The mechanisms and control of genetic recombination in plants EU-Sponsored Research on Safety of Genetically Modified Organisms
EU 9sep99

Background and objectives

Biotechnology relies to a large extent on our ability to introduce foreign genes into cells. A major problem with present day technology is the non-predictability of the integration of such transgenes. DNA introduced into plant cells mostly integrates at random, i.e. at non-predetermined positions of the genome. The biological process ultimately responsible for random integration is known as illegitimate recombination. DNA integrated at random frequently contains multiple copies and often copies are scrambled. Multiple copies also often induce gene silencing and hence instability in the expression of the introduced genes. In addition, the DNA integrates at loci of unknown stability and capacity for expression and randomly integrated copies may induce unpredictable and undesirable mutations in the host genome. Gene targeting is based on homologous recombination and thus allows the integration of DNA at predetermined positions and therefore allows precision manipulation of genes. To make gene targeting commonly available as a tool for plant biotechnology, illegitimate and homologous recombination pathways need to be studied so as to allow the development of reliable and efficient gene targeting methods.

The consortium aims to identify and analyse the control and molecular mechanisms of genetic recombination in plant cells. An understanding of these processes is expected to provide the means to:

1) control the chromosomal location and structure of transgenic loci;
2) control the level of recombination;
3) understand the way in which plants sense chromosomal integrity and DNA damage.

Approach and methodology

Genetic and biochemical methods were used to identify key parameters or key genes that determine the efficiencies of homologous and non-homologous recombination in plant cells. Factors stimulating the homologous pathway were over-expressed and the expression of factors promoting non-homologous recombination was suppressed. The effects on gene targeting were then studied in model systems.

Main findings and outcome

Homologous and non-homologous recombination pathways govern the fate of DNA after transformation. To improve gene targeting, either the non-homologous pathway needs to be suppressed or the homologous pathways must be stimulated. Several avenues were pursued to stimulate the homologous route. Mutants were generated which exhibit elevated levels of homologous recombination and these mutants are currently being analysed. DNA damage repair and homologous recombination share part of their pathways. Therefore mutants sensitive to DNA damaging agents were isolated. One of the genes isolated this way contributes to the maintenance of chromosomes and has also been shown to be important for efficient homologous recombination in plants. Over-expression of a bacterial recombination protein has been shown to stimulate homologous recombination in plants. Despite this, the protein does not increase the frequencies of gene targeting when Agrobacterium is used for transformation, presumably because this recombination protein cannot access its substrate when the Agrobacterium DNA delivery system is used. In line with this observation, once recombination is initiated, however, this protein improves the precision of the recombination reaction. To elucidate the non-homologous pathway of recombination, various plant genes involved are being analysed. To facilitate this study, a yeast system was developed which allows the analysis of the T-DNA transfer process in yeast. This system makes the power of yeast genetics and its repertoire of recombination genes available to plants. A key discovery was that the non-homologous pathway apparently is suppressed in tissues with high rates of homologous recombination. This finding allows a rational approach to the suppression of non-homologous recombination in the cells routinely used for plant transformation.

Conclusions

Although our understanding of the general biology of recombination in plants is constantly improving, we still lack the knowledge for precision engineering of plants' genes. A further investment in basic mechanisms of recombination will be necessary to develop gene targeting as a tool for the genetic manipulation of plants.

Major publications

Albinsky D., Masson J., Bogucki A., Afsar K., Vass I., Nagy F., Paszkowski J., “Plant responses to genotoxic stress are linked to an ABA/salinity signalling pathway”. Plant J., 17, 1999, pp. 73-82.

Goedecke W., Eijpe M., Offenberg H.H., van Aalderen M. and. Heyting C., “MRE11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis”. Nature Genetics, 23, 1999, pp. 194-198.

Hohn B. and Puchta H., “Gene therapy in plants”. Proc. Natl. Acad. Sci. USA, 96, 1999, pp. 8321-8323.

Kovalchuk I., Kovalchuk O., Arkhipov A., Hohn B., “Transgenic Plants are Sensitive Bioindicators of Nuclear Pollution Caused by the Chernobyl Accident”. Nature Biotechnology, 16, 1998, pp. 1054-1057.

Mengiste T., Revenkova E., Bechtold N., and Paszkowski J., “An SMC-like protein is required for efficient homologous recombination in Arabidopsis”. EMBO J, 18, 1999, pp. 4505-4512.

Reiss B., Schubert I., Köpchen K., Wendeler E., Schell J., and Puchta H., “RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium”. Proc. Natl. Acad. Sci. USA, 97, 2000, pp. 3358-3363

Project Information

Project website address
http://www.epsoweb.org/catalog/EU/science/projects/2028.htm

This website provides a comprehensive review of the results of EC-supported research into the safety of Genetically Modified Organisms.

It presents research carried out under successive EC Framework Programmes for Research and Technological Development from 1985 (Biotechnology Action Programme) to 2000 (Fifth Framework Programme).

Contract number BIO4-CT97-2028

Period
October 1997 – September 2000

Coordinators
C. White Université Blaise Pascal Aubière (FR)
B. Reiss Max-Planck-Institut für Züchtungsforschung Köln (DE)
K. Metzlaff University of Ghent (BE)

Partners
B. Hohn, J. Paszkowski Friedrich Miescher Institute Basel (CH)
P. Hooykaas Leiden University (NL)
G. Freyssinet Rhône-Poulenc Agrochimie Lyon (FR)
C. Heyting Wageningen Agricultural University (NL)
H. Puchta Institute for Plant Genetic and Crop Plant Research Gatersleben (DE)
R. Nehls Kleinwanzlebener Saatzucht Institute of Plant Breeding Einbeck (DE)

source: http://europa.eu.int/comm/research/quality-of-life/gmo/01-plants/01-14-project.html 7oct01