Increased resistance to fire blight by expression of the phi-Ea1h phage depolymerase gene with constitutive and inducible promoters, translation enhancer and signal sequence in transgenic apple

One of the strategies to improve fire blight resistance of apple through genetic engineering is transformation of apple with the dpo gene from the bacteriophage phi-Ea1h. This gene encodes a depolymerase that degrades the capsular exopolysaccharide of Erwinia amylovora. Expression of a dpo transgene in apple, driven by CaMV35S promoter in other tabs had shown a slight or no increase in fire blight resistance. In the present research, we generated transgenic M.26 apple lines expressing the dpo gene under the control of the constitutive CaMV35S or the inducible gst1 promoter. The dpo gene was also fused to the AMV translation enhancer and to the signal sequence from the PR1b gene from tobacco. Transgenic diploid apple clones with both promoters were obtained. All transgenic lines were analyzed by western blot and RT-PCR. Resistance to fire blight was evaluated in the growth chamber by inoculation of shoot tips of own-rooted potted plants with Ea273, a virulent strain of E. amylovora. Inoculated plants showed a reduction in necrotic shoot lengths from 94% in non-transformed M.26 to 48% in P35SAMVSPdpo-transgenic lines and 51% in Pgst1Spdpo-transgenic lines. The level of DPO protein expression in P35SAMVSPdpo-transgenic lines was negatively correlated (R=0.73, p<0.01) with severity of infection of inoculated shoots.