Transcript Papaya Ringspot Virus

Papaya Ringspot
Disease
Kate Stokes and Andrew
Tortora
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
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
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 backgroundgreen fruit color
• Can affect vigor of fruit and trees and fruit
quality
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
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
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
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.
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
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
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
Pathogen Derived
Resistance
• Concept conceived in 1980s
• First demonstrated by Beachy’s group in
transgenic tobacco resistant to TMV
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
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
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
Transformation of
Embryogenic Tissue
• Used gene gun newly invented by John
Sanford
• Tissue bombarded with
tungsten particles coated
with the engineered
DNA
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
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
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
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
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
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 23312335, 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
Questions?