SILENCING OF HIPM, THE APPLE PROTEIN THAT INTERACTS WITH HrpN OF ERWINIA AMYLOVORA

Several proteins of Erwinia amylovora, including HrpN, a harpin, have been identified that are required for the bacterium to cause fire blight or that affect disease severity. Harpins are glycine-rich, heat-stable, proteins that lack cysteine and have few aromatic amino acids. HrpN is involved in disease development based on mutational analysis. HrpN elicits the hypersensitive response following infiltration into the leaves of plants. A single protein, encoded by a gene of a cDNA prey library from the important host apple (Malus ×domestica), was found by yeast two-hybrid assay to interact with HrpN. Interaction was confirmed in vitro. The HrpN-interacting protein from Malus (HIPM) was characterized as small (60 amino acids), and it possesses a functional signal peptide and a transmembrane domain. Using RNAi technology, we undertook to silence the HIPM gene in apple and to assess the effects of HIPM on disease development. Assays of in vitro- and greenhouse-grown transgenic plants by RT-PCR and real time PCR for net expression of HIPM indicated silencing in some lines; RNA expression was reduced to 6 to 81% of that of non-transformed 'Galaxy', the parent plant. Some of the transgenic lines also were evaluated for resistance to fire blight by inoculation of shoots of own-rooted potted plants with Ea273, a virulent strain of E. amylovora. The susceptibility to E. amylovora of some lines was reduced by 50%, relative to 'Galaxy'. Silencing of HIPM transcripts, evaluated by the ratio of RT-PCR or Real Time PCR signal intensity between HIPM and the control EF1-, was correlated in 'Galaxy' with the level of resistance in the growth chamber (R=0.55; P<0.01 and R=0.69; P<0.01 for the RT-PCR and Real Time PCR data, respectively).