1.12.21
TRANSFORMATION OF TOBACCO AND POTATO WITH FULL-LENGTH cDNA TO POTATO LEAFROLL VIRUS

H BARKER1, L FRANCO-LARA1, KD McGEACHY1, MA MAYO1 and U COMMANDEUR2

1Scottish Crop Research Institute, Invergowrie, Dundee DD2 SDA, UK; 2Biologisches lnstitut, Stuttgart, Germany

Background and objectives
In infected plants, potato leafroll luteovirus (PLRV) is largely confined to phloem cells, although PLRV can multiply when inoculated into isolated mesophyll protoplasts [1]. We have transformed potato and tobacco plants with a full-length cDNA copy of the PLRV genome driven by a constitutive promoter. Such plants have the potential to express PLRV genome RNA in every cell, and are being used as part of a programme to investigate PLRV movement in plants.

Materials and methods
Tobacco (line SR1) and potato line G8107(1) were transformed by Agrobacterium tumefaciens containing a T1 plasmid that encodes a full-length cDNA copy of the PLRV genome driven by the 35S constitutive promoter. ELISA and tissue printing were used to detect PLRV in transgenic plants, tissue sections and isolated mesophyll protoplasts from T1 plants of tobacco and vegetatively propagated to potato plants. The transgene was detected in transgenic plants by PCR, and transgene RNA transcript was detected by hybridization with a digoxigenin-labelled cDNA probe.

Results and conclusions
Two transgenic tobacco lines and five transgenic potato lines were studied. Extracts of total RNA from the transgenic plants were analysed in Northern blots. Genomic and subgenomic RNAs were found which corresponded in size to those found in extracts from PLRV-infected non-transgenic plants. ELISA detected virus antigen in all the transgenic lines. Virus in transgenic plants was infectious and could be transmitted using aphids or graft inoculation to virus-free test plants. Isolated mesophyll protoplasts and leaf tissue extracts from transgenic tobacco and potato plants were tested by ELISA and compared with those of PLRV-infected non-transgenic control plants. Slightly more PLRV accumulated in leaf extracts of transgenic tobacco than in PLRV-infected non-transgenic plants, although virus concentrations were up to 8-fold greater in transgenic protoplasts than in protoplasts from infected non-transgenic plants. However, in potato protoplasts and leaf tissues, the concentrations of PLRV were up to 100-fold greater in transgenic plants than in PLRV-infected non-transgenic plants.

Tissue printing was used to determine where PLRV-infected cells were located in transgenic plants. In tobacco, transverse sections of stems showed that most infected cells were in the phloem bundles, and that only a few cells of the cortex and other tissues were infected. Leaf tissue prints showed that a few mesophyll cells were infected, but these were not observed in PLRV-infected non-transgenic plants. In potato, transverse sections of stems showed that epidermal cells were often infected, in addition to cells in the phloem bundles. There were many infected cells in the internal phloem bundles, but very few in external bundles. Many mesophyll cells were infected in potato, particularly in older tissue.

A surprising aspect of this work was that the level of virus accumulation was only slightly greater in transgenic than in PLRV-infected non-transgenic tobacco plants, although in potato there was considerably more replication in transgenic plants. This novel model system could provide a valuable tool for investigating several aspects of the biology of PLRV and its interaction with host plants.

References
1. Barker H, Harrison BD, 1982. Plant Cell Reports 1, 247-249.