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The Post-Transcriptional Gene Silencing Pathway and SPVD

The Post-Transcriptional Gene Silencing Pathway and SPVD. Liz Brauer November 2009-May 2010 Centro Internacional de la Papa. Sweet Potato Virus Disease. PTGS is affected RNaseIII seems to be involved But how does it work?

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The Post-Transcriptional Gene Silencing Pathway and SPVD

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  1. The Post-Transcriptional Gene Silencing Pathway and SPVD Liz Brauer November 2009-May 2010 Centro Internacional de la Papa

  2. Sweet Potato Virus Disease • PTGS is affected • RNaseIII seems to be involved • But how does it work? -Normally Potyviruses suppress silencing but not in SPVD, Crinivirus seems to be important

  3. RNA Silencing and siRNA processing Trends in plant science, 2008

  4. RNA Silencing and siRNA processing Defense Counter defense Transcriptional gene silencing RISC-mediated viral RNA degradation Host mRNA degradation Trends in plant science, 2008

  5. Role of RNAse3 Suppression of siRNA mediated defence in Sweetpotato PNAS, 2008

  6. Potential targets • Some viruses currently characterized target PTGS pathway including the AGO1 gene • Deep-sequencing of single and double infection sweet potato plants show 2.9 times more mi168 in SPVD but not in single infections • mi168 targets degradation of AGO1 in other plants

  7. Fits with previous studies about miRNAs affected by AGO1 changes

  8. Hypotheses 1. mi168 is enhanced in SPVD or in RNaseIII overexpressing plants infected with SPFMV 2. Expression of AGO1 is significantly reduced in the above conditions 3. Expression of other PTGS pathway genes or genes controlled by miRNAs are altered in SPVD symptomatic plants

  9. Gene cloning • Used PCR and RACE PCR to sequence in and around contigs from EST database (Rosalina) • Used database to find DCL1, RdR6 and other genes regulated by miRNAs

  10. AGO1 gene characteristics • AGO sequences are not similar within Arabidopsis (10 AGOs) • AGO1 sequences are similar between species

  11. Experimental setup • Two lines: Huachano (wildtype) and RNaseIII overexpressing lines • Four treatments: Healthy, SPFMV, SPCSV, SPVD • Randomized pots (3 plants per treatment)

  12. Dot blot • No results! • Probe detects purified complementary oligo but not any samples isolated from tissue • Not enough sample? (positive control probe did not detect any miRNA either)

  13. qRT PCR (for AGO1) * *

  14. Other genes RDR6 also unfinished…

  15. What does it mean? • The AGO1 expression results support previous observations of mi168 changes • RNaseIII was able to produce the same decreases in AGO1 when in a SPFMV background (necessary and sufficient) • Suggests that AGO1 is a good target for further investigation of viral silencing suppression

  16. Fits with previous literature • With increased mi168, AGO1 expression goes down -production of some miRNAs is compromised, especially mi166/165 and mi159

  17. Future studies • What is the gene in SPFMV that can cause synergism? • Expression analysis of PTGS genes in Arabidopsis expressing RNaseIII

  18. Muchas Gracias!!

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