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Interactions Between Pararetroviruses and Their Plant Hosts

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Interactions Between Pararetroviruses and Their Plant Hosts INTERACTIONS BETWEEN PARARETROVIRUSES AND THEIR PLANT HOSTS Melanie Kalischuk MSc. 2004 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfilment of the Requirements for the Degree DOCTORATE OF PHILOSOPHY, BIOMOLECULAR SCIENCE Department of Biological Sciences University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Melanie Kalischuk, 2015 INTERACTIONS BETWEEN PARARETROVIRUSES AND THEIR PLANT HOSTS MELANIE KALISCHUK Date of Defence: 15 April 2015 Dr. D. Johnson Professor Ph.D. Supervisor Dr. S. Rood Professor Ph.D. Thesis Examination Committee Member Dr. J. Thomas Professor Ph.D. Thesis Examination Committee Member Dr. D. Gaudet Research Scientist Ph.D. Internal Examiner AAFC Dr. K. Eastwell Professor Ph.D. External Examiner WSU Dr. A. Hontela Professor Ph.D. Examination Committee Chair Dedicated To my ever supportive husband Larry, son Nicholas and parents Vic and Ruth. iii Abstract To defend themselves against all types of pathogens, plants have evolved an array of defense strategies to prevent or attenuate invasion by potential attackers. Brassica rapa exposed to 50 ng purified Cauliflo wer mosaic virus (CaMV; Family Caulimoviridae, genus Caulimovirus) virions prior to the bolting stage produced significantly larger seeds and greater CaMV resistance than mock-inoculated treatment. Differences in defense pathways involving fatty acids, primary and secondary metabolites were detected in pathogen resistant and susceptible progeny. To extend the interplay of host and pathogen interactions involving members of the dsDNA plant viruses, the Rubus yellow net virus (RYNV) genome was characterised and contained numerous nucleic acid binding motifs, multiple zinc finger-like sequences and domains associated with cellular signaling. Silencing as a mechanism to combat virus accumulation was indicated by an uneven genome-wide distribution of 22-nt length virus-derived small RNAs with strong clustering to small regions distributed over both strands of the RYNV genome. iv Acknowlegements I would like to extend a thank you to the supervisory committee Dan Johnson, Jim Thomas, Steward Rood and Denis Gaudet. Agriculture and Agri-Food Canada is gratefully acknowledged for providing access into their facilities for carrying out this work. University of Sydney, Austrailia and Washington State University, USA also provided access to facilities for completing a portion of this reasearch and these institutions are also gratefully acknowleged. Acknowlegements for the work entitled "Priming with a double -stranded DNA virus alters Brassica rapa seed architecture and facilitates a disease response" include C. French for providing the CaMV isolate and I. Kovalchuk for providing the original B. rapa CVR018 seed. This work was funded in part by Agriculture and Agri-Food Canada and Alberta Crop Industry Develpment Fund project 2010C001R. The funding agencies did not play a role in the research design, data acquisition, interpretation of the results and submission of the manuscript for publication. Igor Kovalchuk and Larry Kawchuk provided valuable discussion, suggestion and comments influencing the research design and data acquisition for the project. Final experiment design was competed by Melanie Kalischuk. Melanie Kalischuk acquired data, analyzed data and wrote the first draft of the manuscript. Dan Johnson contributed to data analysis. Larry Kawchuk and Dan Johnson reviewed the manuscript before submission and provided valuable comments and suggestions for improvement throughout the peer review process. v Specific acknowlegements for the work entitled "Complete genomic sequence of Rubus yellow net virus and detection of genome-wide pararetrovirus-derived small RNA" include Adriana Fusaro, Peter Waterhouse, Hanu Pappu and Larry Kawchuk provided valuable discussion, suggestions and comments influencing the research design and data aquisition for the project. Final experiment design was completed by Melanie Kalischuk and Larry Kawchuk. Karen Toohey prepared Sanger Sequencing reactions and Jim Lynn cloned the constructs used in preparation of the RYNV antiserum. The remainder of the technical work, data acquisition, data analysis and writing the first draft of the manuscript were completed by Melanie Kalischuk. All authors proof-read the manuscript for scientific principles and grammar before submission. Larry Kawchuk provided assisted during the peer review process. vi Table of Contents Title Page .................................................................................................... i Thesis Committee Member Page.............................................................. ii Dedication Page ......................................................................................... iii Abstract ...................................................................................................... iv Acknowlegements ..................................................................................... v Table of Contents ...................................................................................... vii List of Tables ............................................................................................. ix List of Figures ............................................................................................ x List of Abbriviations .................................................................................. xi 1. Interactions between pararetroviruses and their hosts...................... 1 1.1.Thesis overview ................................................................................. 1 2. Literature review on disease resistance in plants .............................. 6 2.1. Introduction ....................................................................................... 6 2.2. Innate immunity in plants .................................................................. 8 2.2.1. Pattern triggered immunity ............................................................. 8 2.2.2. Gene-for-gene relationship: Effector triggered immunity ............... 10 2.2.3. Systemic acquired resistance ........................................................ 12 2.2.4. Gene silencing ............................................................................... 13 2.2.5. Post-transcriptional gene silencing as an antiviral mechanism ..... 13 2.2.6. Transcriptional gene silencing ....................................................... 18 2.2.7. Transitive silencing ........................................................................ 20 2.2.8. Silencing suppressors .................................................................... 21 2.2.9. Gene silencing and systemic signaling .......................................... 24 2.3. Across-generation resistance ............................................................ 25 2.3.1. Evidence for pathogen resistance as a transgenerational response ........................................................................................ 26 2.3.2. Mechanisms of transgenerational inheritance ................................ 27 2.4. Framework for exploring transgenerational resistance: The host plant Brassica rapa ........................................................................... 30 2.4.1. Priming Brassica rapa with a dsDNA virus ..................................... 31 2.5. Concluding remarks .......................................................................... 34 3. Priming with a double-stranded DNA virus alters Brassica rapa seed architecture and facilitates a disease response1 ................................ 36 1 A version of Chapter 3 has been published ...................................... 36 3.1. Background ....................................................................................... 37 3.2. Material and Methods ....................................................................... 39 3.2.1. Plant material and experimental design ........................................ 39 3.2.2. Examination of stable complex traits and virus resistance ............ 40 3.2.3. cDNA library preparation and sequencing for transcriptome analysis ............................................................................................. 41 3.2.4. Sequence analysis ........................................................................ 42 3.3. Results ............................................................................................. 43 vii 3.3.1. Low dose of cauliflower mosaic virus applied just before bolting .. 43 3.3.2. Small RNA sequencing ................................................................. 48 3.3.3. Resistant and susceptible phenotypes exposed to CaMV have contrasting profiles of differentially expressed loci ........................ 48 3.3.4. Functional annotation of differentially regulated genes ................. 49 3.3.5. Mapping differentially expressed genes to KEGG pathways ......... 55 3.3.6. Common and unique genes among phenotypes ........................... 56 3.4. Discussion ..................................................................................... 59 4. Complete genomic sequence of Rubus yellow net virus and detection of genome-wide pararetrovirus-derived small RNAs2 ............................. 65 2 A version of Chapter 4 has been published ...................................... 65 4.1. Introduction ....................................................................................... 66 4.2. Material and methods ......................................................................
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