1. Role of Arabidopsis RNA- Dependent RNA Polymerase Genes 3, 4 and 5 in Antiviral 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. AnAn AGO DRB SDE5 DCL AGO RDR6 SGS3 HEN1 DRB4 DCL4 RDR AnAn Primary targeting of virus mRNA AGO Secondary targeting of virus mRNA Systemi c spread DRB DCL3 DRB DCL2 Amplification AGO AnAn AnAn RDR1 RDR2 Dicing DRB4 DCL4 DRB DCL3 DRB DCL2 HEN1 Endogenous foldbacks? Viral replication intermediates ? Virus mRNA secondary structure? P02b HC-Pro P38 P19 HC-Pro P19

4. RNA-dependent RNA Polymerases In Arabidopsis: 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 Garcia-Ruiz, et al. Plant Cell. 2010 Feb;22(2):481-96 Col-0 rdr1 rdr2 rdr6 dcl2 dcl3 dcl4 TuMV siRNAs U6

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 Garcia-Ruiz, et al. Plant Cell. 2010 Feb;22(2):481-96 Wt AS9

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 1.rdr3 rdr4 rdr5 in Col-0 2.rdr3 rdr4 rdr5 in rdr1,2,6 single mutants 3.rdr3 rdr4 rdr5 in rdr1,2,6 double mutants 4.rdr null plant 5.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 Wt rdr5-3 RDR5rdr5-3heterozygote

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