This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants plus the addition of a new strategy using gene editing. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. We also are in the process of testing citrus that has been transformed to modify the bs5 gene to enhance resistance to the citrus canker bacterium. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. In late March, 2019, in the field at Fort Pierce in collaboration Dr. Ed Stover, the transgenic material was planted. Citrus canker has developed on plants in the field and the trees were rated for disease in November, 2020 and there was considerable defoliation on all trees including JJ5. We also observed disease on all susceptible Duncan trees, but no evidence on the transgenic JJ5. The disease at this point was difficult to rate on Duncan given the defoliation. I am planning to evaluate in June. As for developing a different transgenic with ProBs314EBE:avrGf2, we have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contain an additional selectable marker that allowed for identifying putative transgenics with a higher success rate. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct is with Vladimir Orbovic, who is in the process of creating additional transformants. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and planted them in the field at Fort Pierce in collaboration Dr. Ed Stover. They were planted in the field in late March and were recently rated in late July. I am in the process of analyzing the data. We have identified additional transgenics from plants received from Dr. Vladimir Orbovic that have been grafted onto rootstocks. They and the original EFR transgenic plants were tested for susceptibility by being inoculated in October by pinprick inoculation. Leaves from all EFR positive grapefruit leaves were susceptble based on the pin-prick inoculatons with typical pustule formation simlar to the wild-type Duncan control. Lesions on leaves of one of the EFR positive sweet orange trees were smaller.We have also tested for EFR gene expression and determined that all EFR grapefruit and orange had similar gene expression levels. We have inoculated young leaves from representative trees of each transgenic event by spray inoculation in February and will rate the leaves for susceptibility. Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying the pepper bs5. We have recently received budwood from UC Berkeley. The budwood was from two transgenic events and a third was from a tree that was run through the transformation process, but that was negative for the gene. The latter was to serve as the negative control as it had undergone the transformation process. We have grafted the buds and several have developed into branches. We are currently growing these. We have recently inoculated leaves from the two CRISPR events, C1 and C2, by pinprock inoculation and assessed the pustule size in December. Pustules on C2 leaves were similar in size to non-edited leaves. Interestingly we observed reduced pustule formation on C1 leaves. We are waiting for new leaves to develop for conducting further testing this spring.