In Planta Transcriptome and Proteome Profiles of Spongospora subterranea in Resistant and Susceptible Host Environments Illuminates Regulatory Principles Underlying Host–Pathogen Interaction

Spongospora subterranea is an obligate biotrophic pathogen, causing substantial economic loss to potato industries globally. Currently, there are no fully effective management strategies for the control of potato diseases caused by S. subterranea. To further our understanding of S. subterranea biology during infection, we characterized the transcriptome and proteome of the pathogen during the invasion of roots of a susceptible and a resistant potato cultivar. A total of 7650 transcripts from S. subterranea were identified in the transcriptome analysis in which 1377 transcripts were differentially expressed between two cultivars. In proteome analysis, we identified 117 proteins with 42 proteins significantly changed in comparisons between resistant and susceptible cultivars. The functional annotation of transcriptome data indicated that the gene ontology terms related to the transportation and actin processes were induced in the resistant cultivar. The downregulation of enzyme activity and nucleic acid metabolism in the resistant cultivar suggests a probable influence of these processes in the virulence of S. subterranea. The protein analysis results indicated that the majority of differentially expressed proteins were related to the metabolic processes and transporter activity. The present study provides a comprehensive molecular insight into the multiple layers of gene regulation that contribute to S. subterranea infection and development in planta and illuminates the role of host immunity in affecting pathogen responses.

Spore Germination of the Obligate Biotroph Spongospora subterranea: Transcriptome Analysis Reveals Germination Associated Genes

For soilborne pathogens, germination of the resting or dormant propagule that enables persistence within the soil environment is a key point in pathogenesis. Spongospora subterranea is an obligate soilborne protozoan that infects the roots and tubers of potato causing root and powdery scab disease for which there are currently no effective controls. A better understanding of the molecular basis of resting spore germination of S. subterranea could be important for development of novel disease interventions. However, as an obligate biotroph and soil dwelling organism, the application of new omics techniques for the study of the pre-infection process in S. subterranea has been problematic. Here, RNA sequencing was used to analyse the reprogramming of S. subterranea resting spores during the transition to zoospores in an in-vitro model. More than 63 million mean high-quality reads per sample were generated from the resting and germinating spores. By using a combination of reference-based and de novo transcriptome assembly, 6,664 unigenes were identified. The identified unigenes were subsequently annotated based on known proteins using BLAST search. Of 5,448 annotated genes, 570 genes were identified to be differentially expressed during the germination of S. subterranea resting spores, with most of the significant genes belonging to transcription and translation, amino acids biosynthesis, transport, energy metabolic processes, fatty acid metabolism, stress response and DNA repair. The datasets generated in this study provide a basic knowledge of the physiological processes associated with spore germination and will facilitate functional predictions of novel genes in S. subterranea and other plasmodiophorids. We introduce several candidate genes related to the germination of an obligate biotrophic soilborne pathogen which could be applied to the development of antimicrobial agents for soil inoculum management.

Evaluation of Effects of Chemical Soil Treatments and Potato Cultivars on Spongospora subterranea Soil Inoculum and Incidence of Powdery Scab and Potato Mop-Top Virus in Potato

Spongospora subterranea is a soilborne plasmodiophorid that causes powdery scab in potato. It also transmits potato mop-top virus (PMTV), which causes necrotic arcs (spraing) in potato tubers. Three field experiments were conducted in naturally S. subterranea-infested soil to investigate the effects of two chemicals, Omega 500F (fluazinam) and FOLI-R-PLUS RIDEZ (biological extract), on powdery scab, PMTV, and changes in S. subterranea inoculum with six different potato cultivars. The efficacy of soil treatment with these two chemicals on tuber lesions, root galling, and pathogen population was also assessed in greenhouse trials. The chemical treatments did not reduce powdery scab, root gall formation, or S. subterranea inoculum in the field or greenhouse trials. Postharvest S. subterranea soil inoculum in fields varied across farms and among potato cultivars but the pathogen population consistently increased by the end of the growing season. The evaluated russet cultivars were more tolerant to powdery scab than the yellow- or red-skinned cultivars but all were susceptible to PMTV. In the field, powdery scab indices and soil inoculum changes were positively correlated, while postharvest S. subterranea inoculum was positively correlated with root galling in both greenhouse trials. Powdery scab and PMTV occurred in noninoculated potting mix, indicating that peat-based potting mix is a source for both pathogens. These results demonstrate that chemical management methods currently used by farmers are ineffective, that S. subterranea and PMTV in potting mix can cause severe epidemics in greenhouses, and that potato cultivar choices impact inoculum increases in soil.

Development of High Resolution DNA Melting Analysis for Simultaneous Detection of Potato Mop-Top Virus and Its Vector, Spongospora subterranea, in Soil

In this study, a set of duplex reverse transcription (RT)-PCR-mediated high resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f.sp. subterranea (Sss), was developed. The infestation of soil by PMTV was detected by using a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel-electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, -CP, and -TGB) were analyzed together with the existing Sss primers using real-time RT-PCR. The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by high resolution DNA melting (HRM) analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV and/or Sss was found in 63-100% samples collected from fields in which PMTV-infected tubers had been observed. In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Between 63%-83% and 100% of the soil samples collected from PMTV-infested fields led to PMTV and Sss infections in the bait tobacco plants, respectively; whereas no PMTV or Sss infected plants were obtained from soil samples collected from PMTV/Sss-free fields.

Optimisation of Sporosori Purification and Protein Extraction Techniques for the Biotrophic Protozoan Plant Pathogen Spongospora subterranea

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.

Metabolomic Fingerprinting of Potato Cultivars Differing in Susceptibility to Spongospora subterranea f. sp. subterranea Root Infection

Plants defend themselves from pathogens by producing bioactive defense chemicals. The biochemical mechanisms relating to quantitative resistance of potato to root infection by Spongospora subterranea f. sp. subterranea (Sss) are, however, not understood, and are not efficiently utilized in potato breeding programs. Untargeted metabolomics using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to elucidate the biochemical mechanisms of susceptibility to Sss root infection. Potato roots and root exudate metabolic profiles of five tolerant cultivars were compared with those of five susceptible cultivars, following Sss inoculation, to identify tolerance-related metabolites. Comparison of the relative metabolite abundance of tolerant versus susceptible cultivars revealed contrasting responses to Sss infection. Metabolites belonging to amino acids, organic acids, fatty acids, phenolics, and sugars, as well as well-known cell wall thickening compounds were putatively identified and were especially abundant in the tolerant cultivars relative to the susceptible cultivars. Metabolites known to activate plant secondary defense metabolism were significantly increased in the tolerant cultivars compared to susceptible cultivars following Sss inoculation. Root-exuded compounds belonging to the chemical class of phenolics were also found in abundance in the tolerant cultivars compared to susceptible cultivars. This study illustrated that Sss infection of potato roots leads to differential expression of metabolites in tolerant and susceptible potato cultivars.