Plant Biotechnology Center and Department of Molecular Genetics, Ohio State University, Columbus, OH 43210, USA

Background and objectives
In the subgroup III geminiviruses (e.g. tomato golden mosaic virus; TGMV) the product of the AL2 gene, transcriptional activator protein (TrAP), is required for expression of the viral coat protein (CP) gene. TrAP stimulates the expression of this gene at the level of transcription [1] by two alternative mechanisms: it activates the CP promoter in mesophyll cells, and acts to de-repress the promoter in phloem tissue [2]. Studies of the mechanisms by which TrAP stimulates transcription are proceeding along several fronts, including a biochemical analysis of TrAP. This small 15-kDa protein contains a transcriptional activation domain and has DNA-binding activity. However, it prefers ssDNA over dsDNA, and binds both in a sequence non-specific manner (unpublished data). This raises the question of how TrAP is targeted to responsive promoters. One possibility is that targeting is achieved through a protein complex consisting of TrAP and a host transcription factor; thus TrAP may be directed to responsive promoters via the sequence-specific DNA-binding properties of a host protein. Consequently, we are seeking host proteins that specifically interact with TrAP. This presentation will discuss the surprising initial results of this search, as well as work to further define the activation domain.

Results and conclusions
We have analysed the ability of substitution and deletion mutants to activate transcription of reporter genes in yeast and mouse fibroblasts (NIH3T3 cells) when tethered to the reporter gene promoter as GAL4 DNA-binding domain fusion proteins. The ability of mutant TrAP proteins to complement TGMV AL2 null mutants in Nicotiana benthamiana protoplasts has also been investigated. Ten different mutants have been analysed to date. The results of these studies are consistent, and delimit the activation domain to a 27-amino-acid region. Further, three amino-acid residues within this domain that are critical for activation activity have been identified. Current efforts are focused on the identification of general transcription factors (GTFs) contacted by the activation domain, and how mutations within the domain affect TrAP:GTF interactions.

The yeast two-hybrid system is being used to identify cellular proteins that interact with TrAP. Screens employ defective TrAP (lacking the activation domain) as bait, and an Arabidopsis cDNA library as prey. In initial screens, a full-length cDNA encoding a protein kinase that interacts with TrAP was retrieved. TrAP also interacts with the yeast homologue of this kinase, which confirms that the interaction is specific. We expect that TrAP is a substrate for the kinase, but it seems unlikely that the TrAP:kinase complex binds TrAP-responsive promoters. However, because the kinase appears to be a key component of the host defence response, we speculated that the TrAP:kinase interaction might modulate host susceptibility and viral pathogenicity. Transgenic plants expressing defective TrAP lacking the activation domain (to avoid toxicity problems and potential synergy with an infecting virus) were prepared. These plants appear normal in all respects, but display an interesting phenotype following inoculation with TGMV: specifically, five of six transgenic lines become infected with viral inoculum doses 100-fold lower than what is required to achieve infection of wild-type plants. We refer to this phenotype as enhanced susceptibility (ES). ES extends to beet curly top geminivirus (subgroup II), and experiments to determine if ES is general for RNA viruses and bacterial and fungal pathogens are under way. Studies to characterize the ES phenotype at the molecular level are also in progress.

1. Sunter G, Bisaro DM, 1992. Plant Cell 4, 1321-1331.
2. Sunter G, Bisaro DM, 1997. Virology 232, 269-280.