experimental approach to investigate horizontal gene transfer between organisms
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
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.
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.
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.
October 1991 September 1993
University of East Anglia
School of Biological Sciences
Centre de Versailles (FR)