5.4.1
TRANSGENIC RICE WITH ENHANCED RESISTANCE TO RICE BLAST BY CONSTITUTIVE EXPRESSION OF THE RICE CHITINASE GENE

Y NISHIZAWA1, Z NISHIO2, M SOMA2, K NAKAZONO2, M UGAKI1 and T HIBI2

1National Institute of Agrobiological Resources, Tsukuba, 305-8602, Japan; 2Department of Agricultural and Environmental Biology, University of Tokyo, Tokyo 113-8657, Japan

Background and objectives
Plant chitinases have been shown to inhibit fungal growth in vitro. We reported that three class I chitinase genes were isolated from rice and that their expression was activated by chitin oligomers [1]. Recently we have shown that transgenic plants (tobacco, strawberry and cucumber) constitutively expressing the rice chitinase gene showed increased resistance to fungal diseases [2]. The objective of the present study is to create transgenic rice plants with enhanced resistance against rice blast, the most important disease in Japan, through reintroduction of the chitinase gene under the control of the enhanced CaMV 35S promoter.

Materials and methods
The coding regions from rice chitinase clones RCC2 or RCG3, with the enhanced CaMV 35S promoter which possesses four tandemly-repeated enhancer regions of its own, and a hygromycin phosphotransferase gene with a CaMV 35S promoter, were cloned into the T-DNA region of a binary vector. Calli induced from scutella of Oryza sativa ssp. japonica were transformed using the Agrobacterium-mediated method. The regenerated hygromycin-resistant plants were inoculated with a conidial suspension of Magnaporthe grisea and disease resistance was evaluated by comparing disease severity with that of the control plants.

Results and conclusions
Several rice cultivars, including a commercially important cv. Koshihikari, were transformed with the rice chitinase gene under the control of the enhanced CaMV 35S promoter. The R0 generation plants, which were confirmed to possess and express the transferred gene by PCR and RT-PCR, showed enhanced resistance to rice blast disease. A few small lesions without further expansion appeared on the inoculated leaves of the transgenic rice and the plants continued normal growth. On the other hand, many lesions formed on the non-transformed control plants and kept expanding. The plants were finally blasted. These results indicate that this approach might be useful to breed blast-resistant transgenic rice plants. The genetic stability and disease resistance of the R1 generation plants are now under investigation.

References
1. Nishizawa Y, Kishimoto N, Saito A, Hibi T, 1993. Molecular and General Genetics 241, 1-10.
2. Asao H, Nishizawa Y, Arai S et al., 1997. Plant Biotechnology 14, 145-149.