Department of Genomics and Biology of Fruit Crops, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all'Adige, Italy.
Department of Agricultural, Food, Environmental and Animal Sciences, Università degli Studi di Udine, Udine, Italy.
Plant Biotechnol J. 2020 Mar;18(3):845-858. doi: 10.1111/pbi.13253. Epub 2019 Oct 3.
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.
韧皮部杆菌，即导致苹果火疫病的病原体，通过 DspA/E 效应子触发感染，该效应子与苹果感病蛋白 MdDIPM4 相互作用。在这项工作中，使用农杆菌递送的 CRISPR/Cas9 系统在两个易感的苹果栽培品种 Malus × domestica 中产生了 MdDIPM4 敲除体。筛选了 57 个转基因系以鉴定 CRISPR/Cas9 诱导的突变。获得了 75%的编辑效率。用病原体接种了 7 个具有功能丧失突变的编辑系。与对照植物相比，观察到易感性显著降低。对五个潜在脱靶位点的测序未发现突变事件。此外，我们的构建体包含一个热休克诱导的 FLP/FRT 重组系统，专门设计用于去除携带 CRISPR/Cas9、标记基因和 FLP 本身表达盒的 T-DNA。对 6 株对病原体易感性降低的植物进行热处理，并通过实时 PCR 进行筛选，以定量计算外源 DNA 的消除情况。在一个植物系中通过测序进一步验证了 T-DNA 的去除。据我们所知，这项工作首次展示了开发和应用 CRISPR/Cas9-FLP/FRT 基因编辑系统用于生产携带最小外源 DNA 痕迹的编辑苹果植物。
