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. In this last quarter, we expressed and purified LexA (CLIBASIA_01645) fused with a His-tag for purification and further analysis of the protein. Mobility shift assays (EMSAs) have now been used to confirm that this repressor protein both binds to its own promoter, as is typical of LexA repressors, and also binds specifically to the primary SC1 lytic prophage early gene promoter. This binding was quite specific to a particular small fragment in the SC1 early gene promoter region and we are now characterizing the region. This finding makes this LexA repressor a promising target for chemical inhibitors, since inhibition of SC1 is likely critical to Las survival in psyllids. Objective 2 is control of HLB using a repressor protein of unknown identity from psyllids as target. This repressor has been was published by us as silencing the Las phage holin gene, expression of which is lethal. We reported earlier that Las-free psyllids carry the repressor, and that similar extracts from Drosophila (fruit fly) do not. The repressor protein from the psyllid extract was immuno-captured using holin promoter DNA immobilized on magnetic DynaBeads. Sufficient protein was captured to enable visualization by Commassie Blue sent for liquid chromatography tandem mass spectrometry (LCMSMS) analyses to attempt to identify the protein. Twenty-five candidate peptide fingerprints were identified, of which two were of bacterial origin. A Wolbachia protein that seems to be unique to Wolbachia found in psyllids was identified that is within the characterized size range of 10- 50 kD (a small protein) and that is also potentially a DNA-binding protein. This protein candidate is not found in Wolbachia sequenced from Drosophila or other insects and is being cloned into an expression vector for use in activity assays with the holin promoter reporter construct used to identify the psyllid repressor activity. Objective 3 is control of HLB using the Las phage peroxidase and Las lytic cycle activator(s) as targets. We are now standardizing the inhibitory dose of hydrogen peroxide against Liberibacter crescens wild type strain BT-1 (lacking peroxidase) and BT-1 transformed with Las peroxidase from prophage SC2. These will be used for high throughput peroxidase inhibition screening assays using two Prestwick combinatorial chemical libraries in a 96-well plate format using a spectrophotometric plate reader. Once an ID50 (50% growth inhibition) value is established, inhibition of SC2 peroxidase by candidate combinatorial library chemicals may be possible. Alternatively, crude cell free protein extracts of wild type and peroxidase containing BT-1 strains will be assayed using an in vitro peroxide/ peroxidase assay kit in a 96-well format screen.