The Antiviral Response in Plants:
RNA Silencing
Melissa Andreas
Carrington Lab
HHMI - Oregon State University
Summer 2007
Plants and Pathogens
•
15% of worldwide food
production is lost to
pathogens
•
•
Garcia-Arenal et.al. (1996) Phytopathology 86: 535
Viruses cause est. $60
billion per year in crop
damages
Antiviral mechanisms in
plants are not fully
understood
Mechanisms of Defense
•
Innate immunity: quarantine of
infected cells
•
•
Not pathogen-specific
Adaptive response: RNA silencing
•
•
Triggered by invading nucleic acids
Response tailored to the pathogen
Project Goals
•
To characterize the antiviral silencing
response in a model organism
•
Infected:
Turnip mosaic virus
Uninfected
Lellis et. al. (2002) Curr.Biol.12: 1046-51
Arabidopsis and Turnip
Mosaic Virus (TuMV-GFP)
• Collect and sequence
small RNA
• Identify origin of RNA
• Describe small RNA
population
Viral Accumulation
Day 1
Day 5
Day 2
Day 6
Day 3
Day 7
Day 4
Control
Control
Viral Accumulation
TuMV-GFP Accumulation Timecourse
Average TuMV-GFP Accumulation
(Signal per 0.5 ug, mock subtracted)
3000
2500
2000
1500
1000
500
0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
-500
Days Post-Infection
•
Is TuMV targeted by RNA silencing
in Arabidopsis?
RNA Silencing Basics
•
•
Mechanism of degrading specific
sequences of RNA
Used mainly for antiviral defense and
gene regulation
RNA Silencing Mechanism
Target RNA
RDR
Double-stranded RNA
|||||||||||||||||||||||||||||||||||
Dicer
|||||||||||
|||||||||||
siRNA*
|||||||||||
AGO
CUAUAACCGCGCCGAGUUAGU
AGO
Target
CUAUAACCGCGCCGAGUUAGU
AGO
Degraded target
RISC
Dicers and Arabidopsis
Target RNA
•
Double-stranded RNA
Four kinds of Dicer-like (DCL)
proteins in Arabidopsis
|||||||||||||||||||||||||||||||||||
Dicer
|||||||||||
|||||||||||
•
|||||||||||
Specialization: nucleotide length
and task
•
AGO
•
CUAUAACCGCGCCGAGUUAGU
•
Target
•
CUAUAACCGCGCCGAGUUAGU
DCL
DCL
DCL
DCL
1:
2:
3:
4:
21 nt – microRNAs
22-23 nt – various
24 nt – heterochromatin formation
21 nt – post-transcriptional silencing
AGO
•
Degraded target
Antiviral roles are unknown
Experimental Setup
•
Arabidopsis plants infected with Turnip
Mosaic Virus (TuMV-GFP)
•
•
•
Mock-infected control plants
Time points: 7 and 10 days post-infection
(dpi)
DCL mutants: dcl1-7 and dcl2 dcl3 dcl4
triple mutant
TuMV-GFP Genome Comparison
Region of Genome
Shown
Diagram of Viral RNA
small RNA arising from
this region of viral RNA
Virus-Derived Small RNA in
Dicer Mutants
Wild-Type TuMV-GFP-infected
7 d.p.i.
dcl 1-7 TuMV-GFP-infected
7 d.p.i.
dcl2 dcl3 dcl4 TuMV-GFP-infected
7 d.p.i.
Wild-Type TuMV-GFP-infected
10 d.p.i.
dcl 1-7 TuMV-GFP-infected
10 d.p.i.
dcl2 dcl3 dcl4 TuMV-GFP-infected
10 d.p.i.
Reads derived from the TuMV-GFP genome
Reads derived from the Arabidopsis genome
Viral RNA in Dicer Mutants
Average TuMV-GFP
Accumulation
(Signal per 1 ug @ 7 dpi)
TuMV-GFP Accumulation in Wild-Type and RNA Silencing Mutants: 7 dpi
10000
8000
6000
4000
2000
0
Wild-Type
dcl
dcl1-7
1-7
dcl234
234
dcl
Genotype
TuMV-GFP Accum ulation in Wild-Type and RNA Silencing Mutants: 10 dpi
Average TuMV-GFP
Accumulation
(Signal per 1 ug @ 10 dpi)
10000
8000
6000
4000
2000
0
Wild-Type
dcl
dcl1-7
1-7
Genotype
dcl
dcl 234
234
Conclusion
•
Turnip Mosaic Virus is a target of RNA
silencing in Arabidopsis.
The Next Step
•
Infect with a TuMV-GFP suppressor mutant
•
Expand to other Dicer mutants
•
•
•
dcl2
dcl4
Examine the dynamics of small RNA
production
Acknowledgments
•
Funding: Howard Hughes Medical Institute
•
Dr. Kevin Ahern
•
Mentors:
•
•
•
Elisabeth Chapman
Kristin Kasschau
Jim Carrington