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

Citation

Balotf, S and Wilson, R and Tegg, RS and Nichols, DS and Wilson, CR, Optimisation of sporosori purification and protein extraction techniques for the biotrophic protozoan plant pathogen Spongospora subterranea, Molecules, 25, (14) Article 3109. ISSN 1420-3049 (2020) [Refereed Article]


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

DOI: doi:10.3390/molecules25143109

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 Details

Item Type:Refereed Article
Keywords:Spongospora subterranea, sporosori, density gradient centrifugation, Ludox®, proteomics, S-Trap
Research Division:Chemical Sciences
Research Group:Medicinal and biomolecular chemistry
Research Field:Proteins and peptides
Objective Division:Plant Production and Plant Primary Products
Objective Group:Horticultural crops
Objective Field:Field grown vegetable crops
UTAS Author:Balotf, S (Mr Sadegh Balotf)
UTAS Author:Wilson, R (Dr Richard Wilson)
UTAS Author:Tegg, RS (Dr Robert Tegg)
UTAS Author:Nichols, DS (Dr David Nichols)
UTAS Author:Wilson, CR (Professor Calum Wilson)
ID Code:139869
Year Published:2020
Funding Support:Australian Research Council (DP180103337)
Web of Science® Times Cited:2
Deposited By:TIA - Research Institute
Deposited On:2020-07-10
Last Modified:2020-08-05
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