of gene expression and silencing in transgenic plants
and objectives (1)
The introduction of novel traits in plants is an extremely powerful tool
in agricultural research and development. Engineering such traits can
be classified into two groups. Either the introduced gene encodes a protein
which itself brings a new structural or regulatory property to the plant,
or the transgene serves as a tool to regulate the expression of an endogenous
gene or gene family. For both strategies, it is essential that the engineered
trait behaves as expected, over several generations and in different genetic
backgrounds. A process known as gene silencing leads to alterations in
gene expression. Extensive selection programmes performed by several biotechnology
companies and in research laboratories, have shown that only a minor fraction
of the transgenic lines obtained, meet the plant breeders' requirements.
The aim of
this project was to develop 'know how' for strategies that maximise the
chances of obtaining stable activity of transgenes, by understanding the
molecular mechanisms (transcriptional and post-transcriptional) that regulate
gene silencing in transgenic plants.
(1) This project was a direct follow-on of the Training
and Mobility of Researchers network Gene silencing in transgenic
plants: Molecular analysis of underlying mechanisms and approaches towards
stabilisation of transgene activity (Contract Number: CHRX-CT94-0530;
Contractual Period: 01.11.1994 - 31.10.1997).
Approach and methodology
To identify the structural and context-specific requirements for the stable
expression of transgenes, we wished to define the influence of transcription,
DNA sequence, DNA methylation, sequence homology and secondary structures
on gene silencing. Likewise, the role of the genetic background and the
spatial arrangement of homologous sequences in the chromosomes and in
the nucleus was also examined. Secondly, to understand and exploit gene
silencing mechanisms, we studied the importance of RNA transcription,
the production of aberrant transcripts and antisense RNA and the spatial
arrangement of homologous sequences and transcripts. Finally, environmental
and physiological conditions that favour stable transgene activity were
defined by conducting a comparative study of factors favouring stable
expression or gene silencing.
Main findings and outcome
As a result of the work of the silencing network, we now understand that
transcriptional silencing is due to chromatin remodelling processes which
package the transgene DNA into a chromatin structure blocking the access
of enzymes required for its transcription. Certain structural features,
especially inverted repeats and other repetitive elements, appear to be
recognised as specific targets for transcriptional silencing.
Post-transcriptional gene silencing (PTGS) is due to specific degradation
of transgene RNA and also targets any other RNA with homology to the transgene,
but does not affect transcription. PTGS therefore blocks the normal processing
pathways of the transgene, and of endogenous genes which share homology
with the transgene. A certain threshold level of transgene RNA is needed
to induce PTGS and our studies have shown that specific structural features,
such as stem loops, strongly enhance the susceptibility of a transgene
to PTGS. This suggests that PTGS requires the production of double-stranded
RNA. These results will be most valuable for the design of transgenes
with highly enhanced PTGS features.
This project includes two research laboratories working on gene silencing
mechanisms in filamentous fungus. Studies in Neurospora crassa
enabled cloning of the first genes that have been identified as encoding
proteins involved in PTGS. Genes with a similar function are probably
responsible for PTGS in plants, making the Neurospora crassa genes
highly valuable for future collaborative work seeking to prevent or enhance
PTGS via modification of the activity of these crucial genes. Other partners
in this project have identified genes involved in transcriptional gene
silencing as well as specific target sequences for transcriptional silencing,
allowing us to make predictions and recommendations to the scientific
community about the likelihood of transcriptional silencing for individual
Other results from the project have demonstrated the importance of considering
genetic background (choice of cultivar) as a factor influencing the level
of silencing of an endogenous gene. Furthermore, it was shown that RNA
signals can trigger promoter methylation and transcriptional silencing
and that viral sequences can be integrated into plant DNA and contribute
to plant genome evolution. From our investigations, it was clear that
gene silencing mechanisms are not specific for transgenes but that they
represent ancient tools which have evolved to protect plants against mobile
elements, such as transposons or retroviruses, and for protection against
This research should assist breeders and farmers to define culture conditions
and propagation techniques that improve the control of gene silencing.
The collaboration of the network partners has not only opened a route
towards improved control and exploitation of gene silencing mechanisms,
it has also provided new insight into fundamental regulatory mechanisms
used by plants, filamentous fungi and, quite probably, most other higher
Cogoni C. and Macino G., Gene silencing in Neurospora crassa
requires a protein homologous to RNA-dependent RNA polymerase.
1999, pp. 166-169.
Cogoni C. and Macino G., Posttranscriptional Gene Silencing
in Neurospora by a RecQ DNA Helicase.
1999, pp. 2342-2344.
Kooter J.M., Matzke M.A. and Meyer P., Listening to the silent
genes: Transgene silencing research identifies new mechanisms of
gene regulation and pathogen control.
Trends Plant. Sci., 4,
1999, pp. 340-347.
Mette M.F., van der Winden J., Matzke M.A. and Matzke A.J.M., Production
of aberrant promoter transcripts contributes to methylation and
silencing of unlinked homologous promoters in trans.
EMBO J., 18, 1999, pp. 241-248.
Metzlaff M., O'Dell M., Cluster P.D. and Flavell R.B., RNA-Mediated
RNA degradation and Chalcone Synthase A silencing in Petunia.
1997, pp. 845-854.
November 1996 - January 2000
University of Leeds (UK)
Plant Genetic Systems
Universiteit Gent (BE)
R. Flavell, R. Hellens
John Innes Centre
University of Nottingham (UK)
Università "La Sapienza"
Austrian Academy of Sciences
O. Mittelsten Scheid
Friedrich Miescher Institute
ZENECA Plant Sciences
Wageningen Agricultural University (NL)
Institut Jacques Monod