Abstract
Phytophthora sojae, the causal agent of soybean root and stem rot, is responsible for enormous economic losses in soybean production. P. sojae secrets various effectors to reprogram host immunity. The plant apoplastic space is a major battleground in plant-pathogen interactions. Here we describe a protocol for purification and isolation of secreted proteins from P. sojae, including precipitation of secreted proteins from P. sojae culture filtrate, chromatographic purification of the secreted proteins and analysis of the proteins by Mass spectrometry. With this protocol, it will be easier to identify potential apoplastic effectors in Phytophthora and will benefit our understanding of plant-microbe interactions.
Keywords: Phytophthora sojae, Soybean root and stem rot, Secreted proteins, Chromatographic purification, ÄKTA, Apoplastic effectors
Background
Purification of proteins secreted from Phytophthora species is essential for understanding Phytophthora pathogenesis. In the past, V8 juice and Plant (tomato, and Lima bean) juice medium have been used to culture Phytophthora and the culture filtrated was used for the subsequent analysis of Phytophthora secreted proteins. The drawback of these protocols is the culturing media contain huge amount of plant proteins, which represent a large proportion of the detected proteins. In this protocol, we made use of the Synthesis Liquid Medium, which does not contain any proteins. This medium significantly reduces the background of the Phytophthora culturing filtrate. Furthermore, making use of the Gel Filtration desalting and sieving columns instead of Ion Exchange columns, allows efficient and large-scale purification of Phytophthora secreted proteins.
Materials and Reagents
Equipment
Note: The materials, reagents and equipment not provided company and catalog number can be ordered from any qualified company for using in this experiment.
Procedure
Data analysis
Based on the molecular mass, secreted proteins were separated by Gel filtration sieving chromatography. Protein with similar molecular mass will form one peak. The area of the peak represents the abundance of purified proteins. Proteins collected in each peak were subsequently used for LC-MS/MS analysis and the data were shown in Ma et al. (2015).
Notes
Recipes
Acknowledgments
The authors would like to thank Dr. Zhenchuan Ma and this work was fund by grants to Yuanchao Wang from the China National Funds for Innovative Research Groups (Grant No. 31721004), the Special Fund for Agro-scientific Research in the Public Interest (201303018), and the China Agriculture Research System (CARS-004-PS14).
Competing interests
The authors declare that there is no conflict of interest.
References
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