Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is ongoing. Detached leaf assays, with CLas+ ACP feeding, have been conducted and lines with the most promising results have begun greenhouse and field studies. Greenhouse studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of Hamlin grafted on Carrizo with all combinations of WT and transgenic.Data collection from the first round of field plantings with Mthionin transgenic Carrizo rootstock (45 plants) with non-transgenic rough lemon continues. Initial results show transgenics maintaining higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. A large second planting of Mthionin transgenics went into the ground in April, including transgenic Carrizo with WT Hamlin scions (81 plants), transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) and WT/WT controls (16 plants) with data collection scheduled in October. Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) on transgenic rootstock are being made for follow up plantings. Mthionin construct transformations have also been completed on 450 Valencia and 300 Ray Ruby explants to provide additional transgenic material of these critical varieties. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, have been conducted on lines representing chimera constructs TPK, PKT, CT-CII, scFv-InvA, scFv-TolC, TBL, BLT, LBP/’74’, `73′, and `188′. Detached leaf feeding assay protocols were adjusted to improve sensitivity and increase transmission rates (See section 4). Multiple lines from several constructs have been moved forward into greenhouse studies based on these results, as noted below.Initial ACP inoculations conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’) showed a statistically significant reduction (13x) in CLas titer for `74′ transgenics vs WT in the CLas+ plants. However, many plants remained CLas negative at 6 months post infestation, indicating a low inoculation efficiency. All ACP inoculations for greenhouse experiments are now using an improved protocol. Through a combination of selecting smaller plants, more aggressively trimming larger plants and close observation, we have been able to extend the caged ACP infestation time from 7 days to 21 without severe mold or cage damage to the plants. We expect this longer infestation period to greatly improve inoculation rates. In June, 150 plants representing the best performing 7 lines of `188′ and 6 lines of `74′ were no-choice caged ACP inoculated using this improved protocol with the first samplings and data collection scheduled for 9.26.19. We are also emphasizing parallel field trials for all phenotyping efforts. Preparations are now being made for a field planting of ~400 `74′ and `188′ transgenics is scheduled for spring 2020. Completed so far are 196 grafts of WT scions (Hamlin, Valencia, and Ray Ruby) onto transgenic Carrizo root stocks. 200 more grafts of `74′ and `188′ transgenic Hamlin on WT rootstocks are underway. All plants will be ready for the post Hurricane planting season.Seven new transformations, totaling over 3000 explants, have been completed to generate Valencia, Ray Ruby, and Hamlin (when not already complete) lines expressing `74′, `188′, TBL, TPK and other advanced chimera constructs. Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types. The best Carrizo lines have been grafted with WT Ray Ruby scions and, with all appropriate permitting now completed, will be moved to the field after hurricane season. An additional 129 rooted cuttings are propagated for follow up plantings.The 3 month data from the 150 plants from the 2nd group of scFv lines (12 lines) that were initially no-choice ACP inoculated showed an insufficient infection rate. These plants have now been graft inoculated with HLB+ RL and qPCR sampling is scheduled for November 2019. The 370 scFv rooted cuttings already propagated for a 3rd round of ACP-inoculations will use the higher pressure 21 day infestation protocol described above. Objective 4, Screening Development and Validation: A protocol using a high throughput ACP homogenate assay for selecting lytic peptides for activity against CLas is now in use. A manuscript on the protocol has been published in Plant Methods (DOI: 10.1186/s13007-019-0465-1) to make it available to the HLB research community. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. The use of a phloem specific endogene would allow for samples to be normalized to phloem cells instead of total citrus cells, more accurately evaluating bacterial titer and potential therapeutic effects with the phloem limited CLas.The best performing lines of Mthionin, chimeras `74′ and `188′ and scFv transgenics have been submitted to Florida Department of Plant Industry for shoot-tip graft cleanup in preparation for future field studies. 3 lines of Hamlin Mthionin transgenics and 2 lines of Carrizo/Mthionin have been returned certified clean. Objective 5, Transgene Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) and both His6 and Flag tagged variants of scFv constructs have been generated and confirmed for transgene expression by RT-qPCR. Experiments are underway using these plants to track the movement and distribution of transgene products in parallel to direct antibody based approaches.