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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image An experimental approach to investigate horizontal gene transfer between organisms

Background and objectives

Horizontal gene transfer is the movement of genetic information between species. There is much evidence that horizontal transfer occurs between different bacteria and in some specialised situations between bacteria and plants. However, there is only circumstantial evidence for horizontal transfer between different eukaryotic organisms (that is higher plants and animals).

Retrotransposons are ubiquitous mobile elements in plant and fungal genomes. The retrotransposons in different species are very similar, which suggests the existence of horizontal gene transfer. This study aimed to detect and measure the frequency of horizontal transfer of retrotransposons between a plant and its fungal pathogen. The discovery of this type of process would have a great impact on the biological species concept and on considerations of the safety of release of genetically modified organisms.


Approach and methodology

The chosen system involved the fungal pathogen, Cladosporium fulvum and its host species, tomato. We created chimaeric retrotransposons containing marker genes, such as antibiotic resistance, whose transfer from the plant to the fungus could be detected. We introduced the chimaeric elements into the tomato and C. fulvum genomes and fungal spores acquiring the marker gene were sought.
We isolated the Tnt1 (tobacco) and the CfT-1 (C. fulvum) retrotransposon elements and assessed the conditions of their expression.

Tobacco plant Tobacco plant.


Main findings and outcome

Expression of CfT-1 in C. fulvum was tested by analysis of fusions between the promoter of the transposon (the LTR) and the reporter gene, GUS. Transformation of C. fulvum with this construct demonstrated that expression of LTR is enhanced during starvation, a condition known to prevail during infection. The plant 35S promoter, used to drive antibiotic resistance, was functional in C. fulvum.

Examination of the possible causes of the high level of sequence identity between retrotransposons established the phylogeny of retrotransposons in C. fulvum in relation to those in Ascomycete fungi and other members of the Cladosporium genus. Phylogenetic analysis of retrotransposons found in C. cladosporioides, CcT-1, and CfT-1 was consistent with vertical transfer, but did not rule out horizontal transfer.

Tnt1 is poorly expressed in the tobacco plant, except in their roots. However, Tnt1 expression was strongly induced during tobacco leaf mesophyll protoplast isolation due to the application of fungal extracts containing cell wall hydrolases. Creation of transgenic plants containing a transcriptional fusion between Tnt1 LTR, carrying the promoter and regulatory sequences, and the GUS reporter gene, showed that Tnt1 expression is strongly induced in tobacco by several factors of microbial origin, such as fungal elicitins produced by Phythophtora species, and culture supernatants of the bacterium Erwinia chyrsanthemi. All the inducing factors tested elicited hypersensitive defence responses in tobacco plants. Tnt1 expression was closely correlated with the necrotic activity of elicitins. In tomato, Tnt1 expression was also strongly induced by fungal extracts and elicitins.

All inducing factors tested elicit plant necrotic defence responses leading to a non-specific incompatible interaction. Thus we attempted determine if Tnt1 induction is restricted to incompatible interactions by studying the gene - gene interactions between tomato and C. fulvum. We found that an interaction between the product of specific elicitors produced by C. fulvum avirulence genes (Avr) and the corresponding plant resistance genes (Cf) was responsible for a plant necrotic response leading to resistance to the pathogen. We, therefore, introduced the Cf9 resistance gene into the LTR-GUS transgenic tomato lines, and tested the effect of the C. fulvum Avr9 gene product. The Avr9 gene product activated Tnt1 expression in tomato lines containing the Cf9 resistance gene, but not those devoid of the Cf9 resistance gene.


Conclusions

The tobacco retrotransposon is expressed in tomato and is therefore a suitable tool for the experimental testing of horizontal transfer between tomato and C. fulvum. Tnt1 expression in tomato and tobacco is linked to the plant response to micro-organisms and may lead to resistance of the plants to the pathogen.
Modifications of the Tnt1 promoter to create constitutively expressed mobile elements may be required to ensure a high level of induction during a compatible reaction.

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imageResearch project
 

Contract number
BIOT-CT91-0287

Period
October 1991 – September 1993

Coordinator
R.P. Oliver
University of East Anglia
School of Biological Sciences
Norwich (UK)

 
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Partner


M.A. Grandbastien
INRA
Centre de Versailles (FR)

 
 
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