Arabidopsis thaliana Response to Tobacco Rattle Virus Jessica Martin, Cory Zoetewey, and Lisa K. Johansen, Department of Biology University of Colorado at Denver and Health Sciences Center Abstract Arabidopsis thaliana is a model plant for genetic studies. RNA interference (RNAi) is a multiple pathway mechanism for gene regulation and genome defense. Several Arabidopsis genes have been identified that operate in an RNAi viral defense pathway. These genes include Dicer-like (DCL) enzymes and RNA dependent RNA polymerases (RDR). Arabidopsis encodes four DCLs and seven RDRs. It has been shown that DCL2, RDR1, RDR2, and RDR6 have a role in the plant defense response to certain viruses. Homozygous mutants which lack a functional RDR have been created for rdr3, rdr4, and rdr5 to examine their possible role in viral defense. Many plant viruses encode silencing suppressors to block the RNAi defense pathway. Tobacco Rattle Virus (TRV) was used to measure the plant virus defense response since it does not encode a strong silencing suppressor. At 14 days post TRV infection, rdr3, rdr4, and rdr5 mutant lines showed increased levels of TRV replication as compared to wild type Arabidopsis. In particular, rdr4 showed a significant reduction in its ability to silence TRV. This indicates that RDR4 plays an important role in the Arabidopsis viral defense pathway against TRV. Figure 2. Schematic of siRNA production and the roles of RDRs and DCLs in virus defense response. When an RNA virus enters the cell, it produces dsRNA during replication of the genome, thus triggering the silencing pathway. Two of the major enzymes involved in RNAi, in Arabidopsis, include RDR and DCL. Once dsRNA is produced during viral replication it is cleaved by a DCL into small RNAs of 21-26nt. These short interfering RNAs (siRNA) have multiple functions: (1) associate with RNA-induced silencing complex (RISC) and cleave an RNA molecule with sequence complementarity, (2) serve as a primer for host RDR-directed dsRNA production, and (3) proposed to move systemically throughout the plant and prevent future virus infections. RNAi works to protect the plant from physical destruction due to a viral infection by stopping viral replication. Figure 1. Arabidopsis chromosome location of rdr and dcl genes Figure 4. Northern blot analysis of TRV infection in Arabidopsis. This blot shows the differences in virus accumulation among the various mutant plant lines in response to Tobacco Rattle Virus (TRV). Mutant lines rdr1-rdr6 and dcl2-dcl4, as well as wild-type Columbia were infected with TRV on day 0. Aerial (shoot and inflorescence) tissue was collected in duplicates (a and b), at both 7 and 14 days post infection. A non-infected control (Non) was run for every plant line, as well as a wild type control. The probe is specific to the coat protein of the TRV and was used to detect the presence of TRV in the systemic tissue. Analysis of the Northern blot revealed that rdr3, rdr4, and rdr5 accumulated a higher concentration of virus as compared to the wild type control. The concentration of TRV virus in the rdr4 mutants was higher at d14 than any of the other plant lines. This means rdr4 mutants have a reduced ability to silence the TRV virus which indicates the RDR4 gene may play an important role in the virus defense pathway against TRV and possibly other viruses Although very exciting, this data is preliminary and is in the process of being repeated. Figure 3. Viral Suppressors: multiple points of suppression Virus-Host Interactions and RNAi Virus infections in a plant can result in many different symptomatic phenotypes. Many of the viral symptoms are really developmental defects caused by the virus interfering with the host gene regulatory systems. Some of these developmental defects are caused by the activity of viral silencing suppressors. These RNAi suppressors are hypothesized to work at unique sites in the RNAi pathway. Each virus appears to have independently evolved a silencing suppressor because of their unique sites of action. We are studying Arabidopsis lines with T-DNA insertions in the RNA-dependent RNA-polymerase genes (rdr genes) and Dicer-like genes (dcl genes). Host RdRps have been proposed to amplify the RNA silencing signal. One role of RNAi is as an adaptive host defense response that results in sequence specific degradation of RNA with homology to the infecting virus. Arabidopsis has seven potential RDRs. There is literature for three of these genes, rdr1, rdr2, and rdr6, for their role in RNA silencing and virus-host interactions. An additional three genes, rdr3, rdr4, and rdr5, belong to a gene family and are at the beginning stages of characterization. Although emphasis is placed on the characterization of the RDRs, the DCLs are being studied concurrently as the RDRs and DCLs are thought to have a common pathway in RNAi We are interested in studying the RNAi host defense mechanism in response to a viral infection. RNAi evolved in plants as a defense mechanism to combat a viral infection. In response, the viruses have evolved mechanisms to suppress the RNAi pathway, thus allowing the virus to propagate. This co-evolution between plant and virus might suggest an explanation for the multiple RDRs found in Arabidopsis. Since there are multiple silencing suppressors that likely act on different points in the RNAi pathway, we will be analyzing viruses that represent multiple virus families with different suppressors. Conclusions rdr4 displayed higher levels of TRV virus accumulate 14 days post infection as compared to the wild type control rdr4 has a reduced ability to silence the TRV virus rdr4 plays a role in the RNAi virus defense pathway Future Directions Develop a more complete picture of the roles of RDRs and DCLs in the RNAi virus defense pathways Repeat virus infection study with TRV Analyze virus susceptibility of the rdr and dcl mutant lines in response to several different viruses encoding diverse silencing suppressors