Transcript Arabidopsis thaliana Response to Tobacco Rattle Virus Jessica

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
Abstract
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 .
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.
ssRNA
PVX p25
cellular RDR
dsRNA
TCV CP
DCR
systemic
signal
TBSV p19/BYV p21
siRNAs
cellular
RDR
siRNA-mRNA duplex
CMV 2b
I
II
**
*
III
*
IV
*
V
DCL1, RDR1
***
Figure 3. Viral Suppressors: multiple points of suppression
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.
DCL2, DCL3, RDR6
RDR2
*
mRNA cleavage
RDR7, RDR3, RDR4, RDR5
**
TEV HC-Pro
RISC
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
rdr 1
Non 7a
rdr 2
7b 14 a 14b Non 7a
rdr 6
7b 14a 14b Non 7a
wt Col
7b 14a 14b Non 7a 7b
Conclusions
14a 14b
DCL4
•
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
Figure 1. Arabidopsis chromosome location of rdr and dcl genes
rdr 3
rdr 4
rdr 5
wt Col
Non 7a 7b 14a 14b Non 7a 7b 14a 14b Non 7a 7b 14a 14b Non 7a 7b
14a 14b
Future Directions
dcl 2
Non 7a
dcl 3
7b 14a 14b Non 7a
dcl 4
7b 14a 14b Non 7a
wt Col
7b 14a 14b Non 7a 7b 14a 14b
• 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 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 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.
virus
TRV
tobacco rattle virus
PVX
Potato virus X
TMV
Tobacco mosaic virus
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.
TCV
Turnip crinkle virus
CMV
Cucumber mosaic virus
PVY
Potato virus Y
TuMV
Turnip mosaic virus
TEV
Tobacco etch virus
family
suppressor
Tobravirus
16K
Potexvirus
p25
Tobamovirus
p30
Carmovirus
CP
Cucumovirus
2b
Potyvirus
HcPro
Potyvirus
HcPro
Potyvirus
HcPro