Fire blight is the major bacterial disease of Maloideae caused by the necrogenic bacterium Erwinia amylovora, which pathogenicity requires a functional type III protein secretion system (TTSS). The protein harpin NEa is secreted through the TTSS and targeted to the intercellular spaces of plant tissues where it acts as an elicitor of plant defense reactions. In order to create novel mechanisms for fire blight resistance in apple, we have generated transgenic apples expressing the hrpN gene from E. amylovora under the control of the constitutive CaMV35S or the pathogen-inducible tobacco Str246C promoters, with or without the tobacco PR1 gene secretion signal peptide. The characterization of these harpin NEa transgenic apples was carried out by: (1) quantification of the constitutive and induced expression of the transgene (by Q-RT-PCR); (2) in planta localization of the harpin NEa protein; (3) evaluation of fire blight resistance after greenhouse inoculation; (4) targeted transcriptomic analysis to identify defenses induced by the presence of the transgene.