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

ABSTRACT: Spongospora subterranea is a soil-borne plant pathogen responsible for economically significant root and powdery scab diseases of potato. However, Tthe obligate biotrophic nature of S. subterranea has made 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 sulfate (SDS) buffer followed by suspension-trapping (S-Trap). Label-free, quantitative proteomics was then used to evaluate the efficiency of sporosori purification and protein preparation methods. The purification protocol produced a highly purified suspension of S. subterranea sporosori without affecting the viability of the spores. 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 compare to using urea and SP3, with 218 and 652 proteins identified using SP3 and S-Trap methods, respectively. 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 proteomics study of obligate biotrophic pathogens such as S. subterranea.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Spongospora Sp.

SUBMITTER: Richard Wilson  

LAB HEAD: Dr Richard Wilson

PROVIDER: PXD019776 | Pride | 2021-09-09

REPOSITORIES: Pride