Role of Arabidopsis RNA-Dependent RNA Polymerase Genes 3, 4 and 5 in Antiviral Defense

1. Role of Arabidopsis RNA-Dependent RNA Polymerase Genes 3, 4 and 5 inAntiviral Defense Nick Lowery Dr. James C. Carrington Dr. Hernan Garcia-Ruiz Oregon State University HHMI 2010

2. RNAi • Major post-transcriptional regulatory pathway • Regulates expression of endogenous genes • Regulates exogenous genetic elements • e.g. Transposons, viruses • Areas for research / commercial potential : • Cancer • Selective gene silencing • Antiviral silencing

3. P0 P38 2b HC-Pro Virus mRNA secondary structure? P19 AGO Primary targeting of virus mRNA Dicing An An AGO DRB4 HEN1 Endogenous foldbacks? DCL4 DRB DCL2 RDR1 SGS3 DRB RDR6 DCL3 SDE5 RDR2 Viral replication intermediates? Amplification RDR An DRB DCL4 DCL2 DRB4 DRB DRB DCL3 HEN1 DCL AGO Systemic spread Secondary targeting of virus mRNA An HC-Pro AGO P19

4. RNA-dependent RNA Polymerases • InArabidopsis: 6 RDR genes. • Functions for RDR1, RDR2 and RDR6 have been characterized for TuMV infections • RDR1 and RDR6 are the primary effectors, acting cooperatively or redundantly • RDR2 plays a minor role in local infection • RDR3, RDR4 and RDR5 currently have no biological function assigned to them

5. RDR3, RDR4, RDR5

6. Basal siRNA Biogenesis TuMV siRNAs U6 Col-0 dcl2 dcl3 dcl4 rdr1 rdr2 rdr6 Col-0 Garcia-Ruiz, et al. Plant Cell. 2010 Feb;22(2):481-96

7. Hypothesis • RDR3, RDR4 and RDR5 play a role in antiviral RNA silencing during TuMV infection of Arabidopsis • May be primary or secondary • One, two or all three genes may be involved

8. Experimental Design • Knock out genes and infect with virus • Compare severity of infection to wild-type plants and dcl triple mutants as controls • Points that must be considered • Viral silencing suppressor, HC-Pro • Masking of effects by other RDR genes • Generation of mutants

9. Viral Infection • TuMV-GFP • Allows for visualization of infection under UV • Monitor for ability to establish infection foci and to move systemically throughout the plant Garcia-Ruiz, et al. Plant Cell. 2010 Feb;22(2):481-96

10. Silencing Suppressor • TuMV encodes a silencing suppressor protein, HC-Pro • Argonaute imposter; sequesters siRNA, so no antiviral response can be mounted • Solution: TuMV-AS9 • Mutant virus lacking functional copy of HC-Pro • Unable to infect wild-type Arabidopsis • Wt infection serves as positive control Wt AS9 Garcia-Ruiz, et al. Plant Cell. 2010 Feb;22(2):481-96

11. Hierarchical Action of RDRs • RDR1,2,6 pathway primary for TuMV infection • Will likely see effects only in absence of primary pathway, either in whole or in part • Strategy: gradually remove RDR1,2,6 pathway in rdr3 rdr4 rdr5 background • rdr3 rdr4 rdr5 in Col-0 • rdr3 rdr4 rdr5 in rdr1,2,6 single mutants • rdr3 rdr4 rdr5 in rdr1,2,6 double mutants • rdr null plant • rdr3,4,5 single mutants in rdr1,2,6 mutants • used to determine which genes are active

12. Generation of Mutants • Single mutants: Salk Institute • Combination Mutants • Crosses • rdr1,2,6 single and combination mutants onto rdr3,4,5 single mutants • Transformation • Required to overcome linkage • Express artificial miRNA to silence genes • rdr3 rdr4 rdr5 triple mutants

13. Progress • All mutant lines generated • Both crosses and dipping result in introduction of one copy of mutant allele • Need to bring to homozygous condition Screen for rdr5-3; rdr5-3 X rdr1-1 rdr2-1 rdr6-15, F2 generation rdr5-3 RDR5 rdr5-3 heterozygote Wt

14. amiRrdr345 Expression

15. Future Projects • Infect with different viruses • Different viruses affect different steps in RNAi pathway during infection • Mapping experiments • RDR null plant incapable of producing secondary siRNA? • Sequence and map primary siRNA back to TuMV genome to determine primary target sites.

16. Acknowledgements • Dr. Hernan Garcia-Ruiz • Dr. James C. Carrington • Steen Hoyer, Noah Fahlgren and everyone in the lab • HHMI, the Cripps scholarship & Dr. Kevin Ahern