Papaya ringspot virus
Papaya ringspot virus (PRSV) is a type of potyvirus that infects papaya and a few closely related species. Potyviruses are the most economically damaging plant viruses. Aphids spread PRSV by feeding off infected and then uninfected plants. The virus is not passed on by the fruit - the offspring of an infected plant are usually virus free until they are inoculated with the virus by aphids carrying PRSV. There are many variants of PRSV that are closely related but have differences in genetic sequence and virulence.
Plants infected by PRSV develop a range of symptoms including ringed spots on the fruit, for which the virus is named. Other symptoms in include mosaic coloration and chlorosis of the leaves, streaks on petioles and the upper part of the trunk, and shape distortion of young leaves. The plants grow more slowly and become stunted. Plant infected withing a few months of planting usually do not produce mature fruit, but those infected later can produce a smaller yield of lower quality papaya.
PRSV has a history of following the papaya industry and causing serious damage to papaya crops in terms of yield, quality, and economic viability.
In the 1960's the papaya intdustry was focused primarily in the Sao Paulo and Rio de Janeiro regions in the southern and more populous part of the country. In 1969 PRSV was detected in the region and quickly spread throughout the area. As the virus spread the industry slowly relocated to more remote areas in the north including Espiritu Snato and Bahia. This allowed for a temporary escape from the virus, but the increased distance from markets increased transportation costs and reduced market fruit quality.
PRSV was discovered in Taiwan in 1974 and spread throughout the island in a few years. Because the entire island was infected, farmers were forced to adapt growing conditions to allow production in spite of the virus. The papaya tree matures quickly and can produce mature fruit less than a year after planting. Plants continue to produce fruit after this time but are often cut down after several years when harvesting of fruit becomes more difficult due to the height of the trees. The spread of the virus in Taiwan caused many farmers to adopt a yearly cycle for papaya and removed infected plants before the next generation was started. The young plants were protected from aphids (and therefore PRSV) using nets for a short period after planting. As the plants matured the nets were removed to allow the plants to receive more sunlight and improve the quality of the fruit. This strategy allowed for papaya production to continue, but hurt the yields and quality of the fruit produced.
The papaya industry began in Hawaii on the island of Oahu in the 1940's. In 1945 the virus was discovered on the island and was having a major impact on the industry in the 1950's. At this time, the largely relocated to the Puna region of the island of Hawaii. The Puna region remained free of PRSV for more than thirty years, with a slight outbreak in 1965 contained by aggressive culling of trees. Barren regions of old lava flows around the area may have acted as ecological barriers to the virus. In 1992 PRSV began to invade the papaya orchards in Puna. Many trees were cut down in an attempt to contain the virus but these attempts were unsuccessful and the virus spread throughout the area within several years. Plans were developed to cull all papaya and curcurbit trees (a wild species related to papaya) from the region in an attempt to eliminate the virus and to simulataneously attempt to relocate the industry to a different region of Hawaii.
A major method of controlling PRSV was simply for papaya growers to move to areas that were PRSV free, as was the case with farmers in Brazil and Hawaii. This is inheritantly a temporary solution and requires constant vigilance to prevent people from bringing in contaminated material and to quickly contain any outbreaks that do occur. Areas that are PRSV free are not neccessary ideal in growing conditions or located near markets, which can drive up the cost of production and reduce fruit quality.
One year cycle and nets on young plants
Though typically grown for several years, papayas can produce mature fruit less than one year. It is only when trees reach a height that makes harvesting the fruit difficult that they are chopped down. One method of controlling PRSV is to grow papaya on a one year cycle and eliminate all the infected trees before planting new ones. The young trees, initially free of the virus, are protect from aphids carrying PRSV by nets. As the trees mature the nets must be removed to allow adequate sunlight for growth. At this point the trees become infected but they are still able to set fruit, although a lower quantity and quality of fruit results. This method was adopted in Taiwan, where relocating papaya farms was not an option.
Traditional breeding approaches were attempted to introduce PRSV resistance into commercial varieties of papaya. No suitable resistant papaya varieties were available despite examining many papaya varieties. Carica, a wild relative of papaya, was found to have resistance against PRSV and resistant hybrids between the two species were created. However, these hybrids were all sterile and so it was not possible to use this method to introduce PRSV resistance into commercial varieties of papaya.
In plant species, infection by a mild virus can sometimes lead to resistance against a similar but more virulent virus. This principle is called cross protection. The immune systems of plants is very different from that of mammals - plants lack antibodies which can result in a similar effect in mammals. The exact mechanism by which cross protection works is unclear, but it may involve RNA silencing. To use cross protection to protect papaya from PRSV, a mild form of the virus was isolated and used to inoculate young trees in areas with high risk for PRSV. Infection of plants with the more virulent PRSV was reduced, but the mild strain did cause symptoms in the plants and the cross protection was primarily effective against only the most closely related viruses. The mild virus worked well in protecting trees in Hawaii from the Hawaii strain (from which the mild form was produced) but was less effective against the strain from Taiwan. Cross protection helped but was not an ideal solution.
Genetically engineered PRSV resistance
Due to fears that PRSV would soon invade the Puna region of Hawaii and severely impact the papaya industry there, researchers from the University of Hawaii and Cornell set out to create a PRSV resistant variety of papaya. Traditional breeding approaches seemed unlikely to work, so a genetic engineering approach was employed.
In the mid 1980's it was found that viral protein coat genes could confer resistance to transgenic plants against specific viruses. This was shown to be the case for several viruses in several species of plants. Researchers attempted to use this "Parasite Derived Resistance" as a mechanism for PRSV resistance in papaya.
A genetic construct was created that contained a GUS reporter gene, a viral coat protein gene from a mild version of PRSV, and a nptII antibiotic resistance gene. This genetic construct was used to transform papaya cells using tungsten particle bombardment. The antibiotic resistance gene and the GUS reporter gene aided the identification of transformed cells. Several transgenetic plants were grown out of the transformed cells and tested for PRSV resistance. One line was identified with complete resistance and several lines with partial resistance. Resistance was found not to correlate with the amount of viral coat protein produced. The exact mechanism of resistance is not precisely known, but is now believed to be through RNA silencing of the viral genes. The original papaya variety that was used was the Sunset variety. The line homoqygous for the resistance gene was named SunUp. A heterzygous line produced by a cross between SunUp and the primary variety grown in Puna, the Kapoho, is called the UH Rainbow variety.
Bot the SunUp and the UH Rainbow variety of papaya have strong resistance to the Hawaiian variety of PRSV. However, these varieties have only partial resistance to other strains of PRSV from other regions of the world. Both of these varieties have been found to have satisfactory fruit characteristics and are suitable to commercial production.
Before transgenic papaya could be commercialized it needed to be deregulated. The researchers involved in creating the strains helped to address the safety concerns and file the applications needed to get the transgenic papaya approved. Several concerns were evaluated including: whether the viral coat protein in plants could lead to the emergence of novel viruses, the presence of the GUS and nptII genes, the vitamin concentration in the transgenic papaya, the concentration of benzyl isothiocyanate in the transgenic papaya, the escape of the genes into related species, and the presence of the viral coat protein in the transgenic papaya.
In evaluating these concerns, the relevant agencies considered the risks of using transgenic papaya as well as the benefits. It was found that the level of viral coat protein was lower in the trangenic papaya that that in papaya infected with PRSV, the fruit of which had been regularly consumed for many years without known side effects. Similarly, because of widespread PRSV already in plants it was deemed that the protein coat gene in papaya did not pose a significant risk in this regard. The vitamin and benzyl isothiocyanate levels were similar between the transgenic and nontransgenic papaya. The presence of the GUS and nptII genes, while not necessary for resistance, were not considered to be harmful for humans or the environment. The concern that the resistance gene could spread to other species or make the papaya into a weedy species was unlikely because athe papaya is not able to produce viable offspring with other species found in Hawaii and papaya itself is not a weedy species even in areas where no PRSV is present. Overall, the transgenic papaya was approved by APHIS and the FDA for use in Hawaii and for human consumption in 1996 and 1997 respectively. The appearance of PRSV in the Puna region in 1992 and successful field trials in the following years likely helped to accelerate the approval process.
To commercialize transgenic papaya, it was necessary to obtain proper licensing from individuals and entities that held patents on the work used to create the PRSV resistant papaya. This effort was spearhead by the Papaya Adminstrative Collective, a group composed primarily of Hawaiian papaya growers. Licensing was succesfully obtained for the commercial use of the transgenic papaya in the state of Hawaii. PRSV resistant papaya became the second commercially used virus resistant transgenic plant in 1997, and remains in use today.
Efforts are underway to create strains of papaya that are resistant to other strains of PRSV so that transgenic papaya can be used in other regions in the world.
Gonsalves, D. (1998). Control of papaya ringspot virus in papaya: A case study. Annual Review of Phytopathology, 36, 415-437.
Gonsalves, D. (2002). Coat protein transgenic papaya: "acquired" immunity for controlling papaya ringspot virus. Current Topics in Microbiology and Immunology, 266, 73-83.
Gonsalves, D. (2006). Transgenic papaya: Development, release, impact and challenges. Advances in Virus Research, 67, 317-354.
Ling, K., Namba, S., Gonsalves, C., Slightom, J. L., & Gonsalves, D. (1991). Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene. Bio/technology (Nature Publishing Company), 9(8), 752-758.
Sakuanrungsirikul, S., Sarindu, N., Prasartsee, V., Chaikiatiyos, S., Siriyan, R., Sriwatanakul, M., et al. (2005). Update on the development of virus-resistant papaya: Virus-resistant transgenic papaya for people in rural communities of thailand. Food and Nutrition Bulletin, 26(4), 422-426.
Tripathi, S., Suzuki, J., & Gonsalves, D. (2007). Development of genetically engineered resistant papaya for papaya ringspot virus in a timely manner: A comprehensive and successful approach. Methods in Molecular Biology (Clifton, N.J.), 354, 197-240.
You, B. J., Chiang, C. H., Chen, L. F., Su, W. C., & Yeh, S. D. (2005). Engineered mild strains of papaya ringspot virus for broader cross protection in cucurbits. Phytopathology, 95(5), 533-540.
Fitch, M., Manshardt, R., Gonsalves, D., Slightom, J., Sanford, J. (1992). Virus resistant papaya plants derived from tissues bombarded with coat protein gene of papaya ringspot virus. Biotechnology, 10, 1466-1472.