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 studies. These 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 (45 plants) rootstock 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 group of Mthionin plants went into the ground in April, including transgenic Carrizo with WT Hamlin scions (81 plants) and transgenic Hamlin on non-transgenic Carrizo rootstock (108 plants) with WT/WT controls (16 plants). Additional grafts of WT Ray Ruby (118 plants) and WT Valencia (118 plants) onto transgenic rootstock are being propagated for future plantings. Mthionin construct transformations have also been completed on 250 Valencia explants to provide sufficient events for this critical variety. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, were conducted on lines representing chimera constructs TPK, PKT, CT-CII, TBL, LBP/’74’, `73′, and `188′. Multiple lines from several constructs were moved forward into greenhouse studies based on these results as noted below. Definitive results for TPK, PKT, CII, and TBL were hindered by low inoculation rates. Assays for these constructs are being repeated to identify which lines of each are best suited for greenhouse studies. Detached leaf feeding assay protocols have also been adjusted to improve sensitivity (See section 4)No-choice caged ACP inoculation has been conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’). Early data from CLas+ plants showed a statistically significant reduction (13x) in CLas titer for transgenics vs WT in the CLas+ plants. However, many plants shown little to no amplification of CLas DNA at 3 and 6 months post inoculation. Amplification by this time would be expected from a successful inoculation, indicating low inoculation efficiency. All plants in this experiment will be re-inoculated by bud grafting with HLB+ rough lemon to allow for continued greenhouse studies. Moving forward, we will be emphasizing parallel field trials for phenotyping efforts and modify the ACP inoculation in greenhouse studies to increase CLas pressure. 475 rooted cuttings were previously made from Hamlin and Carrizo lines expressing constructs `74′ and `188′ and are now of a size appropriate for CLas exposure. 360 of these plants will be grafted with WT scions (for Carrizo lines) or rootstocks (For Hamlin lines) for field trials. The remaining 115 plants will be ACP inoculated for greenhouse studies, with the caged no-choice feeding time period extended from 7 days to 14.Seven new transformations, totaling over 2000 explants, have been completed to generate Valencia, Hamlin and Ray Ruby lines expressing constructs `74′, `188′, and TPK.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 lines have been used as rootstock for WT Ray Ruby scions and will be moved to the field after necessary permit approvals. An additional 129 rooted cuttings are propagated for follow up plantings.Like the `74′ results discussed for objective 2, the 2nd round of ACP-inoculations of scFv plants (150 plants, 12 lines) had a poor infection rate. The plants are to be re-inoculated by budding with HLB+ rough lemon. The 370 scFv rooted cuttings already propagated for a 3rd round of ACP-inoculations will use the higher pressure 14 day feeding protocol described above. Objective 4, Screening Development and Validation: Details of the high throughput ACP homogenate assay, and its use for selecting lytic peptides for activity against CLas, has been submitted for publication and remains in use for early screening of therapeutics in the lab. 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. 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) have been generated and confirmed for transgene expression by RT-qPCR. These plants will be used for generating data on the movement and distribution of transgene products in parallel to antibody based approaches.