Targeted Gene Mutagenesis in Citrus to Produce Citrus Canker Resistance

Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in plants. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering tools developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this study, we describe a detailed protocol for constructing TALEN expression vectors GFP-TALEN:Cslobp, assessing nuclease activities using a citrus protoplast-based transformation system. Citrus protoplast-transformation vector GFP-TALEN:Cslobp was developed to edit the CsLOB1 (lateral organ boundaries 1 of Citrus sinensis) promoter in citrus to thwart the virulence strategy of Xanthomonas citri subspecies citri which causes citrus canker disease. Vector GFP-TALEN:Cslobp has been introduced into citrus protoplast cells using a polyethylene glycol (PEG) system. More than 100 embryos expressing GFP have been recovered. Several transgenic lines have successfully generated, and the detailed analysis is being carried out. This method could have great potential for producing citrus plants resistant to citrus canker and possibly citrus greening or Huanglongbing (HLB) as well once the virulence strategy of CandidatusLiberibacter asiaticus has been identified.