Silencing in Genetically Engineered Prunus domestica Provides Durable and Safe Resistance to Plum pox virus (Sharka Disease)

Originally identified in Bulgaria in 1915, Plum pox virus (PPV) is the most damaging virus of stone fruit trees, including apricot, plum, peach and cherry. PPV steadily spread throughout Europe over the years since its discovery and at the turn of the century (1999-2000) it reached North America (USA and Canada). While many strategies to control the spread of PPV have been undertaken over the decades and many studies have contributed to the characterization of the virus isolates there has been relatively little progress in the development of resistant varieties. With the paucity of natural resistance, transgenic technology, based on the engineering of the virus capsid gene, was investigated as a useful source of resistance. This work identified the C5 plum clone as highly resistant to PPV infection. These findings were supported by detailed molecular studies indicating that post-transcriptional gene silencing (PTGS) is the resistance mechanism with resistance being mediated through the production of small interfering RNA (siRNA). The durability of PPV resistance in C5 (named ‘HoneySweet’) is reflected through more than 10 years of field tests. In total, over 15 years of research with ‘HoneySweet’ have demonstrated that this clone and the resistance mechanism that it represents is: i) an important tool to demonstrate the successful deployment of biotechnology against a quarantine pest; ii) a safe product of biotechnology; and iii) a useful strategy for avoiding the use of pesticides to control natural aphid vectors of PPV. The deregulation of ‘HoneySweet’ in the USA by USDA/APHIS (Federal Register Doc. E7-13649, July 12 2007) and clearance by the U.S. Food and Drug Administration (FDA) corroborate these findings. INTRODUCTION Plum pox potyvirus (PPV) causes severe disease impacts in Prunus (Cambra et al., 2006). Presently, fruit tree growers do not have reliable control measures. Studies of the virus including strain variability, and new serological tools and updated molecular techniques (Real-time, QPCR) (Schneider et al., 2004) have been important in terms of our knowledge of this disease, yet they have had little effect on the spread of the disease throughout Europe and into areas such as North and South America, Asia, and North Africa (EPPO/OEPP Bulletin, 2006). There is neither cure nor treatment against PPV and few Prunus cultivars have been characterized as highly resistant. While the resistant clones that have been identified are being incorporated in breeding programs (Hartmann and Neumüller, 2006) these are long-term efforts and do not solve the present problems associated with PPV spread. This suggests the utility of biotechnology as an alternative or complementary breeding approach to achieving high level resistance to PPV in Prunus. This approach has been well-demonstrated in solving the papaya ringspot virus problem in Hawaii (Gonsalves, 2006). Scorza et al. (1994) engineered the PPV CP gene via A. tumefaciens into plum (P. domestica). They discovered that resistance was the result of post-transcriptional gene silencing (PTGS) (Scorza et al., 2001; Hily et al., 2004) mediated by siRNA produced by 397 Proc. 1 st IS on Biotechnol. of Fruit Species Eds.: M.-V. Hanke et al. Acta Hort. 839, ISHS 2009 a hairpin arrangement of the inserted CP transgene (Hily et al., 2005; Kundu et al., 2008). The resistance of clone C5, later named ‘HoneySweet’ (Scorza et al., 2007) was shown to be of a high level and durable for over 10 years in field tests in Europe (Ravelonandro et al., 2002; Malinowski et al., 2006; Polak et al., 2008; Zagrai et al., 2008). Risk assessment studies of ‘HoneySweet’ and other PPV-CP transgenic lines, showed that virus and viral transgene interactions are not a source of new emerging viruses (Capote and Cambra, 2005; Capote et al., 2008; Ravelonandro, 2006; Fuchs et al., 2007) and that the transgenic trees do not affect the population structures of aphid vectors (Capote et al., 2008). This report reviews how ‘HoneySweet’ plum transformed with the PPV CP transgene is durably resistant to PPV infection. Recognized as the key-factor of PPV resistance, we show how silencing is not only detected in vegetative tissues but also in plum fruits. The last section of the report discusses the safe use of transgenic fruit-trees indicating that such trees can be considered as an important strategy to control quarantine pests such as PPV. MATERIALS AND METHODS Plant Material and Virus Inoculation Transgenic plum clones C-2, C-3, C-4, C-5 and C-6 containing the PPV CP gene (Scorza et al., 1994) were vegetatively propagated onto rootstocks. Virus resistance experiments were initially conducted in greenhouses. Following four artificially-induced dormancy cycles, resistant plums were identified (Ravelonandro et al., 1997). To validate these observations, the same clones were transferred to the field along with relevant nontransgenic controls. Tests were conducted in different European countries including sites in continental (Poland, Romania, Czech Republic) and Mediterranean (Spain) areas (Ravelonandro et al., 2002; Malinowski et al., 2006; Capote et al., 2008; Zagrai et al., 2008). The rationale was to challenge plants in different ecological conditions and with native PPV strains. These factors have been indicated as important parameters for assessing PPV resistance (Kegler et al., 1998). These plums were then assayed either by graftor aphid-inoculation (Ravelonandro et al., 2002; Malinowski et al., 2006; Capote et al., 2008; Zagrai et al., 2008). Biosafety Studies When transgenic plants expressing viral genes began to be extensively developed, potential biological risks were proposed based on the use of CP genes (Hull, 1990). These potential risks were heteroencapsidation (Lecoq et al., 1993; Fuchs and Gonsalves, 2007) and recombination (Aaziz and Tepfer, 1999). ‘HoneySweet’ plum transgene silencing was shown to eliminate the production of CP and therefore the potential for heteroencapsidation in vegetative tissues (Hily et al., 2005). Silencing in fruit tissue was not reported and remained as an area of investigation along with the potential for recombination (in fruit and vegetative tissues) which could result from transgene mRNA production alone. RESULTS AND DISCUSSION siRNA-Induced PTGS Confers the High Level and Durable Nature of Resistance in ‘HoneySweet’ Plum Some of the transgenic clones developed by Scorza et al. (1994) expressed CP (C-2,-3 and -4) and two clones, C-5 (‘HoneySweet’) and C-6 do not. Initial greenhouse results revealed the highly resistant behaviour of clone C-5 (Ravelonandro et al., 1997). It was also observed that plum clones expressing the virus CP gene were susceptible. Scorza et al. (2001) expanded the analysis of the molecular mechanisms involved and found that the resistance to PPV infection was associated with PTGS, a natural regulation system that was initially reported with the silencing of the chalcone synthase gene in petunia