Towards Resistance to Sweetpotato virus Disease (SPVD)

1. Transient studies in Nicotianabenthamiana Towards Resistance to Sweetpotato virus Disease (SPVD) JOAB TUGUME ABL/VIROLOGY

2. RESEARCH SUMMARY • Design of Chimeric gene RNAi constructs for control of SPVD • Optimization of a transient system for Characterization of gene constructs • Characterization of RNAi constructs in transiently and stable transformation • Sweetpotato Begomovirus distribution and variability

3. CIP -Sweetpotato Atalas

4. Factors affecting sweetpotato production Diseases Viruses Fungi Bacterial Deases Drought Low yielding cultivars Weevils Rats Larger mammals like cattle Pests

5. Sweet Potato Virus Disease • Sweet potato virus disease is caused by synergy between two or more viruses • SPVD= • ƳSPCSV +ƿSPFMV+αSPFMMV+ðSPCFV+βSPLCV+ɛCaLV +……. ƸXn • But more importantly, • Sweetpotato feathery mottle virus (SPFMV)-Potyvirus (Family: Potyviridae), • Sweetpotato chlorotic stunt virus ( SPCSV)-Crinivirus (Family: Closteroviridae)

6. 2000 1990 1933 1944 1928 Geminiviruses of sweetpotato could as well be important in future • Recombination and Pseudo recombination Re-emergence of epidemics Scale of damage Time Epidemics controlled • Cultural measures • Resistant cultivars Gemini virus epidemics- Cassava mosaic disease(CMD) in Uganda

7. We confirmed Begomovirus presence in Uganda and Tanzania • Like other viruses they seem to be concentrated around lake Victoria even though an extensive survey has not been done • The 5Ugandan isolates formed separate cluster within the SPLCV-US group and show high variability

8. SPVD Transimission Aphids White flies

9. Sweetpotato virus disease • SPFMV occurs wherever sweetpotato is grown and has been in East Africa for close to a century. • On its own SPFMV cause mild or no symptoms depending on the cultivar • Sweetpotato feathery mottle virus (SPFMV) • Potyvirus, positive sense RNA – translated as a polyprotein

10. Sweet potato virus disease • SPCSV- positive sense single stranded RNA virus • Has bipartite genome ( two separate single strands). Has two strains-the WA and EA . Still the EA-strain is the most severe and has only been confirmed in East Africa . In Uganda 2 serotypes do exist Mukasa., 2004

11. !Plant defenceagainist diseases is a game plant defence Last line of plant defense Pathogen striker RNAase3 of SPCSV is bigger player in this kind of game

12. Strategies to Control SPVD • SPVD remains a problem and there is not true resistance so far • Very difficult to breed for SPVD resistance conventionally because: • Resistance is controlled by recessive genes • Sweetpotato is hexapoloid (6X=90) Strategies have therefore to target both viruses & more importantly the suppressor of silencing(RNase3)

13. Objectives and strategy • The objective is to boost the sweetpotato defence so that it can win the battle by timely increasing the production of siRNA and countering silencing supressor RNase3 • Constructs designed as hairpins- dsRNA • Two regions of the viral genomes used in constructs design: Highly targeted(RNAse3+Nia-Nib) and low (3’UTR) Black bars indicate regions used to make constructs, red bars indicate regions used by Dr. Jan Kreuze

14. Making the constructs RB LB Individual virus fragments were obtained by RT-PCR and fused by regular PCR using a proof reading enzyme