Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci

The application of antisense transgenes in plants is a powerful tool to inh ibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to re sult in strong silencing but often there is no clear correlation between as -RNA levels and the degree of silencing. To obtain insight into these puzzl ing observations, we have analyzed several petunia transformants of which t he pigmentation gene chalcone synthase (Chs) is post-transcriptionally sile nced in corollas by antisense (as) Chs transgenes. The transformants were e xamined with respect to the steady-state as-RNA level, transcription level of the as-transgenes, the repetitiveness and structure of the integrated T- DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the first class contains a sin gle copy (S) T-DNA of which the as-Chs gene is transcribed, although severa l-fold lower than the endogenous Chs genes. As there are not sufficient as- RNAs to degrade every mRNA, we speculate that silencing is induced by doubl e-stranded RNA. The second class contains two T-DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as-Ch s transgenes transcriptionally barely active. The strongest silencing was o bserved with IR loci in which the as-Chs transgenes were proximal to the ce ntre of the IR. Similar features have been described for co-suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisens e IRs also occurs by co-suppression, either via ectopic DNA pairing or via dsRNA.