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Transgenic tobacco plants have been obtained expressing nucleoprotein (N) gene sequences of three different tospoviruses known to affect vegetable crops: tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), and groundnut ringspot virus (GRSV). The chimeric plant transformation vector used comprised the three viral N gene sequences, each with a copy of the CaMV 35S promoter and the nos terminator. Despite the high levels of homology between the different N gene sequences (74-82%) and the presence of repeated promoter and terminator sequences in this construct, unrearranged copies of this triple N gene construct were stably maintained in both Escherichia coli and Agrobacterium tumefaciens plasmids used during the cloning process, as well as in several generations of transgenic tobacco plants. A transgenic tobacco line was obtained that exhibited high levels of resistance to all three tospoviruses, showing the possibility of producing transgenic plants with a broad resistance to tospoviruses by introducing tandemly cloned viral N gene sequences. DNA analysis of this transgenic plant line shows that the multivirus resistance trait is confined to a single genetic locus, which is very convenient for further breeding purposes.

Recently high levels of protection against tomato spotted wilt virus (TSWV), a negative-strand RNA virus infecting plants, have been obtained by transforming tobacco with viral nucleoprotein (N) gene sequences. Here we demonstrate that this protection is primarily due to the presence of N gene transcripts in the cells of transgenic plants, and hence appears to be RNA-mediated. Further, transgenic tobacco plants are only protected to isolates and strains of TSWV and not to other tospoviruses that share considerable nucleotide sequence homology in their N genes to TSWV. In addition to being protected after mechanical inoculation, the transgenic tobacco plants are also resistant to inoculation using viruliferous thrips, i.e. Frankliniella occidentalis (Perg.), one of the most important natural vector species.

To study the control of differential gene expression during plastid biogenesis in Petunia hybrida, we have investigated the in vivo translation and transcription of the rbc L gene, coding for the large subunit of ribulose bisphosphate carboxylase (LSU), and the psa A gene, coding for P700 chlorophyll-a apoprotein (AP700). Differential expression of these plastid-encoded genes was studied in two developmentally different plastid systems, proplastid-like organelles from the green cell suspension AK2401 and mature chloroplasts from green leaves. In vivo translation of rbc L and psa A transcripts was analysed using specific antibodies. Specific transcript levels were analysed using internal fragments of the rbc L and psa A genes. A standardization procedure was used so that a direct correlation could be made between the amount of products and gene copy number. In Petunia hybrida the amount of LSU polypeptides present in both plastid types does not correspond to the amount of specific mRNA for the gene. Although the rbc L transcripts are present in both plastid types, the LSU protein is only present in green leaf plastids and not in cell culture plastids. In vitro translation of isolated rbc L transcripts give similar results, thereby suggesting that differences in the primary structure of the transcripts are responsible for the observed discrepancy. In contrast to this, the amount of AP700 polypeptides does correspond to the amount of the psa A transcripts. Therefore, our results indicate that the expression of chloroplast genes during plastid biogenesis takes place on at least two different levels: expression of the rbc L gene is regulated post-transcriptionally while expression of the psa A gene is regulated at the transcriptional level.

Cytoplasmic differences between male-fertile and male-sterile Brassica napus as well as Raphanus sativus were investigated. Plastids of the male-fertile B. napus were found to differ from those of male-sterile B. napus and R. sativus with respect to DNA restriction enzyme patterns. Differences between male-fertile and male-sterile B. napus mitochondria were detected not only in the restriction fragment patterns of their DNA, but also at the level of expression by in organello translation of mitochondrial polypeptides.The chlorophyll deficiency obtained upon transferral of the male-sterility-conferring radish cytoplasm to a winter variety of B. napus had been corrected earlier through protoplast fusion. The cytoplasmic composition of the corrected lines was analysed using DNA restriction analysis and in organello translation. The stability of the recombined cytoplasm in the corrected lines was confirmed by analysis of the subsequent seed-derived generation.