1.11.2S
MOLECULAR GENETIC STUDIES OF SYMPTOM DEVELOPMENT IN ARABIDOPSIS-GEMINIVIRUS INTERACTIONS

KR DAVIS, K BUCKLEY and D WARE

Plant Biotechnology Center and Department of Plant Biology, Ohio State University, Columbus, OH 43210-1002, USA

Background and objectives
Viruses represent a unique class of pathogenic agents because of the extremely intimate associations formed between the virus and its host. In plant systems, it seems clear that host factors play critical roles throughout the entire virus life cycle, including the expression of viral genes, viral genome replication and the movement of virus throughout the plant. In order to identify specific host factors important for geminivirus infection and symptom development, we have established a model system based on the interaction of beet curly top virus (BCTV) with Arabidopsis [1]. One major molecular genetic approach that we have used to isolate Arabidopsis genes encoding proteins that are required for BCTV infection is the yeast two-hybrid system for identifying protein-protein interactions. Here we report on results demonstrating that the BCTV ORF C4 protein is a major symptom determinant in Arabidopsis, and the identification and characterization of host genes encoding proteins that interact with the C4 protein.

Results and conclusions
Previous studies by others have demonstrated that the BCTV C4 protein is required for symptom development in several hosts. We confirmed that this was also the case in Arabidopsis by generating a series of C4 mutants in BCTV-CFH and testing them in infection assays. All C4 nonsense mutations caused greatly attenuated symptoms that were correlated with significantly reduced levels of viral DNA in the aerial portions of infected plants. To determine if the BCTV C4 protein was capable of inducing symptoms independently of the rest of the viral genome, transgenic Arabidopsis plants containing a transgene composed of the CaMV 35S promoter and BCTV C4 genes were generated. These transgenic plants exhibit a range of phenotypes that include stunting, leaf curling and deformation, abnormal development of inflorescence structures and the inhibition of flowering. Many of these abnormalities are associated with swellings that result from the induction of abnormal cell division. Similar results were obtained by Stanley and co-workers using tobacco [2].

Given the ability of C4 to dramatically affect normal plant development, we initiated a screen for Arabidopsis genes encoding proteins that interact with the C4 protein using the yeast two-hybrid system. These studies have proven successful and resulted in the identification of several genes encoding putative host factors that specifically interact with both the BCTV-Logan and BCTV-CFH proteins. One of these potential host factors has homology to the Shaggy/GSK-3 family of protein kinases. This is particularly intriguing since this family of kinases is involved in regulating the activity of specific transcription factors, some of which are involved in the control of cell division events during specific developmental processes. Our current studies are focused on defining the molecular details of the interaction of wild-type and point mutants of the BCTV C4 protein with these putative host factors.

References
1. Lee S, Stenger DC, Bisaro DM, Davis KR, 1994. Plant Journal 6, 525-535.
2. Latham JR, Saunders K, Pinner MS, Stanley J, 1997. Plant Journal 11, 1273-1283.