This is a new project that is based on several successful outcomes from previously funded research. Las appears to be very well adapted to psyllid hosts, moving through multiple membrane barriers and multiplying in the hemolymph, all without causing obvious disease, and all without expression of any phage genes examined to date. By contrast, Las host range in plants appears to be tenuous, limited to phloem only. Our working hypothesis is that Las acquired key genes for plant adaptation by way of its phage and these phage genes are highly regulated; off in psyllids, and on in plants. We propose targeting specific regulators of key phage encoded virulence genes (such as the Las peroxidase) as well as key regulators of the (lethal) phage lytic cycle. Direct targeting of the Las peroxidase enzyme itself is also proposed. Objective 1 is control of HLB using the putative Las LexA repressor protein, potentially a key phage lytic cycle regulator, and to date we have expressed and purified both a truncated LexA (CLIBASIA_01645, missing C terminal domain) and full length CLIBASIA_01645 fused with a His-tag for purification and further analysis of the protein. We are now using these proteins in mobility shift assays (EMSAs) to verify specific binding to previously cloned SC1 and SC2 promoter fragments as well as its own promoter. Objective 2 is control of HLB using a repressor protein from psyllids as target. A psyllid-sourced repressor of unknown identity silences the Las phage holin gene, expression of which is lethal. We previously reported that the Las holin promoter was strongly active in Liberibacter crescens (Lcr), a culturable proxy for Las, but nearly silent in E. coli. Further, activity of the holin promoter in Lcr was suppressed by aqueous extracts from psyllids applied outside of the Liberibacter cells, indicating cell penetration. The suppressor activity was sensitive to heat and proteinase treatment, indicating a protein, and the molecular size is estimated to be10-50 kDa. Electrophoretic mobility shift assays (EMSAs) were used to demonstrate specific binding of a protein to the Las holin promoter, and small, overlapping holin promoter fragments were used as competitor DNAs to further delineate the target binding sites of the protein. The DNA-binding protein has now been purified by DNA affinity capture, and we provided multiple purifed samples for tandem liquid chromatography mass spectroscopy (LCMSMS) for the purpose of identifying the protein and the source of the gene encoding the protein (the gene could be a psyllid gene or any one of several bacterial endosymbionts living in psyllids). The initial results were inconclusive because of limited amounts of purified protein. This has now been rectified. We have also now determined that Las-free psyllids carry the repressor, and that similar extracts from Drosophila (fruit fly) do not. Objective 3 is control of HLB using the Las phage peroxidase and Las lytic cycle activator(s) as targets. We have acquired two Prestwick combinatorial libraries for use in screening chemicals that may bind to and denature CLIBASIA_01645 (focus of Objective 1) and also the Las phage peroxidase, which we demonstrated was a virulence determinant of Ca. Liberibacter asiaticus. We have also acquired and set up a microplate reader equipped for the spectrophotometry, fluorimetry and luminometry assays needed to screen the combinatorial libraries. A potential lytic cycle activator was identified from Liberibacter crescens extracts using the holin promoter as bait and demonstrated by(EMSAs). The DNA binding site within the holin promoter was delineated using competitive DNA fragments.