Abstract：Rice (Oryza sativa) blast is one of the most important diseases that threatens rice yield stability. In recent years, the incidence and severity of rice blast have been increasing in Northeast China. The inactivation of the function of Bsr-d1 (broad spectrum resistance digul) can weaken the degradation of hydrogen peroxide and improve rice resistance to Magnaporthe oryzae; Bsr-k1 (broad-spectrum resistance Kitaake-1) encodes a TPR protein, and its functional inactivation mutant bsr-k1 leads to the enrichment of OsPAL (phenylalanine ammonia-lyase) family gene mRNA and increased lignin synthesis, thereby enhancing the immune response and giving rice broad-spectrum resistance. The study designed targets for the first exon of the 2 broad-spectrum disease resistance genes Bsr-dl and the third exon of Bsr-kl and constructed gene editing vectors. Agrobacterium tumefaciens-mediated rice transformation technology was used to introduce the gene editing vector into the recipient rice variety 'TH899', and T0 generation plants with various different variants of the Bsr-dl and Bsr-kl genes were obtained. Analysis of the T2 generation gene-edited materials that did not contain transgenic components showed that both single and simultanecous mutations of the Bsr-dl and Bsr-kl genes showed enhanced resistance to rice blast, among which the selected double-gene-edited rice materials had significantly better disease resistance than single-gene-edited materials. In addition, the single-plant yield and seed length-to-width ratio of some gene-edited lines also changed. In summary, this study used gene editing technology to simultaneously edit the Bst-dl and Bsr-kl genes, which was able to significantly enhance the resistance of 'T1899' to rice blast more than single-gene editing, and the yield traits of some mutants were also changed. The results of this study showed that combinatorial editing of rice blast resistance genes regulating different physiological pathways could effectively improve rice disease resistance. This study demonstrates the great potential of multi-gene editing in improving rice disease resistance and provides new material resources for breeding rice blast-resistant japonica rice.
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