Genetic variability and interactions of cymbidium mosaic virus and odontoglossum ringspot virus

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. (2000) Production of transgenic plants expressing virus genes. The Asia Pacific Conference on Plant Tissue Culture and Agribiotechnology. 19-23 Nov., 2000. Singapore. P. A. Ajjikuttira, 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. 17th World Orchid Conference, Shah Alam, Malaysia. P. A. Ajjikuttira, C. L. Lim-Ho, M. H. Woon, K. H. Ryu, C. A. 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: 1943-1954. P. A. Ajjikuttira, Loh C. S. and Wong S. M. (2004) Complementation between Cymbidium mosaic virus and Odontoglossum ringspot virus. (In preparation). P. A. Ajjikuttira, Loh C. S. and Wong S. M. (2004) Synergism between Cymbidium mosaic virus and Odontoglossum ringspot virus. (In preparation). ix Cooper B, Schmitz I, Rao ALN, Beachy RN and Dodds JA. (1996) Cell-to-Cell transport of movement-defective cucumber mosaic and tobacco mosaic viruses in transgenic plants expressing heterologous movement protein genes. Virology 216: 208-213. Davenport GF and Baulcombe DC. (1997) Mutation of the GKS motif of the RNAdependent RNA polymerase form potato virus X disables or eliminates virus replication. J. Gen. Virol. 78: 1247-1251. Dawson WO, Bubrick P and Grantham GL. (1988) Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement and symptomatology. Phytopathology 78: 783-789. De Jong W and Ahlquist P. (1992) A Hybrid plant virus made by transferring the noncapsid movement protein from a rod-shaped to an icosahedral virus is competent for systemic infection. Proc. Natl. Acad. Sci. USA 89: 6808-6812. Deom CM, Oliver MJ and Beachy RN. (1987) The 30-kilodalton gene product of tobacco mosaic virus potentates virus movement. Science 237: 389-394. Derrick PM, Carter SA and Nelson RS. (1997) Mutation of the tobacco mosaic tobamovirus 126- and 183-kDa proteins: effects on phloem-dependent virus accumulation and synthesis of viral proteins. Mol. Plant-Microbe Interact. 10: 589-596. Ding B, Li Q, Nguyen L, Palukaitis P and Lucas WJ. (1995) Cucumber mosaic virus 3a protein potentiates cell-to-cell trafficking of CMU RNA in tobacco plants. Virology 207: 345-353. Domingo E, Holland J, Biebreicher C and Eigen M. (1995) Quasi-species: the concept and the word. In “Molecular Basis of Virus Evolution” (A.J.Gibbs, C.H.Calisher, and F. Garcia-Arenal, Eds.), pp. 181-191. Cambridge University Press, Cambridge. 151 Eigen M. (1993) Viral quasispecies. Sci. Am. July, 42-49. Evans RK, Haley BE and Roth DA. (1985) Photoaffinity labelling of a viral induced protein from tobacco. J. Biol. Chem. 260: 7800-7804. Fedorkin ON, Solovyev AG, Yelina NE, Zamyatnin AA, Zinovkin RA, Makinen K, Schiemann J and Yu Morozov S. (2001) Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. J. Gen. Virol. 82: 449-458. Fedorkin ON, Merits A, Lucchesi J, Solovyev AG, Saarma M, Yu Morozov S and Makinen K. (2000) Complementation of the movement-deficient mutations in Potato Virus X: Potyvirus coat protein mediates cell-to-cell trafficking of C-terminal truncation but not deletion mutant of potexvirus coat protein. Virology 270: 31-42. Fraile A, Malpica JM, Aranda MA, Rodriguez-Cerezo E and Garcia-Arenal F. (1996) Genetic diversity in tobacco mild green mosaic tobamovirus infecting the wild plant Nicotiana glauca. Virology 223: 148-155. Fraile A, Escriu F, Aranda MA, Malpica JM, Gibbs JM and Garcia-Arenal F (1997) A century of tobamovirus evolution in an Australian population of Nicotiana glauca. J. Virology 71: 8316-8320. Frowd JA and Tremaine JH. (1977) Physical, chemical, and serological properties of cymbidium mosaic virus. Phytopathology 67: 43-49. Francki RIB. (1970) Cymbidium mosaic virus. In Descriptions of Plant Viruses No. 27. CMI/AAB, Wellesbourne, Warwick. Fuentes AL and Hamilton RI. (1991) Sunn-hemp mosaic virus facilitates cell-to-cell spread of southern bean mosaic virus in a nonpermissive host. Phytopathology 81: 13021305. 152 Fujiwara T, Giesman-Cookmeyer D, Ding B, Lommel SA and Lucas WJ. (1993) Cell-tocell trafficking of macromolecules through plasmodesmata potentiated by the red clover necrotic mosaic virus movement protein. Plant Cell 5: 1783-1794. Giesman-Cookmeyer D, Silver S, Vaewhongs AA, Lommel SA and Deom CM. (1995) Tobamovirus and dianthovirus movement proteins are functionally homologous. Virology 213: 38-45. Goldberg KB and Brakke MK. (1987) Concentration of Maize Chlorotic Mottle Virus Increased in Mixed Infections with Maize Dwarf Mosaic Virus, Strain B. Phytopathology 77: 162-167. Goelet P, Lomonosoff GP, Butler PJ, Akam ME, Gait MJ and Karn J. (1982) Nucleotide sequence of tobacco mosaic virus RNA. Proc. Natl. Acad. Sci. USA. 79: 5818-5822. Goodman RM and Ross AF. (1974) Independent assembly of virions in tobacco doubly infected by potato virus X and potato virus Y or tobacco mosaic virus. Virology 59: 314318. Goodman RM and Ross AF. (1974b) Enhancement by potato virus Y of potato virus X synthesis in doubly infected tobacco depends on the timing of invasion by the viruses. Virology 58: 263-271. Gorbalenya AE and Koonin RH. (1989) Viral proteins containing the purine NTP-binding sequence pattern. Nucleic Acids Res. 17: 7735-7762. Habili N and Symons RH. (1989) Evolutionary relationship between luteoviruses an other RNA plant viruses based on sequence motifs in their putative RNA polymerases and nucleic acid helicases. Nucleic Acids Res. 17: 9543-9555. 153 Hackland AF, Rybicki EP, Thompson JA. (1994) Coat protein-mediated resistance in transgenic plants. Arch. Virol. 139: 1-22 Hadley G, Arditti M and Arditti J. (1987) Orchids diseases-a compedium. In: Orchid Biology, Reviews and Perspectives IV. Arditti J. (ed.) Cornell University Press, Ithaca pp. 263-322. Hamamoto H, Watanabe Y, Kamada H and Okada Y. (1997) A single amino acid substitution in the virus-encoded replicase of tomato mosaic tobamovirus alters host specificity. Mol. Plant-Microbe Interact. 10: 1015-1018. Hamilton RI and Dodds JA. (1970) Infection of barley by tobacco mosaic virus in single and mixed infection. Virology 42: 266-268. Hoffmann K, Geske SM and Moyer JW. (1998) Pathogenesis of tomato spotted wilt virus in peanut plants dually infected with peanut mottle virus. Plant Dis. 82: 610-614. Holland JJ, DeLaTorre JC and Steinhauer DA. (1992) RNA virus populations as quasispecies. In “ Genetic Diversity of RNA Viruses” (J. J. Holland, Ed.), pp. 1-20. Springer Verlag, Berlin. Holt CA, Hodgson RA, Coker FA, Beachy RN and Nelson RS. (1990) Characterization of the masked strain of tobacco mosaic virus: identification of the region responsible for symptom attenuation by analysis of an infectious cDNA clone. Mol. Plant-Microbe Interact. 3: 417-423. Hu WW, Wong SM, Loh CS and Goh CJ. (1998) Synergism in replication of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts. Arch. Virol. 143: 1265-1275. 154 Huismann MJ, Linthorst HJ, Bol JF and Cornelissen JC. (1988) The complete nucleotide sequence of potato virus X and its homologies at the amino acid level with various plusstranded RNA viruses. J. Gen. Virol. 69: 1789-1798. Jensen DD and Gold AH. (1951) A virus ringspot of odontoglossum orchid: symptoms, transmission and electron microscopy. Phytopathology 41: 648-653. Kahn TW, Lapidot M, Heinlein M, Reichel C, Cooper B, Gafny R and Beachy RN. (1998) Domains of the TMV movement protein involved in subcellular localization. Plant J. 15: 15-25. Karpova OV, Ivanov KL, Rodionova NP, Dorokhov Yu L and Atabekov JG. (1997) Nontranslatability and dissimilar behaviour in plants and protoplasts of viral RNA and movement protein complexes formed in vitro. Virology 230: 11-21. Karyeija RF, Kreuze JF,Gibson RW and Valkonen. (2000) Synergistic Interactions of a Potyvirus and a Phloem-Limited Crinivirus in Sweet Potato Plants. Virology 269: 26-36. Kasschau KD and Carrington JC. (1998) A counter-defensive strategy of plant viruses: Suppression of posttranscriptional gene silencing. Cell 95: 461-470. Khan MA and Demski JW. (1982) Identification of turnip mosaic and cauliflower mosaic viruses naturally infecting collards. Plant Dis. 66: 253-256. Knudson L (1946) A new nutrient solution for germination of orchid seed. American Orchid Society Bulletin 15: 214-217 Krishnamurthy K, Mitra R, Payton ME and Verchot-Lubicz J. (2002) Cell-to-cell movement of the PVX 12K, 8K, or coat proteins may depend on the host, leaf developmental stage, and the PVX 25K protein. Virology 300: 269-281. 155 Kuehnle AR and Sugii N. (1992) Transformation of Dendrobium orchid using particle bombardment of protocorms. Plant Cell Reports 11: 484-488. Kunik T, Salomon R, Zamir D, Navot N, Zeidan M, Michelson I, Gafni Y, Czosnek H. (1994) Transgenic tomato plants expressing the tomato yellow leaf curl capsid protein are resistant to the virus. Bio/Technology 12:500-504. Laemmli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Lauber E, Bleykasten-Grosshans C, Erhardt M, Bouzoubaa S, Jonard G, Richards KE and Guilley H. (1998) Cell-to-cell movement of beet necrotic yellow vein virus: I. Heterologous complementation experiments provide evidence for specific interactions among the triple gene block proteins. Mol. Plant-Microbe Interact. 11: 618-625. Lawson RH and Brannigan M. (1986) Virus diseases of orchids. In Handbook of Orchid Pests and Diseases. American Orchid Society, West Palm Beach, Florida. Lehrach H, Diamond D, Wozney JM and Boedtker H. (1977) RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16: 4743-4751. Lichtsteiner S and Tjian R. (1993) Cloning and properties of the Caenorhabditis elegans TATA-box-binding protein. Proc. Natl. Acad. Sci. USA. 90: 9673-9677. Lim ST, Wong SM, Yeong CY, Lee SC, Goh CJ. (1993) Rapid detection of cymbidium mosaic virus by the polymerase chain reaction (PCR). J. Virol. Methods 41: 37-46. Lister RM. (1997) Coat protein mediated resistance to isolates of barley yellow dwarf virus in oats and barley. Eur. J. Plant Pathol. 103: 695-710. 156 Lomonosoff GP. (1995) Pathogen-derived resistance to plant viruses. Ann. Rev. Phytopathology 33: 323-343. Lough TJ, Shash K, Voconostle-Cazares B., Hofstra K, Beck DL, Balmori E, Forster RLS and Lucas WJ. (1998) Molecular dissection of the mechanism by which potexvirus triple gene block proteins mediate cell-to-cell transport of infectious RNA. Mol. Plant-Microbe Interact. 11: 801-814. Malyshenko SI, Kondakova OA, Taliansky ME and Atabekov JG. (1989) Plant Virus Transport Function: Complementation by Helper Viruses is Non-specific. J. Gen. Virol. 70: 2751-2757. Mansky LM, Andrews Jr, RE, Durand DP and Hill JH. (1990) Plant virus location in leaf tissue by press blotting. Plant Mol. Biol. Reports 8: 13-17. Matthews REF (1991) Plant Virology, 3rd ed. Academic Press, San Diego McGrath PF, Vincent JR, Lei CH, Pawlowski WP, Torbert KA, Gu W, Kaeppler HF, Wan Y, Lemaux PG, Rines HR, Somers DA, Larkins BA and Larkins RM. (1997). Coat protein mediated resistance to isolates of barley yellow dwarf virus in oats and barley. Eur. J. Plant Pathol. 103: 695-710. Meshi T, Watanabe Y, Saito T, Sugimoto A, Maeda T and Okada Y. (1987) Function of the 30kD protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication. EMBO J. 6: 2557-2563. Meshi T, Watanabe Y and Okada Y. (1993) Molecular pathology of tobacco mosaic virus revealed by biologically active cDNAa. In T. M. A. Wilson and J. W. Davies (ed.), Genetic engineering with plant viruses. CRC Press, Boca Raton, Fla. P. 149-186. 157 Morozov S Yu, Gorbulev VG, Novikov VK, Agranovski AA, Kozlov YV, Atabekov JG and Baev AA. (1981) Primary structure of 5’ and 3’ terminal regions of potato virus X genomic RNA. Doklady Akademii Nauk SSR. 259: 723-725. Morozov S Yu, Dolja VV and Atabekov JG. (1989) Probable reassortment of genetic elements among elongated RNA-containing plant viruses. J. Mol. Evol. 29: 52-62. Morozov S Yu, Miroshnichenko NA, Solovyev AG, Fedorkin ON, Zelenina DA, Lukasheva LI, Karasev AV, Dolja VV and Atabekov JG. (1991) Expression strategy of the potato virus X triple gene block. J. Gen. Virol. 72: 2039-2042. Morozov S Yu, Fedorkin ON, Juttner G, Schiemann J, Baulcombe DC and Atabekov JG. (1997) Complementation of potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes. J. Gen. Virol. 78: 2077-2081. Moore PJ, Fenczik CA, Deom CM and Beachy RN. (1992) Developmental-changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus. Protoplasma 170: 115-127. Moya A and Garcia-Arenal F. (1995) Population genetics of viruses: An introduction. In “Molecular Basis of Virus Evolution”. (A.J.Gibbs, C. H. Calisher and Garcia-Arenal F., Eds.) pp. 213-223, Cambridge Univ. Press, Cambridge. Murashige T and Skoog F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant 15: 473-497. Namba R and Ishii M. (1971) Failure of aphids to transmit the ORSV and CymMV to orchid plantlets derived from meristem cultures. Phytopathology 61: 582-583. 158 Nan GL and Kuehnle AR. (1995) Genetic Transformation of Dendrobium (Orchid). In Biotechnology in Agriculture and Forestry, Vol. 34. Plant Protoplasts and Genetic Engineering VI (ed. Y.P.S. Bajaj). Springer-Verlag Berlin Heidelberg 1995 Nan GL and Kuehnle AR. (1998) Transgenic Dendrobium orchid through Agrobacteriummediated transformation. Malayan Orchid Review. 32: 93-96. Noueiry AO, Lucas WJ and Gilbertson RL. (1994) Two proteins of a plant DNA virus coordinate nuclear and plasmodesmatal transport. Cell 76:925-932. Oparka KJ, Roberts AG, Roberts IM, Prior DAM and Santa Cruz S. (1996) Viral coat protein is targeted to but does not gate, plasmodesmata during cell-to-cell movement of potato virus X. Plant J. 10: 805-813. Orman BE, Celnik RM, Mandel AM, Torres HN and Mentaberry AN. (1990) Complete cDNA sequence of a South American isolate of potato virus X. Virus Res. 16: 293-305. Page RDM. (1996) TREE VIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci .12: 357-358. Parrella G, Lanave C, Marchoux G, Finetti Sialer MM, Di Franco A and Gallitelli D. (2000) Evidence for two distinct subgroups of Alfalfa mosaic virus (AMV) from France and Italy and their relationships with other AMV strains. Arch. Virol. 145: 2659-2667. Paul HL, Wetter C, Wittmann HG and Brandes J. (1965) Untersuchungen am odontoglossum ringspot virus, einem verwandten des Tabak mosaik-virus. I. Physikalishe, chemische, serologische und symptomatologische befunde. Z. Vererbungsl. 97: 186-203. Pearson MN and Cole JS. (1991) Further observations on the effects of cymbidium mosaic virus and odontoglossum ringspot virus on the growth of Cymbidium orchids. J. Pathol. 131: 193-198. 159 Pinel A, Guessan PN, Bousalem M, Fargette D. (2000) Molecular variability of geographically distinct isolates of Rice yellow mottle virus in Africa. Arch. Virol. 145: 1621-1638. Poolpol P and Inouye T. (1986) Enhancement of cucumber mosaic virus multiplication by zuchinni yellow mosaic virus in doubly infected cucumber plants. Ann. Phytopathol. Soc. Jpn. 52: 22-30. Pruss G, Ge Xin, Shi XM, Carrington JC and Vance VB. (1997) Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enchancer that transactivates replication of heterologous viruses. The Plant Cell 9: 859-868. Qiu WP and Scholthof KBG. (2001) Genetic identification of multiple biological roles associated with the capsid protein of satellite panicum mosaic virus. Mol. Plant-Microbe Interact. 14: 21-30. Raineri DM, Bottino P, Gordon MP and Nester EW. (1990) Agrobacterium-mediated transformation of rice (Oryza sativa L.). Biotechnology 8: 33-39. Rochow WF and Ross AF. (1955) Virus multiplication in plants doubly infected by potato viruses X and Y. Virology 1: 10-27. Rodriguez-Cerezo E, Moya A and Garcia-Arenal F. (1989) Variability and evolution of the plant RNA virus Pepper mild mottle virus. J. Virology 63: 2198-2203. Richins RD, Donson J, Lewandowsky DJ, Ducasse DA, Dawson WO and Shepherd RJ (1993). Temperature-dependent complementation of movement-deficient mutants of tobacco mosaic virus by gene I of peanut chlorotic streak caulimovirus. Phytopathology 83: 1349- 160 Rao ALN, Bret Cooper and Carl M. Deom. (1998) Defective Movement of Viruses in the Family Bromoviridae is differentially complemented in Nicotiana benthamiana expressing Tobamovirus or Dianthovirus Movement Proteins. Phytopathology 88: 666-672. Ryu KH and Park. (1995) The complete nucleotide sequence and genome organisation of odontoglossum ringspot tobamovirus RNA. Arch. Virol. 140: 1577-1587. Saito Y, Yamanaka K and Okada Y. (1990) Long-distance movement and viral assembly of tobacco mosaic virus mutants. Virology 176: 329-336. Sambrook J, Fritsch EF and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual. Second edition. New York: Cold Spring Harbour Laboratory Press. Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA, Hutchinson CA, Slocombe PM and Smith M. (1977) Nucleotide sequence of bacteriophage phi X 174 DNA. Nature 265: 687-695. Santa Cruz S, Roberts AG, Prior DAM, Chapman S and Oparka K. (1998) Cell-to-cell and phloem mediated transport of potato virus X: the role of virions. Plant Cell. 10: 495-510. Scheets K. (1998) Maize Chlorotic Mottle Machlomovirus and Wheat Streak Mosaic Rymovirus Concentrations Increase in the Synergistic Disease Corn Lethal Necrosis. Virology 242: 28-38. Shi XM, Miller H, Verchot J, Carrington JC and Vance VB. (1997) Mutations in the region encoding the central domain of helper component-proteinase (HC-Pro) eliminate potato virus X/potyviral synergism. Virology 231: 35-42. Scholthof K-B G. (1999) A Synergism Induced by Satellite Panicum Mosaic Virus. Mol. Plant-Microbe Interact. 12: 163-166. 161 Skyrabin KG, Kraev AS, Morozov S Yu, Rozanov MN, Chernov BK, Lukasheva LI and Atabekov JG. (1988) The nucleotide sequence of potato virus X RNA. Nucleic Acids Res. 16: 10929-10930. Solovyev AG, Zelenina DA, Savenkov EI, Grdzelishvili VZ, Morozov S Yu, Lesemann DE, Maiss E, Casper R and Atabekov JG. (1996) Movement of barley stripe mosaic virus chimera with a tobacco mosaic virus movement protein. Virology 217: 435-441. Short MN and Davies JW. (1987) Host ranges, symptoms and amino acid compositions of eight potexviruses. Ann. Appl. Biol. 110: 213-219. Solovyev AG, Zelenina DA, Savenkov EI, Grdzelishvili VZ, Yu Morozov S, Lesemann D-E, Maiss E, Casper R and Atabekov JG. (1996) Movement of a barley stripe mosaic virus chimera with a tobacco mosaic virus movement protein. Virology 217: 435-441. Sonenberg N, Shatkin AJ, Ricciardi RP, Rubin M and Goodman RM. (1978) Analysis of terminal structures of RNA from potato virus X. Nucleic Acids Res. 5: 2501-2512. Srifah P, Loprasert S, Rungroj N. (1996) Use of reverse transcription-polymerase chain reaction for cloning of coat protein-encoding genes of cymbidium mosaic virus. Gene 179: 105-107. Steinhauer DA, Domingo E, Holland JJ. (1992) Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase. Gene 122: 4673-4680. Takamatsu N, Ishikawa M, Meshi T and Okada Y. (1987) Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA. EMBO J. 6: 307-311. 162 Taliansky ME, Atabekova TI, Kaplan IB, Yu S, Malyshenko SI and Atabekov JG. (1982a) A study of TMV ts mutant Ni2519. I. Complementation experiments. Virology 118: 301308. Taliansky ME, Malyshenko SI, Pshennikova ES, Kaplan IB, Ulanova EF and Atabekov JG (1982b). Plant virus-specific transport function. I. Virus genetic control required for systemic spread. Virology 122: 318-326. Taliansky ME, Malyshenko SI, Pshennikova ES, Kaplan IB, Ulanova EF and Atabekov JG. (1982c) Plant virus-specific transport function. II. A factor controlling virus host range. Virology 122: 327-331. Taliansky ME, de Jager CP, Wellink J, van Lent JWM and Goldbach RW. (1993) Defective cell-to-cell movement of cowpea mosaic virus mutant N123 is efficiently complemented by sunn-hemp mosaic virus. J. Gen. Virol. 74: 1895-1901. Taraporewala Z and Culver JN. (1996) Identification of an elicitor active site within the three-dimensional structure of the tobacco mosaic tobamovirus coat protein. Plant Cell 8: 169-178. Thompson JD, Gibson TJ, Plevnik F, Jeanmougin F, Higgins DJ. (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24: 4876-4882. Tomenius K, Clapham D and Meshi T. (1987) Localization by immunogold cytochemistry of the virus-coded 30K protein in plasmodesmata of leaves infected with tobacco mosaic virus. Virology 160: 363-371. Turner DR, Joyce LE and Butler PJG. (1988) The tobacco mosaic virus assembly origin RNA: functional characteristics defined by directed mutagenesis. J. Mol. Biol. 203: 531547. 163 Vance VB. (1991) Replication of Potato Virus X RNA Is Altered in Coinfections with Potato Virus Y. Virology 182: 486-494. Vance VB, Berger PH, Carrington JC, Hunt AG and Shi XM. (1995) 5’ Proximal potyviral sequences mediate potato virus X/potyviral synergistic disease in transgencic tobacco. Virology 206: 583-590. Verwoerd TC, Dekker, BMM and HoekemaA. (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res. 17: 2362. Vaskova D, Petrizk K, Karesova R. (2000) Variability and molecular typing of the woodytree infecting prunus necrotic ringspot ilarvirus. Arch. Virol. 145: 699-709. Verchot J, Angell SM and Baulcombe DC. (1998) In vivo translation of the triple gene block of potato virus X requires two subgenomic mRNAs. J. Virol. 72: 8316-8320. Von Arnim A, Frischmuth T and Stanley J (1993) Detection and possible functions of African cassava mosaic virus DNA B gene products. Virology 193: 264-272. Waigmann E, Lucas WJ, Citovsky V and Zambryski P. (1994) Direct functional assay for tobacco mosaic cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmatal permeability. Proc. Natl. Acad. Sci. USA. 91: 1433-1437. Wanitachacorn R, Harding RM, Dale JL. (2000) Sequence variability in the coat protein gene of two groups of banana bunchy top isolates. Arch. Virol. 145: 593-602. Wang Y-Z, Gaba V, Yang J, Palukaitis P and Gal-On A. (2002) Characterization of synergy between cucumber mosaic virus and Potyviruses in Cucurbit hosts. Phytopathology 92: 51-58. 164 Webster DE, Guy PL, Beck DL, Forster RLS. (1999) Distribution and diversity of New Zealand isolates of ryegrass mosaic virus. Arch. Virol. 144: 2059-2064. Webster RD and Granoff A. (1994) Tobamoviruses. In Encyclopedia of Virology Academic Press, Harcourt Brace & Company Publishers, New York. p 1436-1441. Wisler GC, Zettler FW, Sheehan TJ. (1986) Common questions and misconceptions concerning orchid viruses. Am. Orchid Soc Bull 55: 472-479. Wolf S, Deom CM, Beachy RN and Lucas WJ. (1989) Movement protein of tobacco mosaic virus modifies plasmodesmata size exclusion limit. Science 246: 377-379. Wong SM, Chng CG, Lee YH, Tan Y, Zettler FW. (1994) Incidence of cymbidium mosaic and odontoglossum ringspot viruses and their significance in orchid cultivation in Singapore. Crop Protection 13: 235-239 Wong SM, Mahtani PH, Lee KC, Yu HH, Tan Y, Neo KK, Chan Y, Wu M and Chng CG. (1997) Cymbidium mosaic potexvirus RNA: complete nucleotide sequence and phylogenetic analysis. Arch. Virol. 142: 383-391. Yang J, Lee HJ, Shin DH, Oh SK, Seon JH, Paek KY and Han KH. (1999) Genetic transformation of Cymbidium orchid by particle bombardment. Plant Cell Reports 18: 978-984. Yu H, Yang SH, Goh CJ. (2001) Agrobacterium-mediated transformation of a Dendrobium orchid with the class knox gene DOH1. Plant Cell Reports 20: 301-305. Yu HH. (1999) Molecular manipulation of two orchid viruses. Ph. D. Thesis. National University of Singapore. 165 Ziegler-Graff V, Guilford PJ and Baulcombe DC. (1991) Tobacco rattle virus RNA-1 29K gene product potentiates viral movement and also affects symptom induction in tobacco. Virology 182: 145-155. Zettler FW, Ko NJ, Wisler GC, Elliot MS, Wong SM. (1990) Viruses of orchids and their control. Plant Dis. 74: 621-626. 166 [...]... 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