History and back-ground information
Expression of genes in eukaryotes is controlled by sophisticated and complex mechanisms. Many genes are controlled transcriptionally by transcription factors but there is also post-transcriptional control specified by mRNA stability and rate of synthesis. Although expression of transgenes in genetically modified plants and other organisms is controlled by these mechansims as well, there must be additional steps controlling gene expression. For example, identical transgenes in different transformants are expressed at variable and different levels. More remarkable is the effect of transgenes on the expression of native homologues whereby transgenes and the native homo-  logues are both silenced. The sequence homology between the genes is very important which has led to the common phrase for this phenomenon: Homology Dependent Gene Silencing (HDGS). HDGS can lead to transcriptional gene silencing (TGS) when homologous promoters are involved,
or to post-transcriptional gene silencing (PTGS) when homology is confined to coding sequences.
These unexpected transgene properties are revealing a number of novel and very interesting transcriptional and post-transcriptional processes we were unaware of thus far. These processes
are believed to be defence strategies evolved to combat virus infections and to keep transposons
at bay. Below we describe various features of gene silencing in petunia. For a more detailed description of gene silencing and silencing in other systems, as well as a discussion of the molecular mechanisms, we refer to the research papers and reviews listed in the "silencing reference list"

Silencing of flower pigmentation genes
In our department we are studying the molecular mechanism of silencing of flower pigmentation genes in petunia. These pigmentation genes are required for the synthesis of anthocyanin pigments in flowers and silencing of one or more of these genes is visible by the white flowers produced by these plants. Already in 1988, our group showed that silencing of the pigmentation gene chalcone synthase (Chs) could be achieved by introducing antisense (as) Chs genes. In this instance, gene silencing is assumed to be mediated by asRNA transcribed from the antisense genes and which targets complementary Chs mRNA for degradation. Two years later, we and the group of Richard Jorgensen at DNAP in San Francisco did the amazing discovery that sense transgenes could do the same. Expression of transgenes and all native homologues was repressed, a feature coined by the phrase 'co-suppression'. Since then many genes have been silenced by co-suppression, which is now a valuable tool in studying gene function.

        "Pictures of flower pigmentation phenotypes"
We showed by nuclear run-on transcription assays that co-suppression occurs by a post- transcriptional mechanism. In these assays a silenced gene is transcribed as high as a non-silenced gene and, therefore, the RNAs must be prematurely degraded and importantly, in a sequence-directed manner. But how and why?
Studies by other groups have shown that highly expressed transgenes are more prone to activate PTGS than lowly expressed transgenes, suggesting that activation requires an excessively high mRNA level. This threshold model is also supported by studies of viral transgene-mediated virus resistance in plants. Although attractive, our data indicated that a high level of normal mRNA is not sufficient to induce PTGS because run-on assays indicated that PTGS can be induced by barely active transgenes.

Current projects:

1. PTGS induced by inverted transgene repeats and the role of dsRNA

When plants are transformed with Chs sense transgenes, not all plants show a co-suppression phenotype and we discovered that plants that do, contained two or more transgene copies inserted at the same chromosomal location that were arranged as inverted repeats (IRs). With the constructs we have been using, PTGS is strictly correlated with the presence of IR loci. The same was found for antisense transgenes that were barely expressed but nevertheless induced a strong silencing. It seems therefore that, at least in certain transformants, silencing by sense and antisense transgenes occurs by the same mechanism.
There are two types of IR loci depending on the relative orientation of the transgenes. IR loci with the silencing Chs sequences closest to the point of symmetry (an IRc locus) induce PTGS the strongest. A few examples of IR loci are depicted below.

                            "Picture of IR loci"
We are currently investigating how such IR transgenes, which are sometimes trans- criptionally barely active, induce PTGS.
Two possibilities have been raised. The first is a physical interaction of the IR, by virtue
of its palindromic structure, with a native homologue. This interaction is thought to disturb transcription resulting in truncated transcripts that trigger PTGS. The second possibility we are testing is production of aberrant RNAs by the IR locus itself. These transcripts are most likely double-stranded (ds) because of the palindromic arrangement of the transgenes and the fact that PTGS in other systems, where it is called RNA interference (RNAi), is also induced by dsRNA. dsRNA is then generated by read-through transcription running from one gene into the next.

               'Picture of transcriptional readtrough'
The ability of IR transgenes to induce co-suppression can be exploited to silence genes in a systematic and directed fashion. By driving IR transgenes with appropriate promoters we are testing whether it is conceivable to silence genes in a spatial and temporal manner.
 

2. PTGS and DNA methylation

The cytosine residues of DNA arranged as IRs are usually severely methylated. Sequences near the centre of the IR appear to be more highly methylated than sequences more distal to the centre.

        "Methylation of IR and single copy sequence"
How these specific methylation patterns are established is not known. It could be due the palindromic sequence itself. Theoretically, this sequence can be converted into a cruciform structure, which might be a preferred substrate for DNA-methyltransferases. An alternative possibility is that dsRNA transcribed from the IR locus is triggering DNA methylation. This possibility is based on the surprising discovery of RNA-directed DNA methylation (RdDM) in 1994 by the group of Michael Wassenegger, and more recently by the group of Marjori and Tony Matzke who showed that the effector RNA is most likely double-stranded.
RdDM might also be responsible for the methylation of single copy transgene loci that are sometimes present in IR transformants. The methylation pattern of these loci are very similar to that of the accompanying IR locus. Limited analysis using a methylation sensitive restriction enzyme did not reveal a dramatic increase in methylation of the native silenced Chs genes. We are currently examining this in much greater detail using the bisulfite method whereby the methylation status of every cytosine residue can be assessed.
An interesting question is of course the biological function of RdDM. Is it required to methylate and inactivate repetitive sequences, by which transposons and retroelements are kept silent or is RdDM involved in de novo methylation of more unique and specific sequences?

3. RdDM of specific sequences

Since transgene derived dsRNA can direct methylation of specific DNA sequences in the genome, RdDM could be exploited to hypermethylate specific DNA regions. We are testing whether this is feasible by targeting specific promoter sequences which upon methylation will inactivate the promoter.

4. RNA-directed RNA polymerases (RdRP) and PTGS

RdRP acitvity in plants is known for several decades and Dougherty and coworkers proposed in 1993, while studying transgene induced virus resistance, the involvement of RdRP in PTGS. In collaboration with M. Wassenegger, who cloned the first RdRP gene from tomato, we cloned two RdRP homologues, RdRP1 and RdRP2, from petunia. Petunia plants have been selected that carry a transposon-inactivated RdRP1 or RdRP2 gene and we are currently testing the effect of these mutations on PTGS of flower pigmentation genes. In the meantime, evidence has been obtained from PTGS/RNAi mutants of Neurospora crassa (qde1) and C. elegans (ego1) that RdRP is indeed implicated in PTGS.
Plants turn out to contain several RdRP paralogues. We are examining whether they all have a function in virus and transposon defence by participating in PTGS/RNAi or have other functions,
for example by controlling the expression of particular plant genes.

Relevant publications:
- Van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. M., and Stuitje, A. R. (1990). Flavonoid genes in  petunia: Addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2, 291-299.
- Van Blokland, R., Van der Geest, N., Mol, J. N. M., and Kooter, J. M. (1994). Transgene-mediated  suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. Plant J. 6, 861-877.

- Mol, J. N. M., Van Blokland, R., De Lange, P., Stam, M., and Kooter, J. M. (1994). Post-transcriptional inhibition of gene expression: sense and antisense genes. In Homologous recombination and gene silencing in plants, J. Paszkowski, ed. (Dordrecht, The Netherlands: Kluwer), pp. 309-334.

- De Lange, P., Van Blokland, R., Kooter, J. M., and Mol, J. N. M. (1995). Suppression of flavonoid pigmentation genes in Petunia hybrida by the introduction of antisense and sense genes. In Gene silencing in higher plants and related phenomena in other eukaryotes, P. Meyer, ed. (Berlin: Springer-Verlag), pp. 57-76.

- Fransz, P. F., Stam, M., Montijn, B., Ten Hoopen, R., Wiegant, J., Kooter, J. M., Oud, O., and Nanninga, N. (1996). Detection of single-copy genes and chromosome rearrangements in Petunia hybrida by fluorescence in situ hybridization. Plant J. 9, 767-774.

- Stam, M., De Bruin, R., Kenter, S., Van der Hoorn, R. A. L., Van Blokland, R., Mol, J. N. M., and Kooter, J. M. (1997). Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats. Plant J. 12, 63-82.

- Stam, M., Mol, J. N. M., and Kooter, J. M. (1997). The silence of genes in transgenic plants. Ann. Rev. of Botany 79, 3-12.

- Stam, M., Viterbo, A., Mol, J. N. M., and Kooter, J. M. (1998). Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: Implications for posttranscriptional silencing of homologous host genes in plants. Mol. Cell. Biol. 18, 6165-6177.

- Kooter, J. M., Matzke, M. A., and Meyer, P. (1999). Listening to the silent genes: Transgene silencing research identifies new mechanisms of gene regulation and pathogen control. Trends Plant. Sci. 4, 340-347.

- Stam, M., De Bruin, R., Van Blokland, R., Ten Hoorn, R. A. L., Mol, J. N. M., and Kooter, J. M. (2000). Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci. Plant J. 21, 27-42.

- Sijen, T., and Kooter, J. M. (2000). Post-transcriptional silencing: RNAs on the attack or on the defensive? BioEssays 22, 520-531.

- Sijen, T., Vijn, I., Rebocho, A., Van Blokland, R., Roelofs, D., Mol, J. N. M., and Kooter, J. M. (2001). Transcriptional and posttranscriptional silencing are mechanistically related. Curr Biol 11, 436-440.

- Matzke, M., Matzke, A. J., and Kooter, J. M. (2001). RNA: guiding gene silencing. Science 293, 1080-1083.

- Kooter, JM (2005) RNA interference: Double-stranded RNAs and the processing machinery. In Plant Epigenetics. Edited by Peter Meyer. Blackwell Press.