| Geminiviruses are an emergent group of plant viruses, characterized by a small, singlestranded DNA genome amplified and transcribed in the nuclei of host cells. Disease progression depends on key interactions between virus and host to counter host defense mechanisms and to reprogram the cellular environment to support the viral life cycle. Geminiviruses encode a multifunctional pathogenicity factor called AL2 that is required early in infection to inactivate cellular kinases and suppress RNA silencing and late in the infection to transactivate coat protein (CP) and BR1 movement protein expression. As a transcriptional regulator, AL2 has a C-terminal acidic activation domain, but it does not bind double-stranded DNA, not even sequences known to mediate its activity. This suggests that transcriptional regulation occurs indirectly, through interaction with a host factor(s), which in turn may alter host gene expression. AL2 protein regulates the transcription of late viral genes through two distinct viral sequences. One sequence is located 60 to 125 bp upstream of the CP transcription start site and is required for AL2-mediated activation of the CP promoter in mesophyll cells. The second regulatory sequence is located between 1.2 and 1.5 kb upstream of CP gene and is required for AL2 --mediated derepression of CP promoter in phloem tissue. Binding studies indicate that sequences mediating repression and activation of the Tomato golden mosaic virus and Cabbage leaf curl virus CP promoter specifically bind different nuclear factors common to tobacco, tomato and spinach. In this thesis, a plant specific DNA binding protein, PEAPOD2 was isolated from Arabidopsis thaliana using a yeast one hybrid screen. In vivo and in vitro studies suggest that geminivirus AL2 protein and PPD2 form a complex at the CP promoter to activate CP gene expression. Also, the promoter of a host calmodulin-related protein, rgs-caM (At1g76640) is responsive to geminivirus AL2. Silencing assays demonstrate that rgs-caM is capable of suppressing RNA silencing. My results suggest that this class of proteins may be endogenous silencing suppressors, which are used to shut down the RNA silencing pathway in the absence of virus infection. This research opens up possible targets, such as PPD2 and rgs-caM, for the development of novel disease resistance strategies based on disruption of these key interactions between virus and host. |
