Save the Wine: Expression and Partial Purification of Xylella fastidiosa polygalacturonase
Date
2010-08
Authors
Browning, Jenny L.
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Abstract
Pierce's disease (PD) in grapevines was first identified as problematic in California around 1884, destroying 35,000 acres of grapevines and completely eliminating grape production southeast of Los Angeles. Pierce's Disease, caused by the gram-negative bacterium Xylella fastidiosa (Xf), is transmitted by xylem feeding insects called sharpshooters. X fastidiosa has genes that encode an array of cell-wall-digesting enzymes including polygalacturonases, which catalyze the hydrolysis of the cx.1-4 glycosidic linkages of pectin (polygalacturonic acid).
Polygalacturonase inhibitor proteins (PGIPs) are glycoproteins located in plant cell walls that selectively bind and inhibit pathogen PGs. In order to screen PGIPs for optimum inhibition, we have cloned and expressed functional polygalacturonase in a Drosophila expression system. The polygalacturonase (PG) gene, XfPG, produced by, X. fastzdiosa, was ligated into the expression vector pMT/BiPN5-His A. Positive clones were transfected into Drosophila S2 cells and expression was induced at 24 hours with copper sulfate. PG protein was detected with western blotting and partially purified utilizing a Ni2+ affinity chromatography column. Activity was then detected with a standard reducing sugar assay and radial diffusion assay.
There are few reports on the purification, characterization, and stability of XfPG, however, these properties have been well documented on other species producing polygalacturonases. The results produced from this study will contribute to efforts of characterizing Xf polygalacturonase leading to the creation of transgenic grapevines and suppression of Pierce's Disease. Funding support provided by the Welch Foundation and Texas State University-San Marcos.
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Keywords
polygalacturonase, gram-negative bacteria, Drosophila, Pierce's disease
Citation
Browning, J. L. (2010). Save the wine: Expression and partial purification of Xylella fastidiosa polygalacturonase (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.