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

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Double-stranded RNA

Degraded target

Target RNA

Target

AGOCUAUAACCGCGCCGAGUUAGU

AGOCUAUAACCGCGCCGAGUUAGU

siRNA*

RISC

AGO

Dicer

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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

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Double-stranded RNA

Degraded target

Target RNA

Target

AGOCUAUAACCGCGCCGAGUUAGU

AGOCUAUAACCGCGCCGAGUUAGU

Dicer

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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