The inactivation of MdDIPM4 by CRISPR/Cas9 editing reduces the susceptibility of apple to fire blight disease

Fire blight, caused by the bacterium Erwinia amylovora (E.a.), is one of the most economically important and invasive diseases affecting apple (Malus x domestica). The molecular mechanisms of E.a.-apple interaction are widely elucidated but the management of the disease remains still arduous. Apple protein MdDIPM4 interacts with the DspA/E effector, mandatory for the pathogenesis of Erwinia amylovora, but its biological function is still unknown. In this work, the knock-out of MdDIPM4 has been produced in two Malus x domestica susceptible varieties, by using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty-seven transgenic lines were analyzed using a Next Generation Sequencing in order to identify the CRISPR/Cas9- induced mutations. Some edited plants with a loss of function mutation were selected and inoculated with the pathogen. An increased resistance was observed, demonstrating that MdDIPM4 is involved in plant susceptibility to fire blight. Moreover, with the aim of producing transgene-free plants, we used a heat shock-inducible FLP-FRT recombination system designed specifically to remove the entire T-DNA in those plants with an increased pathogen resistance. For the first time, our data demonstrated the possibility to produce apple varieties more resistant to fire blight and free from exogenous DNA by using the CRISPR/Cas9 technology. These plants may be used in further analysis to better elucidate how MdDIPM4 is involved in the onset of the disease.