1.12.13
GENE SILENCING IN TRANSGENIC TOBACCO PLANTS EXPRESSING THE CP GENE OF SWEET POTATO FEATHERY MOTTLE POTYVIRUS

M NISHIGUCHI1, S SONODA1, H KATO1, 2 and M MORI1

1National Institute of Agrobiological Resources, Kan-nondai, Tsukuba, Ibaraki 305, Japan; 2Present address, Shinshu University, Asahi, Matsumoto, Nagano 390, Japan

Background and objectives
Data on pathogen-derived virus resistance have been accumulated. At least two different mechanisms are involved, one protein-mediated and the other RNA-mediated. Recently post-transcriptional gene silencing has been shown to be related to RNA-mediated virus resistance [1]. We previously reported the genomic analysis of sweet potato feathery mottle potyvirus (SPFMV-S) [2], which is a causal agent of russet crack disease in sweet potato. Using the viral coat protein (CP) gene, we are trying to confer virus resistance to sweet potato. In the course of this study we produced transgenic tobacco plants (Nicotiana benthamiana) with the CP gene including the 3' non-coding region. In this report, we present transgenic plants showing resistance against recombinant PVX carrying the sequence of the CP gene and some features of their gene silencing.

Results and conclusions
The CP gene replaced the GUS gene in pBI121. Agrobacterium with the construct was used to transform N. benthamiana. Nineteen regenerated transgenic plants were inoculated with the recombinant PVX carrying the CP gene sequence of SPFMV-S. Five of them (20%) showed resistance. All of them showed undetectable or a low steady-state level of mRNA by Northern blot analysis. CP accumulation was analysed by Western blot and was under the detection limit in all five lines . The number of loci for the CP transgene in the lines was estimated to be more than two, except in one line, by Southern blot analysis. CP fragments with various deletions were inserted into the PVX vector and used for inoculation to determine which region of the CP gene was required for the virus to be the target of gene silencing. As a result, resistant lines were classified into two groups. One (lines with more than two loci) had a high level of gene silencing where complete resistance depended upon the length of the sequence irrespective of the position within the CP gene. The other (one line with a single locus) was of low level, in which complete resistance was shown only if the whole region was present. Partial resistance in this line was obtained with the 3' terminal region (0.5 kb) of the insert, but not with the 5' terminal (0.7 kb). The CP transgenes in the former group were found to be highly methylated while that in the latter was lower. Further investigation is in progress to characterize the gene silenced trangenic plants in more detail.

References
1. Baulcombe DC, 1996. Plant Cell 8, 1833-1844.
2. Sakai J, Mori M, Morishita M et al., 1997. Archives of Virology 142, 1553-1562.