Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol156.Issue2.2000:

Plant Science 156 2000 235 – 244 Analysis of transgenic grapevine Vitis rupestris and Nicotiana benthamiana plants expressing an Arabis mosaic 6irus coat protein gene Albert Spielmann a, , Stoyanka Krastanova b , Ve´ronique Douet-Orhant a , Paul Gugerli c a Laboratoire de biochimie, Uni6ersite´ de Neuchaˆtel, Emile-Argand 11 , 2007 Neuchaˆtel, Switzerland b ProfigenAgri6itis, Columbia Crest Dri6e, PO Box 188 , Paterson, WA 99345 , USA c Station Fe´de´rale de Recherches en Production Ve´ge´tale de Changins, 1260 Nyon, Switzerland Received 13 September 1999; received in revised form 20 March 2000; accepted 20 March 2000 Abstract A disarmed LBA4404 strain of Agrobacterium tumefaciens harboring a binary vector which contained chimeric genes encoding the neomycin phosphotransferase npt II and the coat protein CP of Arabis mosaic nepo6irus ArMV was used in co-cultivation experiments with leaf discs of Nicotiana benthamiana and somatic embryos of the grapevine rootstock cultivar Vitis rupestris. Transgenic N. benthamiana expressing the ArMV CP gene were regenerated and six independent lines were characterized. Enzyme-linked immunosorbent assay ELISA performed on leaf tissue demonstrated the accumulation of the ArMV CP in five of the six lines analyzed. Immunosorbent electron microscopy ISEM studies revealed the presence of virion-like isometric particles VLPs reacting to a rabbit antiserum specific to ArMV virions. ArMV-CP expressing transgenic N. benthamiana lines showed protection against ArMV expressed as a delay in infection and a reduction of the percentage of infected plants. Four independent transgenic lines of V. rupestris transformed with the ArMV CP gene were regenerated and characterized. In contrast to N. benthamiana, transgenic V. rupestris did not accumulate the ArMV CP at levels detectable by ELISA and no VLPs could be observed by ISEM. Northern blot analysis showed that the ArMV CP mRNA was expressed at lower level in V. rupestris compared with N. benthamiana. The reason for this difference in transgene expression andor mRNA stability between grapevine and N. benthamiana is unclear, but the genetic state of the transgenes homozygous in N. benthamiana versus hemizygous in V. rupestris may have an effect on gene expression. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Coat protein-mediated virus resistance; Nepovirus; Grapevine; Nicotiana benthamiana; Plant virus resistance www.elsevier.comlocateplantsci

1. Introduction

Nepoviruses are plant viruses with polyedric particles which are transmitted via soil-inhabiting longidorid nematodes Xiphinema and Longidorus spp.. They are responsible for economically im- portant diseases, especially of perennial plants such as grapevine and fruit trees. Their genome is composed of two separately encapsidated positive single-stranded RNAs. In grapevine, the most damaging and widespread nepoviruses are Grape6ine fanleaf 6irus GFLV and Arabis mosaic 6 irus ArMV, both responsible for the disease known as infectious degeneration or ‘court-noue´’. The disease is controlled by soil disinfection using nematicides. This procedure is forbidden in Switzerland, Germany, Italy and some States in the USA, due to the high toxicity of the chemical dichlorpropen. Virus-resistant transgenic grapevine plants would be an ecological alterna- tive to control the disease. Since the first report on resistance to Tobacco mosaic 6irus TMV in transgenic tobacco plants expressing TMV coat protein CP [1], similar results have been obtained for a broad spectrum Corresponding author. Tel.: + 41-32-7182222; fax: + 41-32- 7182201. E-mail address : albert.spielmannbota.unine.ch A. Spielmann. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0168-94520000259-4 of plant viruses [2]. CP-mediated protection against nepoviruses has been demonstrated in transgenic Nicotiana benthamiana expressing the ArMV CP gene [3], the Tomato ringspot 6irus CP gene [4], or the GFLV CP gene [5], and in transgenic N. tabacum expressing the Strawberry latent ringspot 6irus CP gene [6], or the Grape6ine chrome mosaic 6irus CP gene [7]. The formation of virus-like particles result- ing from the accumulation of transgenic nepovirus CP in plant tissues was demonstrated for the ArMV CP in N. benthamiana [8]. Genetic transformation of various grapevine tissues and regeneration of transgenic plants have been reported for a number of grapevine rootstocks and cultivars [9]. Recents reports described the regeneration of transgenic grapevine plants expressing a chimeric GFLV CP gene after transformation of somatic embryos of the rootstock cvs 110 Richter [10,11], Gloire de Mont- pellier, 3309 Couderc, Millardet de Grasset 101-14 [11], 41B and SO4, and the Vitis 6inifera variety Chardonnay [12]. In these cases, expression of the GFLV CP transgene leads to the accumulation of the CP in transgenic tissue detected by ELISA, but the presence of virion-like isometric particles VLPs was not reported. In this paper, we describe the expression of a chimeric ArMV CP gene in an annual herbaceous species, N. benthamiana, and a woody perennial species, V. rupestris. The transgenic N. benthamiana lines expressed the ArMV CP gene at high levels and accumulation of ArMV CP resulted in the forma- tion of VLPs in plant tissue. In contrast, none of the transgenic V. rupestris lines accumulated the ArMV CP at a detectable level. Data from northern blot experiments showed that the apparent lack of ArMV CP protein accumulation in V. rupestris tissue was correlated to differences in transgene expression levels andor RNA stability in grapevine tissues compared with N. benthamiana. Little or no transgene mRNA could be detected in transgenic grapevine tissues. Progeny of ArMV CP-expressing transgenic N. benthamiana lines showed resistance against ArMV.

2. Materials and methods