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CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum

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Abstract

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CRISPR/Cas9-mediated editing of Clpsk1 enhanced watermelon resistance to Fusarium oxysporum.

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be an effective genome-editing tool for crop improvement. Previous studies described that Phytosulfokine (PSK) signalling attenuates plant immune response. In this work, we employed the CRISPR/Cas9 system to knockout Clpsk1 gene, encoding the PSK precursor, to confer enhanced watermelon resistance to Fusarium oxysporum f.sp. niveum (FON). Interactions between PSK and FON were analysed and it was found that transcript of Clpsk1 was significantly induced upon FON infection. Meanwhile, application of exogenous PSK increased the pathogen growth. Then, one sgRNA, which targeted the first exon of Clpsk1, was selected for construction of pRGEB32-CAS9-gRNA-Clpsk1 expression cassette. The construct was then transformed to watermelon through Agrobacterium tumefaciens-mediated transformation method. Six mutant plants were obtained and three types of mutations at the expected position were identified based on Sanger sequencing. Resistance evaluation indicated that Clpsk1 loss-of-function rendered watermelon seedlings more resistant to infection by FON. These results indicate that CRISPR/Cas9-mediated gene modification is an effective approach for watermelon improvement.

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Acknowledgements

This work was supported by National Key R&D Program of China (2018YFD0100703), the Natural Science Foundation of Jiangsu Province (BK20171323), the National Industrial Technology System for Watermelon & Melon (CARS-25) and the Earmarked Fund for Jiangsu Agricultural Industry Technology System (JATS [2018]254).

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Authors and Affiliations

  1. Institute of Vegetable, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, Jiangsu, China

    Man Zhang, Qiling Liu, Xingping Yang, Jinhua Xu, Guang Liu, Xiefeng Yao, Runsheng Ren, Jian Xu & Lina Lou

  2. Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing, 210014, Jiangsu, China

    Man Zhang, Qiling Liu, Xingping Yang, Jinhua Xu, Guang Liu, Xiefeng Yao, Runsheng Ren, Jian Xu & Lina Lou

Authors
  1. Man Zhang
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  2. Qiling Liu
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  3. Xingping Yang
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  4. Jinhua Xu
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  5. Guang Liu
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  6. Xiefeng Yao
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  7. Runsheng Ren
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  8. Jian Xu
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  9. Lina Lou
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Contributions

XPY and JHX conceived and designed research. MZ, QLL and LNL performed site-directed mutagenesis and transformation. RSR and XFY performed mutant identification. GL and JX performed FON inoculation. MZ wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Xingping Yang.

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The authors declare that they have no conflict of interest.

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Communicated by Yiping Qi.

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Zhang, M., Liu, Q., Yang, X. et al. CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum. Plant Cell Rep 39, 589–595 (2020). https://doi.org/10.1007/s00299-020-02516-0

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