Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing Guo-qing Song 1,2, *, Kenneth C. Sink 1,2 , Aaron E. Walworth 1,2 , Meridith A. Cook 3,† , Richard F. Allison 3 and Gregory A. Lang 2 1 Department of Horticulture, Plant Biotechnology Resource and Outreach Center, Michigan State University, East Lansing, MI, USA 2 Department of Horticulture, Michigan State University, East Lansing, MI, USA 3 Department of Plant Biology, Michigan State University, East Lansing, MI, USA Received 28 October 2012; revised 18 January 2013; accepted 29 January 2013. *Correspondence (Tel: 517 355 5191 x 1384; fax: 517 432 8853; email: songg@msu.edu) † Current address: Cargill Specialty Seeds & Oils, Fort Collins, CO 80525, USA. Keywords: genetic engineering, interfering RNAS (RNAi), Prunus avium L., Prunus cerasus L., transformation, transgenic plant, woody plant breeding. Summary Prunus necrotic ringspot virus (PNRSV) is a major pollen-disseminated ilarvirus that adversely affects many Prunus species. In this study, an RNA interference (RNAi) vector pART27–PNRSV containing an inverted repeat (IR) region of PNRSV was transformed into two hybrid (triploid) cherry rootstocks, ‘Gisela 6’ (GI 148-1) and ‘Gisela 7’(GI 148-8)’, which are tolerant and sensitive, respectively, to PNRSV infection. One year after inoculation with PNRSV plus Prune Dwarf Virus, nontransgenic ‘Gisela 6’ exhibited no symptoms but a significant PNRSV titre, while the transgenic ‘Gisela 6’ had no symptoms and minimal PNRSV titre. The nontransgenic ‘Gisela 7’ trees died, while the transgenic ‘Gisela 7’ trees survived. These results demonstrate the RNAi strategy is useful for developing viral resistance in fruit rootstocks, and such transgenic rootstocks may have potential to enhance production of standard, nongenetically modified fruit varieties while avoiding concerns about transgene flow and exogenous protein production that are inherent for transformed fruiting genotypes. Introduction The most successful instances of creating viral resistance in fruit crops, to date, are limited to papaya ringspot virus (PRSV) in papaya (Carica papaya L.) and plum pox virus (PPV) in plum (Prunus domestica L.) (Scorza and Ravelonandro, 2006; Souza et al., 2005). In the former case, transformed genotypes have been introduced directly into commercial production following deregulation by APHIS in 1996, with acceptance into the Japanese market occurring as recently as 2011. In the latter case, transformed genotypes were used for conventional breeding of new potential varieties, a long and expensive process, which has resulted in APHIS deregulation in 2010 for the first such introduction, ‘Honeysweet’ plum. However, food and environ- mental safety of genetically modified (GM) fruit crops are two major public concerns. Commercial tree fruit production usually depends on a composite plant, comprised of a commercial fruiting genotype grafted onto a separate rootstock genotype. The potential use of a transgenic rootstock that can be grafted to standard nontransgenic commercial scion varieties to produce nongenetically modified fruits is one of many potential strategies available to address the concerns about transgene flow and exogenous protein production that are associated with the fruit of transformed scion cultivars. Like most important Prunus crops, sweet (Prunus avium L.) and sour (P. cerasus L.) cherries are susceptible to a number of potentially pathogenic viruses, including Prunus necrotic ringspot virus (PNRSV), which can be transmitted by grafting, pruning, pollen, sucking insects like aphids and leaf hoppers, and even microscopic soilborne pests like nematodes (Barbara et al., 1978). Sweet cherry scions generally tolerate PNRSV infection with no or only mild symptoms, but certain viral isolates can cause rugose mosaic disease, which affects fruit quality, ripening time and tree health. Cherry rootstock genotypes range from hypersensitive to tolerant for some of these common viruses such as PNRSV (Lang et al., 1998). However, even if a cherry scion genotype tolerates infection by a mild strain of PNRSV, when the virus reaches the graft union with a sensitive rootstock genotype, a hypersensitive reaction will occur that can cause vascular necrosis at the union, resulting in tree decline and ultimately death. Conventional breeding to achieve virus-resistant cherry culti- vars is uncommon, difficult, and time-consuming due to hetero- zygosity, juvenility and a lack of natural sources of resistance. Genetic engineering can facilitate the incorporation of single or multiple genes into existing sweet cherry genotypes, but concerns about transgene flow and exogenous protein production in the fruit of transformed scion cultivars has hindered such approaches. Additionally, the genetic transformation of cherry, like many woody perennial fruit crops, currently is difficult and far from routine, and the challenge of one-by-one transformation of the dozens of commercially important varieties would be an extremely time-consuming and expensive undertaking (Cheong, 2012; Song et al., 2008). Of more than 30 cherry species, genetic transformation has been reported only for a few commercially Please cite this article as: Song G.-q., Sink K.C., Walworth A.E., Cook M.A., Allison R.F. and Lang G.A. (2013) Engineering cherry rootstocks with resistance to Prunus necrotic ring spot virus through RNAi-mediated silencing. Plant Biotechnol. J., doi: 10.1111/pbi.12060 ª 2013 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd 1 Plant Biotechnology Journal (2013), pp. 1–7 doi: 10.1111/pbi.12060