5.3.17
ENHANCED FIRE BLIGHT RESISTANCE OF APPLE LINES TRANSGENIC FOR ATTACIN E AND T4 LYSOZYME GENES

HS ALDWINCKLE1, K KO2, JL NORELLI1, SK BROWN2 and K DURING3

Departments of 1Plant Pathology, and 2Horticultural Science, Cornell University, Geneva, NY 14456, USA; 3Federal Center for Breeding Research on Cultivated Plants, D-06484 Quedlinburg, Germany

Background and objectives
The apple cultivar Gala has become popular in the USA and elsewhere because of its excellent fruit quality. However, Gala is very susceptible to Erwinia amylovora, the bacterium causing fire blight. The main goal of this research is to genetically transform Gala with genes for lytic proteins known to inhibit bacteria, and to determine their effect on fire blight resistance. New plasmid binary vectors have been constructed to increase the expression of the attacin E (attE) lytic protein [2] and to allow for combined expression of both attE and T4 lysozyme (e) [1] in transgenic plants. These plasmid binary vectors have been transferred to Galaxy, a sport of Gala.

Materials and methods
Genes encoding lytic proteins, attE and e, were cloned into six different plasmid binary vectors for use in Agrobacterium-mediated transformation of apple. The plasmids were: pBINCa2Att (enhanced CaMV 35S promoter (Ca2)/attE/NOS terminator in pBIN19), pCa2AMVAtt (Ca2/untranslated leader sequence of alfalfa mosaic virus (AMV)/attE/NOS terminator in pBI121), pCa2AMVSPAtt (Ca2/AMV/signal peptide of tobacco PR1 protein (SP)/attE/NOS terminator in pBI121), pWIAtt (potato proteinase inhibitor II promoter (WI)/attE/WI terminator in pBI121), pWIAttCa2AMVT4 (WI/attE/WI terminator/Ca2/AMV/e /NOS terminator in pBI121), and pCa2AMVT4 (Ca2/AMV/e/NOS terminator in pBI121). Transformation of Galaxy apple with binary vectors was confirmed by PCR and NPT II ELISA analysis. Western analysis was used to study gene expression in apple. Galaxy transgenic lines were obtained, rooted, acclimated to soil and grown in a growth chamber at 26°C, 80% RH. Vigorously growing shoot tips of 20-cm-tall plants were inoculated by injection of 7.5x107 c.f.u./ml of E. amylovora (Ea 273). Lengths of resultant necrotic lesions were measured at intervals from 4-37 days after inoculation, and expressed as percentage of total shoot length that blighted. The attE coding region was cloned into the pRSET E. coli expression vector for attacin E protein purification. Western analysis was used to confirm attE expression. Polyclonal antibody to attacin was produced by injecting a rabbit with antigen produced in E. coli.

Results and conclusions
All six constructs containing attE, e or both were successfully transferred to Galaxy by Agrobacterium-mediated transformation. 84 Galaxy transgenics have been recovered to date. Expession of attE in transgenic lines was confirmed by Western analysis. Inoculation of transgenic lines indicated that some lines have increased resistance to fire blight. Attacin E was produced using the pRSET E. coli expression vector and purified. Maximum expression of attacin occurred 3-5 h after induction of the pRSET vector. The purified attacin E and T4 lysozyme were used successfully to obtain polyclonal antibodies for protein analysis. Purified attacin E and T4 lysozyme will be used for in vitro assays of antimicrobial activity. Current work is focusing on characterizing the Galaxy transgenics and evaluating the effect of the lytic proteins on disease resistance.

References
1. During K, Porsch P, Fladung M, Lörz H, 1993. Plant Journal 3, 587-598.
2. Norelli JL, Aldwinckle HS, Destefano-Beltran L, Jaynes JM, 1994. Euphytica 77, 123-128.