Silencing of apple proteins that interact with DspE, a pathogenicity effector from Erwinia amylovora, as a strategy to increase resistance to fire blight

The disease specific gene, dspE, of the bacterium Erwinia amylovora encodes a pathogenicity effector of 198 kDa, which is essential to development of the fire blight disease in apple plants. Yeast two-hybrid and in vitro protein-pull-down assays demonstrated that DspE interacts physically and specifically with four similar leucine-rich-repeat (LRR) receptor-like serine/threonine kinases from apple. The genes encoding the four DspE-interacting proteins of Malus (DIPM genes) are conserved in all hosts of E. amylovora tested, but not in tested non-host plants. Interaction between the DIPMs and DspE is thought to be involved in disease development. Ca. 400 bp sense sequences from non-conserved regions of each gene, with homology among each other of <50%, were used to make constructs to be transformed into fire blight susceptible apple cultivars with the aim of silencing the DIPM genes and preventing interaction with DspE. In addition, three constructs containing the four 400 bp sequences in tandem, a full length sense sequence of one gene, and a hairpin sequence of that gene were made. All seven silencing sequences were transferred into 'Galaxy' apple cultivar by Agrobacterium inoculation of leaf pieces. Transgenic lines with all constructs have been recovered. Assays of the trans-genic plants for net expression of the target DIPMs by RT-PCR for their mRNA's have shown evidence of silencing in some lines. Some of the transgenic lines have also been evaluated for resistance to fire blight by inoculation of shoots of own-rooted potted plants with the virulent strain Ea273 of E. amylovora. Preliminary results indicate that some lines have increased resistance. Resistance due to silencing of a native apple gene(s) is likely to be more acceptable to regulators, growers, and consumers than the addition of genes from that organism.