1. Establishment of a System to Replicate, Purify, and Use a Mutant RNA Virus to Study the Antiviral Defense Response in Plants Katie Brempelis Mentors: Dr. James C. Carrington, Dr. Kristin Kasschau, Dr. Hernan Garcia-Ruiz Dr. James C. Carrington Lab HHMI Program, Summer 2008

2. BACKGROUND RNA Silencing Used in antiviral defense and gene regulation mRNA and viral ssRNA degradation, modification of DNA and histones, and translational repression What happens when a plant is infected by a virus? The plant enacts antiviral RNA silencing, producing virus- derived small RNAs (siRNAs) The virus produces suppressors proteins that counteract the plant’s RNA silencing response Experiment Model Arabidopsis thaliana and Nicotiana benthamiana TuMV-GFP ( Turnip mosaic virus with green fluorescent protein )  wild type virus TuMV-GFP-AS9 ( silencing suppression deficient )  mutant virus

3. Components of RNA Silencing Pathways Proteins: DCLs- dsRNA-specific ribonucleases RDRs- RNA-dependent RNA polymerases AGOs- RNAse RISC- RNA Induced Silencing Complex RNA Components: miRNA- gene regulation tasiRNA- gene regulation siRNA- antiviral defense

4. dsRNA Dicer RISC forms Targeted RNA cleaved by DCLs RNA Silencing -hpRNA -viral RNA -Effector complex -Contains AGO protein -Incorporates one strand siRNAs 21-26 nt RDR forms dsRNA - Signal Amplification -mRNA -viral ssRNA

5. Proposed Model The Proposed Three-Phase Model for Antiviral Silencing DCLs- recognition and cleavage AGO- part of RISC RDRs- amplification

6. MOTIVATION Effect of mutating A. thaliana genes is masked by the virus-encoded silencing suppressor A suppressor-deficient virus is needed to provide a clear distinction between plant genes required and dispensable for antiviral RNA silencing upload.wikimedia.org

7. SPECIFIC AIMS Purification of TuMV-GFP-AS9 To determine the requirement of A. thaliana genes in antiviral silencing people.whitman.edu

8. Purification of TuMV-GFP-AS9 Injection with Agrobacterium Established protocol for wild type virus, using N. benthamiana as a host Hypothesis: A highly concentrated inoculum of the mutant virus can be prepared using a similar approach Prediction: A semi-pure mutant virus preparation will be highly infective Method: Purify virus Titrate on dcl2/3/4 mutants www.sgn.cornell.edu www.focuscience.org

9. Maximizing Efficiency of AS9 Harvest What is the best day to harvest AS9-infected N. Benthamiana leaves? Conclusions: GFP does not necessarily indicate virus accumulation Highest viral accumulation at 5 and 6 dpi Leaves were senescing Harvest leaves at 4 dpi

10. Measuring Infectivity What is the infectivity of the AS9 prep? Conclusions: -The AS9 viral prep is infective -Use a 20-fold dilution of the wild type prep for similar infectivity with the mutant AS9 prep

11. Proposed Model The Proposed Three-Phase Model for Antiviral Silencing DCLs- recognition and cleavage AGO- part of RISC RDRs- amplification

12. Determining A. thaliana genes required in antiviral silencing Hypothesis: Both DCL1 and RDR6 proteins are required for antiviral RNA-silencing Prediction: AS9 mutant virus accumulation in dcll-7 and rdr6-15 mutants will be higher than in Col-0 wild type plants Method Inoculate A. thaliana Col-0, dcl1-7, dcl2/3/4, and rdr1/2/6 Collect infected tissue at 7, 10, 15 dpi Western blot detection of TuMV-CP Inoculate A. thaliana Col-0, rdr1-1, rdr2-1, rdr6-15, rdr1/2/6, dcl2/3/4

13. Method for DCL1 Experiment Partial DCL1 activity Infection No infection Lacks RDR1, RDR2, & RDR6 activity Lacks DCL2, DCL3, & DCL4 dicing activity Infection Wild type rdr1/2/6 dcl2/3/4dcl1-7Col-0 A. thaliana Genotype Mutation Predictions AS9 Mutant Wild type Infection

14. RESULTS AS9 did not infect dcl1-7 mutants Probed for TuMV-CP, 7 dpi inflorescence clusters Wt AS9 Wt AS9

15. Method for RDR Experiment Lack activity of a single RDR protein Infection No infection Lacks RDR1, RDR2, & RDR6 activity Infection Wild type A. thaliana Genotype Mutation Predictions dcl2/3/4rdr1/2/6rdr6-15rdr2-1rdr1-1Col-0 AS 9 Mutant Wild type Infection Lacks DCL2, DCL3, & DCL4 dicing activity

16. RESULTS Wt Mutant *Plants with systemic GFP * * 7 dpi AS9 infects all single RDR mutants Conclusions: rdr1, rdr2, and rdr6 are all required in antiviral silencing rdr1 seems to have the largest effect in local leaves

17. ACKNOWLEDGEMENTS Howard Hughes Medical Institute Cripps Scholarship Fund, College of Science Mentors: Dr. James C. Carrington Dr. Kristin Kasschau Dr. Hernan Garcia-Ruiz Dr. Kevin Ahern