Modification of the PthA4 effector binding elements in Type I Cs LOB 1 promoter using Cas9/sg RNA to produce transgenic Duncan grapefruit alleviating XccΔpthA4:dCs LOB 1.3 infection

• Published in: Plant biotechnology journal 2016-05, Vol.14 (5), p.1291-1301
• Main Authors: Jia, Hongge, Orbovic, Vladimir, Jones, Jeffrey B., Wang, Nian
• Format: Article
• Language: English
• ISSN: 1467-7644; 1467-7652
• DOI: 10.1111/pbi.12495

Citrus canker caused by Xanthomonas citri subspecies citri (Xcc) is a severe disease for most commercial citrus cultivars and responsible for significant economic losses worldwide. Generating canker‐resistant citrus varieties will provide an efficient and sustainable solution to control citrus canker. Here, we report our progress in generating canker‐resistant grapefruit by modifying the PthA4 effector binding elements ( EBE s) in the Cs LOB 1 Promoter (EBE PthA4 ‐Cs LOBP ) of the Cs LOB 1 ( Citrus sinensis Lateral Organ Boundaries) gene. Cs LOB 1 is a susceptibility gene for citrus canker and is induced by the pathogenicity factor PthA4, which binds to the EBE PthA4 ‐Cs LOBP to induce Cs LOB 1 gene expression. There are two alleles, Type I and Type II , of Cs LOB 1 in Duncan grapefruit. Here, a binary vector was designed to disrupt the PthA4 EBE s in Type I Cs LOB 1 Promoter ( TI Cs LOBP ) via epicotyl transformation of Duncan grapefruit. Four transgenic Duncan plants with targeted modification of EBE PthA4 ‐T1 Cs LOBP were successfully created. As for Type I Cs LOB 1 promoter, the mutation rate was 15.63% (#D13), 14.29% (#D17), 54.54% (#D18) and 81.25% (#D22). In the presence of wild‐type Xcc, transgenic Duncan grapefruit developed canker symptoms similarly as wild type. An artificially designed d TALE dCs LOB 1.3, which specifically recognizes Type I Cs LOBP , but not the mutated Type I Cs LOBP or Type II Cs LOBP , was developed to infect Duncan transformants. Consequently, #D18 had weakened canker symptoms and #D22 had no visible canker symptoms in the presence of XccΔpthA4:dCs LOB 1.3. Our data suggest that activation of a single allele of susceptibility gene Cs LOB 1 by PthA4 is sufficient to induce citrus canker disease, and mutation in the promoters of both alleles of Cs LOB 1 is probably required to generate citrus canker‐resistant plants. This work lays the groundwork to generate canker‐resistant citrus varieties via Cas9/sg RNA in the future.