1. The Antiviral Response in Plants: RNA Silencing Melissa Andreas Carrington Lab HHMI - Oregon State University Summer 2007

2. 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

3. 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

4. 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

5. Viral Accumulation Day 5 Day 6 Day 7 Control Day 1 Day 2 Day 3 Day 4 Control

6. Viral Accumulation Is TuMV targeted by RNA silencing in Arabidopsis?

7. RNA Silencing Basics Mechanism of degrading specific sequences of RNA Used mainly for antiviral defense and gene regulation

8. RNA Silencing Mechanism Double-stranded RNA Degraded target Target RNA Target AGO siRNA * RISC AGO Dicer RDR

9. 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 AGO Dicer

10. 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

11. TuMV-GFP Genome Comparison Region of Genome Shown small RNA arising from this region of viral RNA Diagram of Viral RNA

12. Virus-Derived Small RNA in Dicer Mutants Reads derived from the Arabidopsis genome Reads derived from the TuMV-GFP genome

13. Viral RNA in Dicer Mutants dcl 1-7dcl 234 dcl 1-7 dcl 234

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

15. The Next Step Infect with a TuMV-GFP suppressor mutant Expand to other Dicer mutants dcl2 dcl4 Examine the dynamics of small RNA production

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