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1. Papaya Ringspot Disease Kate Stokes and Andrew Tortora

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. Symptoms • 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. Distribution • 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

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 • Hawaii • 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?