Manipulating biochemical pathways in rice
The brown planthopper, Nilaparvata lugens, is a pest of rice in tropical regions. Its direct feeding results in loss in yield and plant death ("hopper bum"). Several compounds that stimulate insect attraction have been detected in rice plants colonised by N. lugens, including 1,2-dimethoxybenzene or veratrole. Electro-physiological studies and highresolution gas chromatography have identified veratrole as an attractant of N. lugens. Veratrole is a product of salicylic acid, a derivative of the phenyl propanoid pathway. Salicylic acid is decarboxylated to catechol, a step which is encoded by salicylate hydroxylase. Catechol is subsequently methylated to veratrole, which is released as a volatile compound from rice leaves. Mature scutellum-derived rice calli from (Oryza sativa) cv.Taipei 309 were transformed, using microprojectile bombardment, with pROB5 containing the hpt gene conferring resistance to the antibiotic hygromycin and pSLJ7307 carrying the nahG gene derived from Pseudomonas putida and coding for the enzyme salicylate hydroxylase. Following selection on hygromycin-containing medium, 17 independent transgenic rice plants were regenerated from >3600 bombarded calli, with a transformation frequency of 0.47%. Transgenic plants were confirmed by RT-PCR. Plant lines were classified as high expressors (10 lines) and low expressors (7 lines) depending on salicylate hydroxylase production. All transgenic lines exhibited higher enzyme activity than wild-type plants. Transgenic plants produced had altered metabolism for antioxidant enzymes such as catalase, ascorbate peroxidase and superoxide dismutase and reactive oxygen species such as hydrogen peroxide. Plants unable to accumulate salicylic acid exhibited delayed transcription of pathogenesis related genes and may therefore be compromised in their ability to respond to pathogen attack and mechanical wounding. Enhanced veratrole production was corroborated using gas chromatography of volatiles released from transgenic undamaged and mechanically damaged plants. Bioassays indicated that N. lugens were more attracted to high expressing plants than to wild-type plants, making more visits to areas containing transgenic rice leaves than areas containing non-transformed leaves and spending longer in these areas. Manipulating the production of veratrole by enhancing salicylate hydroxylase activity has therefore modified attraction of the N. lugens for high expressing nahG positive rice plants.