利用CRISPR/Cas9系统敲除葡萄中VviEDR2提高对白粉病的抗性

利用CRISPR/Cas9系统定点编辑葡萄白粉病感病基因VviEDR2（Enhanced disease resistance 2），在VviEDR2的DUF1336结构域设计靶位点VviEDR2-T1，构建CRISPR/Cas9敲除载体，通过农杆菌介导法转化‘无核白’葡萄胚性愈伤组织。对PCR阳性植株进行靶位点扩增测序，结果表明，共有8个转基因植株在靶位点处发生不同类型的双等位基因突变，编辑效率为32%；突变体植株生长势较弱，叶片较小，茎秆丛生、细弱。进一步对突变体植株进行抗病检测，结果表明，接种葡萄白粉菌（Erysiphe necator Schw.）5 d后，突变体植株叶片上白粉菌孢子仅能萌发出少量较短初级菌丝，表皮细胞产生大量明显的H2O2，而野生型叶片中白粉菌萌发出大量初级菌丝、次级菌丝和吸器，无明显H2O2产生。这些结果表明，可以利用CRISPR/Cas9技术编辑葡萄感病基因VviEDR2，提高葡萄白粉菌抗性。

CRISPR/Cas9-mediated Mutagenesis of VviEDR2 Results in Enhanced Resistance to Powdery Mildew in Grapevine（Vitis vinifera）

Grapevine（Vitis vinifera L.）incurs significant yield and quality losses from powdery mildew（Erysiphe necator Schw.）. Here，we used the CRISPR/Cas9 system to precisely edit the susceptibility gene VviEDR2（Enhanced disease resistance 2）and provide reference for disease resistance breeding. We designed a target site in DUF1336 domain of the VviEDR2 gene for the CRISPR/Cas9 vector，and obtained transgenic plants via Agrobacterium-mediated transformation，using somatic embryos of the Thompson Seedless cultivar. Sequencing revealed that 8 out of 25 transgenic plants carried different type of biallelic mutations in target sites and the editing efficiency is 32%. These mutants exhibited reduced growth compared to wild type，such as smaller leaves，thinner and fasciculate stems. Inoculation assays with an adapted grapevine powdery mildew isolate En NAFU1 showed that hyphal growth is significantly restricted on mutant grapevine leaves with obvious H2O2 accumulation in penetrated epidermal cells at 5 days post inoculation. Conversely，massive hyphae and haustoria，but no obvious accumulation of H2O2，were observed in the leaves of wild type. These results indicated that CRISPR/Cas9 technology could be utilized to knockout susceptibility gene VviEDR2 in grapevine，which could improve powdery mildew resistance.