| RNA-directed gene silencing is a primary adaptive response by which plants defend against virus invasion. RNA-silencing pathways are complex, but can be divided into two basic categories: Post-Transcriptional Gene Silencing (PTGS) and Transcriptional Gene Silencing (TGS). PTGS occurs in the cytoplasm and is mediated by small interfering RNAs (siRNAs) 21--22 nucleotides (nt) in length, generated from larger double-stranded RNAs by Dicer-like ribonucleases. One strand of the siRNA is incorporated into another ribonuclease-containing complex known as RISC (RNA-induced silencing complexes), where it targets homologous mRNAs for degradation. In addition to silencing at the site of infection, there is a mobile silencing signal of unknown composition that circulates throughout the plant, is amplified, and triggers systemic silencing. The second pathway, TGS, takes place in the nucleus. In this case 24 nt siRNAs program RISC-like complexes to methylation of cytosine residues in homologous promoter and coding regions of DNA, which encourages the formation of transcriptionally inactive heterochromatin by histone modification. TGS has primarily been thought of as a way to suppress the activity of potentially damaging native sequences such as transposons, but recent evidence has shown that it also can play 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 the expression of late viral genes, and can also activate unknown host genes, whereas L2 is transcriptionally inactive. AL2 can suppress PTGS by two mechanisms. The first requires activation of host gene transcription by AL2 (transcription-dependent mechanism). The second mechanism is shared by AL2 and L2, and involves interaction with an inactivation of adenosine kinase (ADK), which is required for efficient cellular transmethylation activity. It has been previously shown that both AL2 and L2 can suppress silencing in an assay that likely measures the initiation PTGS. This suggests that AL2 and L2 can suppress PTGS initiation via a transcription-independent mechanism. In this thesis AL2, but not L2, is shown to also block systemic spread of PTGS by a transcription-dependent mechanism. In addition, AL2 and L2 are shown to reverse established systemic PTGS, which suggests interference with a maintenance step. However, while both AL2 and L2 reversed silencing in immature plants, silencing could only be reversed by AL2 in plants that had undergone the vegetative to floral transition. This suggests the existence of multiple, developmentally regulated silencing 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 pseudogenes, and cause significant reversal of methylation throughout a plant genome. In this case, TGS reversal of most genes examined could be accomplished by the transcription-independent mechanism, although some require the transcription-dependent mechanism.Taken together, the studies in this thesis 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. |
