CRISPR/Cas9-mediated editing of CsWRKY22 reduces susceptibility to Xanthomonas citri subsp. citri in Wanjincheng orange (Citrus sinensis (L.) Osbeck)

Plant Biotechnology Reports - Tập 13 - Trang 501-510 - 2019
Lijuan Wang1,2, Shanchun Chen1,2, Aihong Peng1,2, Zhu Xie1,2, Yongrui He1,2, Xiuping Zou1,2
1Citrus Research Institute, Southwest University, Chongqing, People’s Republic of China
2Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing, People’s Republic of China

Tóm tắt

CRISPR/Cas9-mediated editing of CsWRKY22 repressed canker development in Wanjincheng orange. Citrus canker, a destructive disease of citrus, is threatening the citrus industry worldwide. Breeding resistant cultivars is the most economical and effective approach to control citrus canker. Recently, CRISPR/Cas9-mediated genome editing was demonstrated to be a powerful tool for the improvement of citrus resistance to the disease. In our previous works, we confirmed that CsWRKY22 is involved in the plant immunity response to citrus canker in Wanjincheng orange (Citrus sinensis (L.) Osbeck). In this study, we targeted this gene to improve the resistance of Wanjincheng orange against citrus canker by CRISPR/Cas9-mediated editing. Sanger sequencing confirmed that CsWRKY22 in Wanjincheng orange contains CsWRKY22G and CsWRKY22C alleles, and the ratio of CsWRKY22G to CsWRKY22C is approximately 2:1. Four sgRNAs, which targeted the first exon of CsWRKY22, were selected for testing. In vitro cleavage activity analysis showed that two (W1 and W2) of the four sgRNAs displayed robust cleavage activities using PCR amplicons from the Wanjincheng orange genome as template. Subsequently, two constructs, pCas9/WRKY22sgRNA-W1 and pCas9/WRKY22sgRNA-W2, were used to modify the CsWRKY22. Three mutant plants were identified from seven independent transgenic plants. Based on Sanger sequencing, the W1-1, W2-2, and W2-3 mutant lines displayed 85.7%, 79.2%, and 68.2% mutation rates, respectively. Resistance evaluation indicated that the mutant plants showed decreased susceptibility to citrus canker. These results indicate that CRISPR/Cas9-targeted gene modification is an efficient approach for enhancing disease resistance in citrus.

Tài liệu tham khảo

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