Faculty of Bioscience Engineering, Ghent University
[en] Pichia anomala (strain Kh6) was isolated from the surface of apple fruits and selected for its high and reliable biocontrol activity against Botrytis cinerea and Penicillium expansum. Its main modes of action have until now been studied using essentially microbiological and molecular approaches. The study continues now using the proteomic approach and considering the in situ P. anomala/B. cinerea/apple interaction. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the most powerful tools used for proteomic analysis. It combines two sequential separation steps, the first dimension via isoelectric focusing (IEF) and the second one by SDS PAGE. Although recent advances in 2-D PAGE, the extraction of the whole proteome and the removal of interfering contaminants still limit its application. Sample preparation constitutes indeed a critical influential step for IEF which in turn affects 2-D gel quality. The objective of the present work was thus to develop an effective protein extraction protocol designed for 2-D PAGE analysis of the proteome of P. anomala strain Kh6. As a starting point, two contrasting protein extraction protocols were chosen to be evaluated in terms of protein yield and one-dimensional (1-D) SDS PAGE and 2-D PAGE gel patterns. The first protocol uses a urea/thiourea-based lysis buffer whereas the second protocol utilizes a hot SDS-based lysis buffer with an additional precipitation step. The comparison model used consisted of apples treated with strain Kh6 alone (K) and apples first treated with Kh6 and then inoculated with B. cinerea conidia (KB). Growth kinetics of strain Kh6 on wounded apples was determined and found to be not affected by the presence of B. cinerea conidia. Proteins were extracted from yeast pellets collected at both the exponential and stationary phases of strain Kh6 growth on apples. The evaluation of both extraction protocols indicates that more proteins were extracted with the SDS protocol and, according to 1-D assays, higher molecular weight proteins were obtained with the ‘urea/thiourea’ protocol and, regardless of the protocol used, more bands were obtained during the exponential phase. 2-D assays are currently underway and the corresponding results will be presented.