|Bar Shimon, M|
Submitted to: Current Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/6/2003
Publication Date: 3/1/2004
Citation: BAR SHIMON, M., YEHUDA, H., COHEN, L., WIESS, B., KOBESHNIKOV, A., DAUS, A., GOLDWAY, M., WISNIEWSKI, M.E., DROBY, S. CHARACTERIZATION OF EXTRACELLULAR LYTIC ENZYMES PRODUCED BY THE YEAST BICONTROL AGENT CANDIDA OLEOPHILA. CURRENT GENETICS. VOL. #45, pp. 140-148. MARCH 2004. Interpretive Summary: The use of biological control agents is one way of addressing consumer demand for decreasing the use of synthetic pesticides in agriculture. In order to identify effective antagonists that have potential for commercial products, research is needed to better understand the genetic and biochemical traits that are responsible for biocontrol activity. In the present study, several enzymes that are capable of disrupting the cell wall of pathogenic fungi were studied in a normal strain of yeast, Candida oleophila, and in strains of the yeast that had been genetically altered to either overexpress or underexpress one particular cell-wall-degrading enzyme (exoglucanase). The results of test tube studies indicated that the transgenic strain that underexpressed the exoglucanase did not control several postharvest pathogens responsible for fruit rots as well as the native strain or the overexpressing strain of the yeast. This indicated that the exoglucanase plays a role in biocontrol activity. On the other hand, when the various strains of the biocontrol agent were used directly on fruit there was no difference in biocontrol activity. All strains performed in a similar manner. This indicated that other cell-wall-degrading enzymes, such as chitinases and other glucanases, were able to compensate for the variable levels of the exoglucanase and that other lytic enzymes must also be involved in biocontrol activity. The results from this study will help us to better understand how biocontrol agents work and this knowledge will enable us to select more effective antagonists and thus reduce dependency on synthetic chemicals.
Technical Abstract: In our efforts to identify genetic traits of the yeast Candida oleophila that potentially affect its biocontrol activity, we have studied the ability of the yeast to produce fungal cell wall-degrading enzymes. This study has focused on four enzymes secreted by C. oleophila: exo-1,3-b-glucanase, endo-1,3-b-glucanase, chitinase and protease. The influences of the type of carbon source on the production of these enzymes by the yeast antagonist were determined by using fluorometric and colorimetric assays. SDS-PAGE and activity gels of partially purified enzyme preparations were used to study the molecular mass. In vivo production of exo-1,3-b-glucanase by the yeast was also determined. In a previous report, a full-length gene coding for a secreted exo-1,3-b-glucanase (CoEXG1) was cloned from this yeast and various transformants characterized (Segal et al., 2002). In the present study, glucanase activity of either a C. oleophila transformant harboring two copies of the CoEXG1 or a knockout-CoEXG1 transformant was characterized. The transformants exhibited either increased (at least two fold) or reduced production of exo-b-1,3-glucanase, respectively. We found that concentrated culture filtrate of C. oleophila transformants overexpressing the exoglucanase inhibited the growth of Penicillium digitatum similar to the wild-type but better than the knockout-transformant in in vitro experiments. In vivo experiments using live yeast cells were also carried out on grapefruit. We found no significant difference in biocontrol activity between C. oleophila wild-type and either of the transformants, probably because other lytic enzymes such as chitinases were involved.