Objective 1, Mthionin Constructs:�Assessment of the Mthionin transgenic lines is continuing apace.� 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 grafted Hamlin on Carrizo with all combinations of WT and transgenic.The first field plantings with Mthionin transgenic Carrizo (45 plants) have been made with leaves of non-transgenic rough lemon on transgenics showing higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months.� Plants for follow up field plantings of transgenic Hamlin on WT Carrizo (112 plants), WT Hamlin on transgenic Carrizo (84 plants), WT Ray Ruby on transgenic Carrizo (118 plants) and WT Valencia on transgenic Carrizo (118 plants) with WT controls are being propagated.Seeds for additional scion variety transformations have been collected and germinated.� Shoots will be developed enough to yield epicotyl tissue for Mthionin construct transformations in 2 (Hamlin), 4 (Ray Ruby) and 6 (Valencia) weeks.��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’).� Three month data has been collected, while many plants are yet to show CLas DNA amplification, there is a statistically significant reduction (13x) in CLas titer for transgenics vs WT in the CLas+ plants.� An additional 475 rooted cuttings have been propagated from chimera constructs (6 lines of `188′, 7 lines of `74′ and 12 lines of `73′) for the next round of ACP inoculation trials.� �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 at the conclusion of the study.� The best lines have been used as rootstock for WT Ray Ruby scions and will be moved to the field once the graft union is strong enough.� An additional 129 rooted cuttings are propagated for additional grafts and field plantings.� ACP inoculations were conducted on 150 more plants from 12 scFv lines.� Data from 3 months post inoculation has been collected, but too few plants are testing positive at this time for a conclusive analysis.� An additional 370 rooted cuttings have been propagated from the remaining scFv constructs/lines to be tested and will soon be mature enough for ACP inoculation.�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.���