Pathogen-induced expression of harpinPss increases resistance in tobacco against fusarium oxysporum f. sp. nicotianae

Abstract

HarpinPss (encoded by the hrpZ gene), a proteinaceous elicitor produced by Pseudomonas syringae pv. syringae, induces cell death in plants through hypersensitive response (HR). With an aim to generate transgenic tobacco resistant to fungal diseases, hrpZ was expressed in a secretable form, tagged with the signal peptide (SP) of PR1a, under the constitutive 35S promoter (P35S) or pathogen-inducible promoters (PIPs) like phenylalanine ammonia lyase (PAL), osmotin (OSM), and hypersensitive-related (HSR) promoters. The constitutive expression of the secretable form of hrpZ did not permit regeneration of transformed cells due to harpinPss-induced cell death. Transformants were recovered at a low frequency (2-6%) from leaf discs infected with Agrobacterium harbouring the SP-hrpZ driven by PIPs due to wound-induced leaky expression of harpinPss. The transgenic lines were confirmed by PCR using transgene-specific primers for SP-hrpZ. The expression of hrpZ under PIPs in transgenic lines was confirmed by Western blotting after challenging the leaves with Fusarium oxysporum f. sp. nicotianae. RT-PCR analysis also confirmed the expression of SP-hrpZ driven by PIPs in transgenic tobacco upon infection with F. oxysporum f. sp. nicotianae. The expression of harpinPss in these transgenic lines was accompanied by expression of defense-response genes such as PR1, PR2, PR3, HSR and HIN1. Transgenic tobacco plants showed enhanced resistance to F. oxysporum f. sp. nicotianae. Our findings suggest the potential use of an elicitor gene (hrpZ), driven by PIPs (PAL, OSM, and HSR) for the development of resistant plants.