Potato virus Y (PVY) - Potyvirus

1. Ensayos de campo, estudios bioquímicos y moleculares en plantas transgénicas de papa de la variedad Spunta, con el fin de liberar al mercado un evento transgénico resistente al virus PVY

2. Potato virus Y (PVY) - Potyvirus • Es uno de los virus más importantes y de mayor prevalencia que afectan al cultivo de la papa. Se han descrito razas de PVY que pueden causar necrosis en hojas y en tubérculos • Es transmitido por áfidos (Myzus persicae) de manera no persistente y también puede transmitirse mecánicamente a través de la maquinaria, las herramientas, etc. S. tuberosum cv. Spunta: > 60%

3. Strategies to develop virus resistance in plants R gene-mediated disease resistance Pathogen-derived resistance in transgenic plants Post transcriptional gene silencing (PTGS)

4. RB pNOS nptII tNOS p35S CP PVY tNOS LB Genetics constructions and plant transformation 400

5. 260 lines 155 lines 105 lines Screening for PVY resistance (greenhouse, mechanical infection)

6. Screening for PVY: resistance field trials

7. Screening for PVY: resistance field trials Summary of the field trial screening procedure used to identify PVY-resistant potato lines during 1998-2001

8. Screening for PVY resistance field trials: SY230, SY233 SY230 and SY233 were chosen for further characterization and possible commercial release

9. Selection of kanamycin resistant lines 400 events Micropropagation and greenhouse growth 140 lines discarded due to poor growth Mechanical infection with PVY in greenhouse 155 lines discarded due to infection Field trial testing for natural PVY infection 83 lines discarded due to virus infection Field trial testing of selected lines Selection of SY230 and SY233 for further characterization Screening for PVY resistant potato plants

10. EcoRV EcoRV EcoRV XbaI RB pNOS nptII tNOS p35S CP PVY tNOS LB Southern blot analysis: Transgene copy number Probe nptII Probe CP PVY Xba I Eco RV Xba I Eco RV SP 230 233 SP 230 233 SP 230 233 SP 230 233 DNA size Marker (bp) 9 000 6000 5000 4000 3000 2000 50 16 1000 850

11. Plants infected Leaves Stem Root A with PVY Wt 230 233 Wt 230 233 Wt 230 233 MW kb 9,5 7,5 4,4 2,4 1,4 0,2 B C D Wt 230 233 YL OL S YL OL S YL OL S MW kb 9,5 7,5 4,4 2,4 1,4 0,2 Transcripts analysis CP-PVY npt-II

12. A PVY / Spunta - 1 - 1 - 1 - 1 - 1 - 1 SP 230 233 5000 M 2500 1000 100 50 10 W kDA 64 49 37 26 19 15 Leaves Tubers 230 233 SP 230 233 SP Mw B 37 NptII 26 Protein levels in lines SY230 and SY233 CP-PVY npt-II

13. Comparative Field Trials

14. Comparative Field Trials

15. Comparative yield studies

16. Tuber characteristics and biochemical analysis

17. Tuber biochemical analysis, cont

18. Conclusions Spunta was transformed with a transgene containing the Potato virus Y (PVY) coat protein Screening for PVY resistance under greenhouse and field conditions allowed identification of two genetically stable PVY-resistant lines: SY230 and SY233 Southern blot analysis showed that SY230 and SY233 are independent transformation events exhibiting multiple transgene insertion Northern blot assays showed low levels of CPPVY transcripts in leaves, stem and root tissues. NptII transcripts was clearly detected Western blot assays failed to detect transgenic CPPVY in SY230 or SY233 leaf extracts. NptII protein was detected in leaves and tubers Biochemical and physical characteristics of SY230 and SY233 tubers are comparable to those of non-transformed Spunta

19. Silvia Cabral María Eugenia Segretin Alejandro Mentaberry Fernando Bravo Almonacid INGEBI-CONICET Valeria Rudoy Bjorn Welin Maximiliano Gomez Fabiana Stolowicz Mariana López Germán Serino Cristina Dos Santos Marcelo Criscuolo BioSidus-TECNOPLANT María Cecilia Bedogni Marcelo Huarte INTA-Balcarce