INVESTIGATION OF THE FUNCTIONS OF P23 AND COAT PROTEIN OF HIBISCUS CHLOROTIC RINGSPOT VIRUS GAO RUIMIN NATIONAL UNIVERSITY OF SINGAPORE 2013 INVESTIGATION OF THE FUNCTIONS OF P23 AND COAT PROTEIN OF HIBISCUS CHLOROTIC RINGSPOT VIRUS GAO RUIMIN (B.Sc., M.Sc., Henan Agricultural University, PRC) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEDGEMENTS My heartiest thank first goes to my supervisor, Prof. Wong Sek Man for his excellent guidance, invaluable instructions, insightful advices and kind support throughout my PhD candidature. I really appreciate Prof. Wong providing me the opportunity to learn plenty of knowledge in the molecular plant virology lab. I have also learnt a lot from Prof. Wong about his wisdom and rich experiences in life which enlightened my life and made me avoiding mistakes. I am grateful of his brilliant minds and warm heart which help me during my hard time and guide me to discover more about my future career. My truthful thanks also go to my PhD quanlify examination committee members, Associate Professor He Yuehui, Dr. He Ying Xin, Cynthia and Dr. Lin Qingsong for their support and kind help during my PhD journey. My sincere thanks to all the members of the Plant Molecular Virology Lab: Dr. Niu Shengniao for her useful research suggestions and experienced technical help. I also thank the undergraduate students Ng Kai Lin Florence, Tan Jia Xin Danica, Wan Zi Yi and Tan Chee Leong Kelvin for helping me in some of the experiments. I also would like to thank my current lab members, Mr. Xie Zhicheng, Ms Wen Yi, Ms Guo Song and Ms Wu Chao and my former lab members Dr. Zhang Xin, Dr. Qiao Yan and Dr. Sunil Kumar Tewary for all of their help and precious friendships during my four years PhD study in the lab. Special thanks also go to all my friends from A/P Pan Shenquan’s lab and Dr. Xu’s lab for their informative discussions and timely help. Special thanks also go to Ms Tong Yan and Ms Foong Choy Mei for their help in my confocal laser microscopy study. I also would like to thank Mr. Chong PL and Madam Loy GL from DBS for their support with electron microscopy work. Last but not the least, I wish to express my deepest appreciations to my family for their encouragement throughout all these years. Special thanks to my husband i Liu Peng, for his continuous support and critical comments. Finally, grateful thanks go to the National University of Singapore for awarding me the NUS research scholarship. ii List of publications 1. Ruimin Gao, Florence Kai Lin Ng, Peng Liu, Sek-Man Wong. (2012) Hibiscus chlorotic ringspot virus coat protein upregulates sulfur metabolism genes for enhanced pathogen defence. Molecular Plant-Microbe Interaction 25: 15741583. 2. Ruimin Gao, Peng Liu, Sek-Man Wong. (2012) Identification of a plant viral RNA genome in the nucleus. PLOS ONE 7(11): e48736. doi:10.1371/journal.pone.0048736 3. Ruimin Gao and Sek-Man Wong. (2013) Basic amino acid mutations in the nuclear localization signal of Hibiscus chlorotic ringspot virus p23 inhibit virus long distance movement. PLOS ONE 8(9): e74000. doi:10.1371/journal.pone.0074000. 4. Ruimin Gao, Danica Jia Xin Tan, Sek-Man Wong. (2013) Upregulation of miR395 targets ATP sulfurylase and sulfate transporter facilitates sulfur enhanced defence after Hibiscus chlorotic ringspot virus infection. Plant Pathology Bulletin 22(2): 107-117 5. Ruimin Gao, Zi Yi Wan, Sek-Man Wong. (2013) Correlation of miRNA fluctuation to plant growth retardation after Hibiscus chlorotic ringspot virus infection (under review). iii Table of Contents ACKNOWLEDGEMENTS . i List of publications iii Summary xi List of Tables . xiii List of Figures xiv Chapter Literature Review . 1.1 Plant virus and its infection . 1.1.1 Plant virus pathogenesis 1.2 Host-virus interaction 1.3 Sulfur enhanced defense 1.4 MicroRNAs and viral microRNAs 1.5 Nuclear localization signal 1.6 Virus movement 1.7 MiRNA related plant development and gene silencing suppressor 1.8 Rationales and objectives of this thesis research 12 Chapter General Materials and methods 14 2.1 Media and buffers 14 2.2 Plant materials and inoculation . 14 2.2.1 Plant materials and growth conditions 14 2.2.2 Plant inoculation 14 2.3 Molecular cloning . 15 2.3.1 Polymerase chain reaction (PCR) .15 2.3.2 Purification of PCR fragments and DNA fragments from agarose gel15 2.3.3 Ligation of DNA inserts into plasmid vectors 15 2.3.4 Preparation of competent E. coli .15 2.3.5 Transformation of bacteria with plasmids .16 2.3.6 Plasmid purification from E. coli 16 2.3.7 DNA sequencing .16 iv 2.3.8 PCR-based mutagenesis 17 2.3.9 Electro-transformation for Agrobacterium 17 2.3.10 Agrobacterium-infiltration 17 2.3.11 TaqMan two-step RT-PCR 18 2.4 Analysis of DNA . 19 2.4.1 Plant genomic DNA extraction .19 2.4.2 Southern blot .20 2.5 Analysis of RNA . 25 2.5.1 Total RNA extraction using TRIZOL reagent 25 2.5.2 Protocol for separating LMW RNAs 26 2.5.3 Detection of Small RNAs by Northern Blot .27 2.6 Analysis of protein 29 2.6.1 Protein extraction from plants .29 2.6.2 Protein expression and extraction from E.coli 29 2.6.3 Enzyme-linked immuno sorbent assay (ELISA) for plant viral proteins30 2.6.4 Western blot 30 2.7 Isolation and transfection of kenaf protoplasts and isolation of HCRSVinfected kenaf cells 31 2.7.1 Isolation of kenaf protoplasts following previous published protocol (Liang et al., 2002a) .31 2.7.2 PEG transfection of protoplasts .32 2.7.3 Isolation of fixed plant cells 33 2.8 Fluorescent in situ hybridization . 34 2.8.1 The fixed cells were attached to coverslips .34 2.8.2 Hybridization .35 2.9 RNA-chromotin-immunoprecipitation (RNA-CHIP) . 36 2.9.1 Tissue collection and nuclear fixation with formaldehyde .36 2.9.2 Sonication 38 2.9.3 Pre-clearing .38 2.9.4 Immunprecipitation .39 2.9.5 RNA analyses 40 v Chapter Nuclear localization of p23 and identification of HCRSV genome in the nucleus where viral miRNAs are produced 42 3.1 Introduction . 42 3.2 Materials and methods 45 3.2.1 Plant materials, plasmid construction and generation of transgenic Arabidopsis .45 3.2.2 Verification of putative transgenic Arabidopsis plants using Southern blot 46 3.2.3 Construction of artificial vir-miRNA Hcrsv-miR-H1-5p 46 3.2.4 Agrobacterium tumefaciens–mediated transient expression .46 3.2.5 Co-immunoprecipitation assay 47 3.2.6 RNA-CHIP analysis 47 3.2.7 Preparation of plant cells and protoplasts for fluorescent in situ hybridization (FISH) and silver/DAPI staining .50 3.2.8 Isolation and verification of highly purified kenaf nuclei and detection of HCRSV RNA .50 3.2.9 Preparation of Total RNA, reverse transcriptase and real-time PCR 51 3.2.10 Prediction and detection of vir-miRNAs .51 3.3 Results . 52 3.3.1 A novel NLS was detected in the p23 .52 3.3.2 Localization of HCRSV RNA in nucleus using fluorescent in situ hybridization (FISH) and highly purified nuclei 59 3.3.3 The NLS of p23 facilitates the entry of HCRSV RNA into nucleus through its binding to impotin α. 63 3.3.4 Prediction and detection of vir-miRNA in total RNA extracted from highly purified kenaf nuclei of HCRSV-infected and agro-infiltrated leaves65 3.4 Discussion . 68 3.4.1 Vir-miRNA Hcrsv-Mir-H1-5p targets the p23 gene of HCRSV 68 3.4.2 The NLS of p23 facilitates Importin α and HCRSV RNA to enter nucleus 70 3.4.3 The presence of viral RNA in the nucleus may unravel novel funcitons in gene regulation .73 vi Chapter Basic amino acid mutations in the nuclear localization signal of Hibiscus chlorotic ringspot virus p23 inhibit virus long distance movement 74 4.1. Introduction 74 4.2 Materials and methods 77 4.2.1 Plant materials and plasmid construction 77 4.2.2 Plant inoculation with in vitro transcripts of p223 and its two mutants79 4.2.3 Preparation of kenaf protoplasts for fluorescent in situ hybridization (FISH) .79 4.2.4 RNA extraction and cDNA synthesis for RT-PCR and qRT-PCR .80 4.2.5 Western blot analysis of HCRSV CP 80 4.2.6 Agrobacterium tumefaciens-mediated transient expression of amiRp23 and amiRSO .81 4.2.7 Inoculation of amiRp23 and amiRSO into apical meristems of HCRSV-infected kenaf leaves .81 4.3. Results 82 4.3.1 Viral replication was unaffected in the two HCRSV mutants 82 4.3.2 Symptoms were only observed in HCRSV wt-inoculated kenaf leaves at 19 dpi 85 4.3.3 Detection of p23 and CP transcript level in the newly emerged leaves of kenaf plants inoculated with wt HCRSV and its two mutants at 19 dpi 87 4.3.4 Less severe symptoms in pGreen-amiRp23-inoculated kenaf plants pre-inoculated with HCRSV 89 4.4. 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Plant Microbe Interact. 19:948-957. 198 Appendix I ® LB medium: 1% Bacto - tryptone, ® 0.5% Bacto - yeast extract, 0.5% NaCl, pH 7.5 LB agar: LB medium with ® 1.5% Bacto - agar, pH 7.5 TAE: 40 mM Tris-acetate, 20 mM sodium acetate, mM EDTA, pH 8.2 TBE: 89 mM Tris-borate, mM EDTA, pH 8.3 TE: 10 mM Tris-HCl, mM EDTA, pH 8.0 20 × SSC: M NaCl, 0.3 M Trisodium citrate Murashige and Skoog (MS) medium: 16 mg/L MnSO •H O, 8.6 mg/L ZnSO •7H O, 6.2 mg/L H BO , 0.83 mg/L KI, 0.25 mg/L, Na MoO , 0.025 mg/L CuSO •5H O, 0.025 mg/L CoCl •6H O 199 [...]... times and large population sizes of viruses impart them with a remarkable evolutionary potential The construction of a molecular model that confines physical interactions established between the components of the host cell and virus is the foundation of the emerging discipline of systems virology (Tan et al., 2007; Bailer and Haas, 2009) Using this model, we can understand how the virus manipulates the. .. p23 in HCRSV, which belongs to Family Tombusviridae Genus Carmovirus, is predicted to be a transcription factor that is indispensable for host-specific replication The overall aim of this study is to investigate the additional functions of p23 and CP of HCRSV The specific aims of this study include: (1) To determine and investigate the subcellular localization of p23 The presence of a positive-strand... Chook, 2009) At this point, Ran-GTP will bind to the importin -protein complex, and its binding will cause the importin to lose affinity for the protein The protein is released, and now the Ran-GTP/importin complex will move back out of the nucleus through the nuclear pore A GTPase activating protein (GAP) in the cytoplasm hydrolyzes the Ran-GTP to GDP, and this causes a conformational change in Ran,... 126 kDa and 183 kDa replicase proteins 7 of TMV (Derrick et al., 1997), helper component proteinase (HC-pro) protein of potyvirus (Cronin et al., 1995), the 2b protein of cucumber mosaic virus (CMV) (Ding et al., 1994) and p19 of tomato bushy stunt virus (TBSV) (Scholthof et al., 1995) have been shown to have specific functions in long-distance movement 1.7 MiRNA related plant development and gene... benefit, and what the actions it achieves for shunting host defenses (Whitham and Wang, 2004; Culver and Padmanabhan, 2007; Dodds and Rathjen, 2010; Elena et al., 2011) Furthermore, the own proteins and small RNAs of a virus interact and compete in replication cycle (Guo et al., 2001), and these interactions may create new paths to communicate separated cellular functions, leading to the appearance of novel... entry into the nucleus through the nuclear envelope, which consists of concentric membranes, the outer, the inner membrane, pores and large nuclear complexes These are the gateways to the nucleus A protein translated with a NLS will bind strongly to importin (aka karyopherin), and the complex will move through the nuclear pore (Fried and Kutay, 2003; Mosammaparast and Pemberton, 2004; Suel and Chook,... kinase (APK), sulfite oxidase (SO) and Hibiscus chlorotic ringspot virus coat protein (HCRSV-CP) gene transcripts in kenaf (Hibiscus cannabinus L.) leaves 10 days post inoculation (dpi) as determined by qRT-PCR 104 Figure 5.2 Gene transcript levels of sulfite reductase (SIR), APS kinase (APK), sulfite oxidase (SO) and Hibiscus chlorotic ringspot virus coat protein (HCRSV-CP) after CP gene was... 8 Conclusion and further work 168 8.1 Conclusion 168 8.2 Future work 171 x Summary Hibiscus cannabinus L (kenaf) was used as a host plant to study a plant virus Hibiscus chlorotic ringspot virus (HCRSV) The p23 is a novel open reading frame in the HCRSV which belongs to Family Tombusviridae Genus Carmovirus The p23 was found to localize in the nucleus and a novel nuclear... CHAPTER 7 147 xiii List of Figures Figure 3.1 Structural organization of HCRSV genomic RNA and its predicted virmiRNAs 43 Figure 3.2 Schematic representations of constructs of HCRSV p23 and its deletion mutants fused with GFP 53 Figure 3.3 Nuclear localization of the p23 protein of HCRSV 54 Figure 3.4 Localization of p23 of HCRSV in nucleus of transgenic Arabidopsis thaliana... countermeasure, viruses and bacteria have evolved VSRs and BSRs to suppress host RNAi machinery and compromise disease resistance in plants Many viruses encode specific proteins that suppress the host antiviral silencing response and thereby benefit viral infection It was reported that suppressor proteins can block host RNA silencing at various stages of the RNA silencing pathways and the molecular mechanisms of . INVESTIGATION OF THE FUNCTIONS OF P23 AND COAT PROTEIN OF HIBISCUS CHLOROTIC RINGSPOT VIRUS GAO RUIMIN NATIONAL UNIVERSITY OF SINGAPORE 2013 INVESTIGATION OF THE FUNCTIONS. FUNCTIONS OF P23 AND COAT PROTEIN OF HIBISCUS CHLOROTIC RINGSPOT VIRUS (B.Sc., M.Sc., Henan Agricultural University, PRC) GAO RUIMIN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. study a plant virus Hibiscus chlorotic ringspot virus (HCRSV). The p23 is a novel open reading frame in the HCRSV which belongs to Family Tombusviridae Genus Carmovirus. The p23 was found