Abstract
The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, MdDIPM4 knockout has been produced in two Malus × domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty-seven transgenic lines were screened to identify CRISPR/Cas9-induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss-of-function mutation were inoculated with the pathogen. Highly significant reduction in susceptibility was observed compared to control plants. Sequencing of five potential off-target sites revealed no mutation event. Moreover, our construct contained a heat-shock inducible FLP/FRT recombination system designed specifically to remove the T-DNA harbouring the expression cassettes for CRISPR/Cas9, the marker gene and the FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat-treated and screened by real-time PCR to quantify the exogenous DNA elimination. The T-DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9-FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA.