I

high level / aberrant transcriptional transcription interference hpRNA formation hpRNA formation high level / aberrant transcriptional transcription interference

nucleolytic degradation / inhibition of translation a b

Fig. 5.1 Transgenes as targets and effectors of gene silencing. Cis-silencing effects are shown in a, trans-effects in b. Transgene integration sites in the plant genome can carry direct repeats, a single-copy insert, or inverted repeats of the transgene (depicted from left to right in a). All configurations may be subject to silencing. For simplicity, cis-silencing effects are only shown for single-copy inserts. Cis-silencing can be induced by integration and position effects, and in these cases involves DNA methylation (dotted arrows) and transcriptional silencing, or by high level and/or aberrant transcription, e.g. from strong or cryptic promoters. The risk of high transcript accumulation may be enhanced in multi-copy transformants such as those with directly repeated inserts (left). The resulting transcripts are recognized and converted into dsRNA by an RNA-dependent RNA polymerase (RDR6 in A. thaliana), and further processed by Dicer-like (DCL) proteins into short interfering RNA (siRNA). In a self-reinforcing amplification loop of RNA silencing, siRNA molecules may anneal to single-stranded primary transcripts (dashed arrow) and thus prime a second round of RDR-mediated dsRNA production followed by processing into secondary siRNAs. Primary and/or secondary siRNAs may also direct DNA methylation to the transgene insert or other homologous target loci (not shown). Transgene loci carrying invertedly repeated inserts (right) produce partially self-complementary hairpin RNA (hpRNA). Because hpRNA is partially double-stranded and a direct substrate of DCL proteins, transcription from inverted repeat transgenes bypasses the upstream events required for siRNA production from single-copy (sense) transgenes and eliminates the requirement for RDR function. Finally, siRNAs effect sequence-specific trans-silencing by guiding an Argonaute protein (AGO)-containing RNA-induced silencing complex (RISC) to complementary single-stranded transcripts derived from one or more transgenic or endogenous target genes (b). Silencing occurs by nucleolytic degradation of technology (see Sect. 5.4). Moreover, several transgene repeat loci have been shown to trans-silence transgenes at non-allelic positions in a promoter homology-dependent manner, e.g. the tobacco loci H9NP (Mette et al. 1999, 2000) and 271 (Vaucheret 1993; Vaucheret et al. 1996; Mourrain et al. 2007). These two silencer loci consist of complex sequence arrangements including inverted and/or direct repeats of transgene promoters and have been shown to produce promoter-derived dsRNA and sRNAs (Mette et al. 2000; Mourrain et al. 2007). Trans--silencing may also be induced by simple transgene inserts. A classic example for which an elaborate phenomenology was developed is cosuppression of Chalcone synthase (Chs) trans- and endogenes in petunia (reviewed by Jorgensen 2003; Jorgensen et al. 2007). Jorgensen and colleagues established that cosuppression does not require the presence of inverted repeat or antisense transgenes but can also be mediated by transgenes that carry only a sense copy of the Chs coding sequence or part thereof. Chs cosuppression by sense transgenes results in flower pigmentation phenotypes that are distinct from those produced by either inverted repeat or antisense transgenes and requires high-level transcription of the transgene (Que et al. 1997; Jorgensen et al. 2007).

The general view that evolved from detailed molecular and genetic characterization of these and other silencing events involving two or more homologous sequences is that silenced genes that share homology within the transcribed region are silenced post-transcriptionally and non-heritably (in the absence of the inducer locus), and those that are homologous to each other within the transgene promoter are silenced at the transcriptional level and in a heritable but often reversible manner (Matzke et al. 2002). Both silencing phenomena involve sRNAs that either direct transcript degradation, or promoter methylation and chromatin condensation, respectively. While dsRNA production from inverted repeat loci by transcription across both repeat units and annealing of complementary transcript regions is thought to be a straightforward process, the production of dsRNA from direct repeats or simple sense transgenes may require complementary RNA strand synthesis by RDRs similar to the processes in anti-viral defense pathways (see Sect. 5.2.1.3; Schubert et al. 2004). Post-transcriptional transgene silencing may also be accompanied by RNA-directed DNA methylation within transcribed regions. Although methylation within transcribed regions often has no obvious effects on the rate of transcription (Elmayan et al. 1998; Mourrain et al. 2007; Lunerova-Bedrichova et al. 2008), there is recent evidence that it is required for the maintenance of post-transcriptional silencing (Eamens et al. 2008b; reviewed by Voinnet 2008).

Fig. 5.1 (continued) the target RNA or inhibition of translation. Filled boxes Genes, gray arrows promoters, gray boxes polyadenylation signals, white (unfilled) boxes repetitive, heterochromatic flanking genomic sequences, white arrow cryptic promoter, black arrows transcription start sites, dark gray box spacer fragment in inverted repeat constructs for intentional production of hpRNA, wavy lines transcripts and RNA processing products, dotted wavy line transcripts produced from cryptic promoters

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