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Transient assay, why?

Transient assay, why?. ANS: Sweetpotato transformation takes a long time and Not reprocuble due to genotype dependence. Intron. Antisense. Sense. mRNA Transcript. Sense. Sense. Hairpin RNA. Intron. Antisense. Antisense. dsRNA. Agroinfiltration of GFP N. benthamiana (C16).

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Transient assay, why?

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  1. Transient assay, why? ANS: Sweetpotato transformation takes a long time and Not reprocuble due to genotype dependence

  2. Intron Antisense Sense mRNA Transcript Sense Sense Hairpin RNA Intron Antisense Antisense dsRNA Agroinfiltration of GFP N. benthamiana (C16) Transformation of Agrobacterium Making culture Pelleting cells 3-4 days 3-5 hrs incubation at RT infiltration GFP silenced plant

  3. Agroinfiltration of GFPN. benthamiana Non silenced GFP silenced GFP

  4. Effect of bacterial concentration on RNA-silencing Systemic silencing at low and high bacterial concentration Molecular data 7.6 X107 Low to high siRNA Loading control 3.04 X108

  5. Time course analysisfor siRNA biogenesis AKK1420-HT AKK1420 -LT DAPI DAPI 1 2 3 4 5 6 7 8

  6. Comparison of siRNA production Sense fragment Vs antisense Local Systemic Vs silencing AKK1420-CONST-I AKK1420-CONST-II • GFP Both local and systemic give siRNA but less from systemic • siRNA for Local silencing but not systemic produced for construct fragment • spread • Both sense and antisense fragments produce siRNA

  7. Comparison of siRNA production AKK1420-LT-CSV siRNA AKK1420 CONST-HT-CSV Loading control • Both fragments are producing siRNA • CSV fragment seem to be producing less amount of siRNA as compared to FMV for both constructs

  8. Peak difference siRNA titer Virus titer Time in days siRNA Peak Virus accumulation peak Transient Protection studies The greater the peak difference the less reliable the results of transient protection

  9. SPCSV/SPFMV -Transmission • Low transmission frequency of 15% for SPCSV • Mechanical transmission for SPFMV • M2-44, C1 and 835 • In 12- 15days symptons begin to show-implying that virus peaks a little bit earlier

  10. Transient protection studies with SPFMV Are siRNA working? Virus challenge done 3dpI with Agrobacteriun containing the construct There seems to be protection against the C1 strain of the virus

  11. Can we reduce the virus titer in the already sick plants? Virus titer 4dpI The titers remain high even after treatment with siRNA-implying that Plant virus silencing requires timely siRNA biogensis

  12. Can we suppress the suppressor? Experiment Control From the high target( HT) targeting the Rnase3 From Low target(LT) not specific to RNase3 We Expect No change Rnase3 Expression We Expect reduced Rnase3 Expression Rnase3 mRNA

  13. Suppressing the suppressor-RNase3 What is the consquence?

  14. What is the RNase3 relationship with other organisms?

  15. SUMMARY • Transient protection studies seem possible with SPFMV but not SPCSV • Plant virus silencing requires timely siRNA biogensis • RNase3 of SPCSV shares less relationship with that of other organism and thus its knockdown is less likely to affect other genes even though we have not confirmed successful knockdown • Constructs are successfully transformed into N.benthamiana and sweetpotato for futher characterization • We have confirmed presence of Begomoviruses in Uganda and Tanzania

  16. Current work& focus • Planning the experiment for the suppression of Rnase3 • Look forward to continue transmission studies with M2-44 and • Test the functionality of siRNA by silencing on spot and during viral infection • Variability among Begomoviruses of sweetpotato

  17. Acknowledgment • Dr. Roger Beachy • Dr. Maria. A. Soto and Brian Kelly • Dr. Jan Kreuze-CIP • Dr.ROM. Mwanga • Dr. B.L Patil • Dr. Beachy Lab & DDPSC • Dr. C. Fauquet and ILTAB members • employees • NACRRI-Sweetpotato and Cassava programs • CIP and its staff • Howard Buffet foundation for funding the project

  18. Thank you very much

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