Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus

Abstract

The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing.

A versatile method to prevent transcriptional gene silencing in Arabidopsis thaliana

“…I transformed the parental line with pPZP200/ RB-masP-BAR-nosT-LB, in which the promoter is replaced with the Agrobacterium tumefaciens mannopine synthase promoter (masP), and found that all of the 295 T1 plants that showed resistance to glufosinate exhibited a level of de-etiolation that was typical of the parental plants (silencing rateϭ0%) (Figure 2). This finding is consistent with the previous observation that TGS can be reduced by using different promoters to drive the expression of each transgene (Neuhuber et al 1994;Thierry and Vaucheret 1996). Then, I replaced the nosT terminator with the Agrobacterium tumefaciens octopine synthase terminator (ocsT) to yield pPZP200/RB-p35S-BAR-ocsT-LB, and performed a similar analysis.…”

“…In such cases, although the possibility cannot be excluded that post-transcriptional gene silencing (PTGS) that is based on homology in mRNA sequences may also be involved, the inactivation of the transgene is most likely due to transcriptional gene silencing (TGS), which occurs in the presence of sequence homology in the promoter regions that are used to overexpress a gene of interest in the first T-DNA and to express a selectable marker gene in the second T-DNA. Although TGS can be substantially reduced by using different promoters to drive the expression of each transgene (Neuhuber et al 1994;Thierry and Vaucheret 1996), a technique that completely prevents TGS is needed in order to facilitate large-scale genetic screens. In this study, I have developed a procedure to block TGS, while designing a large-scale genetic screen to identify downstream components of phytochrome B (phyB) signal transduction.…”

A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production

“…A decade ago, we described a transgene locus that triggers both TGS and PTGS of diVerent targets (Elmayan and Vaucheret 1996;Thierry and Vaucheret 1996). The 271 locus consists of multiple copies of the tobacco nitrite reductase (NIR) sequence in antisense orientation (RIN) driven by the CauliXower Mosaic Virus (CaMV) 35S promoter and the bacterial neomycin phosphotransferase (npt) sequence driven by the CaMV 19S promoter.…”

“…The 271 transgenic tobacco line was regenerated from kanamycin-resistant callus that spontaneously became sensitive to kanamycin and was unable to grow on medium containing nitrate or nitrite as the sole source of nitrogen (Vaucheret et al 1992). The 271 locus was subsequently shown to silence, by TGS, any transgene driven by the 19S or 35S promoter and to silence, by PTGS, endogenous NIR genes as well as NIR transgenes (Elmayan and Vaucheret 1996;Park et al 1996;Thierry and Vaucheret 1996), indicating that the 271 locus is a universal silencer of 19S, 35S, and NIR sequences. The 271 locus, however, was unable to silence npt transgenes by PTGS, but only by TGS when they are driven by the 19S or 35S promoter.…”

In-field frequencies and characteristics of oilseed rape with double herbicide resistance

“…As homology-dependent gene silencing of transgenes under control of the CaMV 35S promoter had previously been reported (e.g. Assaad et al, 1993;Thierry and Vaucheret, 1996), it was conceivable to find reduced expression of one or both HR genes in double-resistant plants. However, although the expression level of pat and cp4 epsps genes in single-resistant as well as in double-resistant plants varied with the developmental state of the plants and with temperature, the presence of an additional transgene in the LL × RR hybrids never resulted in transgene inactivation.…”