RNA-directed gene silencing is an adaptive response by which plants defend against virus invasion. RNA-silencing pathways can be divided into two basic categories: Post-Transcriptional Gene Silencing (PTGS) and Transcriptional Gene Silencing (TGS). PTGS occurs in the cytoplasm and results in mRNA degradation. In addition, a mobile signal circulates throughout the plant, is amplified, and triggers systemic silencing. The second pathway, TGS, takes place in the nucleus and results in methylation of cytosine in DNA, which encourages the formation of transcriptionally inactive heterochromatin by histone modification. TGS suppresses damaging sequences such as transposons, and also plays an important role in defense against DNA viruses.
The AL2 and L2 proteins are related proteins encoded by geminiviruses of the Begomovirus and Curtovirus genera, respectively. AL2 is transcriptionally active, is required for late viral gene expression, and can also activate unknown host genes, whereas L2 is transcriptionally inactive. AL2 can suppress silencing by two mechanisms. The first requires host gene activation by AL2 (transcription-dependent mechanism). The second is shared by AL2 and L2 and involves inactivation of adenosine kinase (ADK), an enzyme required for efficient cellular transmethylation activity. Both AL2 and L2 can suppress local silencing via a transcription-independent mechanism. In this thesis AL2, but not L2, is also shown to block systemic spread of PTGS by a transcription-dependent mechanism. In addition, AL2 and L2 are shown to reverse established PTGS, which suggests interference with a maintenance step. However, while both proteins reversed silencing in immature plants, only AL2 did this in mature plants, suggesting the existence of multiple, developmentally regulated maintenance pathways. Finally, the role of methylation as a host defense was confirmed by studies which demonstrated that both AL2 and L2 can reverse TGS of a transgene and of native loci, and cause significant reversal of methylation throughout a plant genome. In this case, TGS reversal occurred by both transcription-independent and transcription-dependent mechanisms.
These studies further our understanding of viral pathogenesis and the nature of innate host defenses by demonstrating that AL2 and L2 can suppress both TGS and PTGS by multiple mechanisms.