Transcriptomics is essential but not sufficient to unravel complex plant-pathogen interactions.

KEY MESSAGE: A group of genes that were upregulated in a resistant cultivar while downregulated in a susceptible cultivar in a transcriptomics analysis of potato challenged by Spongospora subterranea infection, did not show the same expression pattern at the protein level.

Comparative Proteomic Analysis of Potato Roots from Resistant and Susceptible Cultivars to Spongospora subterranea Zoospore Root Attachment In Vitro.

Potato (Solanum tuberosum L.) exhibits broad variations in cultivar resistance to tuber and root infections by the soilborne, obligate biotrophic pathogen Spongospora subterranea. Host resistance has been recognised as an important approach in potato disease management, whereas zoospore root attachment has been identified as an effective indicator for the host resistance to Spongospora root infection. However, the mechanism of host resistance to zoospore root attachment is currently not well understood. To identify the potential basis for host resistance to S. subterranea at the molecular level, twelve potato cultivars differing in host resistance to zoospore root attachment were used for comparative proteomic analysis. In total, 3723 proteins were quantified from root samples across the twelve cultivars using a data-independent acquisition mass spectrometry approach. Statistical analysis identified 454 proteins that were significantly more abundant in the resistant cultivars; 626 proteins were more abundant in the susceptible cultivars. In resistant cultivars, functional annotation of the proteomic data indicated that Gene Ontology terms related to the oxidative stress and metabolic processes were significantly over-represented. KEGG pathway analysis identified that the phenylpropanoid biosynthesis pathway was associated with the resistant cultivars, suggesting the potential role of lignin biosynthesis in the host resistance to S. subterranea. Several enzymes involved in pectin biosynthesis and remodelling, such as pectinesterase and pectin acetylesterase, were more abundant in the resistant cultivars. Further investigation of the potential role of root cell wall pectin revealed that the pectinase treatment of roots resulted in a significant reduction in zoospore root attachment in both resistant and susceptible cultivars. This study provides a comprehensive proteome-level overview of resistance to S. subterranea zoospore root attachment across twelve potato cultivars and has identified a potential role for cell wall pectin in regulating zoospore root attachment.

Yield-Related QTL Clusters and the Potential Candidate Genes in Two Wheat DH Populations.

In the present study, four large-scale field trials using two doubled haploid wheat populations were conducted in different environments for two years. Grain protein content (GPC) and 21 other yield-related traits were investigated. A total of 227 QTL were mapped on 18 chromosomes, which formed 35 QTL clusters. The potential candidate genes underlying the QTL clusters were suggested. Furthermore, adding to the significant correlations between yield and its related traits, correlation variations were clearly shown within the QTL clusters. The QTL clusters with consistently positive correlations were suggested to be directly utilized in wheat breeding, including 1B.2, 2A.2, 2B (4.9-16.5 Mb), 2B.3, 3B (68.9-214.5 Mb), 4A.2, 4B.2, 4D, 5A.1, 5A.2, 5B.1, and 5D. The QTL clusters with negative alignments between traits may also have potential value for yield or GPC improvement in specific environments, including 1A.1, 2B.1, 1B.3, 5A.3, 5B.2 (612.1-613.6 Mb), 7A.1, 7A.2, 7B.1, and 7B.2. One GPC QTL (5B.2: 671.3-672.9 Mb) contributed by cultivar Spitfire was positively associated with nitrogen use efficiency or grain protein yield and is highly recommended for breeding use. Another GPC QTL without negatively pleiotropic effects on 2A (50.0-56.3 Mb), 2D, 4D, and 6B is suggested for quality wheat breeding.

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.

Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase expression and activity in response to different nitrogen sources in nitrogen-starved wheat seedlings.

The objective of this study was to examine the expression and activity of nitrate reductase (NR, EC 1.7.1.1), nitrite reductase (NiR, EC 1.7.2.2), glutamine synthetase (GS, EC 6.3.1.2), and glutamate synthase (GOGAT, EC 1.4.7.1) in response to potassium nitrate, ammonium chloride, and ammonium nitrate in nitrogen-starved wheat seedlings. Plants were grown in standard nutrient solution for 17 days and then subjected to nitrogen starvation for 7 days. The starved plants were supplied with potassium nitrate ammonium nitrate and ammonium chloride (50 mM) for 4 days and the leaves were harvested. The relative expression of NR, NiR, GS, and GOGAT as well as the enzyme activities were investigated. Nitrogen starvation caused a significant decrease both in transcript levels and in NR, NiR, GS, and GOGAT activities. Potassium nitrate and ammonium nitrate treatments restored NR, NiR, GS, and GOGAT expressions and activities. Ammonium chloride increased only the expressions and activities of GS and GOGAT in a dose-dependent manner. The results of our study highlight the differential effects between the type and the amount of nitrogen salts on NR, NiR, GS, and GOGAT activities in wheat seedlings while potassium nitrate being more effective.

Enzymatic Investigation of Spongospora subterranea Zoospore Attachment to Roots of Potato Cultivars Resistant or Susceptible to Powdery Scab Disease.

For potato crops, host resistance is currently the most effective and sustainable tool to manage diseases caused by the plasmodiophorid Spongospora subterranea. Arguably, zoospore root attachment is the most critical phase of infection; however, the underlying mechanisms remain unknown. This study investigated the potential role of root-surface cell-wall polysaccharides and proteins in cultivars resistant/susceptible to zoospore attachment. We first compared the effects of enzymatic removal of root cell-wall proteins, N-linked glycans and polysaccharides on S. subterranea attachment. Subsequent analysis of peptides released by trypsin shaving (TS) of root segments identified 262 proteins that were differentially abundant between cultivars. These were enriched in root-surface-derived peptides but also included intracellular proteins, e.g., proteins associated with glutathione metabolism and lignin biosynthesis, which were more abundant in the resistant cultivar. Comparison with whole-root proteomic analysis of the same cultivars identified 226 proteins specific to the TS dataset, of which 188 were significantly different. Among these, the pathogen-defence-related cell-wall protein stem 28 kDa glycoprotein and two major latex proteins were significantly less abundant in the resistant cultivar. A further major latex protein was reduced in the resistant cultivar in both the TS and whole-root datasets. In contrast, three glutathione S-transferase proteins were more abundant in the resistant cultivar (TS-specific), while the protein glucan endo-1,3-beta-glucosidase was increased in both datasets. These results imply a particular role for major latex proteins and glucan endo-1,3-beta-glucosidase in regulating zoospore binding to potato roots and susceptibility to S. subterranea.

Multi-omics reveals mechanisms of resistance to potato root infection by Spongospora subterranea.

The pathogen Spongospora subterranea infects potato roots and developing tubers resulting in tuber yield and quality losses. Currently, there are no fully effective treatments for disease control. Host resistance is an important tool in disease management and understanding the molecular mechanisms of defence responses in roots of potato plants is required for the breeding of novel resistant cultivars. Here, we integrated transcriptomic and proteomic datasets to uncover these mechanisms underlying S. subterranea resistance in potato roots. This multi-omics approach identified upregulation of glutathione metabolism at the levels of RNA and protein in the resistant cultivar but not in the susceptible cultivar. Upregulation of the lignin metabolic process, which is an important component of plant defence, was also specific to the resistant cultivar at the transcriptome level. In addition, the inositol phosphate pathway was upregulated in the susceptible cultivar but downregulated in the resistant cultivar in response to S. subterranea infection. We provide large-scale multi-omics data of Spongospora-potato interaction and suggest an important role of glutathione metabolism in disease resistance.

Large-Scale Protein and Phosphoprotein Profiling to Explore Potato Resistance Mechanisms to Spongospora subterranea Infection.

Potato is one of the most important food crops for human consumption. The soilborne pathogen Spongospora subterranea infects potato roots and tubers, resulting in considerable economic losses from diminished tuber yields and quality. A comprehensive understanding of how potato plants respond to S. subterranea infection is essential for the development of pathogen-resistant crops. Here, we employed label-free proteomics and phosphoproteomics to quantify systemically expressed protein-level responses to S. subterranea root infection in potato foliage of the susceptible and resistant potato cultivars. A total of 2,669 proteins and 1,498 phosphoproteins were quantified in the leaf samples of the different treatment groups. Following statistical analysis of the proteomic data, we identified oxidoreductase activity, electron transfer, and photosynthesis as significant processes that differentially changed upon root infection specifically in the resistant cultivar and not in the susceptible cultivar. The phosphoproteomics results indicated increased activity of signal transduction and defense response functions in the resistant cultivar. In contrast, the majority of increased phosphoproteins in the susceptible cultivar were related to transporter activity and sub-cellular localization. This study provides new insight into the molecular mechanisms and systemic signals involved in potato resistance to S. subterranea infection and has identified new roles for protein phosphorylation in the regulation of potato immune response.

Shotgun Proteomics as a Powerful Tool for the Study of the Proteomes of Plants, Their Pathogens, and Plant-Pathogen Interactions.

The interaction between plants and pathogenic microorganisms is a multifaceted process mediated by both plant- and pathogen-derived molecules, including proteins, metabolites, and lipids. Large-scale proteome analysis can quantify the dynamics of proteins, biological pathways, and posttranslational modifications (PTMs) involved in the plant-pathogen interaction. Mass spectrometry (MS)-based proteomics has become the preferred method for characterizing proteins at the proteome and sub-proteome (e.g., the phosphoproteome) levels. MS-based proteomics can reveal changes in the quantitative state of a proteome and provide a foundation for understanding the mechanisms involved in plant-pathogen interactions. This review is intended as a primer for biologists that may be unfamiliar with the diverse range of methodology for MS-based shotgun proteomics, with a focus on techniques that have been used to investigate plant-pathogen interactions. We provide a summary of the essential steps required for shotgun proteomic studies of plants, pathogens and plant-pathogen interactions, including methods for protein digestion, identification, separation, and quantification. Finally, we discuss how protein PTMs may directly participate in the interaction between a pathogen and its host plant.

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.

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.

Quantitative proteomics provides an insight into germination-related proteins in the obligate biotrophic plant pathogen Spongospora subterranea.

The soil-borne and obligate plant-associated nature of S. subterranea has hindered a detailed study of this pathogen and in particular, the regulatory pathways driving the germination of S. subterranea remain unknown. To better understand the mechanisms that control the transition from dormancy to germination, protein profiles between dormant and germination stimulant-treated resting spores were compared using label-free quantitative proteomics. Among the ~680 proteins identified 20 proteins were found to be differentially expressed during the germination of S. subterranea resting spores. Elongation factor Tu, histones (H2A and H15), proteasome and DJ-1_PfpI, involved in transcription and translation, were upregulated during the germination of resting spores. Downregulation of both actin and beta-tubulin proteins occurred in the germinating spores, indicating that the changes in the cell wall cytoskeleton may be necessary for the morphological changes during the germination of the resting spore in S. subterranea. Our findings provide new approaches for the study of these and similar recalcitrant micro-organisms provide the first insights into the basic protein components of S. subterranea spores. A better understanding of S. subterranea biology may lead to the development of novel approaches for the management of persistent soil inoculum.

How exogenous nitric oxide regulates nitrogen assimilation in wheat seedlings under different nitrogen sources and levels.

Nitrogen (N) is one of the most important nutrients for plants and nitric oxide (NO) as a signaling plant growth regulator involved in nitrogen assimilation. Understanding the influence of exogenous NO on nitrogen metabolism at the gene expression and enzyme activity levels under different sources of nitrogen is vitally important for increasing nitrogen use efficiency (NUE). This study investigated the expression of key genes and enzymes in relation to nitrogen assimilation in two Australian wheat cultivars, a popular high NUE cv. Spitfire and a normal NUE cv. Westonia, under different combinations of nitrogen and sodium nitroprusside (SNP) as the NO donor. Application of NO increased the gene expressions and activities of nitrogen assimilation pathway enzymes in both cultivars at low levels of nitrogen. At high nitrogen supplies, the expressions and activities of N assimilation genes increased in response to exogenous NO only in cv. Spitfire but not in cv. Westonia. Exogenous NO caused an increase in leaf NO content at low N supplies in both cultivars, while under high nitrogen treatments, cv. Spitfire showed an increase under ammonium nitrate (NH4NO3) treatment but cv. Westonia was not affected. N assimilation gene expression and enzyme activity showed a clear relationship between exogenous NO, N concentration and N forms in primary plant nitrogen assimilation. Results reveal the possible role of NO and different nitrogen sources on nitrogen assimilation in Triticum aestivum plants.

Analysis of nitrate reductase mRNA expression and nitrate reductase activity in response to nitrogen supply.

Nitrate is one of the major sources of nitrogen for the growth of plants. It is taken up by plant roots and transported to the leaves where it is reduced to nitrite in the. The main objective of this research was to investigate stimulatory effects of sodium nitrate, potassium nitrate, ammonia and urea on the production/generation of the nitrate reductase mRNA in Triticum aestivum plants. The plants were grown in standard nutrient solution for 21 days and then starved in a media without nitrate for seven days. Starved plants were stimulated with various concentrations of sodium nitrate, potassium nitrate, ammonia and urea, and the expression of nitrate reductase mRNA was analyzed by real-time PCR. Our results indicated that starvation caused significant decrease in the production of nitrate reductase mRNA in the plant leaf. Sodium and potassium nitrate were capable of restoring the production of nitrate mRNA in a dose-dependent manner, since 50 mM of each produced the highest level of the mRNA. The stimulatory effect of potassium nitrate was higher than sodium nitrate, while ammonia and urea did not show such activity. At low concentrations, sodium nitrate and potassium nitrate caused significant increase in the nitrate/nitrite mRNA production, whereas high concentrations of these salts suppressed the expression of this gene considerably.