2. Cause Papaya ringspot virus-type P Occurs as 2 strains –Type P infects both papaya and cucurbits such as squash, pumpkin, cucumber, and watermelon –Type W infects watermelon only

3. General Characteristics Member of potyvirus family Long, flexous rod-shaped particles about 800-900 nm in length Non-enveloped filamentous virions Single-stranded linear RNA genome 12 kb total, encapsulated by a coat protein 5’ terminus of RNA has VPg

4. Yellowing and vein-clearing on young leaves Yellow mottling of the leaves, severe blistering and leaf distortion Dark-green streaks and rings in the leafstalks and stems Concentric rings and spots or C-shaped markings, a darker green than the background- green fruit color Can affect vigor of fruit and trees and fruit quality

5. Occurs in nearly every region where papaya is grown except for Africa, including Hawaii, Taiwan, Brazil, Thailand, the Caribbean islands and the Philippines. Particularly severe in Thailand, Taiwan, the Philippines, and the southern region China Distribution

6. Spread Carried from plant to plant by aphids during feeding probes Not spread by other insects and does not survive in soil or dead plant material Also spread by movement of infected papaya plants and cucurbit seedlings Not usually seed-transmitted but there is one case from the Philippines

7. History Brazil –1969 PRSV-p found in two main growing regions, Sao Paulo and Rio de Janeiro –By 1984 73% of the industry had moved to remote regions to evade the virus

8. History HawaiiHawaii –Severely affected the papaya industry in the 1950s. Subsequently, the papaya industry was relocated to Puna district. –In May 1992, PRSV was discovered in Puna, the area where 95% of Hawaii’s papaya was being grown, and was widespread throughout the growing region by 1995. Production steadily dropped from 53 million pounds in 1992 to 26 million pounds in 1998.

9. Control Quarantine measures Restricted distribution Removal of infected plants (rouging) Taiwan –Used protective netting against aphid vectors because the island was too small to effectively isolate plantings by moving Tolerant or resistant cultivars

10. Cross Protection Phenomenon in which plants systematically infected with mild strain of the virus are protected against infection by a more virulent related strain Used to control –Citrus tristeza virus –Tobacco mosaic virus –Zucchini yellow mosaic virus Early attempts failed, but 2 mild strains were eventually isolated after a virulent strain PRSV HA was treated with nitrous acid –PRSV HA 5-1 –PRSV HA 6-1

11. Cross Protection Results –Delay in severe effects of the virus –No complete protection from the virus Not Widely Accepted –Adverse effects of mild strain –Requires extra cultural management –Reluctance of farmers to infect their trees

12. Pathogen Derived Resistance Concept conceived in 1980s First demonstrated by Beachy’s group in transgenic tobacco resistant to TMV

13. Coat Protein Mediated Protection (CPMP) Inhibition of disassembly in initially infected cells –Interferes with release of encapsidated RNA –Re-encapsidation –Overcome by inoculation with naked (+) RNA Coat protein may play a role in replication and expression –Requires high level of expression –Interacts with infection cycle Interference with spread of virus from cell to cell –Blocks movement through vascular tissue Examples –TMV and Alfalfa mosaic virus

14. RNA Mediated Resistance Post transcriptional gene silencing –Homology dependant –Base pairing between the sense RNA transcript of the transgene and the negative strand of the viral RNA –Antisense RNA produced from the transgene could pair with the viral RNA transcript. Duplex RNA is target for degradation Base pairing could inhibit translation Examples –PRSV, PVX and PVY, and all potyviruses

15. Development of Transgenic Papaya Hawaiian papaya industry was in trouble Dennis Gonsalves at Cornell in collaboration with Upjohn, scientists at the University of Hawaii and John Sanford at Cornell try to develop transgenic papaya resistant to PRSV Target was the coat protein gene of PRSV HA 5-1 –97.7% identity to PRSVw from Florida –Gene was engineered a chimeric protein –17 amino acids of CMV at the N-terminus

16. Transformation of Embryogenic Tissue Used gene gun newly invented by John Sanford Tissue bombarded with tungsten particles coated with the engineered DNA

17. Resistant Lines Inoculation tests conducted with transgenic plants and PRSV HA Line 55-1 showed resistance in greenhouse –female rather than hermaphrodite so progeny could not be obtained –Crosses with non-transgenic papaya fallowed by screening provided R1 plants resistant to PRSV

18. Results Experiments showed –Resistance of transgenic papaya to PRSV was not correlated protein expression –R1 plants were highly resistant to Hawaiian strains of PRSV –Line 55-1 had variable levels of resistance to non- Hawaiian strains –Resistance due to RNA mediated mechanism rather than by coat protein

19. Field Trials 1991 APHIS issued a permit for field trials of the new transgenic plants First trails designed to asses resistance to mechanical and aphid inoculations of PRSV Large Scale Tests –Pros Industry desperate Line 55-1 performed well in previous trials Could be done at sufficiently isolated site –Cons Spread of pollen to commercial plants Pilferage Resistant plants may become weeds

20. Trial Results 50% of the non-transgenic control plants infected within four months 100% within seven months Improved performance of transgenic plants Assessments of taste, production, color, size, and packing and shipping qualities were positive for the new plants

21. Deregulation APHIS –Heteroencapsidation – might lead to non-vectored virus to become vector transmissible if co-infection –Recombination – might lead to novel viruses EPA –Transgenic proteins considered pesticides FDA –Nutrition and vitamin content –Presence of GUS and –Presence of benzyl thiocynnate genes

22. References Beachy, R. N., Loesch-Fries, S., Tumer, N. E., 1990. Coat Protein- Mediated Resistance Against Virus Infection. Annu. Review Phytopathol. 28:451-74 Tumer, N. E., Kaniewski, W., Haley, L., Gehrke, L., Lodge, J. K., Sanders, P., 1991. The second amino acid of alfalfa mosaic virus coat protein if critical for coat protein-mediated protection. Proc. Natl. Acad. Sci. USA. Vol. 88 pp 2331- 2335, March 1991 Gonsalves, D., 1998. Control of Papay Ringspot Virus in Papaya: A Case Study. Annu. Review Phytopathol. 36:415-37 2001. Chiang, C., Wang, J., Jan, F., Yeh, S., Gonsalves, D., Comparative reactions of recombinant papaya ringspot viruses with chimeric coat genes and wild type viruses on CP-transgenic papaya, Journal of general Virology. 82, 2827-2836 http://www.cimmvt.org/english/wpp/gen res/ringSpot.htm http://www.aspnet.org/online/feature/ringspot/ http://dpi.qld.gov.au/horticulture/5333.html http://vir.sgmjournals.org/cgi/content/full/ 82/11/2827

23. Questions?