GENETIC ENGINEERING OF AGROBACTERIUM TUMEFACIENS TO TARGET CHLOROPLASTS AND IDENTIFICATION OF A NOVEL NUCLEAR LOCALIZATION SIGNAL OF VIRD2 PROTEIN AKI MATSUOKA B.Sc. University of Lethbridge, 2010 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfilment of the Requirements for the Degree DOCTOR OF PHILOSOPHY IN BIOMOLECULAR SCIENCE Biological Sciences University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Aki Matsuoka, 2015 GENETIC ENGINEERING OF AGROBACTERIUM TUMEFACIENS TO TARGET CHLOROPLASTS AND IDENTIFICATION OF A NOVEL NUCLEAR LOCALIZATION SIGNAL OF VIRD2 PROTEIN AKI MATSUOKA Date of Defence: August 26, 2015 Dr. A. Ziemienowicz Research Scientist Ph.D. Co-supervisor Dr. I. Kovalchuk Professor MD, Ph.D. Co-supervisor Dr. J. Thomas Professor Ph.D. Thesis Examination Committee Member Dr. F. Eudes Research Scientist Ph.D. Thesis Examination Committee Member Dr. S. Rood Professor Ph.D. Internal Examiner Dr. P. Maliga Professor Ph.D. External Examiner Waksman Institute, Rutgers, The State University of New Jersey, USA Dr. T. Burg Associate Professor Ph.D. Chair, Thesis Examination Committee Dedication To my family Kunikazu, Miyo, Tadaaki, Takayuki, Nobuko iii ABSTRACT Agrobacterium tumefaciens has been adopted to become a greatest tool for plant genetic engineering. Since Agrobacteria predominantly integrate single copy of the transgene into the nuclear genome, the transgene expression is relatively low. In contrast, transgene expression from chloroplasts allows high expression. Therefore, my first objective was to develop a new method of chloroplast transformation by modifying Agrobacterium genetics. I attempted to transform tobacco chloroplasts by Agrobacterium carrying the VirD2 protein with inactivated nuclear localization signals (NLSs) fused to a chloroplast transit peptide. However, the transformation of tobacco chloroplasts was unsuccessful. It suggested that the transit peptide was overridden by a novel cryptic NLS of VirD2. My second objective was to determine a novel NLS in VirD2. I discovered the novel NLS in VirD2 by subcellular localization analyses of fusion proteins (fragment of VirD2-green fluorescent protein) in plant cells. My discovery contributed to characterization of VirD2. iv ACKNOWLEDGEMENTS I am truly grateful for what I have experienced under the supervision of Dr. Alicja Ziemienowicz and Dr. Igor Kovalchuk. I cannot describe how much I appreciate their support other than saying thank you. Thank you for giving me the opportunity to be your student. Thank you for all your time, effort, guidance and support in pushing me forward to complete my program. Thank you for being fantastic supervisors. I would like to thank my committee members Dr. François Eudes and Dr. Jim Thomas for their advice and support. I always felt their sincere kindness and enthusiasm towards my success. I sincerely appreciate Dr. Pal Maliga for agreeing to be my external examiner. Because of you, I had a chance to present my research at the Plant Transformation Technologies III conference in Vienna in 2014. Thank you for giving me the amazing opportunity. I would like to thank Dr. Stewart Rood for being my internal examiner. I hope my research is interesting to you. I would like to thank Dr. Theresa Burg for being the chair of my thesis defence. I hope I entertained you during my defence. I am truly thankful to Dr. Henry Daniell at the University of Pennsylvania, Philadelphia for providing me with the chloroplast transformation vector. Your kind gift led my project to a new direction. I am also gratefully thankful to Dr. Jonathan Monroe at James Madison University, Virginia, for providing me with the binary vectors. I was able to identify a novel NLS in VirD2 because of your kind, caring gift. I would like to thank all past and present people at both Kovalchuks’ labs for helpful discussions and technical assistance, including Dr. Andrey Golubov for his suggestion on the transit peptides, and Nina, Rommy and Vladimir for their green thumbs. Special thanks to Dr. Andriy Bilichak for our discussions which helped me to connect the dots in my head. I would like to thank Valentina Titova for correcting my thesis. I would like to thank Neema, Dr. Elizabeth Schultz and Doug Bray for assistance and suggestions with confocal microscopy. I would like to thank the University of Lethbridge and the Alberta Innovates Technology Futures for the financial support, and the University Library for letting me work throughout my undergraduate and graduate programs. Finally, I would like to thank everyone I met in Lethbridge including people on campus and off. I am so fortunate I met such amazing people. They are treasures in my life. Thank you all for being you. v Table of Contents Thesis Exam Committee Members .............................................................................................................. ii Dedication ..................................................................................................................................................... iii Abstract ......................................................................................................................................................... iv Acknowledgements ........................................................................................................................................ v Table of Contents .......................................................................................................................................... vi List of Tables .................................................................................................................................................. x List of Figures ............................................................................................................................................... xi List of Abbreviations .................................................................................................................................. xiv Chapter 1: Introduction ................................................................................................................................ 1 1.1. Agrobacterium-mediated plant transformation .................................................................................... 1 1.1.1. Transformation overview .............................................................................................................. 1 1.1.2. Vir proteins ................................................................................................................................... 3 1.1.2.1. Sensory units: VirA and VirG .............................................................................................. 3 1.1.2.2. Type IV secretion system: VirB1-11 and VirD4 .................................................................. 4 1.1.2.3. Overdrive binding proteins: VirC1 and VirC2 ..................................................................... 5 1.1.2.4. Relaxosome: VirD1 and VirD2 ............................................................................................ 6 1.1.2.5. Single stranded DNA binding protein: VirE2 and VirE1 ..................................................... 7 1.1.2.6. Adaptor and putative transcription activator ........................................................................ 8 1.1.2.7. F-box effector: VirF ............................................................................................................. 8 1.1.2.8. Translocated effector: VirD5................................................................................................ 8 1.1.3. Development of an efficient transformation method: Binary vector systems ............................... 9 1.1.4. Challenges and improvements of Agrobacterium-mediated plant transformation ..................... 10 1.1.4.1. Expansion of the host range ............................................................................................... 10 1.1.4.2. Improvement of transformation efficiency ......................................................................... 11 1.1.4.3. T-DNA integration at the precise genomic location ........................................................... 13 1.2. Chloroplast/plastid transformation ..................................................................................................... 14 1.2.1. Benefit of chloroplast/plastid transformation ............................................................................. 14 1.2.2. Chloroplast/plastid transformation vectors ................................................................................. 15 1.2.3. Chloroplast/plastid transformation methods ............................................................................... 16 1.2.4. Growing research interest in chloroplast/plastid transformation ................................................ 17 1.2.5. Challenges of chloroplast/plastid transformation ....................................................................... 18 1.3. Intracellular protein trafficking .......................................................................................................... 18 1.3.1. Nuclear import ............................................................................................................................ 18 1.3.2. Chloroplast protein import .......................................................................................................... 20 1.4. Agrobacterium VirD2 protein ...........................................................................................................