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Optimisation of sporosori purification and protein extraction techniques for the biotrophic protozoan plant pathogen Spongospora subterranea

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Balotf, S, Wilson, R ORCID: 0000-0003-0152-4394, Tegg, RS ORCID: 0000-0001-7668-7927, Nichols, DS ORCID: 0000-0002-8066-3132 and Wilson, CR ORCID: 0000-0001-5434-3816 2020 , 'Optimisation of sporosori purification and protein extraction techniques for the biotrophic protozoan plant pathogen Spongospora subterranea' , Molecules, vol. 25, no. 14 , pp. 1-13 , doi: 10.3390/molecules25143109.

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Abstract

Spongospora subterranea is a soil-borne plant pathogen responsible for the economically significant root and powdery scab diseases of potato. However, the obligate biotrophic nature of S. subterranea has made the detailed study of the pathogen problematic. Here, we first compared the benefits of sporosori partial purification utilizing Ludox® gradient centrifugation. We then undertook optimization efforts for protein isolation comparing the use of a urea buffer followed by single-pot solid-phase-enhanced sample preparation (SP3) and a sodium dodecyl sulphate (SDS) buffer followed by suspension-trapping (S-Trap). Label-free, quantitative proteomics was then used to evaluate the efficiency of the sporosori purification and the protein preparation methods. The purification protocol produced a highly purified suspension of S. subterranea sporosori without affecting the viability of the spores. The results indicated that the use of a combination of SDS and S-Trap for sample clean-up and digestion obtained a significantly higher number of identified proteins compared to using urea and SP3, with 218 and 652 proteins identified using the SP3 and S-Trap methods, respectively. The analysis of proteins by mass spectrometry showed that the number of identified proteins increased by approximately 40% after the purification of spores by Ludox®. These results suggested a potential use of the described spore purification and protein preparation methods for the proteomics study of obligate biotrophic pathogens such as S. subterranea.

Item Type: Article
Authors/Creators:Balotf, S and Wilson, R and Tegg, RS and Nichols, DS and Wilson, CR
Keywords: Spongospora subterranea, sporosori, density gradient centrifugation, Ludox®, proteomics, S-Trap
Journal or Publication Title: Molecules
Publisher: Molecular Diversity Preservation International
ISSN: 1420-3049
DOI / ID Number: 10.3390/molecules25143109
Copyright Information:

Copyright 2020 the authors. Licensed under Creative Commons Attribution 4.0 International (CC BY 4.0) https://creativecommons.org/licenses/by/4.0/

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