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TuMV, miRNAs and Arabidopsis thaliana

TuMV, miRNAs and Arabidopsis thaliana. Amy Shatswell HHMI Summer Research 2006 Carrington Lab. Some Background . Arabidopsis thaliana is a plant used to study genetics because of its short generation time and sequenced genome.

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TuMV, miRNAs and Arabidopsis thaliana

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  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: 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 TuMV RNA RDR6 DCL4 v-siRNA duplex AGO v-siRNA* AGO RISCv-siRNA

  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. MIRNA gene Pol II AAA DCL1 DCL1 Nuclear export AGO1 RISC components miRNA* AGO1 RISCmiRNA Target AAA ..... ............. AGO1 Cleaved target AAA 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.

  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. TuMV contig Genbank entry: 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.

  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 = -58.8 dG = -117.1

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

  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 infectivitydid 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

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