5.3.6
RNA-MEDIATED POTATO VIRUS Y RESISTANCE IN POTATO AND TOBACCO

AM CHACHULSKA1, B FLIS2, M CHRZANOWSKA2, M MILNER1, A GÓRA-SOCHACKA1, C ROBAGLIA3 and W ZAGÓRSKI1

1Institute of Biochemistry and Biophysics PAS, ul. Pawinskiego 5a, 02-106 Warsaw, Poland; 2Plant Breeding and Acclimatization Institute, Unit Mlochów, 02-832 Rozalin, Poland; 3CEA Centre de Cadarache, 13-108 Saint Paul Lez Durance, France

Background and objectives
Potato virus Y (PVY) is the type member of the Potyviridae family - the largest and economically the most important group of plant viruses. PVY is one of the main viral pathogens of Solanaceae, provoking serious diseases of potato, pepper and tobacco throughout the world. Many attempts to construct PVY-resistant crops have been undertaken. Transgenic plants expressing sense and antisense PVY coat protein RNAs as well as the polymerase (NIb) gene were found to be resistant to virus infection and the mechanism of resistance was studied [1]. In Poland, the most important group, leading to significant crop losses is a subgroup of the necrotic (N) strain. Projects were started to create transgenic potato and tobacco plants resistant to local necrotic PVY isolates.

Materials and methods
A set of constructs, carrying a truncated, untranslatable PVYN polymerase gene (without the C-terminal end) was prepared. The cDNA fragment was cloned in binary vectors in both orientations under two different promoters: 35S CaMV and the tRNA leu gene. Another two constructs contained the PVY 5' and 3' non-translated regions (NTRs) in both orientations under the 35S promoter. As a reference, another plasmid, carrying the translatable LMV CP gene [2], was used in plant transformation experiments.

Constructs were introduced into tobacco plants (cultivars Xanthi XDH8 and SR1) or potato (cultivar Irga) by Agrobacterium-mediated transformation. Transgenic plants were regenerated on selective media, and subsequent generations of tobacco and minitubers and tubers of potato plants were obtained. The viral cDNA insertion, copy number and RNA expression were analysed using the PCR and hybridization methods. Selected transgenic plants were submitted to resistance tests.

Results and conclusions
Over 100 transformed tobacco and potato lines were tested for resistance against phenotypically different PVY isolates. The constructs based on the truncated polymerase-cDNA are more effective in inducing the resistance than the constructs carrying the NTRs. Several transgenic tobacco plants expressing sense or antisense truncated viral polymerase RNA showed complete or partial resistance to PVY after a mechanical inoculation. Several selected potato clones carrying the truncated polymerase gene in the antisense and sense orientations were entirely resistant (immune) to PVY infection in the primary and secondary infections. They are morphologically identical to non-transgenic plants, constituting a new genetic source for agriculture. Molecular analysis (transgenic DNA copy number and the level of RNA expression and accumulation) of resistant versus sensitive transgenic plants carrying various constructs will be presented. The hypothetical mechanism of protection will be discussed in the context of the gene-silencing phenomenon.

References
1. Swaney S, Powers H, Goodwin J et al., 1995. Molecular Plant-Microbe Interactions 8, 1004-1011.
2. Dinant S, Blaise F, Kusiak C et al., 1993. Phytophatology 83, 818-824.