Methylation of geminivirus genomes: Investigating its role as a host defense and evaluating its efficacy as a model to study chromatin methylation in plants

A major finding of this study is that plant hosts methylate geminivirus chromatin as an antiviral epigenetic defense.The first part of this dissertation describes work that establishes methylation as a host defense. We evaluated the susceptibility of methylation-deficient mutant plants to geminiviruses of two distinct genera (Beet curly top virus, BCTV, a monopartite curtovirus, and Cabbage leaf curl virus, CaLCuV, a bipartite begomovirus). We found that Arabidopsis thaliana plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, novel plant polymerases Pol IV and Pol V, RNA-directed DNA methylation pathway components, or adenosine kinase, an enzyme associated with maintaining the methyl cycle, show enhanced symptoms in response to geminivirus infection. We carried out direct biochemical examination of viral chromatin from infected plants for evidence of methylation and it was found that cytosines in geminivirus DNA are methylated in the intergenic region, which contains the origin of replication and promoters for the replication gene and coat protein transcripts. Cytosine methylation in the intergenic region was reduced in the methylation mutants at specific cytosine residues. In addition, histones associated with geminivirus genomes were also found to carry methylation modifications on tail lysine residues that are characteristic of both active and repressed chromatin.We proposed that methylation is a strategy employed by plant hosts to silence invading geminiviruses. Methylation is associated with transcriptional gene silencing as well as post-transcriptional gene silencing. The curtovirus L2 and begomovirus AL2 proteins are silencing suppressors that counter host-mediated silencing. It was previously found that L2 and AL2 can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase. Concurrent work in this lab has also shown that these proteins can reverse established transcriptional gene silencing and genomic methylation.The second part of this dissertation establishes geminiviruses as a model to study chromatin methylation. In infected cells, geminivirus single-stranded DNA replicates through double-stranded intermediates that associate with histones to form minichromosomes. In our investigation, we sought to ascertain which members of the Arabidopsis dsRNA binding protein family (DRB2, 3, or 5) are associated with DCL3, the Dicer-like protein in the methylation arm of silencing. In infection studies with CaLCuV and BCTV, we found that drb3 mutants are uniquely hypersusceptible to geminivirus infection. Methylation analysis of the viral intergenic region showed that methylation is greatly reduced in the drb3 mutant. The drb3 mutant, like ago4 and dcl3, fails to recover from geminivirus disease. We also found that DRB3 interacts with DCL3 and AGO4 in distinct subnuclear locations using bimolecular fluorescence complementation analysis. While the production of 24 nt siRNAs was not affected in the drb3 mutant, the DRB3 protein clearly partners with DCL3 in the methylation pathway, likely by acting downstream of siRNA generation, possibly by facilitating RISC loading of the 24 nt siRNA species into AGO4-containing RISC complexes. This uncovers at least one dsRNA binding protein in Arabidopsis that is important for methylation. It also suggests that geminivirus genomes may be used as convenient probes to identify additional components and elucidate novel functions for known components in the chromatin methylation arm of RNA silencing.The third part of this dissertation further explores methylation as a defense against geminiviruses, specifically, in the context of a phenomenon called 'recovery'. Recovery occurs when axillary shoots that arise following an initial infection with BCTV L2- mutant virus show symptom remission and contain low levels of hypermethylated viral DNA. Work described in earlier parts of this thesis showed that recovery is dependent on DCL3, DRB3 and AGO4. In this study, we found that mutants defective in all non-CG methyltransferases (ddc) lost the ability to recover from infection, indicating that non-CG methylation is required for recovery. We also found that pol V mutants (nrpe nrpd1b) were unable to recover, while pol IV mutants (nrpd nrpd1a) showed delayed recovery. The dcl2/4 mutant, deficient in post-transcriptional gene silencing, showed mixed recovery, where some shoots recover, while others do not. Thus, both post-transcriptional and transcriptional gene silencing are associated with recovery. Interestingly, a dcl2/3/4 triple mutant was found to be methylation-competent, suggesting a possible new role for DCL1 in methylation. Recovery from disease is a keynote manifestation of defense and we report that recovery is mediated by Pol V-DCL3-AGO4-DRM2/CMT3. Further, this study identifies the host recovery assay as a sensitive and definitive tool for the identification of methylation pathway components.