A transgene with repeated DNA causes high frequency, post-transcriptional suppression of ACC-oxidase gene expression in tomato

Gene silencing with sense genes is an important method for down-regulating the expression of endogenous plant genes, but the frequency of silencing is unpredictable. Fifteen per cent of tomato plants transformed with a 35S-ACC-oxidase (ACO1) sense gene had reduced ACC-oxidase activity. However, 96% of plants transformed with an ACC-oxidase sense gene, containing two additional upstream inverted copies of its 5' untranslated region, exhibited reduced ACC-oxidase activity compared to wildtype plants. In the three plants chosen for analysis, there were substantially reduced amounts of both endogenous and transgenic ACO RNA, indicating that this was an example of co suppression. Ribonuclease protection assays using probes spanning intron-exon borders showed that the reduced accumulation of endogenous ACO mRNA occurred post-transcriptionally since the abundance of unprocessed transcripts was not affected. The ACO1 transgene with the repeated 5'UTR also strongly inhibited the accumulation of RNA from the related ACO2 gene in flowers, although there is little homology between the 5'UTRs of ACO1 and ACO2. These results indicate that although repeated DNA in a transgene greatly enhances the probability of gene silencing of an endogenous gene, it also involves generation of a trans-acting silencing signal produced, at least partly, from sequences external to the repeat.