DEVELOPMENT OF OLIGONUCLEOTIDE-BASED MICROARRAYS FOR THE DETECTION OF PLANT VIRUSES AND THE FIRST CHARACTERIZATION OF A PLANT VIRUS BELONGING TO THE FAMILY TOTIVIRIDAE By VEENITA GROVER SHAH Bachelor of Science in Biochemistry University of Delhi Delhi, India 2003 Master of Science in Biosciences Jamia Milia Islamia University Delhi, India 2005 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY December, 2010 DEVELOPMENT OF OLIGONUCLEOTIDE-BASED MICROARRAYS FOR THE DETECTION OF PLANT VIRUSES AND THE FIRST CHARACTERIZATION OF A PLANT VIRUS BELONGING TO THE FAMILY TOTIVIRIDAE Dissertation Approved: Dr. Ulrich K. Melcher Dissertation Adviser Dr. Marilyn J. Roossinck Dr. Michael W. Palmer Dr. Peter Hoyt Dr. Patricia Ayoubi Dr. Mark E. Payton Dean of the Graduate College ii DEDICATION to my Father Mr. Madan Grover and Mother Mrs. Veena Grover iii ACKNOWLEDGMENTS I take this opportunity to thank a number of outstanding individuals and scholars who helped me throughout the period of my graduate program at Oklahoma State University. I will always cherish my memory of all those, to only some of whom it is possible to give particular mention here, who helped in enriching the experiences of my life during this time. This thesis would have not have been possible without the help, support and patience of my major adviser, Dr. Ulrich K. Melcher. I would like to extend my deep gratitude and appreciation to Dr. Melcher for his constant support in guiding me to a deeper understanding of knowledge work, and his invaluable supervision and encouragement during my entire Ph.D. study and research. His mentoring helped me in all the time of research and writing of this thesis. I owe my most sincere gratitude to Dr. Marilyn J. Roossinck for providing me with the opportunity to work at the Noble Foundation, as a part of her group and for her constant guidance, help and motivation throughout the term of my research. I further thank the rest of my thesis committee: Dr. Michael Palmer, Dr. Peter Hoyt and Dr. Patricia Ayoubi for their insightful comments and advice during the course of this work. An expression of gratitude goes to all my friends and fellow lab members at Oklahoma State University and Noble Foundation for their helpful suggestions, encouragements and contribution of humor during my academic endeavors. A special note of thanks is due to the faculty and staff members of the Department of Biochemistry and Molecular Biology at Oklahoma State University and Plant Biology Division at Noble Foundation, Dr. Rick S. Nelson in particular, for iv all their help and assistance. I would like to express my deepest and most sincere gratitude to my parents Mr. Madan Grover and Mrs. Veena Grover, and other family members (Mr. Dhananjay Shah, Mrs. Rashmi Shah, Mr. Vidur Grover, Mrs. Neha Grover, Ms. Heta Shah and Ms. Yuvika Grover) for their incessant love, affection and encouragement which enabled the completion of this degree. Finally, I would like to thank and appreciate my wonderful husband, Mr. Viraj Shah for his tremendous support, endless patience and continual dosage of love and stimulation all through my graduation which made it possible for me to achieve my goal. v TABLE OF CONTENTS Chapter Page I. GENERAL INTRODUCTION ..................................................................................1 II. CHAPTER I: OLIGONUCLEOTIDE-BASED MICROARRAYS FOR DETECTION OF PLANT VIRUSES EMPLYOING SEQUENCE-INDEPENDENT AMPLIFICATION OF TARGETS .......................................................................16 Introduction ............................................................................................................16 Methods and Materials ...........................................................................................20 PCR amplification and purification of DNA targets........................................20 Plant materials, viruses and RNA synthesis ....................................................20 Design of oligonucleotide probes and printing ................................................24 Sample labeling, hybridization and image analysis .........................................26 Results ....................................................................................................................28 Effect of target and probe length variation on hybridization signal intensity..28 Spacer effect.....................................................................................................35 Detection of tymoviruses singly and in mixtures ............................................36 Hybridization with short oligonucleotide probes .............................................38 Hybridization with long oligonucleotide probes..............................................38 Influence of temperature on signal intensities of long oligonucleotide probes ...............................................................................................................44 Discussion ..............................................................................................................46 III. CHAPTER II: GENOMIC APPROACHES TO DISCOVERY OF VIRAL SPECIES DIVERSITY OF NON-CULTIVATED PLANTS ................................................50 Introduction ............................................................................................................50 Genomic approaches ..............................................................................................55 vi Sampling…………. .....................................................................................................55 Individual plants..................................................................................................55 FTA cards...................................................................................................55 Lawnmower ..............................................................................................56 Run-off water .............................................................................................56 Enrichment .......................................................................................................57 dsRNA........................................................................................................58 VLP-VNA ..................................................................................................58 Virus adsorbents.........................................................................................59 Group-specific primers .............................................................................59 siRNA ........................................................................................................61 RDA ...........................................................................................................62 Amplification ...................................................................................................63 SISPA and VIDISCA .................................................................................63 Random PCR .............................................................................................64 RCA ...........................................................................................................65 Macro/microarray targets ...........................................................................66 Detection ..........................................................................................................68 Arrays .........................................................................................................68 Sequencing .................................................................................................73 Future prospects .....................................................................................................77 IV. CHAPTER III: FIRST CHARACTERIZATION OF A PLANT VIRUS BELONGING TO THE FAMILY TOTIVIRIDAE ................................................79 Introduction ............................................................................................................79 Methods and Materials ...........................................................................................83 Plant material ...................................................................................................83 Screening of plants for the presence and absence of the putative totivirus ....83 Mechanical transmission of the virus ..............................................................85 Digoxigenin-labeled RNA probe synthesis .....................................................86 Fixation, dehydration and embedding of plant stem tissues ............................87 Sectioning and tissue section pretreatment ......................................................88 In situ hybridization for localization of plus-strand viral RNA in the infected stem tissues ..............................................................................................................88 Ruellia humilis controls for fungal staining .....................................................90 Localization of fungal endophytes in stem cross-sections using cotton blue stain ..........................................................................................................................90 Isolation of fungal endophytes from stem tissues of virus-infected plants followed by dsRNA extraction........................................................................................91