ISHS Acta Horticulturae 338: VI International Workshop on Fireblight
INCREASING THE FIRE BLIGHT RESISTANCE OF APPLE BY TRANSFORMATION WITH GENES ENCODING ANTIBACTERIAL PROTEINS |
| Authors: | J. Norelli, H. Aldwinckle, L. Destéfano-Beltrán, J. Jaynes |
| Abstract:
Gene transfer systems for agricultural crops will allow the development of improved disease resistant cultivars by strategies that are infeasible by classical breeding. One strategy made possible by biotechnology is to look beyond the plant kingdom for genetic diversity that may be useful for pest control in plants. Insects are capable of a resistance response to bacterial infection, known as humoral immunity, that results in the production of several lytic proteins. Cecropin B and Attacin A are naturally occurring lytic proteins from Hyalophora cecropia, the Giant Silk Moth. Two 38-amino acid peptides, SB-37 and Shiva-1, were designed and chemically synthesized as substitution analogs of Cecropin B. SB-37 is a close analog of the natural peptide, whereas Shiva-1 has only 46% amino acid homology. Attacin A, SB-37, and Shiva-1 are cytotoxic in vitro to a number of different species of plant pathogenic bacteria at micromolar concentrations, with Shiva-1 having the greatest activity. Shiva-1 is also highly cytotoxic to Erwinia amylovora with LC50 values (LC50 is the concentration necessary to kill 50% of the test organism) for three different strains ranging from 28 to 197 nM. Genes encoding these lytic proteins have been synthesized and cloned into Agrobacterium binary vectors under the control of constitutive promoters derived from the 35S promoter of cauliflower mosaic virus and the wound-inducible proteinase II promoter from potato. A. tumefaciens, strain LBA4404, was subsequently transformed with these constructs and tobacco tissue was infected. Beta-glucuronidase (GUS)-positive and kanamycin-resistant plants were obtained indicating effective transformation. Southern analysis has confirmed the presence of single copies of the genes integrated into the genomes of individual plants. Bioassays to test disease resistance of R1 progeny indicate that, compared to transgenic control plants, Shiva-1 seedlings exhibited delayed symptoms and reduced disease severity after inoculation with Pseudomonas solanacearum. We are currently attempting to transform apple with these DNA constructs and to determine the effect of lytic protein production in | |
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