title: Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus.
abstract: 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 CsLOB1Gallele and one copy of the CsLOB1-allele. The promoter of both alleles contains the effector binding element (EBEPthA4), 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 EBEPthA4of 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 EBEPthA4modifications 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 CsLOB1Galone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBEPthA4sequence 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.
authors.affiliation: Citrus Research Institute, Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement, Chongqing, China.|Citrus Research Institute, Southwest University, Chongqing, China.
authors.forename: Aihong
authors.initials: A
authors.lastname: Peng
authors.affiliation: Citrus Research Institute, Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement, Chongqing, China.|Citrus Research Institute, Southwest University, Chongqing, China.
authors.forename: Shanchun
authors.initials: S
authors.lastname: Chen
authors.affiliation: Citrus Research Institute, Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement, Chongqing, China.|Citrus Research Institute, Southwest University, Chongqing, China.
authors.forename: Tiangang
authors.initials: T
authors.lastname: Lei
authors.affiliation: Citrus Research Institute, Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement, Chongqing, China.|Citrus Research Institute, Southwest University, Chongqing, China.
authors.forename: Lanzhen
authors.initials: L
authors.lastname: Xu
authors.affiliation: Citrus Research Institute, Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement, Chongqing, China.|Citrus Research Institute, Southwest University, Chongqing, China.
authors.forename: Yongrui
authors.initials: Y
authors.lastname: He
authors.affiliation: Citrus Research Institute, Southwest University, Chongqing, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Wu
authors.affiliation: Citrus Research Institute, Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement, Chongqing, China.|Citrus Research Institute, Southwest University, Chongqing, China.
authors.forename: Lixiao
authors.initials: L
authors.lastname: Yao
authors.affiliation: Citrus Research Institute, Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement, Chongqing, China.|Citrus Research Institute, Southwest University, Chongqing, China.
authors.forename: Xiuping
authors.initials: X
authors.lastname: Zou
mesh_terms: D064113:CRISPR-Cas Systems; D002957:Citrus / Q000235:genetics* / Q000382:microbiology*; D060467:Disease Resistance / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D006579:Heterozygote; D009154:Mutation; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D014974:Xanthomonas / Q000472:pathogenicity*
chemical_list: D010940:Plant Proteins
keywords: 
  CsLOB1
  ; CRISPR/Cas9; citrus canker; genome editing; resistance