensable help and advise by the molecular and biochemical work. Especial thanks for Islam and Iman for sharing their experiences, success and mistakes with me and the countless scientific discussions. Thanks for your kindness and support. It was a grace to meet unforgettable sincere friends. The Christmas team: Maike van Ohlen, Luise Cramer, Marion Wiggermann, Malte Büttner and Frauke Gumz. I will always remember our happy and joyful conversations and laughter. You were my second family in Germany. Your comprehension, love and respect alleviated my nostalgia. I will be always grateful to the chance meeting and knowing you. I wish you eternal happiness and all the best in your lives. Special Thanks to my friend and colleague Mina Awadalah for the happy joyful discussions and work in the laboratory. Great appreciation and gratitude to my colleagues Nargis Elgahme, Sahar Abdelaziz, Anja Losansky, Ines Bel haj, Ebtesam Ali, Tobias Fiesel, Dennis Reckwell, Maren Lütge, Dibyendu Majumdar and Su Zhang for the friendly working atmosphere and encouragement. Many thanks go to Mrs. Bettina Böttner for the interesting conversations and her diligent work. Thanks a lot for all German language teachers in Egypt and Germany and the workshops held in DAAD-Cairo; they eased the study and life in Germany. I am so grateful to the Egyptian Ministry of Research and High Education and DAAD for co financing and organizing the GERLS program (German Egyptian Research Long- term Scholarship). This program gave me the chance to develop and expand my knowledge, skills and provided me with unforgettable scientific and personal experiences. Many thanks for the German people I met during the language course and in everyday life. I appreciate their hospitality and respect. Words are not enough to express my hearty gratitude, sincere appreciation and great indebtedness to my father, mother, brother and sister for their great care, trust, surveillance, affection, and love. They are the sun which enlightens my life and soul, and the shore where I find always peace and safe. They play a special role in my life. I owe them my life and success. May GOD make their life full of joy and happiness. I appreciate this moment, that I had the chance to express these feelings and to make them everlasting. …………………………To whom I owe my success and Happiness, My Father, Mother Brother, Sister Contents Contents I. Introduction.....................................................................................1 1. Subtribe Pyrinae.......................................................................................1 2. Parasitic diseases of Pyrinae....................................................................1 2.1 Scab.........................................................................................................................1 2.2 Fire blight ...............................................................................................................3 3. Phytoalexins…………………………………………………………...…4 3.1 Phytoalexins of subtribe Pyrinae ............................................................................7 4. Biological activities of biphenyls and dibenzofurans...........................12 5. Biosynthesis of biphenyls and dibenzofurans.......................................13 5.1 Biphenyl synthase (BIS), the key enzyme............................................................13 5.2 Postulated pathway................................................................................................14 5.2.1 Cytochrome P450 (CYP) enzymes...............................................................16 5.2.2 2-oxoglutarate dependent dioxygenase........................................................17 5.2.3 O-methyl