Abstract: 【Objective】The objective of this study is to identify the high efficient method for secreted protein extraction in Verticillium dahliae, provide a technique basis for further functional characterization of V. dahliae secretome, and to construct a platform for functional verification of virulence factors and the mechanism research of V. dahliae. 【Method】Secreted proteins of the high virulent V. dahliae strain VdG1 were extracted by three methods, including ultra-filtration (UF), TCA precipitation and ion-exchange (IEX), followed by LC-MS/MS analysis for protein identification. The bioinformatics softwares WoLFPSORT, SignalP, TMHMM and PHOBIUS were used to predict classical secreted proteins. The classical secreted proteins were annotated by using CAZy (carbohydrate-active enZYmes) and PHI (pathogen-host interaction) databases. In addition, motif searchs was conducted via BLASTp, such as RxLx, LysM and Cys Pattern. The extracted proteins were used to detect the biological activity and pathogenicity on susceptible cotton. 【Result】By the three methods of TCA precipitation, IEX and UF, the extracted efficiency of V. dahliae secretome was 1.18, 0.96 and 0.56 mg?L-1, respectively. The SDS-PAGE result showed that TCA and IEX extracted samples had more efficiency and purity than that of UF. The numbers of classical secreted proteins were 124, 112 and 75, respectively, of which 98, 85 and 57 were possible pathogenic factors. In addition, ion-exchange had a high extraction efficiency for small molecular weight proteins and the extracted protein could induce wilt and necrosis symptom to cotton. 【Conclusion】To extract secreted proteins of V. dahliae by three methods, the lowest efficiency and purity, the minimum amount of classical secreted proteins, the fewest number of potential pathogenic factors were the UF extracted samples, and such method was quite time-consuming for large volume processing. TCA and IEX methods were fit to extract secreted proteins of V. dahliae, but TCA method couldn’t keep the sample’s biological activities and the extracted sample was just for 2-D electrophoresis detection. IEX method was firstly applied in the preparation of pathogenic fungi, and the operation was simple, convenient, all processes were completed by the instrument system. The extracted proteins had biological activity, and could cause cotton leaf wilt and necrotic symptom. In addition, the database and software comparative analysis found that the identified secreted proteins of V. dahliae contain 61 CAZy enzymes, 38 PHI proteins, 13 RxLx motif proteins, 1 LysM motif protein, 15 Cys Pattern motif proteins, and 2 VdNEP family proteins. The results will be used for screening the pathogenic factors and providing an important basis for further functional verification.

Key words: Verticillium dahliae , secreted proteins , extracted methods , LC-MS/MS , comparison