DEVELOPMENT OF PEPTIDE ANTIBIOTIC-BASED CONTROL STRATEGIES FOR XYLELLA FASTIDIOSA

• Author(s): Hammock, Bruce; Kamita, Shizuo; Kirkpatrick, Bruce; Warkentin, Donald;
• Abstract: Peptide antibiotics are short (generally less than 70 amino acid residue-long), pore forming peptides encoded by single genes. Because peptide antibiotics are gene-based they can be produced directly at the target location where they are needed (e.g., grape stock). In this project, we are testing the hypothesis that peptide antibiotics such as cecropins A, B, and/or P1 can be used as an effective means to control or reduce the spread of Xylella fastidiosa-induced disease. During the reporting period, we have established the optimal growth and assay conditions for the X. fastidiosa bacterium. Under these optimal conditions, we found that cecropin A can effectively inhibit X. fastidiosa growth over a two-week period (initial concentration of 0.05microM). Longer-term growth inhibition was seen only when higher concentrations of cecropin A were used suggesting that the cecropin A is being degraded under the conditions of our assay. On the basis of the effectiveness of cecropin A against X. fastidiosa, a synthetic plant codon (i.e., Arabidopsis thaliana) optimized, cecropin A gene was synthesized. The product of this synthetic cecropin A gene was expressed using the baculovirus expression vector system (BEVS) in insect cells. In insect cells roughly 90 mg/liter of culture of biologically active cecropin A was produced by the recombinant baculovirus. Following confirmation of biological activity of the insect cell produced cecropin A, the synthetic cecropin A gene was inserted into the pCAMBIA1305 series of plasmid vectors in order to express the cecropin A in transgenic A. thaliana and eventually grape stock. Four different recombinant pCAMBIA1305 vectors were generated (carrying either the pro or mature cecropin A gene fused to either an authentic insect- or plant- (rice glycine-rich protein) derived signal peptide sequence). We are currently in the process of generating transgenic A. thaliana using these pCAMBIA vectors. We believe that continuous expression (although potentially at relatively low levels) of cecropin A will be effective for reducing or inhibiting the growth of X. fastidiosa within the plant.
• Publication Date: Dec 2004
• Journal: 2004 Pierce's Disease Research Symposium