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 20, 2021and there was moderate disease on Duncan grapefruit trees but none on JJ5. We rated the plots again on July 28 (not June as mentioned in May report) 2021 and there were similar trends as in November 2020 although disease was lower given significant defoliation in the plots. We have also analyzed JJ5 for response to strains from Dr. Nian Wang to determine if those strains with unusual characteristics in terms of targeting the susceptibility gene could overcome the JJ5 resistance. Interestingly we noted unique phenotypes in plants inoculated with these strains although they were not typical disease reactions but more of a watersoaked appearance. We noted that theygrew to higher populations in inoculated tissue, although the disease phenotype was very weak and not typical of canker. We also used a 5′ Race kit to determine the transcription start in our JJ5 construct and observed that all strains tested activated trasncription of our 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. We have created a second construct for Vladimir Orbovic given the first attempt was not successful.The construct was transferred to Vladimir Orbovic, who was tasked with creating additional transformants in sweet orange. He identified several putative transgenic trees that weregrown and tested for disease reaction. Three of the putative transgenics were shown to elicit a hypersensitive reaction when infiltrated with a bacterial suspension of a Xanthomonas citri strain. Currently, we are maintaining the transgenic plants in our greenhouse. We are hopeful that these transgenic trees will be of use in the near future given that these particular ones do not express GFP. We are in the process of publishing this work. 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. The trees were rated for disease in November, 2020 and there was considerable disease on all EFR plants with disease being more severe than on susceptible Duncan control. We rated the plots again on July 28 (not June as mentioned in May report) 2021 and there were similar trends as in November 2020 although disease was lower given significant defoliation in the plots. This research project was complted in 2021. We also conducted greenhouse experiments in which we inoculated by spray inoculation and by pin-prick inoculation. We observed no differences in disease reactions of any of the transgenic trees from different transformation events, when compared to reactions in wild-type Duncan Grapefruit. We are in the process of writing a paper on these results. 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 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 served as the negative control as it had undergone the transformation process. We grafted the buds and several have developed into branches. In our initial tests, one of the transgenic trees when inoculated resulted in reduced canker symptoms, but no differences in bacterial populations when compared to the wild-type Carrizo plamts. The other transgenic tree reacted similarly as the wild-type Duncan grapefruit in symptom development as well as in bacterial populations. We cut back the material several times to stimulate plant growth, but for reasons unknow we were not able to get significant regrowth to confirm earlier results and therefore were unable to make enough progress prior to the grant being terminated to make substantive assessment as to the responses of the transgenic events.