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

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Abstract
The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as‐RNA) are expected to result in strong silencing but often there is no clear correlation between as‐RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is post‐transcriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined 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 single copy (S) T‐DNA of which the as‐Chs gene is transcribed, although several‐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 double‐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‐Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as‐Chs transgenes were proximal to the centre of the IR. Similar features have been described for co‐suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co‐suppression, either via ectopic DNA pairing or via dsRNA.