Signalling switches maintain intercellular communication in the vascular endothelium.

BACKGROUND AND PURPOSE: The single layer of cells lining all blood vessels, the endothelium, is a sophisticated signal co-ordination centre that controls a wide range of vascular functions including the regulation of blood pressure and blood flow. To co-ordinate activities, communication among cells is required for tissue level responses to emerge. While a significant form of communication occurs by the propagation of signals between cells, the mechanism of propagation in the intact endothelium is unresolved. EXPERIMENTAL APPROACH: Precision signal generation and targeted cellular manipulation was used in conjunction with high spatiotemporal mesoscale Ca2+ imaging in the endothelium of intact blood vessels. KEY RESULTS: Multiple mechanisms maintain communication so that Ca2+ wave propagation occurs irrespective of the status of connectivity among cells. Between adjoining cells, regenerative IP3-induced IP3 production transmits Ca2+ signals and explains the propagated vasodilation that underlies the increased blood flow accompanying tissue activity. The inositide is itself sufficient to evoke regenerative phospholipase C-dependent Ca2+ waves across coupled cells. None of gap junctions, Ca2+ diffusion or the release of extracellular messengers is required to support this type of intercellular Ca2+ signalling. In contrast, when discontinuities exist between cells, ATP released as a diffusible extracellular messenger transmits Ca2+ signals across the discontinuity and drives propagated vasodilation. CONCLUSION AND IMPLICATIONS: These results show that signalling switches underlie endothelial cell-to-cell signal transmission and reveal how communication is maintained in the face of endothelial damage. The findings provide a new framework for understanding wave propagation and cell signalling in the endothelium.

Evolution of the Genetic Structure of the Didymella tanaceti Population During Development of Succinate Dehydrogenase Inhibitor Resistance.

Emergence of pathogens with decreased sensitivity to succinate dehydrogenase inhibitor fungicides is a global agronomical issue. Analysis of Didymella tanaceti isolates (n = 173), which cause tan spot of pyrethrum (Tanacetum cinerariifolium), collected prior to (2004 to 2005) and after (2009, 2010, 2012, and 2014) the commercial implementation of boscalid in Tasmanian pyrethrum fields identified that insensitivity developed over time and has become widespread. To evaluate temporal change, isolates were characterized for frequency of mutations in the succinate dehydrogenase (Sdh) B, C, and D subunits associated with boscalid resistance, mating type, and SSR genotype. All isolates from 2004 and 2005 exhibited wild-type (WT) Sdh alleles. Seven known Sdh substitutions were identified in isolates collected from 2009 to 2014. In 2009, 60.7% had Sdh substitutions associated with boscalid resistance in D. tanaceti. The frequency of WT isolates decreased over time, with no WT isolates identified in 2014. The frequency of the SdhB-H277Y genotype increased from 10.7 to 77.8% between 2009 and 2014. Genotypic evidence suggested that a shift in the population structure occurred between 2005 and 2009, with decreases in gene diversity (uh; 0.51 to 0.34), genotypic evenness (E5; 0.96 to 0.67), genotypic diversity (G; 9.3 to 6.8), and allele frequencies. No evidence was obtained to support the rapid spread of Sdh genotypes by clonal expansion of the population. Thus, insensitivity to boscalid has developed and become widespread within a diverse population within 4 years of usage. These results suggest that D. tanaceti can disperse insensitivity through repeated frequent mutation, sexual recombination, or a combination of both.

Mitochondrial ATP Production is Required for Endothelial Cell Control of Vascular Tone.

Arteries and veins are lined by nonproliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis, rather than mitochondrial oxidative phosphorylation, to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells, and little is known about energy production in endothelial cells within the fully formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial respiration disrupts endothelial control of vascular tone. Basal, mechanically activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. Agonist-evoked calcium activity was also inhibited by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for mitochondria in endothelial cell energy production is independent of species, sex, or vascular bed. These data show that a mitochondrial ATP supply is necessary for calcium-dependent, nitric oxide-mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function.

Endothelial PAR2 activation evokes resistance artery relaxation.

Protease-activated receptor-1 & -2 (PAR1 and PAR2) are expressed widely in cardiovascular tissues including endothelial and smooth muscle cells. PAR1 and PAR2 may regulate blood pressure via changes in vascular contraction or relaxation mediated by endothelial Ca2+ signaling, but the mechanisms are incompletely understood. By using single-cell Ca2+ imaging across hundreds of endothelial cells in intact blood vessels, we explored PAR-mediated regulation of blood vessel function using PAR1 and PAR2 activators. We show that PAR2 activation evoked multicellular Ca2+ waves that propagated across the endothelium. The PAR2-evoked Ca2+ waves were temporally distinct from those generated by muscarinic receptor activation. PAR2 activated distinct clusters of endothelial cells, and these cells were different from those activated by muscarinic receptor stimulation. These results indicate that distinct cell clusters facilitate spatial segregation of endothelial signal processing. We also demonstrate that PAR2 is a phospholipase C-coupled receptor that evokes Ca2+ release from the IP3 -sensitive store in endothelial cells. A physiological consequence of this PAR2 signaling system is endothelium-dependent relaxation. Conversely, PAR1 activation did not trigger endothelial cell Ca2+ signaling nor relax or contract mesenteric arteries. Neither did PAR1 activators alter the response to PAR2 or muscarinic receptor activation. Collectively, these results suggest that endothelial PAR2 but not PAR1 evokes mesenteric artery relaxation by evoking IP3 -mediated Ca2+ release from the internal store. Sensing mediated by PAR2 receptors is distributed to spatially separated clusters of endothelial cells.

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.

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.

Abundance of Poleroviruses within Tasmanian Pea Crops and Surrounding Weeds, and the Genetic Diversity of TuYV Isolates Found.

The genus Polerovirus contains positive-sense, single-stranded RNA plant viruses that cause significant disease in many agricultural crops, including vegetable legumes. This study aimed to identify and determine the abundance of Polerovirus species present within Tasmanian pea crops and surrounding weeds that may act as virus reservoirs. We further sought to examine the genetic diversity of TuYV, the most commonly occurring polerovirus identified. Pea and weed samples were collected during 2019-2020 between October and January from thirty-four sites across three different regions (far northwest, north, and midlands) of Tasmania and tested by RT-PCR assay, with selected samples subject to next-generation sequencing. Results revealed that the presence of polerovirus infection and the prevalence of TuYV in both weeds and pea crops varied across the three Tasmanian cropping regions, with TuYV infection levels in pea crops ranging between 0 and 27.5% of tested plants. Overall, two species members from each genus, Polerovirus and Potyvirus, one member from each of Luteovirus, Potexvirus, and Carlavirus, and an unclassified virus from the family Partitiviridae were also found as a result of NGS data analysis. Analysis of gene sequences of the P0 and P3 genes of Tasmanian TuYV isolates revealed substantial genetic diversity within the collection, with a few isolates appearing more closely aligned with BrYV isolates. Questions remain around the differentiation of TuYV and BrYV species. Phylogenetic inconsistency in the P0 and P3 ORFs supports the concept that recombination may have played a role in TuYV evolution in Tasmania. Results of the evolutionary analysis showed that the selection pressure was higher in the P0 gene than in the P3 gene, and the majority of the codons for each gene are evolving under purifying selection. Future full genome-based analyses of the genetic variations will expand our understanding of the evolutionary patterns existing among TuYV populations in Tasmania.

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.

Genomic Characterisation of an Isolate of Brassica Yellows Virus Associated with Brassica Weed in Tasmania.

Brassica yellows virus (BrYV), a tentative species in the genus Polerovirus, of the Solemoviridae family, is a phloem-restricted and aphid-transmitted virus with at least three genotypes (A, B, and C). It has been found across mainland China, South Korea, and Japan. BrYV was previously undescribed in Tasmania, and its genetic variability in the state remains unknown. Here, we describe a near-complete genome sequence of BrYV (genotype A) isolated from Raphanus raphanistrum in Tasmania using next-generation sequencing and sanger sequencing of RT-PCR products. BrYV-Tas (GenBank Accession no. OM469309) possesses a genome of 5516 nucleotides (nt) and shares higher sequence identity (about 90%) with other BrYV isolates. Phylogenetic analyses showed variability in the clustering patterns of the individual genes of BrYV-Tas. Recombination analysis revealed beginning and ending breakpoints at nucleotide positions 1922 to 5234 nt, with the BrYV isolate LC428359 and BrYV isolate KY310572 identified as major and minor parents, respectively. Results of the evolutionary analysis showed that the majority of the codons for each gene are evolving under purifying selection, though a few codons were also detected to have positive selection pressure. Taken together, our findings will facilitate an understanding of the evolutionary dynamics and genetic diversity of BrYV.

Small-world connectivity dictates collective endothelial cell signaling.

Every blood vessel is lined by a single layer of highly specialized, yet adaptable and multifunctional endothelial cells. These cells, the endothelium, control vascular contractility, hemostasis, and inflammation and regulate the exchange of oxygen, nutrients, and waste products between circulating blood and tissue. To control each function, the endothelium processes endlessly arriving requests from multiple sources using separate clusters of cells specialized to detect specific stimuli. A well-developed but poorly understood communication system operates between cells to integrate multiple lines of information and coordinate endothelial responses. Here, the nature of the communication network has been addressed using single-cell Ca2+ imaging across thousands of endothelial cells in intact blood vessels. Cell activities were cross-correlated and compared to a stochastic model to determine network connections. Highly correlated Ca2+ activities occurred in scattered cell clusters, and network communication links between them exhibited unexpectedly short path lengths. The number of connections between cells (degree distribution) followed a power-law relationship revealing a scale-free network topology. The path length and degree distribution revealed an endothelial network with a "small-world" configuration. The small-world configuration confers particularly dynamic endothelial properties including high signal-propagation speed, stability, and a high degree of synchronizability. Local activation of small clusters of cells revealed that the short path lengths and rapid signal transmission were achieved by shortcuts via connecting extensions to nonlocal cells. These findings reveal that the endothelial network design is effective for local and global efficiency in the interaction of the cells and rapid and robust communication between endothelial cells in order to efficiently control cardiovascular activity.

Subversion of Phytomyxae Cell Communication With Surrounding Environment to Control Soilborne Diseases; A Case Study of Cytosolic Ca2+ Signal Disruption in Zoospores of Spongospora subterranea.

Ca2+ signaling regulates physiological processes including chemotaxis in eukaryotes and prokaryotes. Its inhibition has formed the basis for control of human disease but remains largely unexplored for plant disease. This study investigated the role of Ca2+ signaling on motility and chemotaxis of Spongospora subterranea zoospores, responsible for root infections leading to potato root and tuber disease. Cytosolic Ca2+ flux inhibition with Ca2+ antagonists were found to alter zoospore swimming patterns and constrain zoospore chemotaxis, root attachment and zoosporangia infection. LaCl3 and GdCl3, both Ca2+ channel blockers, at concentrations ≥ 50 µM showed complete inhibition of zoospore chemotaxis, root attachment and zoosporangia root infection. The Ca2+ chelator EGTA, showed efficient chemotaxis inhibition but had relatively less effect on root attachment. Conversely the calmodulin antagonist trifluoperazine had lesser effect on zoospore chemotaxis but showed strong inhibition of zoospore root attachment. Amiloride hydrochloride had a significant inhibitory effect on chemotaxis, root attachment, and zoosporangia root infection with dose rates ≥ 150 µM. As expected, zoospore attachment was directly associated with root infection and zoosporangia development. These results highlight the fundamental role of Ca2+ signaling in zoospore chemotaxis and disease establishment. Their efficient interruption may provide durable and practical control of Phytomyxea soilborne diseases in the field.

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.

Mitochondria regulate TRPV4-mediated release of ATP.

BACKGROUND AND PURPOSE: Ca2+ influx via TRPV4 channels triggers Ca2+ release from the IP3 -sensitive internal store to generate repetitive oscillations. Although mitochondria are acknowledged regulators of IP3 -mediated Ca2+ release, how TRPV4-mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+ -induced Ca2+ release at IP3 receptors to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. EXPERIMENTAL APPROACH: TRPV4-evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. KEY RESULTS: TRPV4 activation with GSK1016790A (GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+ . The ATP synthase inhibitor oligomycin did not potentiate TRPV4-mediated Ca2+ signals. GSK-evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the PLC blocker U73122 and the inositol trisphosphate receptor blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. CONCLUSION AND IMPLICATIONS: These results highlight a previously unknown role of mitochondria in shaping TRPV4-mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3 -evoked Ca2+ release.

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.

Moments of weaknesses - exploiting vulnerabilities between germination and encystment in the Phytomyxea.

Attempts at management of diseases caused by protozoan plant parasitic Phytomyxea have often been ineffective. The dormant life stage is characterised by long-lived highly robust resting spores that are largely impervious to chemical treatment and environmental stress. This review explores some life stage weaknesses and highlights possible control measures associated with resting spore germination and zoospore taxis. With phytomyxid pathogens of agricultural importance, zoospore release from resting spores is stimulated by plant root exudates. On germination, the zoospores are attracted to host roots by chemoattractant components of root exudates. Both the relatively metabolically inactive resting spore and motile zoospore need to sense the chemical environment to determine the suitability of these germination stimulants or attractants respectively, before they can initiate an appropriate response. Blocking such sensing could inhibit resting spore germination or zoospore taxis. Conversely, the short life span and the vulnerability of zoospores to the environment require them to infect their host within a few hours after release. Identifying a mechanism or conditions that could synchronise resting spore germination in the absence of host plants could lead to diminished pathogen populations in the field.