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Oral: The Function and Mechanism of CRISPR and Its Applications


Development of citrus canker resistant varieties by manipulation of CsLOB1, the citrus susceptibility gene to Xanthomonas citri, using Cas9/sgRNA
N. WANG (1), H. Jia (1) (1) University of Florida, U.S.A.

Citrus canker caused by Xanthomonas citri subspecies citri (Xcc) is a severe disease for most commercial citrus cultivars. Generating canker resistant citrus varieties will provide an efficient and sustainable solution to control citrus canker. PthA4 is the dominant pathogenicity gene of Xcc and recognizes the corresponding promoter sequence in the host and activates the expression of susceptibility gene CsLOB1. Here, we report our latest progress in generating canker resistant citrus by modifying the CsLOB1 gene and the effector binding elements (EBEs) of PthA4 in the CsLOB1 Promoter (EBEPthA4-CsLOBP) of the CsLOB1 gene via Cas9/sgRNA technology. Cas9/sgRNA system has been successfully employed for genome modification in multiple plant species. We have successfully mutated the EBEs of one allele (Type I), but not the second allele (Type II) in Duncan grapefruit. The mutated Duncan grapefruit is susceptible to wild type Xcc, but resistant to XccΔpthA4:dCsLOB1.3, a pthA4 mutant of Xcc expressing an artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by PthA4 is enough to induce citrus canker disease and mutation of the promoters of both alleles of CsLOB1 is required to generate citrus canker resistant plants. We will also present our progress in generating canker resistant citrus by modifying CsLOB1.