Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Transgenic Duncan grapefruit and sweet orange lines carrying either EFR alone or EFR plus an XA21-EFR chimera were tested for canker resistance in the greenhouse. The two most promising Duncan grapefruit lines carrying EFR were selected for further testing in the field in collaboration with Dr. Ed Stover at the USDA ARS. Some new Duncan grapefruit transformants carrying EFR, XA21, or both genes have been produced at the Core Citrus Transformation Facility at UF Lake Alfred, and any that survive will be analyzed for canker resistance. No transformants were obtained for CSPR, and this construct has been abandoned. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement Our gene editing target is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we chose to mutate the native citrus Bs5 alleles while simultaneously introducing the effective resistance allele as a transgene, rather than to attempt precise gene editing. Two editing constructs were created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. The constructs were transformed into Carrizo citrange, and transformants are currently being analyzed at UC Berkeley. To date, we have identified one plant with mutations knocking out both alleles of the native Bs5 gene and several other candidate plants that may also have a loss of function of both alleles. More plants remain to be analyzed. Any plants confirmed to have biallelic loss of function mutations in Bs5 plus the introduced bs5 resistance allele will be propagated and shipped to Dr. Jeff Jones’ lab at UF Gainesville for canker testing.