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A major finding of this study is that plant hosts methylate geminivirus chromatin as an antiviral epigenetic defense. 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) was evaluated. It was found that Arabidopsis thaliana plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed DNA methylation pathway components, or adenosine kinase, an enzyme required for maintaining the methyl cycle, show enhanced susceptibility to geminivirus infection. We found that cytosines in geminivirus DNA are methylated in the intergenic region, which contains the origin of replication and promoters for bidirectional transcription, and that methylation levels are reduced in mutants that show enhanced susceptibility. In addition, methylation-deficient mutant plants are unable to recover from geminivirus infection, and do not display symptom remission characteristic of wild-type plants. We concluded that viral genome methylation, leading to transcriptional gene silencing, is a strategy employed by plant hosts to silence invading geminiviruses. Genetic analysis revealed that genes important for methylation-based antiviral defense were also critical for repression of endogenous transposons. Thus, we proposed that geminiviruses may be used as a model to study chromatin methylation. In this investigation, we sought to ascertain which members of the Arabidopsis dsRNA binding protein family (DRB 2, 3, 4 or 5) are associated with DCL3 and AGO4, key components in the chromatin methylation pathway. We found that drb3 mutants are uniquely hypersusceptible to geminivirus infection, and that viral methylation levels are greatly reduced in the drb3 mutant. The drb3 mutant, like ago4 and dcl3, fails to recover from geminivirus disease. In addition, DRB3 physically interacts with DCL3 and AGO4 in distinct subnuclear locations. This uncovers a function for DRB3 in methylation, downstream of 24 nt siRNA biogenesis. It also suggests that geminivirus genomes may be used as convenient probes to identify additional pathway components and elucidate novel functions for known components in the chromatin methylation arm of RNA silencing. Finally, we expanded our analysis of host genes required for recovery from geminivirus disease in order to better define their roles in the methylation pathway. In this study, we discovered that non-CG methylation, conditioned by the cytosine methyltransferases CMT3 and DRM2, is required for recovery. Of the two plant-specific RNA polymerases involved in methylation, Pol V is required for recovery. Recovery was delayed in pol IV mutants suggesting that Pol II may compensate for Pol IV activity, and that Pol IV might also be involved in the spread of antiviral silencing. An investigation of DCL2/4-mediated post-transcriptional gene silencing (PTGS) revealed that the dcl2/4 mutants showed a mixture of recovered and non-recovered shoots suggesting that PTGS, although non-essential, plays a role in recovery. Interestingly, a dcl2/3/4 triple mutant was found to be methylation-competent, suggesting a possible new role for DCL1 in methylation. In summary, this work identifies methylation as a host defense against geminiviruses, and introduces the geminivirus model to study chromatin methylation.

RNAi-induced gene silencing plays a role in plant DNA methylation and defense. While most gene silencing studies have been performed on annuals, little is known about the expression of key components of this process (like ARGONAUTE proteins) in ornamentals. Using a combination of polymerase chain reaction techniques, an ARGONAUTE4 gene, PhAGO4, was isolated from Pelargonium. PhAGO4 encodes a predicted product of 934 amino acids that contains the PAZ and PIWI domains typical of ARGONAUTE (AGO) proteins. Phylogenetic analyses indicate that PhAGO4 clusters with other plant AGO4 proteins. Organ expression patterns of the AGO4 genes in Pelargonium and Arabidopsis show intriguing differences. AGO4 RNA levels decline with leaf age in both Arabidopsis and Pelargonium. In contrast AGO4 RNA levels in roots relative to leaves are higher in Pelargonium than in Arabidopsis. Both Arabidopsis and Pelargonium AGO4 showed higher RNA levels in flowers than leaves or roots. Even though flowers show higher levels of PhAGO4 RNA when compared to leaves and roots, protein gel blot analysis shows that at the protein level, the reverse is true. RNA interference (RNAi) is a regulatory mechanism found in all eukaryotes that occurs at either the transcriptional or post-transcriptional level. Two key players in Arabidopsis transcriptional gene silencing are small RNAs (sRNAs) and ARGONAUTE (AGO) proteins, specifically AGO4. Therefore, a combination of microarray-based genome-wide transcript profiling, a potential sRNA binding profile, and methylome datasets was used to identify candidate AGO4-regulated protein coding target genes. The highest number of AGO4-associated small RNAs with the potential for binding protein-coding genes showed a preference for either the upstream 1000 bp portion (promoter) or intronic regions. Microarray studies identified 243 up-regulated genes in ago4-1 mutant plants when compared to wild type. Interestingly, 33 up-regulated genes correlated with loci containing potential AGO4-associated sRNA binding sites. AGO4 is thought to recruit enzymes catalyzing non-CG methylation. The majority of potential sRNA binding sites did show a correlation with the non-CG methylation pattern. Taken together, these 33 protein-coding genes are good candidates for direct AGO4 regulation.