Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Putative transgenics are currently being verified by PCR in the Jones lab, and three PCR positive plants have been identified so far. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we will initiate more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Constructs are being created in the Staskawicz lab to express Bs2 under the 35S promoter and under a resistance gene promoter from tomato. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. Three CRISPR constructs are being created in the Staskawicz lab: 1) A construct targeting two sites that will produce a deletion in Bs5 in both Carrizo and Duncan (the bs5 transgene will be added); 2) A construct targeting a site overlapping the two conserved leucines, containing a bs5 repair template for Carrizo that will not be cut; and 3) a construct targeting the same site, with a repair template for Duncan grapefruit.