Transgenic expression of a viral EPS-depolymerase is potentially useful to induce fire blight resistance in apple

Fire blight caused by Erwinia amylovora is the most destructive bacterial disease of pear and apple. The goal of this research was to improve fire blight resistance of apple by transformation with a gene encoding for an extracellular polysaccharide (EPS)-depolymerase from the E. amylovora phage phi-Ea1h. The EPS-depolymerase gene (dpo) driven by the constitutive promoter CaMV35S was transferred into apple scion cv. 'Pinova' through Agrobacterium tumefaciens-mediated leaf disk transformation using a binary vector and strains EHA105 or LBA4404. The integration of the dpo gene and the marker gene nptII was confirmed by polymerase chain reaction (PCR) and by Southern hybridisation. From 4560 leaves inoculated in 23 different transformation experiments, 33 independent shoots, regenerated from a single transformation event, were identified as transgenic. Nine of the shoots were randomly selected and vegetatively propagated to establish transgenic clones. The transgenic clones were diploid as was the non-transgenic genotype 'Pinova' analysed by flow cytometry. Both transgenes (dpo and nptII) were transcriptionally active determined by reverse transcription (RT)-PCR and quantitative RT-PCR. EPS-depolymerase was expressed in all transgenic clones showing substantial differences in its activity for different clones concerning EPS degradation. Resistance to E. amylovora was evaluated by infection of in vitro leaves as well as by artificial shoot inoculation of ex vitro plants in the greenhouse. No correlation was obtained for the transgenic lines between the level of dpo gene expression and both the level of depolymerase activity and the disease resistance in vitro, whereas, depolymerase activity did correlate positively with resistance to fire blight in vitro. Seven clones had less disease than the non-transformed genotype when measured in greenhouse, but no statistically significant differences were found, except for line T373. One line showed the highest depolymerase activity and the least susceptibility to fire blight in vitro as well as in greenhouse (T187).