Our transgenic efforts have evolved greatly in the three years of this project. As data accumulated and new ideas came to the forefront, efforts were focused on those aspects of the research proving most valuable, while several of the initial objectives were deemphasized.� A new project was approved by CRDF (18-022) that is the next series of steps following up on the successes of project 15-026.�To accelerate screening for CLas-killing transgenics, a detached leaf inoculation method was developed via CLas+ ACP no-choice infestation. This high throughput lab based method can test plants at small seedling stage, is non-destructive, and provides guiding information on assessment 6-12 month earlier than greenhouse based tests. To evaluate AMPs for potential use in CLas-killing transgenes, we have developed an�in vitro procedure for directly measuring their ability to disrupt CLas cells.� A homogeneous CLas suspension is recovered by macerating CLas+ ACP in a specially developed extraction buffer and removing cellular debris through spin filter centrifugation.� Homogenates are exposed to AMPs vs controls for 4 hours. CLas cell integrity is determined by use of a photo-reactive DNA binding dye. Small plant destructive inoculation assays, where all plant tissues are weighed and sampled after no-choice CLas+ ACP feeding, now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This method is being used in our transgenic efforts to validate the detached leaf assay results.�� �A modified plant Thionin (Mthionin), was designed by G. Gupta using biophysical modeling.� Transgenic expression of this peptide in Carrizo citrange showed a marked and highly statistically significant decrease in symptoms when challenged with�Xanthomonas citri, the causal agent of citrus canker.� When transgenic plants were challenged through graft inoculation with HLB infected material, both transgenic Carrizo tissues and non-transgenic scion (Rough Lemon) tissue showed reduced CLas titer up to 12 month (latest time-point) post graft when compared to control plants, with root CLas titer 1800 times greater in wild-type Carrizo.� We are testing Mthionin transgenics in the field.� Several of our best lines are at DPI for cleanup and broader field trialing, ideally head-to-head with transgenics from other programs��Newer generations of AMPs, categorized as 2nd and 3rd generation, were designed (also by G. Gupta) using citrus native thionin as the foundation combined with other citrus genes with high affinity for CLas membranes.� Numerous lines and events of 2nd and 3rd gen AMP transgenic citrus have been subject to the detached leaf assessments.� Some showed promising CLas clearance; indicated by transgenic Carrizo leaves showing significantly lower CLas titer compared to wild type controls after a 7-day ACP infestation.� Interestingly, bacterial quantity in the ACP bodies was also lower after feeding on the transgenic leaves, suggesting an uptake of AMPs into the insect body and disruption of gut bacterial cells. Several of our best lines are also at DPI for cleanup and broader field trialing.� � � � �ScFv sequences targeting CLas outer membrane proteins were developed by J. Hartung and used for the creation of transgenes disrupting the CLas infection process. Transgenic plants are showing a consistent and statistically significant decrease in Clas titer twelve months after no choice CLas+ ACP inoculation (up to 250x reduction, measured by qPCR) and have a much higher incidence of plants with no measurable bacterial DNA amplification. Approximately 120 additional rooted cuttings were propagated for field trials, with the primary focus being rootstocks to protect conventional scions.� �We sought (with W. Belknap and J. Thomson) to identify highly expressed genes in the citrus phloem, reasoning that their promoters might be useful for transgenics combatting HLB. Through this work, a citrus gene family was identified and characterized encoding a group of Small Cyclic Amphipathic Peptides (SCAmpPs) with highly conserved gene structure, but considerable variation in the ultimate gene products. Variants of a tissue-specific SCAmpP promoter were tested using GUS as a reporter gene and resulted in excellent phloem-specific expression: 500x greater expression in leaf midribs/petioles compared to laminar area and visibly greater GUS gene product activity in midribs and vascular tissue compared to GUS driven by D35S.� These citrus promoters are being used in all new transgenics from our program, usually with a parallel set of transgenics driven by D35S, to combat gram-negative pathogens in other citrus tissues.����