Engineering canker‐resistant plants through CRISPR/Cas9‐targeted editing of the susceptibility gene CsLOB1 promoter in citrus

Summary

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 Cs LOB1 promoter in citrus. Wanjincheng orange ( Citrus sinensis Osbeck) harbours at least three copies of the Cs LOB1 ^G allele and one copy of the Cs LOB1 ⁻ allele. The promoter of both alleles contains the effector binding element ( EBE _{P thA4}), which is recognized by the main effector PthA4 of Xcc to activate Cs LOB1 expression to promote citrus canker development. Five pCas9/Cs LOB1sg RNA constructs were designed to modify the EBE _{P thA4} of the Cs LOB1 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 _{P thA4} modifications were identified from 38 mutant plants. Four mutation lines (S2‐5, S2‐6, S2‐12 and S5‐13), in which promoter editing disrupted Cs LOB1 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 Cs LOB1 ^G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE _{P thA4} sequence from both Cs LOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9‐mediated promoter editing of Cs LOB1 is an efficient strategy for generation of canker‐resistant citrus cultivars.