Efficiency of transcriptional gene silencing of GBSSI in potato depends on the promoter region that is used in an inverted repeat

Transcriptional gene silencing (TGS) of the endogenous GBSSI promoter in potato was induced by inverted repeat constructs containing different regions of the GBSSI promoter. Clear differences in silencing efficiency were observed. The 35SGBP-IR construct, containing sequences from -766 to -168 bp relative to the transcription initiation site (TIS), induced weak silencing effects in 57-60% of the transformants. Weak silencing effects were also induced by the ASP-IR construct harbouring allele-specific sequences covering the region from -531 to -330 bp relative to the TIS, but only in a low percentage (4-5.5%) of the transformants. These percentages are too low to distinguish effects between the two potato cultivars. Therefore, this approach cannot be used to induce allele-specific TGS. Strong silencing effects were obtained in 49% of the transformants harbouring the full promoter inverted repeat construct. This construct contained sequences from -766 to +194 bp relative to the TIS. In the strongly silenced transformants no GBSSI mRNA could be detected by Northern blot analysis. This was accompanied by the accumulation of GBSSI promoter-specific small interfering RNAs. Methylation studies revealed that, in the weakly silenced 35SGBP-IR transformants, the HpaII site at -213 bp relative to the TIS was methylated. Apparently, methylation of this sequence does not result in strong silencing effects. In the full promoter transformants, both CG methylation and CNN methylation were detected. We show that, to obtain strong TGS, it is important to include sequences in the vicinity of the TIS.