The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data During the project period, we conducted the following activities. Objectives (1) to (4) were accomplished. Objective (5) was delayed due to the Covid-19 pandemic. We will continue collecting filed trial data, although the project has been terminated. (1) We previously generated three HLB-tolerent transgenic lines, ‘Hamlin’ 13-3, 13-29, and Duncan ’57-28′. The transgenic plants carried both the HLB-causing bacterium CLas and the flowering-promoting CTV vector. To remove CTV and CLas, the plants were treated in a growth chamber with alternating temperatures of 25 and 42 degree C every 4 hours for a total of 60 to 90 days. New shoots formed on the treated plants were tested for the presence of CLas and CTV by quantitative PCR (qPCR) with specific primers. The alternating temperature treatment is known to be highly efficient for removing CTV. We found that it is also effective for eliminating CLas. The new shoots obtained were free of both CTV and CLas. (2) & (3) Budwoods from the new shoots were grafted onto ‘Swingle’ rootstocks. A total of 83 transgenic plants were produced using CTV- and CLas-free budwoods of the above mentioned transgenic lines (28 for 13-3, 31 for 13-29, and 28 for 57-28). Moreover, 13 plants propagated from a new NPR1 transgenic line generated through mature tissue transformation, 17 plants from an EDS5 transgenic line, and 7 plants from an ELP3 transgenic line were produced. All these transgenic lines showed HLB tolerance in the greenhouse. In addition, a total of 27 transgenic rootstock plants were produced. These transgenic plants include eight transgenic ‘Carrizo’ lines that express three different disease resistance genes. The transgenic rootstocks were replicated and grafted with ‘Valencia’. All plants were grown and maintained in the greenhouse for two to three years. (4) The transgenic plants were planted in The Picos Farm in 2019 and 2021 (no plants were planted in 2020 due to Covid-19). A total of 69, 98, and 27 plants were planted on May 9, 2019, May 20, 2021, and October 8, 2021, respectively. (5) This objective is still ongoing with funding from other resources. The transgenic plants transplanted on May 9, 2019 and May 20, 2021 were examined on April 24, 2022. The plants grow well in the field and one plant from the 2019 planting has shown HLB symptoms. Leaf tissues were collected on April 24, 2022 and analyzed for CLas titers. We will continue monitor the transgenic plants in the field and periodically analyze leaf samples in the coming years. Besides the proposed objectives, we continue working on development of techniques that are able to produce consumer friendly citrus products. These techniques include CTV-delivered gene silencing, transgene-free CRISPR, and cisgenesis or intragenesis (cis/intragenesis). Our CTV and CRISPR projects are supported by USDA. The cis/intragenesis project is partially supported with the CRDF funds. We have previously created an intragenic vector. Unfortunately, the efficiency of the vector is extremely low. Our goal is to develop a strategy to significantly improve the efficiency. We are using the citrus 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutant (called EPSPS TIPS) that provides tolerance to glyphosate as a selective marker to increase the intragenic vector efficiency. A strong citrus promoter is needed to drive the EPSPS TIPS gene, we are building a system to identify such citrus promoters. Once the efficiency of the intragenic vector is improved, it can be used to either silence or overexpress a target gene. We are using CTV-delivered gene silencing to identify targets for silencing, which is supported by USDA. We are also screen for genes for overexpression through cis/intragenesis. We recently discovered two nicotinamide adenine dinucleotide (NAD)-binding receptors in the model plant Arabidopsis, which when overexpressed, increase resistance to bacterial pathogens. With the support of the CRDF funds, we generated transgenic citrus expressing the Arabidopsis NAD receptors. The transgenic citrus plants were inoculated with CLas-infected psyllids and are maintained in the greenhouse for HLB symptom development. We are grafting the transgenic scions onto sweet orange rootstocks for easier detection of HLB resistance or tolerance. Furthermore, the citrus genome encodes several putative NAD-binding receptors. NAD-binding activities of two of the putative receptors were tested. These citrus receptors will be overexpressed using the intragenic vector to create HLB tolerance. We also developed highly efficient citrus microRNA (miRNA) vectors. These vectors will be combined with the intragenic vector to specifically silence negative immune regulators to created HLB tolerance in citrus. In summary, we have accomplished four of the five proposed objectives and will finish field data collection (Objective 5) in the coming years using funding from other sources.