Transgenic grapevine rootstock clones expressing the coat protein or movement protein genes of Grapevine fanleaf virus: Characterization and reaction to virus infection upon protoplast electroporation Laure Valat a, * , Marc Fuchs b,1 , Monique Burrus a,2 a Laboratoire de Stress, De ´fense et Reproduction des Plantes, Unite ´ de Recherche Vigne et Vin de Champagne, UPRES EA 2069, Universite ´ de Reims Champagne-Ardenne, BP1039, 51687 Reims Cedex 2, France b Laboratoire de Virologie, Unite ´ Mixte de Recherche Vigne et Vins d’Alsace, Institut National de la Recherche Agronomique, Universite ´ Louis Pasteur, 28 rue de Herrlisheim, 68021 Colmar, France Received 22 June 2005; received in revised form 2 November 2005; accepted 12 November 2005 Available online 5 December 2005 Abstract The reaction to Grapevine fanleaf virus (GFLV) infection in 42 independent transgenic grapevine rootstock 41B clones expressing the coat protein (CP) or movement protein (MP) gene of GFLV was assayed by protoplast electroporation. Two of the 26 transgenic clones expressing the CP gene did not support the accumulation of GFLV MP to detectable levels, 12 accumulated substantially lower levels of MP, and 12 accumulated equivalent levels of MP relative to protoplasts of nontransformed controls at 72 h post-electroporation, as shown by Western blots with anti-MP g- globulins. Interestingly, inhibition of MP accumulation was achieved against virions but not viral RNAs, and was dependent on the inoculum dose. No interference was observed with the multiplication of Arabis mosaic virus, which is closely related to GFLV, likely due to low nucleotide identity between the CP genes. Also, one of the 16 transgenic clones expressing the MP gene significantly reduced the accumulation level of GFLV CP at 72 h post-electroporation, as shown by DAS-ELISAwith anti-GFLV g -globulins. The potential of protoplast electroporation as rapid identification of GFLV-resistant grapevine clones at the cell level will be discussed relative to field screening for resistance at the plant level by nematode- mediated GFLV transmission. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Transgenic grapevine; Protoplast electroporation; Grapevine fanleaf virus; Coat protein; Movement protein; Resistance 1. Introduction Fanleaf degeneration is one of the most important viral diseases of grapevines worldwide [1]. It causes a significant reduction in crop yield (up to 80%) and a progressive decline that reduces plant longevity or can even lead to plant mortality. Fanleaf degeneration is caused by several virus species from the genus Nepovirus in the family Comoviridae. The most important of them is Grapevine fanleaf virus (GFLV), which is vectored by the ectoparasitic nematode Xiphinema index [1]. The viral genome of GFLV is composed of two single stranded positive-sense RNAs, denoted RNA1 and RNA2, which carry a small covalently linked viral protein (VPg) at their 5 0 extremities and a poly(A) stretch at their 3 0 ends [1]. Each genomic RNA codes for a polyprotein, which is proteolytically processed into functional proteins. RNA1 codes for the proteins implicated in RNA replication and for the viral proteinase [1]. RNA2 codes for protein 2A, which is required for RNA2 replication, the movement protein (MP), and the coat protein (CP) [1]. www.elsevier.com/locate/plantsci Plant Science 170 (2006) 739–747 * Corresponding author at: Laboratoire de Physiologie Ve ´ge ´tale, Faculte ´ des Sciences, Unite ´ Mixte de Recherche A 408, Universite ´ d’Avignon et des Pays de Vaucluse, 74 rue Louis Pasteur, 84029 Avignon Cedex 1, France. Tel.: +33 4 90 14 44 53; fax: +33 4 90 14 44 49. E-mail address: laure.valat@univ-avignon.fr (L. Valat). 1 Present address: Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456, USA. 2 Present address: Evolution et Diversite ´ Biologique, Unite ´ Mixte de Recherche 5174, Universite ´ Paul Sabatier, Bat 4R3, 118 Route de Narbonne, 31062 Toulouse Cedex 4, France. 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.11.005