RNA silencing refers to a set of mechanistically related and evolutionarily conserved processes including post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). In plants, TGS is often accompanied by RNA-directed DNA methylation (RdDM). Both PTGS and TGS use specialized double stranded RNA binding proteins (DRBs) in conjunction with specific ribonucleases (Dicer-like proteins; DCL) to cleave 21, 22, or 24 nucleotide (nt) small interfering RNAs (siRNAs) from large double stranded RNAs (dsRNA). Cytoplasmic PTGS utilizes 21 and 22 nt siRNAs to target mRNA for degradation. The current model for TGS/RdDM involves a complex nuclear pathway where evolutionarily related forms of RNA polymerase II (Pol II), known as Pol IV and Pol V, transcribe DNA and work together with RNA dependent RNA polymerase 2 (RDR2) to produce long dsRNAs that are cleaved into 24 nt siRNAs by Dicer-like 3 (DCL3). These siRNAs associate with a complex that contains the ribonuclease argonaute 4 (AGO4) to recruit histone and DNA methyltransferases that subsequently target homologous DNA for methylation-mediated silencing. A feature that enhances PTGS and TGS defense is the ability of the silencing signal to spread from cell-to-cell and systemically throughout the plant. An antiviral role is well established, and the importance of PTGS as a host defense is clear from the fact that virtually all plant viruses encode proteins that target and suppress different aspects of this pathway. By contrast, while TGS has long been known to suppress potentially damaging DNA such as transposons, it has only recently been shown to target geminivirus DNA for repressive methylation.
Geminiviruses are circular single-stranded DNA (ssDNA) viruses that replicate in the nucleus through a double-stranded DNA (dsDNA) intermediate that associates with histones to form minichromosomes. Geminiviruses depend on host cellular machinery for both replication and transcription, thus providing excellent models to study the epigenetic regulation of these processes. Unlike most plant viruses (which have RNA genomes) geminiviruses must combat both PTGS and TGS defense pathways in order to be successful. We have shown that two related geminivirus-encoded silencing suppressors, AL2 and L2, suppress both PTGS and TGS by interacting with and inhibiting adenosine kinase (ADK), a methyl cycle co-factor. However, recent work has identified an additional suppression function of AL2 that is independent of ADK inhibition and requires AL2ߣs ability to activate transcription of host genes. Chapter 2 of this thesis characterizes the transcription activation-dependent suppressor function of AL2 in TGS reversal and the inhibition of systemic spread of silencing. Using ADK knock-down, it was determined that methyl cycle inhibition has no impact on systemic spread and that AL2, but not L2 or AL2 lacking its transcriptional activation domain (AL2 1-114), can prevent the spread of the silencing signal. Thus inhibition of systemic silencing likely depends on the ability of AL2 to activate transcription. Subsequent studies analyzing TGS reversal in vegetative and reproductive tissues of Nicotiana benthamiana plants revealed that only AL2 could reverse TGS in reproductive tissue in an ADK and transcription-independent manner. Thus, a third, previously unknown suppressor function of AL2 that is independent of transcription activation and ADK inhibition was identified. These studies show that unraveling the mechanisms that geminivirus suppressors use to inhibit various silencing pathways will lead to a better understanding of these complex host defenses.
Since TGS targets geminivirus dsDNA for repressive methylation and aspects of the RdDM pathway remain elusive, the studies described in this thesis also use geminiviruses as sensitive models to identify new pathway components and to unveil interesting roles of well-established pathway proteins. In Arabidopsis thaliana, it is well known that DRB proteins interact with specific DCL proteins to produce the canonical 21, 22, and 24 nt siRNAs for PTGS/TGS. Arabidopsis contains five DRB (DRB1-DRB5) proteins and four DCLs (DCL1-DCL4), each of which functionally partner in particular RNAi pathways. Previous work has shown that DRB1 and DCL1 functionally interact to cleave long hairpin dsRNA in the microRNA pathway, while DRB4 and DCL4 often target long dsRNA derived from replication intermediates of RNA viruses. Chapter 3 of this thesis uses the geminivirus model system to identify the DRB protein that functions with AGO4 and DCL3 in the RdDM pathway. It is shown that like dcl3 deficient plants, drb3 mutant plants are hypersusceptible to geminiviruses. Moreover, neither dcl3 or drb3 mutants are able to hypermethylate the viral genome, indicating that drb3 plants are deficient in the methylation-mediated defense pathway. DRB3 is also shown to physically interact with both AGO4 and DCL3 in the nucleus. Furthermore, analysis of geminivirus derived siRNAs found that drb3 mutants are able to produce 24 nt siRNAs similar to wild-type plants, indicating that DRB3 is not required for siRNA biogenesis. Altogether the data demonstrate that DRB3 associates with AGO4 and DCL3 in the RdDM pathway that is crucial for defending against viral DNA. Furthermore, this work emphasizes the sensitivity of the geminivirus model system to study RdDM pathway components.
Although it is believed that Pol IV and Pol V are required to initiate methylation, a key unanswered question is how these polymerases are targeted to specific DNA sequences. Due to their ssDNA nature and amplification by rolling-circle replication, nascent geminivirus genomes evade methylation and thus provide a unique transient model system to study de novo RdDM. Chapter 4 of this thesis shows that Pol IV and V, as well as their associated chromatin remodelers, play critical roles in the methylation-mediated defense against geminiviruses. However, promoter regions of geminivirus DNA continue to be methylated in the absence of both Pol IV and V, suggesting that another polymerase may initiate DNA methylation. Subsequent experiments found that Pol IV and V initially associate with the promoter region of the viral genome and during later infection spread to a coding region. Alternatively, Pol II constitutively associated with both the promoter and coding region. DNA methylation analysis of this coding region found hypermethylation only during late stages of infection, concurrent with the appearance of Pol IV and V. This supports previous work by others indicating that Pol IV and V are important for spread of RdDM. Finally, analysis of virus-derived siRNAs in pol IV, pol V, and pol IV/V infected mutant plants showed that Pol IV and V are not required for the biogenesis of viral siRNAs. Taken together, this data indicates that Pol IV and V play important roles in amplifying, maintaining, and spreading DNA methylation, but are not required for the initiation of RdDM. This suggests that another polymerase, possibly Pol II, initially transcribes DNA leading to the production of siRNAs and ultimately resulting in the initiation of DNA methylation.
Altogether the work in this thesis furthers our understanding of geminivirus pathogenesis by unveiling new suppressor functions of AL2. Moreover, it provides novel information on the host defenses by placing DRB3 in the RdDM pathway, and re-defining the roles of Pol IV and Pol V. Together these studies illustrate the utility of the geminivirus model system for analyzing epigenetic regulation and host defense pathways.