The Antiviral Response in Plants: RNA Silencing

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The Antiviral Response in PlantsRNA Silencing

• Melissa Andreas
• Carrington Lab
• HHMI - Oregon State University
• Summer 2007

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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
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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

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Project Goals

• To characterize the antiviral silencing response
  in a model organism

• Arabidopsis and Turnip Mosaic Virus (TuMV-GFP)
• Collect and sequence small RNA
• Identify origin of RNA
• Describe small RNA population

Infected Uninfected
Turnip mosaic virus
Lellis et. al. (2002) Curr.Biol.12 1046-51
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Viral Accumulation
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Viral Accumulation

• Is TuMV targeted by RNA silencing in Arabidopsis?

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RNA Silencing Basics

• Mechanism of degrading specific sequences of RNA
• Used mainly for antiviral defense and gene
  regulation

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RNA Silencing Mechanism
Target RNA
RDR
Double-stranded RNA
Dicer
siRNA
AGO
RISC
AGO
Target
AGO
Degraded target
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Dicers and Arabidopsis
Target RNA

• 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
Dicer
AGO
Target
AGO
Degraded target
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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

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TuMV-GFP Genome Comparison
Region of Genome Shown
Diagram of Viral RNA
small RNA arising from this region of viral RNA
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Virus-Derived Small RNA in Dicer Mutants
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Viral RNA in Dicer Mutants
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Conclusion

• Turnip Mosaic Virus is a target of RNA silencing
  in Arabidopsis.

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The Next Step

• Infect with a TuMV-GFP suppressor mutant
• Expand to other Dicer mutants
• dcl2
• dcl4
• Examine the dynamics of small RNA production

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Acknowledgments

• Funding Howard Hughes Medical Institute
• Dr. Kevin Ahern
• Mentors
• Elisabeth Chapman
• Kristin Kasschau
• Jim Carrington