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

Abstract

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