ISHS Acta Horticulturae 489: VIII International Workshop on Fire Blight
GENE TRANSFER FOR FIRE BLIGHT RESISTANCE IN PEAR |
| Authors: | E. Chevreau, F. Mourgues, J.P. Reynoird, M.N. Brisset |
| Keywords: | gene transfer, antibacterial peptides, Erwinia amylovora, pear |
| Abstract:
A pear genetic engineering program was begun in 1992 at INRA Angers, with the aim of increasing fire blight resistance of several important European cultivars that are very susceptible to this disease. Several lytic peptides from insects are highly bactericidal. Our team has demonstrated that Cecropin B and its analogs (SB-37 and Shiva-1) have a high antibacterial activity against Erwinia amylovora, without inhibiting the growth of pear cell suspensions. However, Cecropin B is partially degraded after incubation with pear extracellular fluids. Attacin E, another lytic peptide of similar origin, previously reported to be active in transgenic apple, has also been used in our program. A transformation protocol based on co-culture of in vitro leaves with Agrobacterium tumefaciens hypervirulent strain EHA101 has been developed for several pear cultivars. Transformation experiments with the antibacterial genes SB-37, Shiva-1 and Attacin E, under the control of the constitutive promoter CaMV35S, with or without a signal peptide, produced a total of 37 transgenic clones from the cultivar Passe Crassane and 24 from cv. Conférence. Integration and expression of the transgenes in all Passe Crassane transformants have been checked by PCR and RT-PCR, respectively. Semi-quantitive RT-PCR revealed important differences in expression among clones. These differences correlated very well with the differences of transgenic Attacin E accumulation revealed by western blot analysis. In vitro fire blight inoculation tests were performed on all cv. Passe Crassane transgenic clones, and enabled the identification of several promising clones with each transgene. All transgenic clones have been successfully acclimatized and greenhouse fire blight inoculations are underway. Future directions of this program include the use of other antibacterial genes (T4 lysozyme, lactoferrin), alone or in combination, and the search for promoters specifically induced by bacterial infection. | |
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