Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker.
Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker.
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DOI：
10.1111/pbi.12677
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发表时间：
2017-07
影响因子：
13.8
通讯作者：
Wang N
中科院分区：
文献类型：
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作者：
Jia H;Zhang Y;Orbović V;Xu J;White FF;Jones JB;Wang N
柑橘是世界范围内价值很高的木本作物，但与此同时，柑橘生产面临着许多生物挑战，包括细菌性溃疡病和黄龙病（HLB）。选育抗病品种是控制植物病害最有效和可持续的方法。由于多倍体、多胚、延长幼期和长杂交周期等多种限制，柑橘品种的传统育种具有挑战性。靶向基因组编辑技术有可能缩短某些性状的品种发育，包括抗病性。在这里，我们利用CRISPR/Cas9/sgRNA技术来修饰邓肯柚子的溃疡病易感基因CsLOB1。生成了 6 个独立行：DLOB2、DLOB3、DLOB9、DLOB10、DLOB11 和 DLOB12。对6个株系的靶向二代测序结果显示，各株系细胞的DLOB2、DLOB3、DLOB9、DLOB10、DLOB11和DLOB12的突变率分别为31.58%、23.80%、89.36%、88.79%、46.91%和51.12%。当接种病原体柑橘黄单胞菌亚种时，DLOB2 和 DLOB3 显示出与野生型葡萄柚类似的溃疡病症状。柑橘 (Xcc)。使用 Xcc 接种后 4 天 (DPI) 时，在 DLOB9、DLOB10、DLOB11 和 DLOB12 上未观察到溃疡症状。在后期的DLOB9、DLOB10、DLOB11和DLOB12上都观察到了由Xcc引起的脓疱，与野生型柚子相比，脓疱减少了很多。 DLOB9和DLOB10上的脓疱没有发展成典型的溃疡症状。在突变植物中没有检测到副作用和脱靶突变。这项研究表明，使用 CRISPR 技术进行基因组编辑将为培育抗病柑橘品种提供一条有前景的途径。
Citrus is a highly valued tree crop worldwide, while, at the same time, citrus production faces many biotic challenges, including bacterial canker and Huanglongbing (HLB). Breeding for disease‐resistant varieties is the most efficient and sustainable approach to control plant diseases. Traditional breeding of citrus varieties is challenging due to multiple limitations, including polyploidy, polyembryony, extended juvenility and long crossing cycles. Targeted genome editing technology has the potential to shorten varietal development for some traits, including disease resistance. Here, we used CRISPR/Cas9/sgRNA technology to modify the canker susceptibility gene CsLOB1 in Duncan grapefruit. Six independent lines, DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12, were generated. Targeted next‐generation sequencing of the six lines showed the mutation rate was 31.58%, 23.80%, 89.36%, 88.79%, 46.91% and 51.12% for DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12, respectively, of the cells in each line. DLOB2 and DLOB3 showed canker symptoms similar to wild‐type grapefruit, when inoculated with the pathogen Xanthomonas citri subsp. citri (Xcc). No canker symptoms were observed on DLOB9, DLOB10, DLOB11 and DLOB12 at 4 days postinoculation (DPI) with Xcc. Pustules caused by Xcc were observed on DLOB9, DLOB10, DLOB11 and DLOB12 in later stages, which were much reduced compared to that on wild‐type grapefruit. The pustules on DLOB9 and DLOB10 did not develop into typical canker symptoms. No side effects and off‐target mutations were detected in the mutated plants. This study indicates that genome editing using CRISPR technology will provide a promising pathway to generate disease‐resistant citrus varieties.