Dissecting molecular mechanisms of disease in the wheat pathogen, Parastagonospora nodorum

Dissecting molecular mechanisms of disease in the wheat pathogen, Parastagonospora nodorum Parastagonospora nodorum is a wheat specific pathogen that causes annual losses to the Australian wheat industry in excess of $100 million AUD. This necrotrophic fungus kills the host tissue generating necrotic lesions within which fruiting bodies develop, spreading spores and continuing the disease cycle. This polycyclic infection cycle leads to field epidemics resulting in the losses to growers. Sporulation and virulence are the two crucial aspects for disease development in the P. nodorum-wheat pathosystem and form the basis of this project. A forward genetics approach was employed to discover novel mechanisms by which P. nodorum facilitates infection on wheat. A library of random P. nodorum insertion mutants was generated, and subsequently screened for gain and loss of virulence phenotypes on non-susceptible and susceptible wheat cultivars. From a library of 950 transformants seven displayed a consistent avirulent phenotype on the susceptible wheat cultivar. To identify the disrupted loci leading to avirulence, genomes of the seven avirulent P. nodorum strains were sequenced elucidating a Catechol-1,2-dioxygenase and a Copper dependent amine oxidase. To complement the virulence investigation, a combined transcriptomics and metabolomics approach was employed to decipher sporulation in this pathogen. This is of particular interest as the canonical sporulation pathways in other, model fungi, were previously shown to be not applicable in P. nodorum. A differential gene analysis of fungal material collected at critical developmental time points identified several key genes involved in initiating a sporulation cascade. Notably, a WetA homolog was identified, along with an uncharacterised Aquaporin-like protein and a Pr1-like protein. Metabolomics and subsequent sporulation assays revealed a polyamine pathway plays an integral role in initiating and coordinating asexual development of P. nodorum

Constellations of Capital: Applying Architecture’s Spatial Language to Global Networks of Food, Armed Conflict, and Reverberations of Violence

Architecture, as the art and/or practice of designing and constructing buildings, offers an often overlooked array of representational devices that, as tested within this senior project, prove significant in reframing geopolitical perspectives in a manner that circumvents the limitations of metaphorization. The physical products and results of architectural design are the built objects that facilitate and coordinate socio-political spaces, yet it is the spatially descriptive process of architectural design itself in representing built relationships that can be valuable in constructing provocative and experimental investigations of present and future spatial environments.This project focuses on a tripartite of present-day global issues: food sovereignty in the hierarchical landscape of a globalized economy, the delicate network that carries these commodified and crucial foodsorts, and the embedded interrelationship of armed conflict and war. Focusing on the Tigray War in northern Ethiopia and its intertwined relationship with the Russian Invasion of Ukraine, these two case studies become the heart of a project that aims to visualize geopolitical relationships through a translation into the spatial language of architecture

Characterising the role of GABA and its metabolism in the wheat pathogen Stagonospora nodorum

A reverse genetics approach was used to investigate the role of γ-aminobutyric acid metabolism in the wheat pathogenic fungus Stagonospora nodorum. The creation of mutants lacking Sdh1, the gene encoding succinic semialdehyde dehydrogenase, resulted in strains that grew poorly on γ-aminobutyric acid as a nitrogen source. The sdh1 mutants were more susceptible to reactive oxygen stress but were less affected by increased growth temperatures. Pathogenicity assays revealed that the metabolism of γ-aminobutyric acid is required for complete pathogenicity. Growth assays of the wild-type and mutant strains showed that the inclusion of γ-aminobutyric acid as a supplement in minimal media (i.e., not as a nitrogen or carbon source) resulted in restricted growth but increased sporulation. The addition of glutamate, the precursor to GABA, had no effect on either growth or sporulation. The γ-aminobutyric acid effect on sporulation was found to be dose dependent and not restricted to Stagonospora nodorum with a similar effect observed in the dothideomycete Botryosphaeria sp. The positive effect on sporulation was assayed using isomers of γ-aminobutyric acid and other metabolites known to influence asexual development in Stagonospora nodorum but no effect was observed. These data demonstrate that γ-aminobutyric acid plays an important role in Stagonospora nodorum in responding to environmental stresses while also having a positive effect on asexual development.The work was supported by Australian Research Council and Grains Research and Development Corporation

SnPKS19 encodes the polyketide synthase for alternariol mycotoxin biosynthesis in the wheat pathogen Parastagonospora nodorum

Alternariol (AOH) is an important mycotoxin from the Alternaria fungi. AOH was detected for the first time in the wheat pathogen Parastagonospora nodorum in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829 (SnPKS19), a close homolog of Penicillium aethiopicum norlichexanthone (NLX) synthase gene gsfA, is required for AOH production. We further validate that SnPKS19 is solely responsible for AOH production by heterologous expression in Aspergillus nidulans. The expression profile of SnPKS19 based on previous P. nodorum microarray data correlated with the presence of AOH in vitro and its absence in planta. Subsequent characterization of the ΔSnPKS19 mutants showed that SnPKS19 and AOH are not involved in virulence and oxidative stress tolerance. Identification and characterization of the P. nodorum SnPKS19 cast light on a possible alternative AOH synthase gene in Alternaria alternata and allowed us to survey the distribution of AOH synthase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future

Wound healing and regeneration in the reef building coral Acropora millepora

Branching scleractinian corals are niche-constructing organisms, providing continuously-growing, structural foundation for spectacularly biodiverse coral reef ecosystems. A large part of their success lies in the ability to quickly regenerate following mechanical damage. Even now, when the corals undergo great decline due to anthropogenic weather and storm extremes, it is surprising how little is known about molecular mechanisms governing regeneration in these iconic organisms. In this study, we used RNA-seq to identify genes involved in the regeneration of Acropora millepora, starting with the initial wound closure up to complete rebuilding of lost structures. Many of the differentially expressed genes we found in the wound healing steps are homologues of genes known to be involved in wound healing and regeneration of bilaterian and other cnidarian species, prominently including multiple components of FGF and Wnt signalling pathways. Comparison between genes involved in wound healing and continuous growth of the colony demonstrates both similarity and distinctiveness of the genetic programmes controlling these processes. A striking example is specific expression of c-Fos, a transcription factor with conserved role in early injury response, during the earliest stages of wound healing of A. millepora. By comparing results obtained in diverse experimental conditions including a closed-loop, recirculating aquarium and a flow-through system of marine station, we have demonstrated feasibility of using zooxanthellate scleractinian corals as experimental models in fundamental biology research, including studies of regeneration

Engineering a Model Cell for Rational Tuning of GPCR Signaling.

G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.BBSR

The genome sequence of the brown trout, Salmo trutta Linnaeus 1758

publishedVersio

Colloquium on Solid State Devices

[no abstract

Volatile Molecules Secreted by the Wheat Pathogen Parastagonospora nodorum Are Involved in Development and Phytotoxicity

Septoria nodorum blotch is a major disease of wheat caused by the fungus Parastagonospora nodorum. Recent studies have demonstrated that secondary metabolites, including polyketides and non-ribosomal peptides, produced by the pathogen play important roles in disease and development. However, there is currently no knowledge on the composition or biological activity of the volatile organic compounds (VOCs) secreted by P. nodorum. To address this, we undertook a series of growth and phytotoxicity assays and demonstrated that P. nodorum VOCs inhibited bacterial growth, were phytotoxic and suppressed self-growth. Mass spectrometry analysis revealed that 3-methyl-1-butanol, 2-methyl-1-butanol, 2-methyl-1-propanol, and 2-phenylethanol were dominant in the VOC mixture and phenotypic assays using these short chain alcohols confirmed that they were phytotoxic. Further analysis of the VOCs also identified the presence of multiple sesquiterpenes of which four were identified via mass spectrometry and nuclear magnetic resonance as β-elemene, α-cyperone, eudesma-4,11-diene and acora-4,9-diene. Subsequent reverse genetics studies were able to link these molecules to corresponding sesquiterpene synthases in the P. nodorum genome. However, despite extensive testing, these molecules were not involved in either of the growth inhibition or phytotoxicity phenotypes previously observed. Plant assays using mutants of the pathogen lacking the synthetic genes revealed that the identified sesquiterpenes were not required for disease formation on wheat leaves. Collectively, these data have significantly extended our knowledge of the VOCs in fungi and provided the basis for further dissecting the roles of sesquiterpenes in plant disease.MM-G is a recipient of an Australian Government Endeavour Award and a Mexican CONACYT scholarship. Y-HC is Australian Research Council Future Fellow (FT160100233)

HPV, tumour metabolism and novel target identification in head and neck squamous cell carcinoma

Background Metabolic changes in tumour cells are used in clinical imaging and may provide potential therapeutic targets. Human papillomavirus (HPV) status is important in classifying head and neck cancers (HNSCC), identifying a distinct clinical phenotype; metabolic differences between these HNSCC subtypes remain poorly understood. Methods We used RNA sequencing to classify the metabolic expression profiles of HPV+ve and HPV−ve HNSCC, performed a meta-analysis on FDG-PET imaging characteristics and correlated results with in vitro extracellular flux analysis of HPV−ve and HPV+ve HNSCC cell lines. The monocarboxylic acid transporter-1 (MCT1) was identified as a potential metabolic target and tested in functional assays. Results Specific metabolic profiles were associated with HPV status, not limited to carbohydrate metabolism. There was dominance of all energy pathways in HPV-negative disease, with elevated expression of genes associated with glycolysis and oxidative phosphorylation. In vitro analysis confirmed comparative increased rates of oxidative phosphorylation and glycolysis in HPV-negative cell lines. PET SUV(max) scores however were unable to reliably differentiate between HPV-positive and HPV-negative tumours. MCT1 expression was significantly increased in HPV-negative tumours, and inhibition suppressed tumour cell invasion, colony formation and promoted radiosensitivity. Conclusion HPV-positive and negative HNSCC have different metabolic profiles which may have potential therapeutic applications