GENETIC VARIABILITY AND INTERACTIONS OF CYMBIDIUM MOSAIC VIRUS AND ODONTOGLOSSUM RINGSPOT VIRUS PRABHA ARUNA AJJIKUTTIRA, M.Sc., M.Phil. A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENTS I would like to thank my supervisors Associate Professor Sek-Man Wong and Associate Professor Chiang-Shiong Loh for their guidance, advice and encouragement during the course of my candidature. I would also like to thank my Ph.D. committee member Associate Professor Eng-Chong Pua for his constructive advice during the committee meetings. Special thanks go to Mrs. Ang, Madam Loy and Mr. Ping-Lee Chong for their technical assistance and to Mr. Yip and Mr. Ong for the help in photography. I take this opportunity to express my sincere gratitude to my former fellow student Miss Li-Huan Koh, for the advice and help she extended to me during the first year of my studies. I also appreciate the help rendered to me during my project from the following members of my laboratory, past and present: Dr. Kian-Chung Lee, Dr. Hai-hui Yu, Dr. Hai-He Wang, Dr. Dora Koh, Mr. Srinivasan K.G., Miss Aileen Lim, Miss Stella Tan and Dr. Theiingi Maw. To all the members of the lab, Chun-Ying, Lena, Luo Quiong and Yong-Mei, thank you for the friendship and the help rendered at one time or the other. I thank the National University of Singapore for awarding me a research scholarship. My immense gratitude to my dear husband and mother for the unfailing advice, love and encouragement to help me endure these arduous years and to dad for being my inspiration. i CONTENTS Acknowledgements i Contents ii List of Publications ix List of Figures x List of Tables xv List of Abbreviations xvi Summary xx CHAPTER 1.1. INTRODUCTION Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) 1.1.1. Economic significance and incidence of CymMV and ORSV 1.1.2. Host range and symptomatoglogy 1.1.3. Mode of transmission 1.1.4. Molecular structure and composition 1.2. Sequence Variability in the CP genes 1.3. Regeneration of transgenic orchids 11 1.4. Synergism in CymMV and ORSV 12 1.5. Complementation of MP and/or CP genes 14 1.6. Molecular Biology of PVX 15 1.7. Molecular Biology of TMV 18 1.8. Objectives of this research 20 ii CHAPTER MATERIALS AND METHODS 22 2.1. Bacterial strains 22 2.2. Plasmids 22 2.3. Media 22 2.4. Synthesis of Oligonucleotides 23 2.5. Preparation of DEPC-treated reagents 23 2.6. Preparation of water-saturated phenol 23 2.7. Plant materials 23 2.8. Virus purification 24 2.8.1. Purification of CymMV 24 2.8.2. Purification of ORSV 25 2.9. RNA extraction 25 2.10. PCR 25 2.11. Isolation of plasmid DNA from E. coli 26 2.12. DNA purification 27 2.13. DNA ligation 27 2.14. 27 Transformation of E. coli and A. tumefaciens 2.15. PCR sreening of transformants 28 2.16. 28 Automated DNA sequencing 2.16.1. Cycle sequencing 28 2.16.2. Preparation of polyacrylamide gel for DNA sequencing 29 2.16.3. Loading of DNA samples and electrophoresis 29 2.17. 30 Point mutation by PCR iii 2.18. In vitro transcription 30 2.19. Generation of non-radioactive DIG-labelled cRNA probes 31 2.20. Extraction of total RNA 31 2.21. 32 Northern blot hybridization 2.21.1. Electrophoresis of RNA 32 2.21.2. Transfer, Probing and Detection of RNA 32 2.22. Extraction of total protein 33 2.23. SDS-PAGE and Western Blot Analysis 33 CHAPTER GENETIC VARIABILITY IN THE COAT PROTEIN GENES OF CYMBIDIUM MOSAIC VIRUS AND ODONTOGLOSSUM RINGSPOT VIRUS 35 3.1. Plant Materials 35 3.2. Bacterial strains 35 3.3. Transformation of E. coli 35 3.4. Plasmids 35 3.5. Synthesis of oligonucleotides 36 3.6. Virus detection 36 3.6.1. ELISA 36 3.6.2. Small scale virus purification and TEM 37 3.7. Preparation of template for RT-PCR 37 3.8. RT-PCR 38 3.9. PCR purification and ligation 39 iv 3.10. Automated DNA sequencing 39 3.11. Phylogenetic Analyses 39 3.12. 40 Results and Discussion CHAPTER REGENERATION OF TRANSGENIC ORCHIDS 55 4.1. Plant Materials 55 4. 2. Bacterial Strains and plasmids 56 4.3. Cloning of genes of interest into pBI121 vector 56 4.3.1. Coat protein gene of CymMV 56 4.3.2. Coat protein gene of ORSV 57 4.4. Preparation of electrocompetent Agrobacterium LBA 4404 57 4.5. Electroporation of Agrobacterium LBA 4404 60 4.6. Agrobacterium cell suspension 60 4.7. Agrobacterium-mediated transformation 61 4.8. Results and Discussion 61 CHAPTER COMPLEMENTATION BETWEEN CYMBIDIUM MOSAIC VIRUS AND ODONTOGLOSSUM RINGSPOT VIRUS 5.1. Materials and Methods 65 66 5.1.1. Plant Materials 66 5.1.2. Bacterial strains and plasmids 66 5.1.3. Cloning of the genes of interest into pBI121 vector 67 v 5.1.3.1. Movement protein gene (TGB123) of CymMV 67 5.1.3.2. Movement protein gene of ORSV 68 5.1.4. Preparation of electrocompetent A. tumefaciens LBA 4404 68 5.1.5. Electroporation and cell suspension of A. tumefaciens LBA 4404 71 5.1.6. Generation of transgenic plants 71 5.1.7. Transformation of N. benthamiana 72 5.1.8. Hardening of in vitro grown plants 73 5.1.9. Generation of non-radioactive DIG labelled cDNA probes for Southern Blot Analysis 73 5.1.10. Generation of non-radioactive DIG labelled cRNA probes for Northern blot analysis 78 5.1.11. Southern blot hybridization 79 5.1.11.1. Electrophoresis of DNA 79 5.1.11.2. Southern blot analysis 79 5.1.12. Mutagenesis of cloned DNA 79 5.1.12.1. Point mutation by PCR 80 5.1.12.2. Site-directed mutagenesis 82 5.1.13. Replication and infectivity of the RNA transcripts generated from the mutant cDNA clones 5.1.13.1. 82 Linearization of mutant cDNA clones and generation of in vitro transcripts 82 5.1.13.2. Protoplast isolation from N. benthamiana 82 5.1.13.3. Electroporation of RNA into protoplasts 83 5.1.13.4. Extraction of RNA from protoplasts 84 vi 5.1.13.5. Infectivity of in vitro transcripts of mutant cDNA on N. benthamiana 84 5.1.14. Inoculation of in vitro transcripts on F1 transgenic N. benthamiana 84 5.1.15. Tissue-printing hybridization 85 5.1.16. Leaf Immuno-blot 85 5.2. 86 Results 5.2.1. Protoplast Isolation 86 5.2.2. Replication and infectivity of the RNA transcripts generated from the mutant cDNA clones 86 5.2.3. Molecular analysis of transgenic plants 90 5.2.4. Complementation of movement function of p18Cy13inaTGB by transgenic plants expressing ORSV MP 93 5.2.5. Complementation of movement function of pOT2inaMP by transgenic plants expressing CymMV TGB123 102 5.2.6. Complementation of encapsidation of pOT2inaCP by transgenic plants expressing CymMV CP 104 5.2.7. Complementation of encapsidation of p18Cy13inaCP by transgenic plants expressing ORSV CP 109 5.2.7.1. RT-PCR and DNA sequencing 109 5.2.7.2. Tissue printing analysis 110 5.2.7.3. Whole leaf and immunoblot 113 5.2.8. Electron microscopy 113 5.2.9. Infectivity of crude sap 116 vii 5.3. Discussion CHAPTER 116 SYNERGISM BETWEEN CYMBIDIUM MOSAIC VIRUS AND ODONTOGLOSSUM RINGSPOT VIRUS 6.1. 124 Materials and Methods 125 6.1.1. Plant material and inoculations 125 6.1.2. Samples for RNA extraction 128 6.1.3. RNA extraction and Northern blot hybridization 128 6.1.4. Samples for extraction of proteins 128 6.1.5. Protein extraction and Western blot analysis 128 6.1.6. TEM of singly and doubly infected N. benthamiana tissues 129 6.1.7. Analysis of ORSV RNA accumulation in CymMV transgenic plants 129 6.2. 129 Results 6.2.1. Accumulation of CymMV RNA 129 6.2.2. Accumulation of ORSV RNA 130 6.2.3. Accumulation of CymMV and ORSV coat proteins 132 6.2.4. TEM of singly and doubly infected N. benthamiana tissues 132 6.2.5. Analysis of ORSV RNA in CymMV CP transgenic plants 135 6.3. 139 Discussion CHAPTER REFERENCES GENERAL DISCUSSION AND FUTURE PROSPECTS 143 147 viii LIST OF PUBLICATIONS P. A. Ajjikuttira, C. S. Loh and S. M. Wong. 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CymMV cymbidium mosaic virus MCMV maize chlorotic mottle virus ORSV odontoglossum ringspot virus PCV peanut clump virus PMMoV pepper mild mottle virus PeMoV peanut mottle virus PMV panicum mosaic virus PNRSV prunus necrotic ringspot virus PSbMV pea seed-borne mosaic virus PVX potato virus X PVY potato virus Y RMV ryegrass mosaic virus RCNMV red clover necrotic mosaic virus RYMV rice yellow mottle virus. .. F0 and F1 generations 70 Table 5.3 Primers used to construct the point mutations 123 xv LIST OF ABBREVIATIONS Viruses AMV alfalfa mosaic virus BBTV banana bunchy top virus BNYVV bean necrotic yellow vein virus BPMV bean pod mottle virus BSMV barley stripe mosaic virus CarMV carnation mottle virus CaMV cauliflower mosaic virus CMV cucumber mosaic virus CPMV cowpea mosaic virus CTV citrus tristeza virus. .. mosaic virus RYMV rice yellow mottle virus SHMV sun-hemp mosaic virus SPMV satellite panicum mosaic virus TMV tobacco mosaic virus TMGMV tobacco mild green mottle virus xvi ToMV tomato mosaic virus TSWV tomato spotted wilt virus WClMV white clover mosaic virus WMV watermelon mosaic virus WSMV wheat streak mosaic virus ZYMV zucchini yellow mosaic virus Others amino acid aa Amp ampicillin A tumefaciens... volume w/v weight/ volume xix SUMMARY Two of the most prevalent plant viruses infecting orchids worldwide- Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) were studied Genetic variability of the coat protein gene in the viruses from different geographical areas were investigated The results indicated that the coat protein genes could be ideal candidates in a pathogen-derived resistance... the major genera of orchids are cultivated in Singapore The orchid industry is affected by reduction in flower yield and quality caused by various pests and diseases Of more than 50 viruses infecting orchids, Cymbidium Mosaic Potexvirus (CymMV) and Odontoglossum Ringspot Tobamovirus (ORSV) are reported to be the most prevalent and economically important (Zettler et al., 1990) Both these viruses have been... reports of variability studies in the sequences of potexviruses and tobamoviruses In a comparative study of the CP sequences of eight potexviruses infecting different host ranges, an overall similarity in aa composition was observed, with variation of structurally important aa such as lysine, arginine, leucine and proline (Short et al., 1987) This could not lead to classification of the viruses Members of. .. vectors or seeds (Namba and Iishi, 1971) 1.1.4 Molecular structure and composition CymMV belongs to the potexvirus group of viruses Potato Virus X is the type member of this group Viruses of this group are typically flexouous and filamentous, and the particles are 450-550 nm long (Francki, 1970) CymMV particles measure 480 nm in 2 Table 1.1 Species of plants susceptible to CymMV and ORSV infection CymMV... 4807-5718) overlaps with the 3’-terminus of the RdRp by 94 nt and is required for cell-to-cell movement of the virus (Ryu and Park, 1995) A putative origin of assembly (Oa) of ORSV-S1 is located whin the MP gene The secondary structure of the Oa has been determined to possess two loops and a XXG 8 repeat motif and are necessary for binding and initiation of assembly of the coat protein (Turner et al., 1988;... double infections and displayed symptoms highly similar to doubly inoculated plants These results demonstrated that the CymMV coat protein is capable of inducing the synergism effect when co-inoculated with ORSV xxi CHAPTER 1 INTRODUCTION 1.1 Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) 1.1.1 Economic significance and incidence of CymMV and ORSV Many members of the orchid family... protein genes of CymMV and ORSV 1.4 Synergism in CymMV and ORSV Multiple virus infections are commonly seen in the plant kingdom Doubly or multiply infected plants show symptom intensification in the host plants The phenomenon of severity of symptoms and higher amounts of virus accumulation of one or both viruses involved is referred to as synergism Many synergistic interactions involving a potyvirus with . GENETIC VARIABILITY AND INTERACTIONS OF CYMBIDIUM MOSAIC VIRUS AND ODONTOGLOSSUM RINGSPOT VIRUS PRABHA ARUNA AJJIKUTTIRA, M.Sc., M.Phil. A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT. Chang, C. S. Loh and S. M. Wong. (2002) Genetic variability in the coat protein genes of two orchid viruses: Cymbidium mosaic virus and Odontoglossum ringspot virus. Archives of Virology 147:. mottle virus CaMV cauliflower mosaic virus CMV cucumber mosaic virus CPMV cowpea mosaic virus CTV citrus tristeza virus CymMV cymbidium mosaic virus MCMV maize chlorotic mottle virus ORSV odontoglossum