Molecular analysis of hibiscus chlorotic ringspot virus coat protein mediated suppression of gene silencing

MOLECULAR ANALYSIS OF HIBISCUS CHLOROTIC RINGSPOT VIRUS COAT PROTEIN MEDIATED SUPPRESSION OF GENE SILENCING MENG CHUNYING (B.Sc Hons.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements My first thanks go to my supervisors, Prof Wong Sek Man and Assoc Prof Peng Jinrong, for their inspiration, guidance and encouragement to support my accomplishment of the project, especially for their enlightenment in the research career I would like to extend my special appreciation to Prof Wong Sek Man who provided me with very good training opportunities in every aspect and extended his consideration during my time in his lab My sincere thanks also go to my PhD committee members, Asst Professor Low Boon Chuan and Asst Prof Yu Hao for their helpful discussion and advice during the committee meetings I sincerely wish to thank Dr Chen Jun from IMCB for his special help and suggestions in the experiments Heartily thanks also goes to Prof Ding Shouwei and Dr Li Hongwei from University of California, Riverside, for their patience and good suggestions on my experiments Special thanks to all the members of the Plant Molecular Virology Lab: Xiaoxing, Shishu, Weimin, Cheng Ao for the cDNA clone pGST-CP, Chinchin, Yang Jing, Yiyang, Soonguan and Anabelle Special appreciation goes to Haihe, Luo Qiong, Srini, Yuhong, Yunfeng, Yifeng, Zhou Jing, Lailai, Wenqing, Hock Chun, Xiaobo, Zhuolei, Wang Yu, Li Mo and Hongbin, thanks to you guys for the help and also the precious friendships Sincere thanks go to Mr Chong PL and Madam Loy GL of DBS for their administrative help and Mr Lin QW of Temasek Life Sciences Laboratory for his help with SEM work I wish to pay special tributes to my parents and my parents-in-law for their encouragement and understanding throughout all these years Special thanks to my husband, Kong Lesheng, for his full support, love and continuous encouragement Finally, grateful thanks go to the National University of Singapore for awarding me the NUS research scholarship i Table of Contents Acknowledgements i Table of Contents ii Figures viii Tables ix Abbreviation x Summary xiii Publications xv Chapter Introduction 1.1 Gene silencing 1.1.1 Discovery of gene silencing 1.1.2 Induction of gene silencing 1.1.3 Mechanisms of gene silencing 1.1.4 Systemic silencing signal 14 1.1.5 Natural roles of RNA silencing 16 1.2 Gene silencing suppressors 19 1.2.1 Discovery of viral gene silencing suppressor 19 1.2.2 Plant virus-encoded suppressors 21 1.2.3 Animal virus-encoded suppressors 25 1.2.4 Molecular basis of silencing suppression 27 1.3 MiRNA 30 1.3.1 miRNA biogenesis 30 ii 1.3.2 Mechanism of miRNA function: translational repression and mRNA cleavage 33 1.3.3 miRNA function 34 1.4 Ta-siRNAs 38 1.5 HCRSV 39 1.5.1 HCRSV genome organization and recent research progress on this virus 39 1.6 Rationales and objectives of this project 43 1.6.1 The gap of current knowledge 43 1.6.2 The objectives of the research 44 Chapter General materials and methods 46 2.1 Media and buffers 46 2.2 Plant materials and inoculation 46 2.2.1 Plant materials and growth conditions 46 2.2.2 Plant inoculation 47 2.3 Molecular cloning 47 2.3.1 Polymerase chain reaction (PCR) 47 2.3.2 Purification of PCR fragments and isolation of DNA fragments from agarose gel 47 2.3.3 Ligation of DNA inserts into plasmid vectors 48 2.3.4 Site-directed mutagenesis 48 2.3.5 Preparation of competent Escherichia coli cells 49 2.3.6 Transformation of bacteria with plasmids 49 2.3.7 Small scale plasmid purification from E coli 50 iii 2.3.8 DNA sequencing 50 2.3.9 Preparation of Agrobacterium electro-competent cells 50 2.3.10 Transformation of Agrobacterium with binary plasmids 51 2.3.11 Agro-infiltration 51 2.3.12 GFP imaging 52 2.3.13 Transformation of Arabidopsis by Agrobacterium flower dip transformation method 52 2.4 In vitro transcription of DNA with T7/T3 RNA polymerases 53 2.4.1 Preparation of digoxin (DIG)-labeled RNA probes 53 2.4.2 Preparation of radio-labeled RNA probes 54 2.4.3 Preparation of infectious transcripts 54 2.5 Analysis of RNA from plants 2.5.1 Isolation of total RNA from plants 55 2.5.2 Northern blot 55 2.5.3 Isolation of low molecular weight (LMW) RNA from plants 57 2.5.4 Detection of siRNA 58 2.5.5 Detection of miRNA 59 2.6 Protein expression and purification 60 2.6.1 Plasmid construction and transformation 60 2.6.2 Induction of bacterial expression 60 2.6.3 Protein purification 61 2.6.4 Sodium dodecylsulfate-polyacrylamide electrophoresis (SDS-PAGE) 61 2.6.5 Immunodetection of proteins 62 2.7 Protein extraction from virus-infected plants 62 iv 2.8 Isolation and transfection of kenaf protoplasts 63 2.8.1 Isolation of kenaf protoplasts 63 2.8.2 PEG transfection of protoplasts 64 2.9 Reverse Transcription-PCR 64 Chapter Host-induced avirulence of HCRSV mutants correlates with reduced gene silencing suppression activity 66 3.1 Introduction 66 3.2 Materials and methods 68 3.2.1 Construction of plasmids for agroinfiltration assay and PVX expression 68 3.2.2 Plant materials 71 3.2.3 Agrobacterium co-infiltration and GFP imaging 72 3.2.4 Isolation and detection of HMW RNA and LMW RNA 72 3.2.5 In vitro transcription and plant inoculation 73 3.2.6 Cell Culture, transfection, and analyses 73 3.3 Results 74 3.3.1 HCRSV was a strong gene silencing suppressor and various domain deletion mutants failed to suppress PTGS effectively 74 3.3.2 p27 and its isoforms failed to suppress gene silencing 80 3.3.3 Avirulent mutant CPm1+2+3 lost its ability to suppress PTGS 83 3.3.4 HCRSV CP enhances symptom severity in N benthamiana 87 3.3.5 HCRSV CP failed to suppress dsGFP-induced local and systemic PTGS 90 v 3.3.6 HCRSV CP failed to rescue the Nodamura virus genome RNA accumulation caused by antiviral silencing (in collaboration with Dr Li Hongwei at University of California, Riverside, USA) 94 3.4 Discussion 96 Chapter HCRSV CP affects microRNA and ta-siRNA accumulation in transgenic Arabidopsis and causes abnormal development of leaves and flowers 101 4.1 Introduction: 101 4.2 Materials and methods 104 4.2.1 Plant materials 104 4.2.2 Generation of CP and ΔCP transgenic Arabidopsis 105 4.2.3 Genetic crossing 105 4.2.4 Isolation and detection of HMW RNA and small (~25 nt) RNA (siRNA, miRNA and ta-siRNA) 106 4.2.5 Phenotypic analysis 106 4.2.6 Real time PCR analysis 106 4.2.7 Histochemical staining for GUS activity 108 4.3 Results 108 4.3.1 Generation of HCRSV CP transgenic Arabidopsis 108 4.3.2 HCRSV CP could inhibit PTGS induced by sense-RNA transgene and virusderived amplicon transgene 110 4.3.3 HCRSV CP transgenic Arabidopsis showed rdr6-like developmental defects 114 4.3.4 CP affects miRNA accumulation: miR172 118 vi 4.3.5 HCRSV CP affects siR255 accumulation 121 4.3.6 HCRSV CP changes the accumulation of gene silencing related genes 122 4.4 Discussion 126 4.4.1 Transgene-expressed CP suppresses genetic and epigenetic gene silencing 126 4.4.2 How does HCRSV CP affect normal Arabidopsis development? 127 Chapter Interaction of HCRSV CP with host proteins and siRNAs 130 5.1 Introduction 130 5.2 Material and methods 132 5.2.1 Plasmids 132 5.2.2 Agrobacterium transient expression assay 133 5.2.3 TAP purification 133 5.2.4 Expression and Purification of HCRSV CP and TBSV p19 134 5.2.5 Gel mobility shift assay 135 5.3 Results and discussion 135 5.3.1 Isolation of HCRSV CP-interacting protein(s) 135 5.3.2 HCRSV CP could bind siRNA in vitro 139 Chapter Conclusions and future work 143 6.1 Conclusions 143 6.2 Future work 144 References 148 Appendix Media and buffers 171 vii Figures Figure 1.1 Schematic representation of HCRSV genome organization…………………40 Figure 3.1 Phylogenetic tree based on the CP amino acid sequence of Tombusviridae 69 Figure 3.2 HCRSV viral genome organization and binary constructs used for the agroinfiltration assay ……………………………………………………………………75 Figure 3.3 Suppression of transgene-induced PTGS by HCRSV CP ………….……….78 Figure 3.4 p27 and its isoforms failed to suppress PTGS ………………………………81 Figure 3.5 Schematic representation of CP mutant constructs cloned into pBI121…… 84 Figure 3.6 Suppression of PTGS by CP mutants originated from serial passage in HCRSV local host………………………………………………………………….…….85 Figure 3.7 Effect of CP when constitutively expressed in pP2C2S vector in systemically infected N benthamiana…………………………………………………………………88 Figure 3.8 HCRS CP could not suppress dsGFP induced PTGS in 16c and wt N benthamiana …………………………………………………………………………….91 Figure 3.9 Suppression of the RNAi antiviral response by Nodamura virus (NoV) B2 but not HCRSV CP in Drosophila S2 cells ……………………………………………… 95 Figure 4.1 Making of HCRSV CP transgenic Arabidopsis…………………………….109 Figure 4.2 HCRSV CP inhibits PTGS induced by sense-RNA transgenes and amplicon silenced PTGS………………………………………………………………………… 111 Figure 4.3 Developmental defects of CP transgenic Arabidopsis…………………… 116 Figure 4.4 Blot analyses of miRNA accumulation in CP transgenic Arabidopsis and rdr611 and zip-1 in the col-0 background………………………………………………… 119 Figure 4.5 Reduced accumulation of siR255 in CP6 flowers……………………….….123 viii Figure 4.6 Real-time PCR analyses for the comparison of gene accumulation of Col-0 and CP6 transgenic Arabidopsis……………………………………………………… 125 Figure 5.1 Purification of HCRSV interacting host proteins………………………… 136 Figure 5.2 EMSA of HCRSV CP and synthesized siRNAs in vitro……………………140 Figure 6.1 Proposed model of how HCRSV CP interferes with the gene silencing related pathways……………………………………………………………………………… 145 Tables Table Genes involved in gene silencing across various organisms…………………… Table RNA-silencing suppressors encoded by plant, insect and vertebrate viruses… 22 Table Similarities and differences of miRNA and siRNA…………………………… 31 Table Oligos used for the miRNA and ta-siRNA detection….………………………107 Table Primers used for the real-time PCR analysis………………………………… 107 ix Lewis, B.P., Shih, I., Jones-Rhoades, M.W., Bartel, D.P., and Burge, C.B (2003) Prediction of mammalian microRNAs targets Cell 115, 787-798 Li, H.W., Li, W.X., 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responses Virus- encoded gene silencing suppressors offered an excellent tool for dissection of the gene silencing process Gene silencing suppressors... 1.1 Gene silencing 1.1.1 Discovery of gene silencing 1.1.2 Induction of gene silencing 1.1.3 Mechanisms of gene silencing 1.1.4 Systemic silencing. .. 1.2.4 Molecular basis of silencing suppression Although many gene silencing suppressors have been found, there is little knowledge of how these gene silencing suppressors inhibit the gene silencing