Silencing of β-1,3-glucanase genes in tobacco correlates with an increased abundance of RNA degradation intermediates

Post-transcriptional gene silencing of beta-1,3 glucanase genes in the transgenic tobacco line T17 is characterised by an increased turnover and, as a consequence, reduced levels of gn1 transgene and endogenous beta-1,3 glucanase mRNAs, Here, additional gn1 RNAs, both larger and smaller than the full-length messenger, are shown to accumulate in silenced plants of the transgenic tobacco line T17, The longer-than-full-length gn1 RNAs are the result of cryptic processing of the gn1 messenger, The small gnl RNAs in silenced plants correspond to distal and proximal parts of the mature gn1 messenger. The proximal RNA products are intact at their 5' extremity, but terminate at different positions at the 3'-end. The distal RNA products contain a poly(A) tail and are truncated to various positions at the 5'-end, These observations indicate that degradation of the mature gnl transcript does not start at the 5'- or 3'-end, but rather are consistent with degradation of the gnl transcript starting with an endonucleolytic cleavage followed by internal exonuclease digestion. Importantly, the truncated products are more abundant in silenced plants than in expressing plants, This suggests, together with the previously reported silencing-related increased gnl mRNA turnover and the similar rates of gnl transcription in silenced and expressing T17 plants, that the predominant decay route for the gnl transcripts differs between silenced and expressing conditions

Post-transcriptional β-1,3-glucanase gene silencing involves increased transcript turnover that is translation-independent

In tobacco line T17, basic beta-1,3-glucanases are silenced when a beta-1,3-glucanase (gn1) transgene of Nicotiana plumbaginifolia is present in a homozygous condition. Cosuppression of endogenous and transgenic basic beta-1,3-glucanases occurs via a post transcriptional process. This study shows that silencing correlates with a reduced stability of the transcripts encoded by gn1 and the co-suppressed endogenous glucanase genes. The observed decreases in mRNA stability at least partially explain the reductions in mRNA level. Furthermore, the results show that the enhanced, silencing-related turnover of gn1 mRNA does not require continued protein synthesis. The implications of these findings for the mechanism of gene silencing are discussed

Sequences throughout the basic β-1,3-glucanase mRNA coding region are targets for homology dependent post-transcriptional gene silencing

In the transgenic tobacco line T17, plants homozygous for the gn1 transgene display developmentally regulated post-transcriptional silencing of basic beta-1,3-glucanase genes. Previously, it has been shown that silencing involves a markedly increased turnover of silencing-target glucanase mRNAs. Using a two-component viral reporter system facilitated a comparison, in a quantitative manner, of the relative silencing efficiencies of various sequences derived from the gn1 transgene. The results show that target sites for the silencing mechanism are present throughout the coding region of the gn1 nnRNA. Similar-sized coding region sequences along the entire gn1 mRNA display a similar susceptibility to the silencing mechanism. The susceptibility to silencing increases as the coding region elements increase in size. Relative to internal sequences, the 5' and 3' terminal regions of the gn1 mRNA are inefficient targets for the silencing machinery. Importantly, sequences of the gn? transgene that are not part of the mature gn1 mRNA are not recognized by the silencing machinery when expressed in chimeric viral RNAs. These results show that the glucanase silencing mechanism in T17 plants is primarily directed against gn1 mRNA-internal sequences and that terminal sequences of the gn1 mRNA are relatively unaffected by the silencing mechanism