Articles
GENETIC ENGINEERING OF PEAR FOR INCREASED FIRE BLIGHT RESISTANCE
Article number
538_113
Pages
639 – 643
Language
Abstract
Fire blight caused by Erwinia amylovora is a major disease of the European pear (Pyrus communis L.). The pear genetic engineering program which began in 1993 at INRA Angers led to the development of an efficient transformation protocol based on co-culture of in vitro leaves with Agrobacterium tumefaciens hypervirulent strain EHA101 and bud regeneration in presence of kanamycin.
Two types of strategies have been chosen: 1) a direct lytic action on the pathogen with transgenes encoding lytic peptides from insect origin (attacin, cecropin) or lysozyme isolated from the T4 bacteriophage; 2) a more specific inhibition of pathogenicity factors using a lactoferrin gene of bovin origin (for its possible competition with the bacterial siderophores) or a depolymerase gene (able to degrade specifically the exopolysaccharides). Transformation experiments with these various genes produced so far a total of 81 transgenic clones from the cultivar ‘Passe Crassane’. Integration of the transgenes has been checked by PCR. In vitro fire blight inoculation tests enabled the identification of several promising clones with each transgene.
A method of semi-quantitative RT-PCR revealed important differences among clones expressing the Attacin E gene.
These differences correlated very well with the differences of transgenic attacin E accumulation revealed by western blot analysis.
However, no clear correlation between transgene expression and level of resistance has been established.
Most of the transgenic clones have been successfully acclimatized and greenhouse fire blight inoculations are underway.
This program is now focusing on two directions.
In the first one, several antibacterial constructions are being evaluated.
They include the use of the lactoferrin and depolymerase genes and the simultaneous expression of the T4-lysozyme and the Attacin E genes to take advantage of a synergistic effect.
In order to obtain a more efficient and targetted expression of these transgenes, the search for inducible promoters is now our second line of research.
Two types of strategies have been chosen: 1) a direct lytic action on the pathogen with transgenes encoding lytic peptides from insect origin (attacin, cecropin) or lysozyme isolated from the T4 bacteriophage; 2) a more specific inhibition of pathogenicity factors using a lactoferrin gene of bovin origin (for its possible competition with the bacterial siderophores) or a depolymerase gene (able to degrade specifically the exopolysaccharides). Transformation experiments with these various genes produced so far a total of 81 transgenic clones from the cultivar ‘Passe Crassane’. Integration of the transgenes has been checked by PCR. In vitro fire blight inoculation tests enabled the identification of several promising clones with each transgene.
A method of semi-quantitative RT-PCR revealed important differences among clones expressing the Attacin E gene.
These differences correlated very well with the differences of transgenic attacin E accumulation revealed by western blot analysis.
However, no clear correlation between transgene expression and level of resistance has been established.
Most of the transgenic clones have been successfully acclimatized and greenhouse fire blight inoculations are underway.
This program is now focusing on two directions.
In the first one, several antibacterial constructions are being evaluated.
They include the use of the lactoferrin and depolymerase genes and the simultaneous expression of the T4-lysozyme and the Attacin E genes to take advantage of a synergistic effect.
In order to obtain a more efficient and targetted expression of these transgenes, the search for inducible promoters is now our second line of research.
Authors
E. Chevreau, M. Malnoy, F. Mourgues, M.N. Brisset, J.P. Reynoird
Keywords
gene transfer, fire blight, antibacterial peptides, Erwinia amylovora, pear
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