This RNA-based immune system is also activated in trans- genic plant lines exhibiting gene silencing. The surveillance system that detects viral RNA in the infected plant is also able to detect the transgene or its RNA. The immune system is then targeted against the transgene RNA and the transgene protein accumulates at only a low level in these plants. Endogenous RNAs can also be targeted if the transgene is similar to a plant gene. In this situation, there is cosup- pression of both the transgene and the endogenous gene.

By extrapolation from our work with viral systems, we predicted that there would be antisense RNA in these trans- genic plants that confers specificity to this immune system. We have subsequently found antisense RNA in many examples of transgene silencing and virus infection4. In each example, the antisense RNA was present as a short, 25 nt, RNA species. In tomato exhibiting co-suppression of the ACO ethylene biosynthetic gene, the ACO antisense RNA was the same size as the PVX antisense RNA in PVX-infected plants. Similar 25 nt RNAs have also been found in petunia, tobacco and Arabidopsis plants and, in each instance, they corresponded to the target of gene silencing or of a virus.

Production of these short antisense RNAs in a gene silencing system depends on sense transcription of the transgene4. Our interpretation of this result is that the sense RNA is the template for antisense RNA production and that the synthetic enzyme is an RNA-dependent RNA polym- erase. However, we do not yet know whether these RNAs are produced directly as 25 nt molecules or whether they are processed from a larger precursor. Nor do we know

what the role is of these molecules in the mechanisms of gene silencing and immunity against virus infection. We favour the possibility that they are the specificity deter- minant of the RNA targeting mechanism, and the properties of these molecules are consistent with that proposal. To confirm this suggestion, we will purify the 25 nt RNA together with any associated host proteins, and identify the function of these proteins.

Note in proof: Analyses of Drosophila cell culture extracts support our proposed model and indicate -25 nt RNAs are involved in gene silencing in plants and animals

References

1. RatcliffF. Harrison BD, Baulcombe DC (1997) Science 376, 1558-1560.

2. RatcliffF, MacFarlane S. Baulcombe DC (1999) Plant Cell 11, 1207-1316.

3. Baulcombe DC (1999) Arch. Virol. Suppl. 15, 189-201.

4. Hamilton AJ, Baulcombe DC (1999) Science 386, 950-952.