CURRENT STATUS OF TRANSGENIC APPROACH FOR THE CONTROL OF RINGSPOT

Shyi-Dong Yeh Department of Plant Pathology National Chung-Hsing University Taichung 402, Taiwan ROC

ABSTRACT

Production of papaya has been limited in many areas of the world due to the disease caused by the (PRSV). The coat protein (CP) gene-mediated transgenic resistance has become the most effective method to prevent crops from this virus infection. In the late 1980s, the group of Gonsalves at Cornell University and Hawaii started a research project to develop transgenic papaya resistant to PRSV by biolistic transformation method. By May 1998, PRSV-CP gene transgenic papaya Rainbow and SunUp were deregulated by the U.S. Animal and Plant Health Inspection Service and Environmental Protection Agency, and granted the approval from the Food and Drug Administration (FDA) for commercial application. This is the first successful case of transgenic fruit tree commercialized in the world. Although the transgenic varieties are not resistant to most other PRSV strains from different geographic areas, the breakdown of the resistance in Hawaii has not been observed. Other than in Hawaii, a CP gene of a native Taiwan strain PRSV YK was used to transform Taiwan papaya cultivars by Agrobacterium-mediated transformation. The transgenic lines obtained showed various levels of resistance, ranging from delay of symptom development to complete immunity. However, during the four-year field test period, the PRSV CP-transgenic papaya lines were found susceptible to an unrelated Papaya leaf-distortion mosaic virus (PLDMV) when evaluated under greenhouse conditions. Therefore, in Taiwan PLDMV is considered as a serious threat to papaya production once PRSV CP-transgenic papaya has been widely applied for the control of PRSV. Through the posttranscriptional-gene-silencing mechanism, transgenic papaya plants carrying a chimeric transgene including parts of the CP genes of PRSV and PLDMV were developed to confer resistance against both PRSV and PLDMV under greenhouse conditions. These transgenic papaya plants with double resistance are considered having a great potential for the control of PRSV and PLDMV in Taiwan. During the field test period, a super strain PRSV-519 was found able to breakdown the resistance of PRSV YK-CP transgenic lines. The breakdown of the transgenic resistance was found controlled by a strong gene-silencing suppressor of the super strain in a transgene-homology independent manner. It is suggested that a chimeric construct targeting multiple viral genes including the gene determining viral virulence and gene silencing suppression, such as the HC-Pro gene of a potyvirus, may minimize the chance of emergence of a super virus in overcoming the transgenic resistance.

Keywords: Papaya, PRSV, transgenic resistance

PAPAYA AS AN IMPORTANT CASH CROP IN THE TROPICS AND SUBTROPICS Central America and brought to Caribbean countries and Southeast Asia during the Spanish exploration in the sixteenth century There are many common names for papaya (Storey 1996). It then spread rapidly to India, (Carica papaya L.), such as papaw or paw Africa, and today it is widely distributed paw (Australia), mamao (Brazil), tree melon throughout the tropical and subtropical areas (China), etc. The species is believed to be a of the world. native of southern Mexico and neighboring

1 A papaya plant has a single, erect and fruit is a good source of vitamin A and C treelike herbaceous stem, with a crown of large, (Manshardt 1992). Ripe fruits are largely used palmately and deeply lobed leaves. The main as fresh desert fruits, and green fruits are stem is cylindrical, hollow, with prominent leaf often used as salad, and pickled or cooked as scars and spongy-fibrous tissue. Leaves are vegetable. Papain, a proteolytic enzyme spirally arranged, with petioles extending present in the latex, collected mainly from horizontally up to 1 m long. Trees contain green fruits, has various usage in beverage, white latex in all parts. Flowers are male, food and pharmaceutical industries, e.g. chill- female, or hermaphrodite, found on separate proofing beer, tenderizing meat, and drug trees, and are borne in the axils of the leaves. preparations for digestive ailments (Chan and The modified cymose inflorescences structure Tang 1978). It is also used in bathing hides, allows the flowers to be easily pollinated by softening wool, and as soap for washing cloth. wind and insects. The type of flowers Papaya grows relatively easily and quickly produced may change on the same tree, from seeds. It can grow up to 10 or 12 feet depending on age and environmental factors in height. Fruits are ready to be harvested 9- such as drought and broad temperature 12 months after planting and a tree can fluctuations. Hermaphroditic trees consistently continue producing fruits for about 2-3 years produce male flowers, with few female flowers up to when plant height is too tall for efficient that produce fruits during warmer or cooler harvesting. Since plant sex can not be seasons, whereas, female trees are more stable distinguished before flowering, 3-5 seedlings and always produce pistillate flowers under are normally planted together and only the these conditions. most vigorous hermaphrodite ones during Papaya fruits are fleshy berry and flowering are selected and cultivated. In 2004, superficially resemble melons. Fruits from the FAO estimated that about 3.7 hundred female trees are spherical, whereas, those from thousand harvested hectares, and about 6.5 hermaphroditic trees are pyriform, oval, or million metric tons of fruits were harvested cylindrical with grooved surface. Since the (Table 1). Brazil, Mexico, Nigeria, India, and female fruits contain thinner flesh and more Indonesia yield more than 70 percent of the seeds in the central cavity, the hermaphrodite total world production. The extensive fruits are more demanded by consumers. The adaptation of this plant and wide acceptance

Table 1. World papaya production, 2004a

Hectares Metric Tons Country (x1,000) (x1,000)

Brazil 36.0 1600 Mexico 26.3 956 Nigeria 91.0 755 India 80.0 700 Indonesia 10.0 650 Ethiopia 11.0 230 Congo 12.5 211 Venezuela 11.0 170 Peru 13.0 170 China 6.2 165 Cuba 7.0 125 Thailand 10.5 125 Colombia 4.0 102 Philippines 6.6 79 Malaysia 6.5 65 Others 34.2 401 TOTAL 365.8 6504 a Data from Food and Agriculture Organization (FAO), Statistical Division, 2004 (http://faostat.fao.org/faostat/).

2 of the fruit offer considerable promise for (Martelli and Russo 1976) in the cytoplasm of papaya as a commercial crop for local and host cells. The former consists of a protein of export purposes. Like banana, pineapple, and 70 kDa (cylindrical inclusion protein CIP; Yeh mango, papaya is one of the most important and Gonsalves 1984) and the latter consists of cash crops in the tropics and subtropics. a protein 51 kDa (amorphous inclusion protein, However, the production of this economically AIP; de Mejia et al. 1985a & b). In papaya, important fruit crop is limited by the PRSV causes severe mosaic and distortion on destructive disease caused by the Papaya leaves, ringspots on fruits, and water-soaking ringspot virus (PRSV), and the fragile and oily streaks on upper stems and petioles. It perishable fruit traits unfavorable for large-scale stunts the plant and drastically reduces the exportation make papaya lag far behind banana size and the quality of the fruit. and pineapple in the world market. ABSENCE OF EFFECTIVE CONTROL WORLDWIDE THREAT BY PRSV INFECTION MEASURES

Production of papaya has been limited in many Although tolerant selections of papaya have areas of the world due to the disease caused been described (Cook and Zettler 1970; by PRSV (Purcifull et al. 1984). Papaya Conover 1976; Conover et al. 1986), resistance ringspot disease is the major obstacle to large- to PRSV does not exist in the species of C. scale commercial production of papaya (Yeh papaya, making the conventional breeding and Gonsalves 1984). PRSV was first reported approach difficult (Cook and Zettler 1970; in Hawaii in the 1940s (Jensen 1949a), and Wang et al. 1978). Tolerance to PRSV has became prevalent in Florida (Conover 1964), been found in some particular papaya lines and Caribbean countries (Adsuar 1946; Jensen introduced into commercial varieties, but their 1949b), South America (Herold and Weibel horticultural properties are still not commercially 1962), Africa (Lana 1980), India (Capoor and desirable (Mekako and Nakasone 1975; Conover Varma 1948; Singh 1969), the Far East (Wang and Litz 1978). Other control methods for PRSV et al. 1978), and Australia (Thomas and including agricultural practices such as Dodman 1993). To date, most of the major roughing, quarantine, intercropping with corn papaya plantation areas of the world suffer as a barrier crop, and protecting transplanted from devastation by this noxious virus. seedlings with plastic bags, provide only temporary or partial solutions to the problems CHARACTERISTICS OF PRSV (Wang et al. 1987; Yeh and Gonsalves 1994). PRSV HA 5-1, a cross-protecting mild PRSV is a member of the genus Potyvirus mutant strain of PRSV that was selected (Purcifull et al. 1984; Murphy et al. 1995), is following nitrous-acid treatment of a severe transmitted nonpersistently by , and is strain (HA) from Hawaii (Yeh and Gonsalves sap-transmissible in nature. PRSV genome 1984), was tested extensively in the field and contains a single-stranded positive sense RNA has been used commercially in Taiwan and of about 40 S (dela Rosa and Lastra 1983; Yeh Hawaii since 1985 in order to achieve a good and Gonsalves 1985). Strains of PRSV from economic return from papaya production (Wang Hawaii (Yeh et al. 1992) and Taiwan (Wang et al. 1987; Yeh et al. 1988; Yeh and Gonsalves and Yeh 1997) have been completely 1994). However, using the approach of sequenced, both containing 10,326 nucleotides deliberately infecting a crop with a mild virus in length. The viral RNA encodes a strain to prevent economic damage by more polypolyprotein that is proteolytically cleaved virulent strains has several drawbacks, to generate 8-9 final proteins including the coat including the requirement for a large-scale protein for encasidation of viral genome (Yeh inoculation program, the reduction in crop et al. 1992). The virus has a single type of yield, and losses of cross-protected plants due coat protein (CP) of 36 kDa (Purcifull and to superinfection by virulent strains (Stubbs Hiebert 1979; Gonsalves and Ishii 1980). It 1964; Gonsalves and Garnsey 1989; Yeh and induces cylindrical inclusion (CI) (Purcifull and Gonsalves 1994). Edwardson 1967) and amorphous inclusion (AI)

3 CONTROL OF PRSV BY 1999; Hamilton and Baulcombe 1999; Gonsalves TRANSGENIC APPROACH 2002). However, the resistance is affected by the sequence identity between the CP The concept of “pathogen-derived resistance” transgene and the CP coding region of the (Sanford and Johnston 1985) proposes that challenge virus (Tennant et al. 1994). For transforming plants with a pathogen’s gene example, Rainbow (a CP-hemizygous line would generate resistance to the infection of derived from SunUp crossed with non- the corresponding pathogen. By this concept, transgenic Kapoho) is susceptible to PRSV Powell-Abel et al. (1986) first demonstrated that isolates outside Hawaii, and SunUp (a CP- transgenic tobacco plants expressing the coat homozygous line of 55-1) is resistant to a protein (CP) gene of Tobacco mosaic virus wider range of isolates from Jamaica and Brazil, (TMV) confer resistance to TMV infection. but susceptible to isolates from Thailand and The CP gene-mediated transgenic resistance Taiwan (Gonsalves 1998; Tennant et al. 2001; has been proven effective for protecting Gonsalves 2002). This characteristic of tobacco, tomato, potato, and other crops from sequence homology-dependent resistance limits infection by many different (Beachy the application of CP-transgenic papaya for 1990; Lomonossoff 1995; Goldbach et al. 2003). controlling PRSV in other geographic regions Thus, the transgenic approach has become the other than Hawaii (Gonsalves 2002). most effective method to prevent crops from The field trial of the homozygous line virus infection. SunUp and hemizygous line Rainbow indicates In order to solve the problems caused by that both of them offer a good solution to the PRSV, in the late 1980s the group of PRSV problem in Hawaii (Ferreira et al. 2002). Gonsalves at Cornell University and Hawaii By May 1998, Rainbow and SunUp were started a research project to develop transgenic deregulated by the U.S. Animal and Plant papaya. Ling et al. (1991) first demonstrated Health Inspection Service and Environmental that the expression of the PRSV HA 5-1 CP Protection Agency, and granted the approval gene in tobacco affords a broad-spectrum from the Food and Drug Administration (FDA) protection against different . for commercial application (Gonsalves 2002). However, effective gene transfer systems This is the first successful case of transgenic require reliable and efficient procedures for fruit tree being commercialized in the world. plant regeneration from cells. Fitch and Manshardt (1990) reported that somatic TRANSGENIC PAPAYA GENERATED embryogenesis from immature zygotic embryos IN TAIWAN of papaya can be integrated into a useful gene transfer technology. By the same year, Fitch Other than Hawaii, a CP gene of a native et al. (1990) successfully incorporated the CP Taiwan strain PRSV YK was used to transform gene of HA 5-1 into papaya via microprojectile Taiwan papaya cultivars by Agrobacterium- bombardment and obtained plants resistant to mediated transformation (Cheng et al. 1996). infection by the severe Hawaii HA strain. The transgenic lines obtained showed various Among their transgenic papaya lines, line 55-1 levels of resistance, ranging from delay of was virtually immune to infection by HA. symptom development to complete immunity (Bau et al. 2003). Several lines highly resistant SUCCESSFUL APPLICATION OF TRANSGENIC to the homologous strain (PRSV YK) provide PAPAYA IN HAWAII wide-spectrum resistance to three different geographic strains from Hawaii, Thailand, and The plants of transgenic papaya line 55-1 are Mexico (Bau et al. 2003). During four repeats highly resistant to Hawaiian PRSV isolates of field trials from 1996 to 1999, the transgenic under greenhouse and field conditions (Fitch et papaya exhibited high degrees of protection al. 1992; Lius et al. 1997). The resistance is against PRSV in Taiwan (Bau et al. 2004). triggered by the posttranscriptional gene Unfortunately, 18 months after plantation in the silencing (PTGS) – an RNA-mediated specific fourth field trial, unexpected symptoms of degradation process of innate nature of plants severe distortion on fully expended leaves, against pathogens (Baulcombe 1996; Baulcombe stunning on apex, water-soaking on petioles

4 and stem, and yellow ringspot on fruit were were found (Tripathi et al. 2004). The noticed on PRSV CP-transgenic papaya plants. nucleotide identity between the transcript of The causal agent was distinguished from PRSV the CP transgene and PRSV 5-19 RNA is less by host reactions and serological properties divergent than those between the CP transgene (Bau 2000) and later identified as Papaya leaf- and other PRSV geographic strains that are not distortion mosaic virus (PLDMV), a potyvirus able to overcome the transgenic resistance which originated from Okinawa, Japan, in 1954 (Tripathi et al. 2004), indicating that the (Maoka et al. 1996). All of the PRSV CP- breakdown of the transgenic resistance is not transgenic papaya lines were susceptible to correlated to the sequence divergence between PLDMV infection when evaluated under the infecting virus and the transgene. In order greenhouse conditions. Therefore, in Taiwan, to analyze the role of the gene-silencing PLDMV is considered as a serious threat to suppressor HC-Pro of this super strain, the papaya production once PRSV CP-transgenic virus recombinant was constructed by replacing papaya is widely applied for the control of a HC-Pro region of PRSV YK with that of 5-19 PRSV. and the resistance against recombinant was evaluated on transgenic papaya. Results MULTIPLE AND DURABLE RESISTANCE showed that heterologous HC-Pro region of 5- AGAINST DIFFERENT VIRUSES 19 alone provides the ability to breakdown the transgenic resistance in a transgene sequence- In order to control two or more viruses, homology independent manner, even though transgenic plants with multiple resistances have the sequences of the transgene transcript been generated by combining the entire CP shares 100 percent identity with the genome of gene of more than one virus, with each gene the infecting virus (S. D. Yeh, unpublished driven by a promoter and a terminator (Fuchs results). The breakdown of the transgenic and Gonsalves 1995). Transgenic lines resistance by a strong gene-silencing expressing these chimeric CP constructs were suppressor of a super strain has strong resistant to the corresponding viruses and impacts on the application of transgenic crops protected from mixed infection such as for virus control. It is suggested that a Cucumber mosaic virus, Watermelon mosaic chimeric construct targeting multiple viral genes virus, and Zucchini yellow mosaic virus (Fuchs including the gene determining viral virulence and Gonsalves 1995; Tricoli et al. 1995; Fuchs and gene silencing suppression, such as the et al. 1998). Furthermore, the novel approach HC-Pro gene of a potyvirus, may minimize the proposed by Jan et al. (2000) described that chance of emergence of a super virus for transgenic plants with resistance to a potyvirus overcoming the transgenic resistance. and a tospovirus can be obtained through the PTGS mechanism by fusing a segment of TRANSGENIC PAPAYA GENERATED tospoviral N gene to a segment of potyviral IN OTHER GEOGRAPHIC AREAS CP gene. The same strategy was used to develop double resistance to both PRSV and Because of the apparent homology dependence PLDMV. An untranslatable chimeric construct of PRSV CP transgene-associated resistance, that contained the truncated PRSV CP and the utilization of a CP gene of a local PLDMV CP genes was then transferred to prevalent strain is a prerequisite to obtain papaya. Through the PTGS mechanism, effective PRSV resistance in transgenic papaya transgenic papaya plants carrying this chimeric lines for a particular geographic region, as long transgene indeed conferred resistance against as genetic variation among virus strains in that both PRSV and PLDMV under greenhouse region is not a limiting factor (Gonsalves 2002). conditions (S. D. Yeh, unpublished results). Using the CP genes of local PRSV isolates to These transgenic papaya plants with double transform local papaya cultivars have been resistance are considered to have a great successfully reported in different countries. potential for the control of PRSV and PLDMV Lines et al. (2002) used an untranslatable PRSV in Taiwan. CP coding region as a transgene to develop In a four-year field trial, a super PRSV two Australian transgenic papaya cultivars strain 5-19 infected transgenic papaya lines which showed immunity to the local PRSV

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