UNIVERSITY OF ADELAIDE STRUCTURED PROGRAMME FOR PHD STUDENTS RESEARCH PROPOSAL: DARYL R.

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1 UNIVERSITY OF ADELAIDE STRUCTURED PROGRAMME FOR PHD STUDENTS RESEARCH PROPOSAL: DARYL R. WEBB Localization by in situ hybridization of members of viroid subgroups using transmission electron microscopy and confocal laser scanning microscopy Daryl R. Webb Department of Plant Science Faculty of Agricultural and Natural Resource Sciences Funded by the Special Research Centre in Basic and Applied Plant Molecular Biology Supervised by: Prof. Robert H. Symons and Dr. Peter Langridge. Table of Contents 1. Introduction 2. Research plan 2.1 project outline 2.2 project aims 3. Significance 4. Methodology 5. Proposed timetable 6. Budget considerations 7. References 1.Introduction. Viroids are single stranded RNA molecules endowed with special features: small size ( nucleotides), high degree of self complementarity and a circular structure. They are believed to form a stable rod-like structure in vivo (Diener 1987). Viroids are currently classified into two major groups on the basis of comparative sequence homology; the avocado sunblotch viroid (ASBV) group and the potato spindle tuber viroid (PSTV) group. The PSTV group is further subdivided into the PSTV, apple scar skin viroid (ASSV) and the coleus blumei viroid (CbV) sub-groups. Sub-groups are defined by the presence of very highly conserved sequences in the central part of the molecule (Koltunow and Rezaian 1985). There is essentially no homology between the ASSV and PSTV sub-groups in this region. This central conserved region (CCR) is critical for replication and, probably, modulation of pathogenicity (Visvader et al 1985, Diener 1993). It is not known if the differences in PSTV sub-group CCR reflect differences in viroid-host interactions. Viroids are the smallest known plant pathogen and are responsible for a number of economically important fruit and vegetable crop diseases (Singh and Singh 1995), such as Coconut cadang cadang, Potato spindle tuber and Avocado sunblotch. Diseases associated with the ASSV sub-group include Apple scar skin, Pear blister canker and a number of citrus diseases. Members of the ASSV sub-group of viroids have also been shown to be the causal agent of Grapevine yellow speckle disease. Grapevines may host viroid infections from both the PSTV and ASSV sub-groups. Individual plants often contain mixed populations of four or more viroids with representatives from both sub-groups present (Y. Wan Chow Wah pers com.). The economic importance of these grapevine infections is unknown. Whilst viroids have been formally recognised for nearly 25 years, very little is currently known about the biology of viroid infection. Their apparent inability to encode any protein means they must rely entirely on host cell enzymes and processes for replication. Viroids are believed to exert their pathogenic effects by binding to and interfering with the proper functioning of host snrnas and/or proteins (Diener et al 1993). In addition, while the host range of viroids is known to vary, the basis of host specificity remains unknown. Much of our knowledge of viroids has been obtained from in vitro studies. It is extremely difficult to extrapolate in vitro behaviour back to natural systems. One of the largest gaps in our knowledge is that of tissue and cellular localization. Recently Bonfiglioli and colleagues (1996) have localized citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV), both members of the PSTV sub-group, to vascular tissue and mesophyll cells. Sub cellular localization in mesophyll cells revealed CEV to be distributed throughout the nucleus; in contrast CCCV is concentrated within the nucleolus. Why two members of the same sub-group should exhibit different ultrastructural localization is unknown. At this time little research of any nature, let alone localization, has been done on ASSV sub- 1

2 group viroids. Knowledge of the localization of all viroid sub-groups at the tissue and ultrastructural levels is of great importance in pathogenicity studies, as well as studies of replication strategies and processing. If viroids do interact with plants, by binding to and interfering with the proper functioning of host cell components, then accurate and precise knowledge of their localization will greatly assist in unravelling many of the remaining mysteries of viroid biology. 2. Research Plan 2.1 Project outline This research is primarily directed at determining the site of viroid replication and accumulation within infected plant cells and tissues. This knowledge is currently only available for 3 out of 22 viroids. The mixed viroid infections found in grapevine offer a wonderful opportunity to examine any differences or interactions between viroids from within these two sub-groups. Many of the viroids infecting grapevine have alternate hosts and this attribute will permit host-specific effects on replication and accumulation to be examined. The use of infectious multimeric cdna clones will also permit the examination of the effect of sequence variation on viroid localization, movement, and processing. Accurate in situ localization of viroids is of fundamental importance to studies of replication strategies and of the pathogenic processes of these organisms. It is, in addition, crucial to developing a fuller understanding of the basic biology of viroids. 2.2 Project aims. - To develop techniques which permit the accurate in situ localization of a range of viroids, in a wide range of host plants. - To use these techniques to examine the in situ localization of viroids, at both the tissue and ultrastructural level, paying particular attention to ASSV group members. - To examine changes in the localization of viroids and their replicative intermediates over the course of infection. -To compare the differences in localization and movement between sub-groups and sequence variants. -To investigate the effect of multiple co-infection on the localization and possible competitive interactions in situ. - To apply knowledge gained from in situ localization studies to viroid pathogenicity, replication and evolution. 3. Significance The studies of Bonfiglioli et al (1994, 1996) and Harders et al (1989) show that viroids within the PSTV sub-group are the only known form of pathogen to target the nucleolus, with ASBV one of two pathogens known to target the chloroplast. How viroids achieve and maintain this localization is unknown. The unexpected finding that two viroids from the same sub-group (CEV and CCCV) exhibit different sub-nuclear localization raises significant questions regarding differences in replication strategy and modes of pathogenesis. Results obtained from the study of other viroids and sub-groups in this research will provide basic localization information This is crucial in order permit a detailed analysis of the significance of localization differences, and to identify common sequence or structural features which may regulate viroid behaviour. 4. Methodology The small size of viroids presents significant problems for microscopical localization in infected tissue. It will be necessary to develop new tissue fixation protocols, nucleic acid in situ hybridization techniques and blocking procedures for grapevine, tomato and other plant tissues. Amplification procedures such as in situ RT-PCR (Reverse Transcription - Polymerase Chain Reaction) and PRINS (PRimed IN-Situ 2

3 labelling), will need to be adapted for use in this study, to permit the detection of low titre viroid infections in situ. Signal amplifying reporter systems will also need to be modified in order to obtain maximum sensitivity and specificity. Extensive use will be made of fluorescent probe technology and confocal laser scanning microscopy to generate images suitable for 3-dimensional reconstruction. The combination of these techniques, culminating in a 3-dimensional image, will permit clear and unequivocal tissue level localization and, with modification, permit co-localization of two or more viroids in the one host. Ultrastructural transmission electron microscopy studies will define more precisely the intracellular localization of the various viroids and require adaptation of the above procedures to the specific requirements of electron microscopy. Infectious multimeric cdna clones of viroids will be constructed as required to permit the unique infection of host plants with specific viroids of interest. Non infectious clones will be constructed and inserted into transcription vectors to provide a ready supply of probes. All viroids studied will require plus (+) and (-) minus sense clones. Naturally infected field plant tissue from the field will be used once probe specificity is established on laboratory infected material. Along with tissue studies I will, in collaboration with other workers in our laboratory, be developing techniques based upon protoplast culture. Synchronously infected protoplasts provide an opportunity to examine early events in the infection process and to quantitatively follow the changes in viroid titre and the ratio of positive (+) to negative (-) strand RNA forms during the course of infection. I would hope to take advantage of my previous experience and access the Flow cytometry facilities at the Hansen Centre for Cancer Research, IMVS, or Flinders Medical Centre in order to confirm synchronous infection and analyze large numbers of protoplasts rapidly. Techniques developed for confocal microscopy will readily adapt to flow techniques. 5. Proposed Timetable Research item Date Collaborative CEV and CCCV studies June Mar Concise outline June 1995 Development of confocal microscopy skills 1994 and ongoing Updating electron microscopy skills 1995 and ongoing Construction, sequencing and testing of HSV cdna clones Dec Sept 1995 HSV optical studies Oct Feb 1996 HSV ultrastructural studies Nov April 1996 Preparation of data for CCCV and CEV paper Nov. - Dec Structured Program Sept. - Dec 1995 Preparation of conference poster Jan. - Feb Analysis and preparation of HSV data for publication May July

4 Development of probes for GYSV-1, GYSV-2, AGV Mar July 1996 GYSY-1, GYSV-2 and AGV localization in grapevine May July Development of PRINS for low titre studies Mar July 1996 Development of in situ RT-PCR May Sept Analysis and preparation of preliminary Grapevine data for pub Dec Mar. Preparation of sequence variant clones and probes Aug May Examination of sequence variant effects Nov July Studies involving protoplasts Dec Aug. Data collation and analysis Jan - Oct Writing up July - Dec 6. Budget considerations ($) Item Travel and conference fees Stationary and photocopying Thesis Other Operating Glasshouse (costs covered under agreement with Campus registrar, S. Graebner) Containment facility Watering and pest control Soil and pots Confocal microscope FOC CEMMSA (EM) Flow cytometry Consumables

5 Data storage media Photo and Comp. Graphics Microscopical Molecular Other Minor equipment and maintenance Micro pipette set Disk drive Computing General Total References Bonfiglioli, R. G., McFadden, G. I. and Symons, R. H.: In situ hybridisation localises avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleolus. Plant J , 99 Bonfiglioli, R. G., Webb, D. R., and Symons, R. H: Tissue and Intra-Cellular Distribution of Coconut Cadang Cadang Viroid and Citrus Exocortis Viroid determined by In situ Hybridisation and Confocal Laser Scanning and Transmission Electron Microscopy. Plant J accepted for publication Diener, T. O.: Biological properties. In The Viroids T.O.Diener Ed, Plenum Press NY USA 1987 p9 Diener, T. O., Owens, R. A., and Hammond, R.W.: Viroids- the smallest agents of infectious disease - how do they make plants sick. Intervirology Harders, J., Luk cs, N., Robert-Nicoud, M., Jovin, T. M. and Riesner, D.: Imaging of viroids from tomato leaf tissue by in situ hybridisation and confocal laser scanning microscopy. EMBO J , 3941 Koltunow, A. M. and Rezaian, M. A.: A scheme for viroid classification. Intervirology, , 194. Singh M., and Singh R. P.: Potato spindle tuber viroid group. In Pathogenesis and host specificity in plant diseases. Histopathology, biochemical, genetic and molecular bases. Vol. III Viruses and Viroids. Singh R. P., Singh Y. S., and Kohmoto K. Eds. Permagon/Elsiever science Ltd. Oxford. UK 1995 pp Visvader, J. E., Forster, A. C., and Symons R. H.: Infectivity and in vitro mutagenesis of monomeric cdna clones of citrus exocortis viroid indicate the site of processing of viroid precursors. Nucl. Acids Res Reproduced on the ACUE site with the kind permission of the author. Entry last updated 7 September

Viroids. NOMENCLATURE Replication Pathogenicity. Nabil Killiny

Viroids. NOMENCLATURE Replication Pathogenicity. Nabil Killiny Viroids NOMENCLATURE Replication Pathogenicity Nabil Killiny A VIROID is A VIR(virus) OID(like) particle. Viroids are sub-viruses composed exclusively of a single circular strand of nucleic acid (RNA)