Exploiting the Las and Lam phage for potential control of HLB

Exploiting the Las and Lam phage for potential control of HLB

Report Date: 02/11/2019
Project: 15-009   Year: 2018
Category: Horticultural & Management
Author: Dean Gabriel
Sponsor: Citrus Research and Development Foundation

Two Las repressors from Objective 1, and a Wolbachia repressor from Objective 2 were all confirmed as functional transcriptional regulators of Las phage genes and with DNA binding sites within key Las phage promoter regions. These three repressors are therefore potential chemical targets for inhibitors that may control HLB. In addition, a likely Las virulence effector, a secreted peroxiredoxin enzyme, was identified in Objective 3. This enzyme appears to prevent citrus host phloem cells from killing Las and also blocks systemic host responses to Las. This secreted enzyme is also a high value potential chemical target Two publications have appeared and a third is in press covering these Objectives. . Despite repeated attempts, the fluorimetric assay proved unusable for chemical library screening of the three small transcriptional regulatory proteins. All three repressors are very small DNA binding proteins with little potential to form folded structures, which is necessary for the thermal denaturation assay. An alternative approach was evaluated using Micro Scale Thermophoresis (MST), a biophysical technique that measures the strength of binding between two molecules by fluorescence. MST was used in an attempt to evaluate binding between commercially synthesized C2 protein and DNA from the promoter region previously demonstrated to bind C2 by gel retardation assays. Attempts to use MST failed, indicating that the C2 protein was unstable in the buffer conditions needed for MST. A high throughput fluorimetric thermal denaturation screen was first used to identify chemicals that bound to the (large) Las peroxiredoxin target. A total of 320 phytochemicals were screened, resulting in the identification of fourteen (14) lead candidates for phytochemical control of HLB. Several of the lead candidates are generally recognized as safe (GRAS) and are not pharmaceutical drugs. The larger library of 1,600 chemicals, including drugs, was then further screened using a direct enzymatic activity inhibition assay to independently verify the results of the fluorimetric assay and also potentially identify additional inhibitors that directly affect the secreted Las peroxiredoxin. Peroxiredoxins react with hydrogen peroxide and both aliphatic and aromatic hydroperoxide substrates. Of 1,600 chemicals screened, 28 exhibited a strong inhibitory effect on the Las peroxiredoxin. Based on possible commercial value as being both GRAS and relatively inexpensive, 7 chemicals were selected for further study. Three of the 7 chemicals were confirmed repeatedly as having a strong inhibitory effect. One of these was confirmed inhibitory by both fluorimetry and direct enzyme inhibition assays. Field trials in commercially grown, Las infected citrus failed to demonstrate a practical level of reduction of Las titer in heavily infected citrus. We speculate that Las peroxiredoxin is required for initial colonization and early establishment of infections, but once an infection is established, reduction in peroxiredoxin activity did not lead to clearing of existing infections. This compound will be further evaluated for efficacy in preventing new Las infections in commercial citrus replants.


Your browser does not support pdfs, click here to download the file.