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Title: CLONING AND CHARACTERIZATION OF A RHAMNOGALACTURONAN HYDROLASE GENE FROM BOTRYTIS CINEREA

Author
item CHEN, HUEY - UNIVERSITY OF MARYLAND
item Smith, David
item STARRETT, DAVID - SE MISSOURI STATE UNIV.
item ZHOU, DINGBO - UNIVERSITY OF MARYLAND
item Tucker, Mark
item SOLOMOS, THEOPHANES - UNIVERSITY OF MARYLAND
item Gross, Kenneth

Submitted to: Molecular Plant Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Much fresh produce is never consumed due to postharvest decay. A significant portion of this loss is attributable to postharvest fungal decay by Botrytis cinerea, an important pathogen of fresh fruits and vegetables. Because of the economic importance of postharvest decay, substantial research has been conducted to alleviate the problem commercially and to understand how fungi are able to degrade the plant cel wall, an important barrier to fungal invasion of fruit and vegetable tissues. In this research project, we have isolated and characterized a rhamnogalacturonase gene from B. cinerea and studied the ability of the enzyme produced by this gene to degrade cell wall sugars found in fruit and vegetable cell walls. This information will be used by us and other scientists to screen for naturally occurring, non-toxic inhibitors for reducing action of this enzyme, thereby reducing decay without using fungicides.

Technical Abstract: Rhamnogalacturonan hydrolase, also known as RG-hydrolase (RGase A), cleaves a1,2 linkages between non-esterified galacturonosyl and rhamnosyl residues of the pectic backbone. To evaluate the role of RGase A in relation to cell wall degradation during fungal decay of fruits and vegetables by Botrytis cinerea, an important postharvest pathogen, we identified a cDNA clone for RGase A. A 1.9 kb RGase A cDNA clone (BCRHGA) was isolated from a Botrytis cinerea cDNA library using a PCR-amplified Aspergillus aculeatus RGase A probe. The 1.7 kb open reading frame, within the BCRHGA cone, shared 62 percent identity at the amino acid level with A. aculeatus RGase A. Northern blot analysis of B. cinerea total RNA probed with teh BCRHGA cDNA revealed a 2 kb band, showing the BCRHGA clone is a full or nearly-full length cDNA clone. To determine the expression profile of the rhgA gene, we grew B. cinerea spores in Richmond media containing 0.5 percent apple pectin, 0.5 percent rhamnogalacturonan-I (RG-I) and 1 percen glucose as a carbon source. Northern blot analyses revealed that the rhgA gene was constitutively expressed in cells grown on all three carbon sources. With 0.5 percent apple pectin and RG-I substrate, maximum expression was reached in three-day-old cells and then dropped after day three. A different gene expression pattern was obtained when cells were grown on 1 percent glucose, in which rghA expression increased progressively from day one to six. Botrytis cinerea RGase A appears to be coded for by a single or low copy number gene based on Southern blot analysis.