In Objectives 1 and 2, we proposed targeting specific regulators of key phage encoded virulence genes (such as the Las LexA-like repressor, LC1, a second downstream repressor, LC2, controlled in part by LC1), and a key exogenous regulator of the (lethal) phage lytic cycle encoded by Wolbachia, an important psyllid endosymbiont that is always found when Las is present. These results have so far resulted in three full length manuscripts and numerous. LC1, LC2 and the Wolbachia repressor have all been confirmed to be transcriptional repressors. All three are therefore prime targets for chemical interference. The Wolbachia protein has been shipped to both the De La Fuente lab in Auburn, and Duan lab at USDA-Ft. Pierce, our collaborators on a separate culturing project. In an attempt to duplicate a method previously used to identify a drug (tolefemic acid) that interfered with binding of a Las regulatory protein (PrbB) (Gardner et al., 2016), purified Wolbachia, C2 and C1 repressor proteins were subject to thermal denaturation analyses using two different florescent markers: SYPRO Orange and Nile Red. Commercially obtained citrate synthase was used as the control. The assays were performed using 5, 20 and 40uM purified protein in 14 different buffers. However, except for the citrate synthase control, none of the target Las repressor proteins yielded thermal shifts amenable for high throughput chemical screens, despite multiple attempts under differing conditions. A newly developed alternative to thermal denaturation screens that we are now evaluating is Protein Induced Fluorescence Enhancement (PIFE). This is also a high throughput method to identify chemicals that interfere with DNA binding proteins. The 3 regulatory protein targets were previously demonstrated to bind specific promoter DNAs. In the PIFE method, the promoter DNAs are labeled by covalently attaching the fluorescent tag Cy3 to one end and biotin at the other. The biotin tag is used to anchor the DNA to 96 well microtiter plates, and the bound DNA fluoresces due to the Cy3 tag. The unlabeled target protein is then applied, and binding to the target promoter should result in a decrease in fluorescence. Binding of an interfering chemical to the target protein can result in a change in fluorescence (usually, an increase) in fluorescence. These experiments are well underway and should be completed in a few months. We have identified a new secreted Las virulence factor that is likely critical for Las pathogenicity in citrus: peroxiredoxin CLIBASIA_00485. Expression of this gene occurs only in the citrus host and is nearly undetectable in psyllids. This pattern of expression is similar to the phage SC2 peroxidase. CLIBASIA_00485 and a second, non-secreted peroxidase, CLIBASIA_00980, were both functionally confirmed as active in the cultured Las proxy, L. crescens (Lcr). Lcr cells expressing CLIBASIA_00980 driven by the lacZ promoter were 65-fold more resistant to hydrogen peroxide. Lcr carries genes with ca. 50% predicted protein similarity to CLIBASIA_00980 and CLIBASIA_00485. Importantly, Lcr with CLIBASIA_00485 were 36-fold more resistant to hydrogen peroxide but a striking 214-fold more resistant to tert-butyl hydroperoxide (tBOOH, an organic peroxide). Transient over-expression of CLIBASIA_00485 in tobacco suppressed (a) H2O2-induced activation of RbohB, the key gatekeeper of plant defense signaling cascade, and (b) tBOOH-induced lipid peroxidation of plant cell membranes. Suppression of lipid peroxidation prevents biosynthesis of antimicrobial oxylipins, and subsequent transcriptional activation of plant defense genes. This likely essential pathogenicity enzyme is also being evaluated as a potential control target.