Multi-omics analysis of Spongospora subterranea spore germination and interaction with its potato host

The obligate biotroph Spongospora subterranea is an important pathogen of potato, the world‚ÄövÑv¥s third most valuable food crop for human consumption. Resting spores are essential for the spread and survival of S. subterranea and can remain viable in the soil in their dormant states for decades rendering fields unsuitable for planting. In the presence of potato roots, resting spores germinate and release zoospores that subsequently infect potato roots. A better understanding of the molecular basis of resting spore germination of S. subterranea and the regulatory principles underlying Spongospora-potato interactions could be important for developing novel disease interventions. However, as a soilborne and obligate biotroph pathogen, the application of omics techniques for the detailed study of the pre- and post-infection processes in S. subterranea has been problematic. This thesis developed a method for the partial purification of S. subterranea resting spores utilizing Ludox\\(^¬¨vÜ\\) gradient centrifugation. A series of preliminary experiments were then undertaken for S. subterranea protein preparation. Protein profiles between dormant and germination stimulant-treated resting spores were compared using label-free quantitative proteomics. A deep RNA sequencing approach was then employed to analyse S. subterranea resting spores' reprogramming during the transition to zoospores in an in vitro model. Further, to expand our understanding of S. subterranea biology during infection, the transcriptome and proteome of the pathogen during the invasion of roots of a susceptible and a resistant potato cultivar was characterized in planta. Later an integrated transcriptomic and proteomic dataset was employed to uncover these mechanisms underlying S. subterranea resistance in potato roots. In relation to this, the proteome and phosphoproteome of potato leaves were also profiled to explore potato resistance mechanisms to S. subterranea at the post-transcriptome levels. Without affecting the viability of the spores, the Ludox\\(^¬¨vÜ\\) purification protocol produced a semi- purified and concentrated suspension of S. subterranea resting spores and improved protein identification by approximately 40%. The transcriptome and proteome analysis of S. subterranea spore germination introduced several candidate genes and proteins related to the germination of the pathogen, including those belonging to transcription and translation, transport, energy metabolic processes, stress response, and DNA repair. The in planta analysis of S. subterranea transcriptome and proteome indicated that transportation, metabolic processes and cytoskeletal processes were induced in the resistant cultivar. Enzyme activity and nucleic acid metabolism were decreased in this cultivar, suggesting a probable influence of these processes in the virulence of S. subterranea. The transcriptomic and proteomic datasets of potato roots infected by S. subterranea uncovered the critical role of glutathione metabolism and lignin metabolic process in potato resistance to S. subterranea. In addition, this experiment also confirmed that the inositol phosphate pathway might be related to the susceptibility of potato plants to the root disease by S. subterranea. The proteome and phosphoproteome analysis of potato leaves after infection by S. subterranea showed that oxidoreductase activity, electron transfer, and photosynthesis were significant processes that differentially changed in the proteome of resistant cultivar. The phosphoproteomics results indicated increased activity of signal transduction and defence response functions in resistant cultivars but not in the susceptible cultivar. In contrast, the majority of increased phosphoproteins in the susceptible cultivar were related to transporter activity and subcellular localisation. This thesis provides a comprehensive overview of the changes in transcriptome and proteome during the germination of S. subterranea resting spores and extends our knowledge on the spore germination in plasmodiophorids. In planta study and potato defence response analysis contributes to the significant progress in our understanding of the interaction between the obligate biotrophic pathogens and their host plants. More than that, the study increased the availability of omics data in such a complex interaction and can allow the exploitation of this knowledge for the benefit of agriculture.