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Proquest Dissertations INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMl films the text directly from the original or copy submitted. Thus, som e thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMl a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMl directly to order. Bell & Howell Information and Learning 300 North Zeeb Road, Ann Artror, Ml 48106-1346 USA 800-521-0600 UMl MOLECULAR ANALYSIS OF SYMPTOM DEVELOPMENT AND VIRAL GENE EXPRESSION IN GEMINIVIRUS-INFECTED ARABIDOPSIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jingyung Hur, B.S. and M.S. ***** The Ohio State University 2000 Dissertation Committee: Approved by Dr. David M. Bisaro Dr. Randall L. Scholl Adviser Dr. Keith R Davis, Adviser Department of Plant Biology UMl Number: 9994879 UMl UMl Microform 9994879 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17. United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT The gemini viruses are a group of single-stranded DNA viruses with a twin icosahedrai capsid. The Geminividae family is composed of three different genera - Masirevinises, Curtovinises, and Begomovinises grouped - according to their genomic structure, insect vector and their plant host. This group of viruses causes economically important disease in crop plants worldwide. Biologically they are interesting because they use the host replication and transcription machinery to replicate and express their genome without virus encoded polymerase activities. In this study, we looked at the interaction between one of these geminiviruses. the curtovims beet curly top virus (BCTV), with our model plant system, Arabidopsis. Symptoms caused by BCTV infection ofArabidopsis are characteristic of those observed in other hosts and include ectopic cell divisions in tissues and disruption of normal development. These symptoms are similar to phenotypes in Arabidopsis associated with mutations in genes involved in plant hormone transport. To address the question of whether disruption of hormone transport in virus infected plants is correlated with symptom development, we used cell-cycle marker-gene promoter driven ii reporter gene transgenic lines and auxin-up-regulated gene promoter driven reporter gene transgenic lines for viral infection studies. The results show that both cell-cycle maker- gene and auxin responsive gene were concomitantly expressed in the same region of symptomatic tissues. The auxin polar transport rate in virus infected plants was measured and this rate was significantly reduced in virus infected plants. Auxin-related mutants Arabidopsis of were also used to see if a normal response to changed auxin concentration and the normal downstream signal transduction pathway of auxin is needed for symptoms to develop in virus infected plants. To further address the interaction between BCTV and Arabidopsis, viral promoter activity in plants was studied using transgenic plants expressing the GUS reporter gene under control of virus sequences. The promoter region of BCTV ORF Cl was identified using reporter gene fusions in transgenic Arabidopsis. Unlike other geminiviruses, the intergenic region was not sufficient to promote Cl expression in transgenic plants. As the promoter region was extended into the coding region of Cl, strong expression of the reporter protein was observed in vascular tissues of transgenic plants. This suggests that important transcriptional activator elements for Cl expression reside in the Cl coding area itself. Transgenic plants expressing the reporter gene under control of the complete C l promoter were inoculated with virus to find out if any viral protein, especially Cl, is down-regulating Cl expression. The results suggested that BCTV Cl protein does not auto-regulate its own expression and that Cl expression 111 is controlled differently than other geminiviruses. In the case of virion sense gene expression, expression from the less virulent Logan promoter was stronger than expression from the CFH promoter. The viral promoters were active in seedlings, in actively dividing tissues such as root tips and in apical meristems. As plants matured, promoter activity diminished. Infection of mature transgenic plants by virus restored reporter gene expression, especially in transgenic plants containing Logan virion sense- gene promoter constructs. An interesting 30 bp motif that is tandemly repeated three times in the Logan promoter but only once in CFH was identified in this study. When this motif was removed from the Logan promoter one by one, the promoter activity in transgenic plants was greatly reduced. CFH promoters with one or two extra copies of this motif did not show increased virion sense promoter activity in transgenic plants but did have increased promoter activity in the complementary sense direction. This motif has previously been identified as a late conserved element and is known to be involved in late gene expression via AL2 protein transactivation. Since BCTV does not have a functional homologue of AL2 and this motif showed different types of control of transcription in our reporter gene fusion experiment, this motif must be involved in both early and late gene expression via a control mechanism distinct from that seen in the bipartite viruses. IV ACKNOWLEDGEMENTS I sincerely thank my adviser, Dr. Keith Davis for his unconditional support and guidance throughout my graduate studies. He always offered me intellectual advice and constant encouragement when I need them most. I also thank other members of my advisory and dissertation committee, Dr. Randall Scholl, Dr. David Bisaro, Dr. Desh Pal Verma and Dr. Zhenbiao Yang for their valuable suggestions and comments. I thank Dr. Kenneth Buckley for his help and sharing his profound knowledge in gene cloning techniques, for letting me use his vast variety of cloning vectors, and for putting up with my constant whining in the laboratory. I also would like to express my deepest thanks to Dr. Doreen Ware for her unconditional support and constant encouragement whenever I felt down. She is my real guardian angel. I am also grateful to all the other members of Davis lab and the Arabidopsis Biological Stock Center, Dr. Rao Mulpuri, Dr. Greg Bertoni, Dr. Jennifer Koch, Dr. Emma Knee, Staci Putney, and Jeff Cotrill, for their help and friendship. I thank my family, especially my parents, for their love, support, and supreme confidence in me since I was a little baby. VI VITA April 30, 1968 .......................................Bom - Jinhae, Republic of Korea 1991......................................................... B. S. Biology, Sogang University, Seoul, Korea 1992-1994 ................................................Laboratory supervisor. Quality analysis laboratory, Sunkyung Pharmaceutical Co. Ltd., Seoul, Korea. 1995 - 1998 ............................................ M.S. Department of Plant Biology, The Ohio State University. Columbus OH, U.S.A. 1995 - 2000 .......................................... Graduate Teaching and Research Associate, Department of Plant Biology, The Ohio State University, Columbus OH, U.S.A. PUBLICATIONS 1. Hur, J., Lee, S. and Davis, K. R., Molecular analysis of symptom development in BCTV infected Arabidopsis thaliana. Manuscript in preparation. 2. Hur, J. and Davis, K. R, Identification of the BCTV early gene promoter region using transgenic Arabidopsis. Manuscript in preparation. 3. Hur, J. and Davis, K. R., Regulation of BCTV late-gene expression and identification of a motif involved in this regulation. Manuscript in preparation. Vll POSTERS PRESENTED AT SCIENTIHC MEETINGS. 1. Hur, J. and Davis, K.R., Molecular analysis of symptom development in Beet Curly Top Virus \x\ïtcied Arabidopsis thaliana. 1997, 8th International conference on Arabidopsis research, Madison, Wisconsin. 2. Buckley, K.J., Ware, D.H., Hur, J. and Davis, K.R., BCTV ORF L4 as symptom determinant in Arabidopsis. 1997, 8th International conference on Arabidopsis research, Madison, Wisconsin. 3. Hur, J and Davis, K.R., Study of gemini virus Beet Curly Top Virus (BCTV) viral promoter-reporter gene constructs. 1998, 1st Annual Plant Biology Research Symposium, Columbus, Ohio. 4. Hur, J. and Davis, K. R., Analysis of Beet Curly Top Virus (BCTV) viral promoter activity. 1998, Plant Molecular Biology and Biotechnology
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