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, inserted into a plasmid (pROK‐2) and replicated in Escherichia coli XL1–blue. The recombinant plasmid was mobilised into Agrobacterium tumefaciens LBA 4404 and inoculated into tobacco leaf discs. The presence of the expressed chitinase in foliar tissue of kanamycin‐resistant plantlets of three Nicotiana tabacum cultivars (CF80, K326 and Xanthi‐nc) was inferred using immunoblotting, and enzyme activity was confirmed using a fluorometric assay. Confocal laser scanning microscopy with immunofluorescent staining of foliar sections from N. tabacum Xanthi‐nc expressing the viral chitinase indicated that the enzyme was restricted to the vascular tissue. Heliothis virescens larvae fed on leaf tissue expressing chitinase were not impaired either in their development to pupation or in their feeding behaviour, in comparision with their counterparts that had consumed similar amounts of untransformed tobacco leaf tissue. By contrast, when tobacco leaves were mechanically inoculated with Alternaria alternata, very few brown spots were observed at inoculation sites in chitinase‐expressing tissue, whereas large and spreading lesions formed in untransformed tobacco tissue. Of all lines that were transformed, as determined by kanamycin resistance, 59% had fewer symptoms of disease (smaller disease indices) than those for untransformed controls.