title: Genome editing of the disease susceptibility gene CsLOB1 in citrus confers resistance to citrus canker.
abstract: Citrus is a highly valued tree crop worldwide, while, at the same time, citrus production faces many biotic challenges, including bacterial canker and Huanglongbing (HLB). Breeding for disease-resistant varieties is the most efficient and sustainable approach to control plant diseases. Traditional breeding of citrus varieties is challenging due to multiple limitations, including polyploidy, polyembryony, extended juvenility and long crossing cycles. Targeted genome editing technology has the potential to shorten varietal development for some traits, including disease resistance. Here, we used CRISPR/Cas9/sgRNA technology to modify the canker susceptibility gene CsLOB1 in Duncan grapefruit. Six independent lines, DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12, were generated. Targeted next-generation sequencing of the six lines showed the mutation rate was 31.58%, 23.80%, 89.36%, 88.79%, 46.91% and 51.12% for DLOB2, DLOB3, DLOB9, DLOB10, DLOB11 and DLOB12, respectively, of the cells in each line. DLOB2 and DLOB3 showed canker symptoms similar to wild-type grapefruit, when inoculated with the pathogen Xanthomonas citri subsp. citri (Xcc). No canker symptoms were observed on DLOB9, DLOB10, DLOB11 and DLOB12 at 4 days postinoculation (DPI) with Xcc. Pustules caused by Xcc were observed on DLOB9, DLOB10, DLOB11 and DLOB12 in later stages, which were much reduced compared to that on wild-type grapefruit. The pustules on DLOB9 and DLOB10 did not develop into typical canker symptoms. No side effects and off-target mutations were detected in the mutated plants. This study indicates that genome editing using CRISPR technology will provide a promising pathway to generate disease-resistant citrus varieties.
authors.affiliation: Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, FL, USA.
authors.forename: Hongge
authors.initials: H
authors.lastname: Jia
authors.affiliation: Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, FL, USA.
authors.forename: Yunzeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, FL, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Orbović
authors.affiliation: Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, FL, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Plant Pathology, IFAS, University of Florida, Gainesville, FL, USA.
authors.forename: Frank F
authors.initials: FF
authors.lastname: White
authors.affiliation: Department of Plant Pathology, IFAS, University of Florida, Gainesville, FL, USA.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Jones
authors.affiliation: Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences (IFAS), University of Florida, Lake Alfred, FL, USA.
authors.forename: Nian
authors.initials: N
authors.lastname: Wang
mesh_terms: D002957:Citrus / Q000235:genetics* / Q000382:microbiology*; D060467:Disease Resistance / Q000235:genetics*; D004198:Disease Susceptibility / Q000378:metabolism; D000072669:Gene Editing; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D010935:Plant Diseases / Q000235:genetics* / Q000382:microbiology*; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000382:microbiology*; D018749:RNA, Plant / Q000235:genetics
chemical_list: D018749:RNA, Plant
keywords: 
  Citrus paradisi
  ; 
  Xanthomonas citri
  ; Cas9; sgRNA