Studies on interactions between HCRSV coat protein and host proteins from kenaf

STUDIES ON INTERACTIONS BETWEEN HCRSV COAT PROTEIN AND HOST PROTEINS IN KENAF Zhang Xin (B.Sc, CAU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS My first thank goes to my supervisor, Prof. Wong Sek Man for his excellent instructions and constant support. I really appreciate the research study in the molecular virology lab and the opportunity to learn under his guidance. I have also learnt a lot from Prof. Wong about his wisdom and social experience which have enlightened me. I really appreciate his patience and warm heart to help me to overcome difficulties in my studies. My sincere thanks also go to my PhD committee members, Associate Professor Yu Hao and Assistant Professor Cynthia He for their helpful discussions and advice during the committee meetings. Special thanks to all the members of the Plant Molecular Virology Lab: Li Weimin for the clone of pGBKT7-CP and L10a, Cheng Ao for the cDNA clone pGST-CP, Niu Shengniao for her technical advice. I also thank the undergraduate students in the lab Fiona Setiawan, Sharon Kok, Tan Shihao, Lin Bitong and Xie Zhicheng. I also would like to thank my former lab members Lim Chin Chin, Meng Chunying, Xie Juntao and current lab members Sunil Kumar Tewary, Zhuang Linjie, Yi Wen and Gao Ruimin for all of their help and encouragement during my PhD study in the lab. Special appreciation goes to Wang Cheng, for his continuous care and love; special thanks also go to Tu Haitao, Zhang Jingfeng, for their help and the precious friendship. i Sincere thanks go to Mr Chong PL and Madam Loy GL of DBS for their help with transmission electron microscopy work. I also want to thank Ms Tong Yan for her help in my confocal laser microscopy study. I wish to pay special tributes to my family members for their encouragement throughout all these years. Special thanks to my former teachers in China Agricultural University, Associate Professor Cheng Yuqin, Professor Fan Zaifeng, Professor Li Huaifang for their continuous encouragement and care. Finally, grateful thanks go to the National University of Singapore for awarding me the NUS research scholarship. ii Table of Contents ACKNOWLEDGEMENTS . i Table of Contents . iii LIST OF TABLES x LIST OF FIGURES xi ABBREVIATIONS . i SUMMARY viii PUBLICATIONS . x CHAPTER Introduction 1.1 Symptoms and developmental abnormalities 1.1.1 Virus infection affected host gene expression and proteins 1.1.2 Virus infection initiated plant response . 1.1.3 Virus infection altered hormone metabolism 1.1.4 Virus infection triggered plant defense mechanism 1.2 Virus-host interaction . 12 1.2.1 Virus-host interactions that coordinate replication or translation . 12 1.2.2 Virus-host interactions that coordinate movement . 14 1.2.3 Virus-host interaction that suppresses resistance of plant . 16 iii 1.3 Protein-protein interaction study 18 1.3.1 Yeast Two-Hybrid (Y2H) Screening . 19 1.3.2 Other methods to study protein-protein interaction 23 1.4 Virus-induced silencing of genes in plant 27 1.5 The functions of viral coat protein in virus infection and virus-host interaction . 28 1.6 Studies on HCRSV and HCRSV CP 30 1.6.1 HCRSV . 30 1.6.2 Kenaf . 33 1.6.3 Interaction studies on HCRSV CP 33 1.7 Rationales and objectives of this project . 34 CHAPTER General materials and methods 37 2.1 Media and buffers 37 2.2 Plant materials and inoculation 37 2.2.1 Plant materials and growth conditions 37 2.2.2 Plant inoculation . 37 2.3 Molecular cloning 38 2.3.1 Polymerase chain reaction (PCR) . 38 2.3.2 Purification of PCR fragments and DNA fragments from agarose gel . 38 2.3.3 Ligation of DNA inserts into plasmid vectors 38 iv 2.3.4 Preparation of competent E. coli . 39 2.3.5 Transformation of bacteria with plasmids 39 2.3.6 Plasmid purification from E. coli 40 2.3.7 DNA sequencing . 40 2.4 Y2H study 41 2.4.1 Preparation of yeast competent cells (LiAC method) . 41 2.4.2 Transformation of plasmids into the yeast cells 42 2.4.3 Purification of plasmids from yeast cells 42 2.5 In vitro transcription of DNA with T7/T3 RNA polymerases 43 2.5.1 Preparation of infectious transcripts . 43 2.5.2 Inoculation of in vitro transcription product onto true leaves . 44 2.6 Analysis of RNA from plants . 44 2.6.1 Isolation of total RNA from plants 44 2.6.2 Real-time PCR 45 2.7 Protein expression and purification . 46 2.7.1 Plasmid construction and transformation 46 2.7.2 Induction of protein expression 47 2.7.3 Protein purification . 47 2.7.4 Protein concentration measurement 48 2.7.5 Sodium dodecylsulfate-polyacrylamide electrophoresis (SDS-PAGE) 48 2.7.6 Staining of SDS gel by coomassie brilliant blue 49 v 2.7.7 Immunodetection of proteins 50 2.7.8 In vitro translation of DNA with T7 RNA polymerases . 50 2.7.9 Detection of the in vitro translation product . 51 2.8 Protein extraction from virus-infected plants 52 2.9 Isolation and transfection of kenaf protoplasts 53 2.9.1 Isolation of kenaf protoplasts 53 2.9.2 PEG transfection of protoplasts 54 2.9.3 Confocal study of protoplasts with fluorescence 54 2.10 Reverse Transcription-PCR 54 CHAPTER Screening and identification of host proteins interacting with HCRSV CP 56 3.1 Introduction 56 3.2 Materials and methods . 57 3.2.1 Construction and screening of kenaf cDNA library 57 3.2.2 Sequencing and cloning 59 3.2.3 Confirmation of the interaction . 59 3.2.4 5‟ RACE PCR to amplify the complete sequence of C2 domain containing protein . 61 3.3 Results 62 3.3.1 Kenaf cDNA Library constructed . 62 3.3.2 Sulfite oxidase in kenaf (HcSO) . 62 vi 3.3.3 Identification of interaction between HCRSV CP and HcSO and mapping of the interacting domains 66 3.3.4 C2 domain-containing protein in kenaf 68 3.3.5 Putative CsP 13.9 70 3.4 Discussion 72 CHAPTER Plant sulfite oxidase plays important roles in the symptom development of Hibiscus chlorotic ringspot virus in Kenaf . 75 4.1 Introduction 75 4.2 Material and Methods 77 4.2.1 Plant materials and construction of plasmids 77 4.2.2 Co-localization of HCRSV CP and HcSO using BiFC . 80 4.2.3 Protein expression and pull down assay of CP with HcSO 81 4.2.4 Determination of HcSO transcript level and sulfate level in mock and HCRSV infected kenaf leaves 82 4.2.5 Biochemical assays of SO and H2O2-generating activities . 83 4.2.6 Transmission electron microscopy (TEM) and immuno-EM study . 83 4.2.7 Gene-silencing study of HcSO 85 4.3 Results 86 4.3.1 HcSO interacts with HCRSV CP in kenaf protoplasts 86 4.3.2 HCRSV CP bound to HcSO in vitro . 89 4.3.3 HCRSV infection induces peroxisome proliferation and aggregation in vii kenaf cells . 91 4.3.4 HCRSV infection leads to an up-regulation of HcSO gene transcript and SO activity 93 4.3.5 4.4 HcSO was successfully silenced by TCV silencing vector . 98 Discussion 103 CHAPTER Hibiscus chlorotic ringspot virus coat protein interacts with plant 60S ribosomal protein RPL10A 109 5.1 Introduction 109 5.2 Materials and methods . 111 5.2.1 Preparation of Hibiscus stem sap proteins 111 5.2.2 Preparation of HCRSV virions and coat protein subunits 111 5.2.3 HCRSV CP affinity chromatography 112 5.2.4 Sequence analysis of putative CP-interacting protein 112 5.2.5 Molecular cloning of L10A protein from kenaf plant . 113 5.2.6 Rapid amplification of cDNA ends (RACE) and nested PCR 113 5.2.7 Yeast two-hybrid analysis . 116 5.2.8 In vitro translation . 116 5.2.9 Pull down assay . 117 5.3 Results 118 5.3.1 Identification of RPL10A . 118 5.3.2 Sequence analysis of putative L10A protein in kenaf plant 118 viii 5.3.3 Amplification of RPL10A gene using two degenerate primers generates a band with a molecular size at around 300bp . 121 5.3.4 Y2H study . 129 5.3.5 Pull down assay . 131 5.4 Discussion 134 CHAPTER Conclusions and future work 136 6.1 Conclusion and discussion . 136 6.2 Future work 138 References 140 ix by hooking up with Argonautes. 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Posters and presentations: 1 Zhang Xin, Sharon Kwok and Wong Sek Man 2007 Screening of interactions between HCRSV CP and proteins in kenaf using yeast-two-hybrid system Poster presentation for the 2nd Asian Conference on Plant Pathology, Indonesia 2 Zhang Xin and Wong Sek Man 2008 Screening of interactions between HCRSV CP and proteins in kenaf using yeast-two-hybrid system Poster presentation for the... presentation for the 9th International Conference of Plant pathology, Italy 3 Zhang Xin and Wong Sek Man 2009 Studies on interactions between HCRSV coat protein and host proteins in kenaf Oral presentation for the 14th Biological Sciences Graduate Congress, Bangkok, Thailand x CHAPTER 1 Introduction Virus infection may disrupt host gene expression and physiology, causing disease symptoms and developmental abnormalities... on host physiology appear to be derived from the interaction of virus and host 11 components and not from general metabolic perturbations caused by the overproduction of viral components and competition for host resources 1.2 Virus -host interaction Virus -host interaction involve in the whole process of virus infection, including replication and translation within the host cells, locally spreading and. .. spreading and long distance movement and suppression of the host defense 1.2.1 Virus -host interactions that coordinate replication or translation Viral replication depends on compatible interactions between a virus and its host Several host translation machineries have been suggested to play essential roles in the replication and translation of viral RNAs in plants For example, the translation eukaryotic... hybrid screening for interaction between viral proteins and host proteins and the significance of the interaction 20 Table 1.2 Y2H method used to study interacting partners of viral proteins 22 Table 3.1 Primers for cDNA library construction and RACE PCR of C2 domain containing protein 60 Table 3.2 Clones with cDNA sequences matching putative proteins from NCBI database ... infection affected host gene expression and proteins The virus diverts a substantial amount of plant metabolites for the production of virus specific proteins and nucleic acids, and alters host gene expression to create a more favorable environment for infection This diversion of resources and alterations in host gene expression profile can either cause significant changes in plant physiology and development... (HCRSV) infection can cause severe chlorotic ringspot symptoms and stunted growth on Hibiscus which is an ornamental plant grown in many parts of the world HCRSV coat protein (CP) is an important viral gene product required for encapsidation and viral systemic movement To better understand the roles of HCRSV CP in viral infection and its interactions with host proteins, a kenaf cDNA library was constructed... 5.2 Interaction between HCRSV CP and L10A and identification of domains of CP that are involved in the interaction in yeast cells 130 x LIST OF FIGURES Figure 1.1 Schematic representation of HCRSV genome organization 32 Figure 3.1 Amino acid sequence alignment of the SO from kenaf (HcSO) and other plants 65 Figure 3.2 Interaction between HCRSV CP and HcSO and identification of domains... library was constructed and screened using a yeast two-hybrid (Y2H) system to identify CP-interacting proteins Using the Y2H system, several putative proteins that interact with HCRSV coat protein were identified These proteins include sulfite oxidase (SO), a putative major latex-like protein, a putative chaperon P13.9, a C2 domain-containing protein, a ricin domain-containing protein and putative alpha-D-xylosidase... interaction in yeast cells 67 Figure 3.3 Amino acid sequence alignment of C2 domain containing protein from kenaf (HcSO) and similar proteins in other plants 69 Figure 3.4 Alignment of CsP13.9 with putative chaperon P13.9 [Castanea sativa], and putative bundle sheath defective protein [Oryza sativa Japonica Group] 71 Figure 4.1 BiFC co-localization of HCRSV CP and HcSO in kenaf . The functions of viral coat protein in virus infection and virus -host interaction 28 1.6 Studies on HCRSV and HCRSV CP 30 1.6.1 HCRSV 30 1.6.2 Kenaf 33 1.6.3 Interaction studies on HCRSV CP. 2.7 Protein expression and purification 46 2.7.1 Plasmid construction and transformation 46 2.7.2 Induction of protein expression 47 2.7.3 Protein purification 47 2.7.4 Protein concentration. STUDIES ON INTERACTIONS BETWEEN HCRSV COAT PROTEIN AND HOST PROTEINS IN KENAF Zhang Xin (B.Sc, CAU) A THESIS SUBMITTED