the antiviral response in plants: rna silencing melissa andreas carrington lab hhmi - oregon state...
DESCRIPTION
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 pathogenTRANSCRIPT
The Antiviral Response in Plants:RNA Silencing
Melissa AndreasCarrington Lab
HHMI - Oregon State UniversitySummer 2007
Plants and Pathogens• 15% of worldwide food
production is lost to pathogens• Viruses cause est. $60
billion per year in crop damages
• Antiviral mechanisms in plants are not fully understood
Garcia-Arenal et.al. (1996) Phytopathology 86: 535
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: Uninfected Turnip mosaic virus
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 5
Day 6
Day 7
Control
Day 1
Day 2
Day 3
Day 4
Control
Viral Accumulation
• Is TuMV targeted by RNA silencing in Arabidopsis?
TuMV-GFP Accumulation Timecourse
-500
0
500
1000
1500
2000
2500
3000
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Days Post-Infection
Average TuMV-GFP Accumulation (Signal per 0.5 ug, mock subtracted)
RNA Silencing Basics
• Mechanism of degrading specific sequences of RNA
• Used mainly for antiviral defense and gene regulation
RNA Silencing Mechanism
|||||||||||||||||||||||||||||||||||
Double-stranded RNA
Degraded target
Target RNA
Target
AGOCUAUAACCGCGCCGAGUUAGU
AGOCUAUAACCGCGCCGAGUUAGU
siRNA*
RISC
AGO
Dicer
||||||||||| ||||||||||| |||||||||||
RDR
Dicers and Arabidopsis• Four kinds of Dicer-like (DCL)
proteins in Arabidopsis• Specialization: nucleotide
length and task• DCL 1: 21 nt – microRNAs• DCL 2: 22-23 nt – various• DCL 3: 24 nt – heterochromatin
formation• DCL 4: 21 nt – post-transcriptional
silencing
• Antiviral roles are unknown
|||||||||||||||||||||||||||||||||||
Double-stranded RNA
Degraded target
Target RNA
Target
AGOCUAUAACCGCGCCGAGUUAGU
AGOCUAUAACCGCGCCGAGUUAGU
Dicer
||||||||||| ||||||||||| |||||||||||
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
small RNA arising from this region of viral RNA
Diagram of Viral RNA
Virus-Derived Small RNA in Dicer Mutants
Reads derived from the Arabidopsis genome
Reads derived from the TuMV-GFP genome
Wild-Type TuMV-GFP-infected 7 d.p.i.
Wild-Type TuMV-GFP-infected 10 d.p.i.
dcl 1-7 TuMV-GFP-infected 7 d.p.i.
dcl 1-7 TuMV-GFP-infected 10 d.p.i.
dcl2 dcl3 dcl4 TuMV-GFP-infected 7 d.p.i.
dcl2 dcl3 dcl4 TuMV-GFP-infected10 d.p.i.
Viral RNA in Dicer Mutants
TuMV-GFP Accumulation in Wild-Type and RNA Silencing Mutants: 10 dpi
0
2000
4000
6000
8000
10000
Wild-Type dcl 1-7 dcl 234
Genotype
Average TuMV-GFP Accumulation
(Signal per 1 ug @ 10 dpi)
dcl 1-7 dcl 234
TuMV-GFP Accumulation in Wild-Type and RNA Silencing Mutants: 7 dpi
0
2000
4000
6000
8000
10000
Wild-Type dcl 1-7 dcl 234
Genotype
Average TuMV-GFP Accumulation
(Signal per 1 ug @ 7 dpi)
dcl 1-7 dcl 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