1.12.8
PLANT PROTECTION AGAINST PLUM POX VIRUS BY TRANSGENE DERIVED FROM THE VIRUS

K WYPIJEWSKI, H KORCZ-SZATKOWSKA and J AUGUSTYNIAK

Institute of Molecular Biology and Biotechnology, A. Mickiewicz University, Miedzychodzka 5, 60-371 Poznan, Poland

Background and objectives
Since 1986 when the first TMV-resistant Nicotiana tabacum plants were genetically engineered by Powell-Abel et al. [1], the search for molecular mechanisms involved in the induction of virus resistance in transgenic plants was initiated [2, 3]. Plum pox virus (PPV), a member of the Potyviridae family of plant viruses, is responsible for a severe disease (sharka) of the genus Prunus. Its occurrence has been reported from many European countries as well as from Egypt. Because in Poland PPV causes significant losses in orchards, there is a necessity both to study the mechanisms of transgenic resistance in model plants (e.g. N. benthamiana) and for obtaining virus-resistant or tolerant cultivars producing high quality fruit.

Results and conclusions
A cDNA of the capsid protein gene of plum pox virus, Skierniewice the isolate (PPV-S) was prepared. By using appropriate primers for PCR amplification, the PPV-CP cDNA was made translatable and untranslatable. Both constructs, ligated to the plasmid pBI121 between the 35S CaMV promoter and the NOS termination signal, were used for transformation of N. benthamiana plants with the Agrobacterium tumefaciens system. In the regenerated plants, grown on kanamycin-containing medium, the presence of the PPV-CP sequences was confirmed by PCR. The transformed plants were also tested for the presence of agrobacterial DNA impurities. As real transformants were recognized, only those plants that were free of agrobacterial DNA other than of T-DNA were selected. After self-pollination several R1 plant lines were obtained, in which the percentage of plants containing T-DNA was measured. The data obtained show that most of the transformants contained a single T-DNA insert and only two plant lines had more than one insert locus. The R1 plants were tested for PPV resistance. Only plant lines that contained more than one copy of the PPV-CP transgene were resistant to PPV infection. One of the resistant lines contained a translatable transgene construct, while the other contained an untranslatable PPV-CP sequence. This result clearly indicates that no transgene-coded protein is necessary for inducing the resistance against PPV, and that the observed resistance is mediated by RNA transcribed from the transgene. These results agree with those obtained for RNA virus-resistant plants by many other groups. They also fit well to the post-transcriptional gene silencing models proposed in the past few years by Baulcombe et al. and Smith et al. [2, 4].

References
1. Powell-Abel P, Nelson RS et al., 1986. Science 232, 738-743.
2. Baulcombe DC, 1996. Plant Cell 8, 1833-1844.
3. Sijen T, Wellink J, Hiriart JB, van Kammen A, 1996. Plant Cell 8, 2277-2294.
4. Smith HA, Powers H, Swaney S et al., 1995. Phytopathology 85, 864-870.