This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Tomlinson, Katherine Title: Use of infectious clones to gain insights into Cassava brown streak virus gene function General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. 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Use of infectious clones to gain insights into Cassava brown streak virus gene function Katie Rose Tomlinson A dissertation submitted to the University of Bristol in accordance with the requirements for award of the degree of Doctor of Philosophy in the Faculty of Science School of Biological Sciences September 2018 80,767 words I Abstract Cassava is the second most important food staple in terms of per capita calories consumed in Africa and offers unique climate change adaptation opportunities. Unfortunately, production in sub- Saharan Africa is currently constrained by cassava mosaic disease (CMD) and cassava brown streak disease (CBSD), which together are estimated to cause annual losses worth US$1 billion and adversely affect food security across the entire region. Compared with CMD, relatively little is currently understood about CBSD infections and there are no cassava varieties with high levels of CBSD resistance available to farmers. CBSD is caused by at least two potyviral species: Cassava brown streak virus and Ugandan cassava brown streak virus, collectively referred to as U/CBSVs. Little is known about U/CBSV gene function; to date only the silencing suppressor activity of the UCBSV P1 protein has been reported. Viral infectious clones are highly valuable tools that can be used to screen for viral resistance and to gain fundamental insights into viral infections. Unfortunately, until recently the construction of U/CBSV ICs has been circumvented by sequence instability during propagation in Escherichia coli. In 2014 -16, two U/CBSV ICs were constructed at the University of Bristol, as part of a larger project related to this work. The main aim of this study was to test the tractability of these ICs and use them to characterise U/CBSV gene functions. The UCBSV IC exhibited high levels of sequence instability during propagation in all tested E. coli strains. In contrast, the CBSV IC which contains introns, demonstrated sequence stability during propagation in the E. coli strains ccdB and TOP10. This permitted the insertion of GFP at two genome positions. N. benthamiana infections with CBSV_GFP ICs enabled visualisation of viral movement in planta. U/CBSV genomes encode Ham1-like peptides with conserved ITPase pyrophosphohydrolase motifs, which have only been reported in Euphorbia ringspot virus. Eu/prokaryotic ITPase proteins selectively hydrolyze non-canonical nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby reduce mutation rates. In vitro pyrophosphohydrolase assays, demonstrated that U/CBSV Ham1 proteins have significantly higher activities with non-canonical nucleotides compared with canonical nucleotides. However, no significant differences were found in the number of viral variants during N. benthamiana infections with full-length CBSV IC and a CBSV IC containing Ham1 deletion, indicating that CBSV Ham1 may not reduce viral mutation rates. Additionally, mutation of the CBSV IC demonstrated that a Ham1 pyrophosphohydrolase motif is associated with the development of severe necrosis during N. benthamiana infections and a chimera IC, consisting of the CBSV genome with UCBSV coat protein replacement caused systemic N. benthamiana infections; raising the possibility for transcapsidation to occur during mixed U/CBSV infections in the field. These insights into CBSV gene function demonstrates the utility of U/CBSV ICs and should aid future studies into these important viruses. II Dedication and acknowledgements First and foremost, I would like to thank my supervisors Dr. Andy Bailey and Prof. Gary Foster for their continued support throughout my PhD. I really appreciate the time and energy they have invested to improve my confidence, nurture my scientific abilities and provide opportunities that have expanded my horizons from the beyond the lab to the real world. I have loved being a part of the Molecular Plant Pathology group at the University of BristoI. I am especially grateful to Dr. Alice Banks, Dr. Sarah Nanyiti and Dr. Anna Tiley, who spent time training me at the start of my PhD. I also would like to thank my good friend Dr. Luis Pablo- Rodriguez, who taught me techniques, talked about viruses (a lot) and made working in the lab fun, especially the singing and dancing! I am extremely thankful for my experiences visiting the National Crops Resources Research Institute in Uganda in 2016. I would like to thank Dr. Titus Alicai and his team who generously welcomed me into the Root Crops Research Programme during my internship. Seeing the fantastic work being done at NaCRRI has inspired me to continue working in this research area. I would also like to thank my friend Everline Odrumuru and her lovely family, who kindly made me feel at home in Uganda. I would like to give thanks to my parents Peter and Rosie Tomlinson. They have shown endless love and support right from an early age but also now as an adult. Their encouragement continues to be a great source of strength that I am extremely grateful for. I would also like to thank my sister Amy for her love and encouragement; I am so proud of you! I have also had some incredible support from friends at the University of Bristol including Dora, Donald, Fiona, Noriane, Ashley and Francesca de la Torre, with whom I have shared good times over coffee, cake and beer. I would really like to thank Dr. Kelly Atkins who has been there through the highs and lows and never fails to make me smile. I am so excited to see your career move in a new direction and cannot wait to celebrate with you! I would like to dedicate my thesis to my dear friend Nina Trott, who sadly passed away in 2017. I was very lucky to have grown-up under her friendship; her passion for life continues to inspire me. Finally Joe, I cannot put into words how much I love you. These last few years have not always been easy, but you have always been there to listen, encourage and support me through even the hardest days. I am so excited to share the next stages of our lives together and to start some new adventures with you! III Author’s declaration I declare that the work in this dissertation was carried out in accordance with the requirements of the University’s Regulations and Code of Practice for Research Degree Programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate’s own work. Work done in collaboration with, or with the assistance of, others, is indicated as such. Any views expressed in the dissertation are those of the author. SIGNED: ……… …….. DATE: …16/01/2019……………………… Katie Rose Tomlinson IV Table of Contents Chapter 1: Introduction .............................................................................................................. 1 1.1 Global food security ..................................................................................................... 1 1.2 Plant diseases ............................................................................................................... 2 1.3 Plant viruses ................................................................................................................. 3 1.4 Cassava ........................................................................................................................ 3 1.4.1 Uses of cassava for food and industrial products ................................................. 6 1.5 Constraints on cassava production in Africa ........................................................ 6 1.5.1 Bacterial diseases ................................................................................................. 6 1.5.2 Fungal diseases .................................................................................................... 6 1.5.3 Pests ..................................................................................................................... 7 1.5.3.1 Bemisia tabaci ............................................................................................... 7 1.5.4 Viral diseases .......................................................................................................