Arabidopsis thaliana Response to Tobacco Rattle Virus

1
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
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.
Figure 3. Viral Suppressors
multiple points of suppression

• 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

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.