5.3.S
INCREASED FUNGAL RESISTANCE OF TRANSGENIC PLANTS BY HETEROLOGOUS EXPRESSION OF BACTERIOPHAGE T4 LYSOZYME GENE

DJ STAHL1, A MASER1, J DETTENDORFER1/sup>, B HOLTSCHULTE2, JE THOMZIK3, R HAIN3 and R NEHLS1

1PLANTA GmbH, Grimsehistrasse 31, D-37574 Einbeck, Germany; 2KWS AG, Grimsehistrasse 31, D-37574 Einbeck, Germany; 3Bayer AG, PF-E/Fu, Institut fur Biotechnologie, D-51368 Leverkusen, Germany

Background and objectives
In recent years increasing progress has been made in improving fungal resistance of plants by genetic engineering. High-level synthesis of antifungal proteins, such as chitinases, glucanases and ribosome-inactivating proteins, the production of low molecular-weight fungitoxic compounds, such as resveratrol, or the generation of a hypersensitive cell death at the infection site, resulted in plants with enhanced fungal resistance. Here we report that the expression of the bacteriophage T4 lysozyme gene represents a novel approach for engineering fungal disease resistance in plants. Lysozymes constitute a widespread family of bacteriolytic enzymes which are found in all living species including plants [1]. Effective use of bacteriophage T4 lysozyme gene in transgenic potato plants to achieve enhanced bacterial resistance has been reported [2].

Results and conclusions
The T4 lysozyme gene was introduced into winter- and summer-type oilseed rape and into tomato plants by Agrobacterium-mediated transformation. In the transformation vector used the expression of the lysozyme gene was under control of the 1' end of the mannopine synthase promoter (MAS). Transcripts of the lysozyme gene could not be detected in leaves of transgenic oilseed rape by RNA blot analysis. However, RT-PCR analysis revealed that the phage gene is expressed at a low level in plants.

Resistance assays were performed under greenhouse conditions to evaluate the resistance level of the T4 lysozyme plants. Transgenic plants derived from different cultivars of summer- and winter-type oilseed rape exhibited an increased tolerance to Phoma lingam, the causal agent of blackleg disease. Resistance was evaluated by scoring the severity of stem cankers 2, 4, 6 and 8 weeks after inoculation. The resistance mechanism conferred by the T4 lysozyme gene seemed to be active all the time as shown by the temporal development of disease symptoms. Transgenic tomato plants showed an enhanced tolerance to Phytophthora infestans. The number of lesions and the spread of the fungus were reduced significantly on inoculated tomato leaves under high infection pressure.

These results indicate that the introduction of the T4 lysozyme gene into plants is a promising approach to improve fungal disease resistance. However, the molecular mechanism of resistance is still unknown. Experiments are in progress to describe the kind of resistance at the cellular and molecular level.

Reference
1. During K, 1996. Molecular Breeding 2, 297-305.
2. During K, Porsch P, Fladung M, Lbrz H, 1993. Plant Journal 3, 587-598.