Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter...

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease-susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9-targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1^G allele and one copy of the CsLOB1^- allele. The promoter of both alleles contains the effector binding element (EBE_PthA4 ), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE_PthA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%-64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE_PthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2-5, S2-6, S2-12 and S5-13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2-6 and S5-13 lines. Promoter editing of CsLOB1^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE_PthA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9-mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker-resistant citrus cultivars.