The expression of a baculovirus-derived chitinase gene increased resistance of tobacco cultivars to brown spot (Alternaria alternata)

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase gene coding region was amplified using the polymerase chain reaction, inse rted into a plasmid (pROK-2) and replicated in Escherichia coli XL1- blue. The recombinant plasmid was mobilised into Agrobacterium tumefaciens LBA 44 04 and inoculated into tobacco leaf discs. The presence of the expressed ch itinase in foliar tissue of kanamycin-resistant plantlets of three Nicotian a tabacum cultivars (CF80, K326 and Xanthi-nc) was inferred using immunoblo tting, and enzyme activity was confirmed using a fluorometric assay. Confoc al laser scanning microscopy with immunofluorescent staining of foliar sect ions from N. tabacum Xanthinc expressing the viral chitinase indicated that the enzyme was restricted to the vascular tissue. Heliothis virescens larv ae fed on leaf tissue expressing chitinase were not impaired either in thei r development to pupation or in their feeding behaviour, in comparision wit h their counterparts that had consumed similar amounts of untransformed tob acco leaf tissue. By contrast, when tobacco leaves were mechanically inocul ated with Alternaria alternata, very few brown spots were observed at inocu lation sites in chitinase-expressing tissue, whereas large and spreading le sions formed in untransformed tobacco tissue. Of all lines that were transf ormed, as determined by kanamycin resistance, 59% had fewer symptoms of dis ease (smaller disease indices) than those for untransformed controls.