Project description:In order to gain a first insight into the Mycosphaerella graminicola global transcriptome in different nutritional environments, we performed initial experiments on two in vitro growth conditions during log-phase growth and on infected plant material twenty-eight days after inoculation. In vitro log phase growth in nutrient-rich Potato Dextrose Broth (PDB) was used as a control in independent comparisons with 1) log phase growth in nutrient-limiting Czapek-Dox Broth (CDB) and 2) twenty-eight days of plant infection. Growth in PDB results in a rapid budding type growth of the M. graminicola sporidia. Growth in CDB is phenotypically similar in that the fungus continues to grow as budding sporidia but this occurs at approximately 20% of the rate in PDB. In contrast, late stage infected plant material contains fungus growing as filamentous hyphae, generating pycnidia and sporulating. At this stage the plant material is completely senesced and the RNA isolated from this tissue is entirely of fungal origin. The complete lack of plant RNA enabled the microarray comparison to be made against growth in PDB. In order to generate statistically significant data for further analysis sixteen independent microarray blocks were hybridised for each experiment. Within these sixteen replicates were three biological repeats. For data analysis we employed limits of a two-fold cut-off in expression based upon statistical analysis of the replicate hybridisations (P <0.01).
Project description:Z. tritici is a fungal pathogen causing the disease septoria tritici blotch, one of the most economically devastating foliar diseases in wheat. The molecular basis underlying Z. tritici growth, development and pathogenicity is not fully understood yet. Compared to the genomic investigations in this fungus, little is known about the protein expression at a systematic level. The aim of the project is to construct a comprehensive protein database of Z. tritici growing in nutrient-limiting and rich media and in vivo at a late stage of wheat infection by using 1D gel-based and SCX-based proteomics and subproteomics (intracellular and extracellular) approaches.
Project description:Verticillium dahliae is a soilborne fungus that causes wilt disease in plants. The microsclerotia of V. dahliae produce infectious hyphae that give rise to primary infections. In this study, RNA-seq libraries were prepared from microsclerotia (MS)-producing cultures of V. dahliae (ave = 52.23 million reads), and those not producing microsclerotia (NoMS, ave = 50.58 million reads) and analyzed for differential gene expression.
Project description:Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that over a third of the genes induced after 6 h of exposure to wheat straw were also induced during 6 h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. Eight samples in total consisting of duplicate shake flask Aspergillus niger cultures from four conditions: 48h glucose, 6 h starvation, 6 h wheat straw, 24 h starvation
Project description:Fusarium graminearum (teleomorph Gibberella zeae) is a prominent pathogen that infects major cereal crops, such as wheat, barley, and maize. FSS1 contains a Zn(II)2Cys6 fungal-type DNA-binding domain and localized exclusively to nuclei responding to sodium, suggesting that FSS1 is a TF required for sodium tolerance. By RNA-seq and genetic studies, we found a P-type ATPase pump (FgENA5) that is under control of FSS1 and is responsible for phenotypic defects of fss1 mutants. The wild-type, fss1 deletion, fss1 overexpression mutant strains were incubated in potato dextrose broth (PDB) with or without 1 M NaCl supplementation for an hour. 6 samples examined: 1 h after inoculation of Fusarium graminearum wild-type, Δfss1(Δfss1::gen), and fss1 overexpression mutant (fss1::gen-Pef1a-fss1) strains in potato dextrose broth with or without 1 M NaCl supplementation
Project description:Fusarium head blight (FHB) is a devastating fungal disease of cereal crops causing substantial reductions in grain quality and yield, impacting Canada’s economy. Fusarium graminearum, the causative agent of FHB in wheat, also produces harmful mycotoxins, including deoxynivalenol (DON), 3-acetyl DON (3ADON), 15-acetyl DON (15ADON), and nivalenol (NIV), which impact livestock and human health upon consumption of contaminated grains in feed and processed foods, respectively. Critically, over the past 15 years, surveillance programs have defined the synthesis of a novel trichothecene, 3ANX, and the deacetylated variant, NX, with increased virulence compared to DON. In this study, we assessed the impact of 3ANX from dual proteome perspectives of wheat and fungi to identify proteins and pathways activated in response to the emerging chemotype. We defined a core wheat proteome detected across all isolates (15ADON- and 15ADON+3ANX-producing, and untreated controls), and explored changes in protein abundance associated with defense response, seed storage and grain development, and reduced photosynthesis upon infection. Conversely, we identified 32 wheat proteins produced only in the presence of 15ADON+3ANX isolates, providing further insight into chemotype-specific responses of wheat. Assessment from the fungal perspective reported 119 proteins exclusive to the 15ADON+3ANX isolates, including those associated with virulence and mycotoxin production. Lastly, investigation of isolate-specific proteome changes showed a significant reduction in mycotoxin protective mechanisms in wheat upon exposure to two 15ADON+3ANX isolates, as well as a novel connection between elevated ergosterol biosynthesis and 3ANX production in F. graminearum. Together, we our study characterizes distinct protein production profiles in wheat and F. graminearum in response to 3ANX treatment and provides evidence that these molecular changes influence fungal virulence and host defense responses
Project description:Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.
Project description:In this study, we performed a comprehensive proteomic analysis of mango leaves inoculated with the leaf spot pathogen A. alternata. Down-regulated proteins during pathogen invasion and colonization were primarily associated with photosynthesis, the phenylpropanoid and flavonoid biosynthesis pathways, and phenylalanine metabolism. In contrast, significantly up-regulated proteins were involved in tyrosine metabolism and the MAPK signaling pathway, highlighting their critical role in host resistance to the leaf spot pathogen. These findings provide valuable data on protein expression changes, offering potential targets for developing novel management strategies to enhance control of leaf spot disease.
Project description:Fungi are ubiquitous and are often confronted with the need to secure utilisable carbon from their external growth milieu through the use of extracellular proteins to scavenge for carbon from a vast array of complex polymeric carbon sources. This attribute is conserved across evolution in fungi. To understand how filamentous fungi extracellular proteins are modulated in response to the presence polymeric carbons in the environment, we have typed the array of the main extracellular proteins involved and their dynamics using a known hypercellulolytic fungus – Penicillium funiculosum (NCIM 1228), through multiplexed quantitative proteomics
Project description:The induction of genes in response to exposure of T. reesei to wheat straw was explored using genome-wide RNA-seq and compared to published RNA-seq data and model of how A. niger senses and responds to the lignocellulose. After 24 h of exposure to straw, transcript levels of known and predicted lignocellulose-degrading enzymes increased to around 8% of total cellular mRNA in T. reesei, which was much less when compared to A. niger. The bulk of enzymes used to deconstruct wheat straw is similar in both fungi. Other, non-plant cell wall-degrading enzymes which may aid in lignocellulose degradation were also uncovered in T. reesei and similar to those described in A. niger. Antisense transcripts were also shown to be present in T. reesei and their expession can be regulated by the respective growth condition. Triplicate samples of T. reesei cultivated in each of the three following conditions were taken: 1) After 48 h growth in glucose-based minimal media; 2) After transfer of mycelia from glucose-based media into media containing wheat straw as a sole carbon source and 3) 5 h after addition of glucose to straw cultures.