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

Abstract

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.

Size and positional effects of promoter RNA segments on virus-induced RNA-directed DNA methylation and transcriptional gene silencing

“…15 Similar results were also observed for the potato granule-bound starch synthase I gene promoter. 16 These results imply that the efficiency of TGS induction may depend on the length of an RNA trigger of RdDM and/or TGS. However, only a couple of RNA triggers have been tested in these studies and length-or positional dependency of promoter-targeted silencing has not been fully understood.…”

Induction of RNA-directed DNA methylation and heritable transcriptional gene silencing as a tool to engineer novel traits in plants

“…Transgenes in plant genome can be easily silenced, and the silenced state is heritable in the presence or absence of the silencing inducer (Jones et al 2001). On the other hand, several previous reports have shown that endogenous genes can be silenced only in the presence of the silencing inducer (Cigan et al 2005;Heilersig et al 2006;Sijen et al 2001). The Potato virus X (PVX) and Tobacco rattle virus (TRV) vectors have been shown to induce heritable RNA-mediated TGS against transgenes such as the green fluorescent protein (GFP) and β-glucuronidase (GUS) genes (Jones et al 1999;, but no RdDM and RNA-mediated TGS against an endogenous gene was induced (Jones et al 1999).…”

Epimutant Induction as a New Plant Breeding Technology

“…The results obtained indicated that the silencing efficiency of the different promoter sequences varied. Maximal efficiency was induced with an inverted-repeat construct containing the full promoter sequence (Heilersig et al 2006b). …”