1. TuMV, miRNAs and Arabidopsis thaliana Amy Shatswell HHMI Summer Research 2006 Carrington Lab

2. Some Background Arabidopsis thaliana is a plant used to study genetics because of its short generation time and sequenced genome. TuMV stands for Turnip Mosaic Virus, a plant virus that infects Arabidopsis. Upon TuMV infection of Arabidopsis, virus-derived siRNAs are produced from the viral RNA siRNAs bind complementary viral RNA and inhibit translation (siRNAs act in cis) miRNAs (microRNAs) are produced from microRNA genes, bind to complementary messenger RNAs (targets) and inhibit translation (miRNAs act in trans) MicroRNAs regulate many genes involved in important developmental pathways and cell growth (Sullivan and Ganem, 2006).

3. siRNAs as an Antiviral Defense Mechanism RDR6 DCL4 AGO v-siRNA* RISC v-siRNA TuMV RNA v-siRNA duplex RDR6: enzyme responsible for making RNA double stranded. DCL4: enzyme responsible for dicing RNA into short fragments that can be recognized by RISC. AGO: incorporates siRNA and degrades viral RNA target. AGO-siRNA is known as RISC

4. How miRNAs are formed miRNAs are derived from single-stranded RNAs that fold back on themselves to form an imperfectly matched hairpin structure. These foldback RNA structures are processed by enzymes to form the 21 to 24 nucleotide-length miRNAs. The newly formed miRNAs can repress gene expression after transcription (Mallory and Vaucheret, 2006).

5. DCL1: a dicer enzyme that is necessary for the cleavage of all miRNA precursors. RISC: the protein complex that is responsible for carrying the miRNA to the target so it can be silenced. MIRNA gene Nuclear export AAA Pol II DCL1 AGO1 AAA.................. AAA DCL1 miRNA* RISC components RISC miRNA Target Cleaved target

6. More Background Some viruses encode miRNAs that interact with the host’s mRNAs, resulting in up-regulation or down- regulation of certain genes. Virus-encoded miRNAs enhance replication in some viruses (Sullivan and Ganem, 2006). www.omafra.gov.on.ca/.../ facts/88- 091f7.jpg

7. Hypothesis TuMV encodes siRNAs that act like miRNAs (act in trans) to enhance virus infectivity through regulating the expression of host genes.

8. Predictions based on hypothesis siRNAs encoded by TuMV are complementary to Arabidopsis mRNAs Those siRNAs arise from a microRNA precursor- like structure TuMV infection will be compromised in plants mutant for miRNA processing factors Expression of targeted host genes is down- regulated in infected plants Targeted host genes are involved in viral defense

9. Experiment 1- Test the prediction that siRNAs encoded by TuMV are complementary to Arabidopsis mRNAs Sequence through our lab’s strain of TuMV. Generate all possible siRNAs from TuMV sequence using bioinformatics. Generate list of host mRNAs complementary to TuMV siRNAs using bioinformatics. Results TuMV cDNA clone sequenced with four-fold redundancy (contig submitted to Genbank) Bioinformatics generated 10594 possible TuMV siRNAs and a list of Arabidopsis transcripts putatively targeted by TuMV siRNAs.

10. LOCUS bankit843647 10644 bp mRNA circular VRL 27-SEP-2006 DEFINITION Turnip mosaic virus-GFP mRNA, complete cds. ACCESSION 843647 VERSION KEYWORDS. SOURCE Turnip mosaic virus ORGANISM Turnip mosaic virus Viruses; ssRNA positive-strand viruses, no DNA stage; Potyviridae; Potyvirus. REFERENCE 1 (bases 1 to 10644) AUTHORS Lellis,A.D., Kasschau,K.D., Whitham,S.A. and Carrington,J.C. TITLE Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for eIF(iso)4E during potyvirus infection JOURNAL Current Biology 12, 1046-1051 (2002) REFERENCE 2 (bases 1 to 10644) AUTHORS Chapman,E.J., Shatswell,A.L., Lellis,A.D. and Carrington,J.C. TITLE Direct Submission JOURNAL Submitted (27-SEP-2006) Center for Genome Research and Biocomputing and Department of Botany and Plant Pathology, Oregon State University, 3021 ALS, Corvallis, OR 97331, USA COMMENT Vector Explanation: Turnip mosaic virus cDNA was engineered to contain GFP reporter gene Bankit Comment: Sequence of mRNA encoded by binary plasmid pCBTuMV-GFP; transcript represents Turnip mosaic virus (strain UK1) carrying a soluble modified red-shifted GFP reporter. TuMV contig Genbank entry:

11. Experiment 2- Test the prediction that siRNAs siRNAs arise from a microRNA precursor-like structure Predict secondary structure of TuMV RNA using mFOLD Compare TuMV to TEV, a related virus known to form foldback structure, using ClustalW Results 2 foldback structures were identified in TuMV TuMV and TEV are 58% identical in these regions

12. Stem-loop structures identified in TuMV. They are very similar to those found in TEV (Haldeman-Cahill et al., J. Virol. 1998). dG = -117.1 dG = -58.8

13. Comparison of TEV Sequence and TuMV sequence

14. Experiment 3: test the prediction that TuMV infection will be compromised in plants mutant for miRNA processing factors Infect RNA pathway mutants with TuMV that has been labeled with a GFP marker. Infect wildtype control plants. Examine plants under UV light for several weeks and record infection progress. Compare infection in mutant plants to wildtype plants. Determine whether infection is compromised in miRNA processing mutants. Photo courtesy of Andrew Lellis

15. TuMV-GFP Infection 4 dpi 0 25 50 75 100 Col-0rdr6-15/dcl4-2dcl1-7 % infected n = 31 n = 30 n = 23n = 34n = 33n = 24 n = 23 5 dpi 0 25 50 75 100 Col-0rdr6-15/dcl4-2dcl1-7 none 1:10 1:100 inoculum dilution 6 dpi 0 25 50 75 100 Col-0rdr6-15/dcl4-2dcl1-7 % infected 9 dpi 0 25 50 75 100 Col-0rdr6-15/dcl4-2dcl1-7 Results of Experiment 3

16. Summary TuMV-GFP sequence was determined and submitted to Genbank Bioinformatics generated 10594 possible TuMV siRNAs and a list of putative targets in Arabidopsis. Secondary structures of TuMV RNA were predicted. Virus infectivity did not appear to depend upon Arabidopsis genotype.

17. Future Directions Test prediction that expression of predicted targets is down-regulated in infected plants (analyze microarray data) Test prediction that targeted host genes are involved in viral defense

18. Acknowledgements The Howard Hughes Medical Institute Elisabeth Chapman Kristin Kasschau Jim Carrington