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United States Department of Agriculture

Agricultural Research Service

Title: Cloning and Analysis of Coexg1: a Secreted 1,3-B Glucanase of the Yeast Biocontrol Agent Candida Oleophila

Authors
item Segal, E. - MIGAL GALILEE TECH CTR
item Segal, E. - MIGAL GALILEE TECH CTR
item Yehuda, H. - MIGAL GALILEE TECH CTR
item Yehuda, H. - MIGAL GALILEE TECH CTR
item Droby, S. - ARO, THE VOLCANI CENTER
item Droby, S. - ARO, THE VOLCANI CENTER
item Wisniewski, Michael
item Wisniewski, Michael
item Goldway, M. - MIGAL GALILEE TECH CTR
item Goldway, M. - MIGAL GALILEE TECH CTR

Submitted to: Current Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 15, 2002
Publication Date: October 1, 2002
Citation: SEGAL, E., YEHUDA, H., DROBY, S., WISNIEWSKI, M.E., GOLDWAY, M. CLONING AND ANALYSIS OF COEXG1: A SECRETED 1,3-B GLUCANASE OF THE YEAST BIOCONTROL AGENT CANDIDA OLEOPHILA. CURRENT GENETICS. Vol. 40, pgs. 282-287, October 2002.

Interpretive Summary: Developing biological methods of controlling postharvest diseases of fruits and vegetables is a critical area of research that addresses the public demand for decreased use of synthetic chemicals in agriculture. Previous research has identified several yeasts that displace pathogens from wounds and can be used to control postharvest rots. Some of these yeasts have been developed into commercial products by industry. Developing a better understanding of the underlying mechanisms in yeast responsible for biocontrol activity can lead to the selection of superior biocontrol agents and improve the efficacy of already identified biocontrol agents. One method that the biocontrol agents use to control pathogenic fungi is to secrete enzymes which can degrade the cell walls of the pathogens thus incapacitating them. The current research uses previous technology developed in our laboratories to clone the gene for a cell-wall degrading enzyme (glucanase) from a yeast antagonist and then insert it into baker's yeast in order to characterize it and determine if it is functional. The development of this system can allow us to determine which genes play an important role in biocontrol activity. Future research will be conducted to re-insert this gene into the antagonistic yeast so that it will be expressed at a higher level. We will then determine if higher levels of this enzyme increase the effectiveness of the antagonist. This research should enable us to select more effective antagonists and thus reduce dependency on synthetic chemicals.

Technical Abstract: Lytic enzymes are considered to have an important role in the biological control activity of fungi. The yeast biocontrol agent Candida oleophila is an excellent candidate to research this subject matter. In the present study, CoEXG1, encoding for a secreted 1,3-B-glucanase, the first gene cloned from C. oleophila, was analyzed. CoEXG1 was isolated from a partial genomic library. Its open reading and its putative promoter that we examined in frame was expressed in the baker's yeast Saccharomyes cerevisiae. The reading frame expressed under the inducible GAL1 promoter, resulting in an increased secretion of B-glucanase and the putative promoter region activated the lac-Z reporter gene to which it was fused. Sequencing analysis revealed that CoEXG1 carries the signature pattern of the 5 glycohydrolases family, has a putative secretion leader, and has a high degree of identity to yeast 1,3-B-glucanases.

Submitted to: Current Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 15, 2002
Publication Date: October 1, 2002
Citation: SEGAL, E., YEHUDA, H., DROBY, S., WISNIEWSKI, M.E., GOLDWAY, M. CLONING AND ANALYSIS OF COEXG1: A SECRETED 1,3-B GLUCANASE OF THE YEAST BIOCONTROL AGENT CANDIDA OLEOPHILA. CURRENT GENETICS. Vol. 40, pgs. 282-287, October 2002.

Interpretive Summary: Developing biological methods of controlling postharvest diseases of fruits and vegetables is a critical area of research that addresses the public demand for decreased use of synthetic chemicals in agriculture. Previous research has identified several yeasts that displace pathogens from wounds and can be used to control postharvest rots. Some of these yeasts have been developed into commercial products by industry. Developing a better understanding of the underlying mechanisms in yeast responsible for biocontrol activity can lead to the selection of superior biocontrol agents and improve the efficacy of already identified biocontrol agents. One method that the biocontrol agents use to control pathogenic fungi is to secrete enzymes which can degrade the cell walls of the pathogens thus incapacitating them. The current research uses previous technology developed in our laboratories to clone the gene for a cell-wall degrading enzyme (glucanase) from a yeast antagonist and then insert it into baker's yeast in order to characterize it and determine if it is functional. The development of this system can allow us to determine which genes play an important role in biocontrol activity. Future research will be conducted to re-insert this gene into the antagonistic yeast so that it will be expressed at a higher level. We will then determine if higher levels of this enzyme increase the effectiveness of the antagonist. This research should enable us to select more effective antagonists and thus reduce dependency on synthetic chemicals.

Technical Abstract: Lytic enzymes are considered to have an important role in the biological control activity of fungi. The yeast biocontrol agent Candida oleophila is an excellent candidate to research this subject matter. In the present study, CoEXG1, encoding for a secreted 1,3-B-glucanase, the first gene cloned from C. oleophila, was analyzed. CoEXG1 was isolated from a partial genomic library. Its open reading and its putative promoter that we examined in frame was expressed in the baker's yeast Saccharomyes cerevisiae. The reading frame expressed under the inducible GAL1 promoter, resulting in an increased secretion of B-glucanase and the putative promoter region activated the lac-Z reporter gene to which it was fused. Sequencing analysis revealed that CoEXG1 carries the signature pattern of the 5 glycohydrolases family, has a putative secretion leader, and has a high degree of identity to yeast 1,3-B-glucanases.

Last Modified: 12/18/2014
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