ABSTRACT NI, SIHUI. RNA Translocation into Chloroplasts. (Under the direction of Dr. Heike Inge Sederoff). Plastids and mitochondria are known as semi-autonomous organelles because they can encode and synthesize proteins that are essential for metabolism. They are both derived from endosymbiotic events. The bacterial ancestor of plastids was cyanobacteria, while the bacterial ancestor of mitochondria was a proteobacteria. During their evolution, most of the ancestral bacterial DNA was translocated into the nuclear genome DNA. The current plastid genome encodes proteins for the photosynthetic apparatus, the transcription/translation system, and various biosynthetic processes. The current mitochondrial genome mainly encodes proteins for electron transport, ATP synthesis, translation, protein import and metabolism. Plastids and mitochondria are not self-sufficient; they import the majority of their proteins, which are synthesized in the cytosol. For plastids, a transit peptide at the N-terminus of protein precursors can be recognized by TOC/TIC complex and mediate their import. Similarly, for mitochondria, a piece of β-signal at the N-terminal of protein precursors can be recognized by TOM/TIM complex and mediate the import of the protein precursors. It worth noting that plastids also encode a suite of transfer RNAs while mitochondria need to import certain types of tRNAs. In most angiosperms, plastids and mitochondria are inherited maternally and can only be passed down from the maternal plant organs through seed to the next generation. Maternal inheritance has benefits for organellar genetic engineering such as stable inheritance of genes over generations, absence of out-crossing, absence of contamination by pollen and minimal pleiotropic effects. The tradition methodology of plastid genomic engineering is biolistic transformation, where DNA covered gold particles parent cell walls of tissue or protoplasts at high velocity, leading to integration of DNA into chromosome(s) through recombination. However, biolistic based integration of double-stranded DNA is relatively random. Regeneration of transformed plants is complicated and often require a species-specific protocol before a homoplasmic plants can be established. The biolistic transformation of mitochondria has not yet been accomplished. For chloroplast genetic engineering, new methods are needed. The central idea of our proposed method is to import two components into chloroplasts to engineer the organellar genomic DNA and to generate homoplasmic plants. First, the reverse transcriptase is needed to generate double-stranded transgene DNA in the chloroplast in vivo. Second, the cas9 endonuclease associated with single guide RNAs is needed to degrade untransformed chromosomes to generate homoplasmic plants. Enzymes can be imported as proteins by being fused to a transit peptide for plastids and a β-signal for mitochondria. While, the mechanism of RNA translocation into organelles is not yet well understood. We tested two candidates, the derived Eggplant Latent Viroid (ELVd) and the eucaryotic initiation factor 4E (eIF4E), which have the potential to mediate the translocation of RNA into chloroplasts. To detect successful translocation, enhanced green fluorescent protein (eGFP) was used as a marker. The chimeric complementary DNA (cDNA) of the derived ELVd and eGFP was transformed into Nicotiana benthamiana using agroinfiltration and into Arabidopsis thaliana using floral dip. EGFP was detected in N. benthamiana by eGFP fluorescence and confocal microscopy but not by western blots. EGFP was not detected in A. thaliana either by confocal microscopy or western blots. The chimeric cDNA containing the eIF4E and eGFP construct was transformed into N. benthamiana using agroinfiltration. EGFP was not detected either by confocal microscopy or western blots. More research, such as in situ hybridization, is needed to determine the cause of the reason for the absence of eGFP expression. © Copyright 2018 by Sihui Ni All Rights Reserved RNA Translocation into Chloroplasts by Sihui Ni A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Plant Biology Raleigh, North Carolina 2018 APPROVED BY: ________________________________ ______________________________ Heike Inge Sederoff Richard L. Blanton Chair of Advisory Committee _______________________________ Deyu Xi ii BIOGRAPHY Sihui Ni was born in China. The very first motivation for her working in plant biology is to develop a reliable and nutritious food source in Africa using transgenic technologies. Fortunately, she was introduced to Dr. Heike Sederoff by Dr. Mari Chinn and therefore had a chance to explore the world of plant biology. Sihui gained her bachelor’s degree in Functional Materials at Lanzhou University in China, which helped her build a background in chemistry and physics. Driven by the strong motivation of working in plant biology, she started to work in plant biology labs since the second year in undergraduate school. During this period, she obtained various experimental skills in molecular biology. iii ACKNOWLEDGMENTS Funding: NCSU Chancellor’s Innovation Fund Advisors, Committee, Mentors, Graduate Student Support: Dr. Heike Sederoff, Professor, Department of Plant & Microbial Biology (PMB) at North Carolina State University (NCSU); Dr. Larry Blanton, Professor, Director of Graduate Programs, Department of PMB at NCSU; Dr. Deyu Xie, Professor, Department of PMB at NCSU; Sue Vitello, Executive Assistant, Department of PMB at NCSU; Dwayne Barnes, Graduate Services Coordinator, Department of PMB at NCSU; Catherine Freeman, Executive Assistant, Department of PMB at NCSU； Dr. Jay Cheng, Professor, Department of Biological and Agricultural Engineering (BAE) at NCSU. Colleagues & Collaborators: Dr. Imara Perera, Research Professor, Department of PMB at NCSU; Dr. Eva Johannes, Director of Cellular and Molecular Imaging Facility at NCSU; Dr. Mari Chinn, Professor, Department of BAE at NCSU; Colin Murphree, Graduate student, Department of PMB at NCSU; Jacob Dums, Doctor of Philosophy, Department of PMB at NCSU; Danielle Young, Graduate student, Department of Plant Biology at Michigan State University; Sathya Jali, post doctorate, Department of PMB at NCSU; Christophe La Hovary, post doctorate, Department of Crop and Soil Sciences at NCSU; Eli Hornstein, Melodi Charles, Nathan Wilson and Samuel Acheampong, Graduate students, Department of PMB at NCSU; Brianne Edwards, technician, Department of PMB at NCSU; Avery Ashley and Nikki Khoshnoodi, Undergraduate students, Department of PMB at NCSU Other moral support: Juanying Shen, Jianhua Ni, Guangming Wang, Xiting Liu, Lora J. Gary, Jing Wu, Wenbin Zhou, Yue Zhu, Huangchao Yu, Tao Jiang and Katherine Speight. iv TABLE OF CONTENTS LIST OF TABLES .................................................................................................................... v LIST OF FIGURES ................................................................................................................. vi LIST OF ABBREVIATIONS ................................................................................................. vii CHAPTER 1: RNA Transport into Organelles ................................................................... 1 1.1 Organellar Genomes in Plant Cells ...................................................................... 2 1.2 Plastids Genome Engineering ............................................................................ 14 1.3 RNA Translocation into Plastids ....................................................................... 20 1.4 Mitochondrial Genome Editing ......................................................................... 23 1.5 RNA Translocation into Mitochondria .............................................................. 24 REFERENCES ........................................................................................................... 28 CHAPTER 2: RNA Transport into Chloroplasts Mediated by ELVd ............................ 39 Introduction ................................................................................................................. 40 Methods and Materials ................................................................................................ 45 Results ......................................................................................................................... 50 Plasmid Constructions .................................................................................... 50 Tobacco Transformation ................................................................................. 52 EGFP Was Not Detected in Arabidopsis ........................................................ 54 Discussion ................................................................................................................... 60 REFERENCES ........................................................................................................... 65 APPENDICES ........................................................................................................................ 69 Appendix A: Recipes and Protocols ........................................................................... 70 Appendix B: DNA Segments Sequences and Primers ................................................ 73 v LIST OF TABLES Table 1. Successful cases of plastid gene transformation using biolistics. …………………..15 vi LIST OF FIGURES CHAPTER 1 Figure