Papaya Ringspot Disease Kate Stokes and Andrew Tortora Cause вЂў Papaya ringspot virus-type P вЂў Occurs as 2 strains вЂ“ Type P infects both papaya and cucurbits such as squash, pumpkin, cucumber, and watermelon вЂ“ Type W infects watermelon only General Characteristics вЂў Member of potyvirus family вЂў Long, flexous rod-shaped particles about 800-900 nm in length вЂў Non-enveloped filamentous virions вЂў Single-stranded linear RNA genome 12 kb total, encapsulated by a coat protein вЂў 5вЂ™ terminus of RNA has VPg Symptoms вЂў Yellowing and vein-clearing on young leaves вЂў Yellow mottling of the leaves, severe blistering and leaf distortion вЂў Dark-green streaks and rings in the leafstalks and stems вЂў Concentric rings and spots or C-shaped markings, a darker green than the backgroundgreen fruit color вЂў Can affect vigor of fruit and trees and fruit quality Distribution вЂў Occurs in nearly every region where papaya is grown except for Africa, including Hawaii, Taiwan, Brazil, Thailand, the Caribbean islands and the Philippines. вЂў Particularly severe in Thailand, Taiwan, the Philippines, and the southern region China Spread вЂў Carried from plant to plant by aphids during feeding probes вЂў Not spread by other insects and does not survive in soil or dead plant material вЂў Also spread by movement of infected papaya plants and cucurbit seedlings вЂў Not usually seed-transmitted but there is one case from the Philippines History вЂў Brazil вЂ“ 1969 PRSV-p found in two main growing regions, Sao Paulo and Rio de Janeiro вЂ“ By 1984 73% of the industry had moved to remote regions to evade the virus History вЂў Hawaii вЂ“ Severely affected the papaya industry in the 1950s. Subsequently, the papaya industry was relocated to Puna district. вЂ“ In May 1992, PRSV was discovered in Puna, the area where 95% of HawaiiвЂ™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. Control вЂў вЂў вЂў вЂў Quarantine measures Restricted distribution Removal of infected plants (rouging) Taiwan вЂ“ Used protective netting against aphid vectors because the island was too small to effectively isolate plantings by moving вЂў Tolerant or resistant cultivars Cross Protection вЂў Phenomenon in which plants systematically infected with mild strain of the virus are protected against infection by a more virulent related strain вЂў Used to control вЂ“ Citrus tristeza virus вЂ“ Tobacco mosaic virus вЂ“ Zucchini yellow mosaic virus вЂў Early attempts failed, but 2 mild strains were eventually isolated after a virulent strain PRSV HA was treated with nitrous acid вЂ“ PRSV HA 5-1 вЂ“ PRSV HA 6-1 Cross Protection вЂў Results вЂ“ Delay in severe effects of the virus вЂ“ No complete protection from the virus вЂў Not Widely Accepted вЂ“ Adverse effects of mild strain вЂ“ Requires extra cultural management вЂ“ Reluctance of farmers to infect their trees Pathogen Derived Resistance вЂў Concept conceived in 1980s вЂў First demonstrated by BeachyвЂ™s group in transgenic tobacco resistant to TMV Coat Protein Mediated Protection (CPMP) вЂў Inhibition of disassembly in initially infected cells вЂ“ Interferes with release of encapsidated RNA вЂ“ Re-encapsidation вЂ“ Overcome by inoculation with naked (+) RNA вЂў Coat protein may play a role in replication and expression вЂ“ Requires high level of expression вЂ“ Interacts with infection cycle вЂў Interference with spread of virus from cell to cell вЂ“ Blocks movement through vascular tissue вЂў Examples вЂ“ TMV and Alfalfa mosaic virus RNA Mediated Resistance вЂў Post transcriptional gene silencing вЂ“ Homology dependant вЂ“ Base pairing between the sense RNA transcript of the transgene and the negative strand of the viral RNA вЂ“ Antisense RNA produced from the transgene could pair with the viral RNA transcript. вЂў Duplex RNA is target for degradation вЂў Base pairing could inhibit translation вЂў Examples вЂ“ PRSV, PVX and PVY, and all potyviruses Development of Transgenic Papaya вЂў Hawaiian papaya industry was in trouble вЂў 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 вЂў Target was the coat protein gene of PRSV HA 5-1 вЂ“ 97.7% identity to PRSVw from Florida вЂ“ Gene was engineered a chimeric protein вЂ“ 17 amino acids of CMV at the N-terminus Transformation of Embryogenic Tissue вЂў Used gene gun newly invented by John Sanford вЂў Tissue bombarded with tungsten particles coated with the engineered DNA Resistant Lines вЂў Inoculation tests conducted with transgenic plants and PRSV HA вЂў Line 55-1 showed resistance in greenhouse вЂ“ female rather than hermaphrodite so progeny could not be obtained вЂ“ Crosses with non-transgenic papaya fallowed by screening provided R1 plants resistant to PRSV Results вЂў Experiments showed вЂ“ Resistance of transgenic papaya to PRSV was not correlated protein expression вЂ“ R1 plants were highly resistant to Hawaiian strains of PRSV вЂ“ Line 55-1 had variable levels of resistance to non- Hawaiian strains вЂ“ Resistance due to RNA mediated mechanism rather than by coat protein Field Trials вЂў 1991 APHIS issued a permit for field trials of the new transgenic plants вЂў First trails designed to asses resistance to mechanical and aphid inoculations of PRSV вЂў Large Scale Tests вЂ“ Pros вЂў Industry desperate вЂў Line 55-1 performed well in previous trials вЂў Could be done at sufficiently isolated site вЂ“ Cons вЂў Spread of pollen to commercial plants вЂў Pilferage вЂў Resistant plants may become weeds Trial Results вЂў 50% of the non-transgenic control plants infected within four months вЂў 100% within seven months вЂў Improved performance of transgenic plants вЂў Assessments of taste, production, color, size, and packing and shipping qualities were positive for the new plants Deregulation вЂў APHIS вЂ“ Heteroencapsidation вЂ“ might lead to non-vectored virus to become vector transmissible if co-infection вЂ“ Recombination вЂ“ might lead to novel viruses вЂў EPA вЂ“ Transgenic proteins considered pesticides вЂў FDA вЂ“ Nutrition and vitamin content вЂ“ Presence of GUS and вЂ“ Presence of benzyl thiocynnate genes References вЂў Beachy, R. N., Loesch-Fries, S., Tumer, N. E., 1990. Coat Protein- Mediated Resistance Against Virus Infection. Annu. Review Phytopathol. 28:451-74 вЂў 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 23312335, March 1991 вЂў Gonsalves, D., 1998. Control of Papay Ringspot Virus in Papaya: A Case Study. Annu. Review Phytopathol. 36:415-37 вЂў 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 вЂў http://www.cimmvt.org/english/wpp/gen res/ringSpot.htm вЂў http://www.aspnet.org/online/feature/ringspot/ вЂў http://dpi.qld.gov.au/horticulture/5333.html вЂў http://vir.sgmjournals.org/cgi/content/full/ 82/11/2827 Questions?