EXPRESSION PROFILING IN RESPONSE TO ASCOCHYTA RABIEI INOCULATIONS IN CHICKPEA A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY BY BANU AVCIOĞLU DÜNDAR IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY SEPTEMBER 2008 Approval of the thesis: EXPRESSION PROFILING IN RESPONSE TO ASCOCHYTA RABIEI INOCULATIONS IN CHICKPEA submitted by BANU AVCIOĞLU DÜNDAR in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biotechnology Department, Middle East Technical University by, Prof. Dr. Canan Özgen ________________ Dean, Graduate School of Natural and Applied Sciences Prof. Dr. Gülay Özcengiz ________________ Head of Department, Biotechnology Prof. Dr. Mahinur Akkaya Supervisor, Chemistry Dept., METU ________________ Assist. Prof. Dr. Mücella Tekeoğlu Co-Supervisor, Faculty of Agriculture, Ondokuz Mayıs University ________________ Examining Committee Members: Prof. Dr. Gülay Özcengiz _____________________ Biology Dept., METU Prof. Dr. Mahinur Akkaya _____________________ Chemistry Dept., METU Prof. Dr. Sara Dolar _____________________ Plant Protection Dept., Ankara University Assoc. Prof. Dr. Sertaç Önde _____________________ Biology Dept., METU Assoc. Prof. Dr. Müge Türet Sayar _____________________ Molecular Biology and Genetics Dept., Boğaziçi University Date: September 12, 2008 I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work. Name, Last name : Banu Avcıoğlu Dündar Signature : ABSTRACT EXPRESSION PROFILING IN RESPONSE TO ASCOCHYTA RABIEI INOCULATIONS IN CHICKPEA Avcıoğlu Dündar, Banu Ph.D., Department of Biotechnology Supervisor: Prof. Dr. Mahinur Akkaya Co-Supervisor: Assist. Prof. Dr. Mücella Tekeoğlu September 2008, 256 pages In this study, it was aimed to identify chickpea (Cicer arietinum) genes or gene fragments expressed upon Ascochyta rabiei infection using a tolerant chickpea cultivar ILC195 and fungal isolates with varying level of pathogenicity. PCR amplification of resistance gene analogs (RGA) and disease related genes, and mRNA differential display reverse transcription (DDRT) were used to get these expressed gene fragments in chickpea. The constitutively or differentially expressed PCR product fragments were cloned and sequenced. Out of nearly 300 clones, 160 sequences (expressed sequence tags, ESTs) could be analyzed and these sequences were disclosed in this study. About 100 of these ESTs were classified according to predicted “molecular function”, “biological process” and “cellular component”. The most common ppredicted functions of the products coded by these ESTs were “Protein Fate”, “Metabolism”, “Cell Rescue, Defense and Virulence”, “Transcription”, “Transport”, “Energy”, and “Cell Fate”. Six ESTs were subjected to Real-Time quantitative RT-PCR analysis to compare the response of ILC195 infected by one A.rabiei isolate with another resistant chickpea genotype (FLIP84-92C)/A.rabiei pathotype system. Some of these genes were differentially expressed among different chickpea/A.rabiei isolate combinations. Highly upregulated ESTs in all these combinations were a formate dehydrogenase (metabolism and detoxification), a serine carboxypeptidase iv (protein fate and communication) and a hypothetical protein probably similar to acyl-CoA synthetases. A genetic mapping study was carried out with EST specific primers and two EST markers were assigned in the current chickpea genetic map. However, no genetic linkage of them was detected with known chickpea quantitative trait loci for A.rabiei resistance. Key words: Chickpea, Ascochyta rabiei, Differential Display, Resistance Gene Analogs, Genetic Mapping. v ÖZ ASCOCHYTA RABIEI İNOKULASYONLARINA KARŞI NOHUTTA OLUŞAN EKSPRESYON PROFİLİ Avcıoğlu Dündar, Banu Doktora, Biyoteknoloji Bölümü Tez Yöneticisi: Prof. Dr. Mahinur Akkaya Ortak Tez Yöneticisi: Yrd. Doç. Dr. Mücella Tekeoğlu Eylül 2008, 256 sayfa Bu tezde, tolerant nohut (Cicer arietinum) çeşidi ILC195 ve çeşitli düzeyde patojeniteye sahip fungus izolatları kullanılarak, Ascochyta rabiei enfeksiyonuna karşı ifade gösteren nohut gen veya gen parçalarının tanımlanması amaçlanmıştır. Nohutta ifade olan gen parçalarının elde edilmesi için dayanıklılık geni analoglarının (RGA) ve hastalıkla ilişkili genlerin PCR yöntemiyle çoğaltılması ve mRNA farklılık gösterimi ters transkripsiyonu (DDRT) kullanılmıştır. Sabit veya farklı şekilde ifade olan PCR ürün parçaları klonlanarak dizi analizleri yapılmıştır. Yaklaşık 300 klon içinden, 160 dizi (ifade olan dizi etiketleri, ETS) analiz edilebilmiş ve bu diziler çalışmada verilmiştir. Bu EST’lerden yaklaşık 100 adedi tahmini “moleküler işlev”, “biyolojik işlem” ve “hücre bileşeni”ne göre sınıflandırılmıştır. Bu EST’lerin kodladığı ürünlere ait en çok görülen tahmini işlevler “Protein Yazgısı”, “Metabolizma”, “Hücre Kurtarma, Savunma ve Virulans”, “Transkripsiyon”, “Taşıma”, “Enerji” ve “Hücre Yazgısı”dır. Bir izolatla enfekte edilmiş ILC195’nin tepkisinin, başka bir dirençli nohut genotipi (FLIP84-92C)/A.rabiei patotipi sistemiyle karşılaştırılması amacıyla, altı EST gerçek zamanlı kantitatif RT-PCR analizine tabi tutulmuştur. Bu genlerden bazıları değişik nohut/A.rabiei izolatı kombinasyonlarında farklı şekilde ifade olmuştur. Tüm bu kombinasyonlarda yüksek derecede ifade olan EST’ler bir format dehidrogenaz (metabolizma ve detoksifikasyon), bir serin karboksipeptidaz (protein yazgısı ve haberleşme) ve alkil-CoA sentetaz olabilecek bir vi varsayımsal proteindir. EST’lere özgü primerlerle bir genetik haritalama çalışması yapılmış ve iki EST etiketi güncel nohut genetik haritasına yerleştirilmiştir. Ancak, bunların A.rabiei dayanıklığına ilişkin bilinen nohut kantatif karakter lokuslarıyla genetik bağlantısı olmadığı görülmüştür. Anahtar kelimeler: Nohut, Ascochyta rabiei, Farklılık Gösterimi, Dirençlik Geni Analogları, Genetik Haritalama vii To Nameless People Who Have Been Slogging Away Scientific Research for Years viii ACKNOWLEDGMENTS I wish to express my gratitude to my supervisor Prof. Dr. Mahinur S. Akkaya for her support, guidance, advice and encouragement throughout this research. I would like to appreciate to Prof. Dr. Sara Dolar for her support and guidance for the infection studies in Ankara University Department of Plant Protection; and Dr. Harun Bayraktar and Dr. Muharrem Türkkan for their help during these studies. I wish to thank to post-docs of Prof. Dr. Mahinur S. Akkaya; Assist. Prof. Dr. Şenay Vural Korkut and Dr. Semra Hasançebi, for their suggestions and comments on gaining laboratory skills in my work. I like to thank to Assist. Prof. Dr. Mücella Tekeoğlu for accepting to be my co- supervisor. I would like to thank to Dr. Kevin McPhee and Prof. Dr. Patricia Okubara for their support and insight, and Dr. P.N. Rajesh for his advices and criticism for the research done in WSU USDA-ARS Grain Legume Genetics and Physiology Unit. I would like also to express my thanks to Sheri Rynearson, Tony Chen and Sheri McGrew for their technical assistance. I wish to thank to other members of this unit and friends for their help and friendship throughout this time period of the study. I am also thankful to my parents and relatives for their support; to my husband Dr. Halil Dündar, my sister Başak Avcıoğlu, to my friends Assist. Prof. Dr. Aslıhan Günel, Dr. Adnan Al-Asbahi, İlay Çelik, Beray Ünsal and other members of Akkaya’s laboratory for their friendship and help. This study was supported by METU grant BAP-2004-07-02-00-62; and partly by the State Planning Organization (DPT) Grant No: DPT2004K750120. The author was supported by TÜBİTAK scholarship number 2214 for the research during the period of March-June 2008 done in USA. ix TABLE OF CONTENTS ABSTRACT………………………………………………………………………. iv ÖZ………………………………………………………………………………… vi ACKNOWLEDGEMENTS ……………………………………………………… ix TABLE OF CONTENTS ………………………………………………………… x LIST OF TABLES ……………………………………………………………….. xiii LIST OF FIGURES ………………………………………………………………. xv ABBREVIATIONS ………………………………………………………………. xvii CHAPTER 1.INTRODUCTION ……………………………………………………………… 1 1.1. The Chickpea ………………………........................................................... 1 1.2. Pathogens of Chickpea ................................................................................. 2 1.3. Ascochyta Blight in Chickpea...................................................................... 3 1.4. Management of Ascochyta Blight in Chickpea ........................................... 5 1.5. Understanding Molecular Basis of Ascochyta Blight Resistance in Chickpea ....................................................................................................... 6 1.5.1 Resistance Mechanisms of Plants Against Pathogens ........................ 6 1.5.2 Findings Related to the Resistance of Chickpea Against Ascochyta Blight ................................................................................................. 19 1.5.2.1 Pathogenic Variability of Ascochyta rabiei ........................... 19 1.5.2.2 Identification of Quantitative Trait Loci and Mapping for Resistance to Ascochyta Blight .............................................. 20 1.5.2.3 Resistance Gene Analog (RGA) and Gene Specific Markers for Genetic Mapping .............................................................. 26 1.5.2.4