Project description:Mycosphaerella graminicola is the causal agent of Mycosphaerella graminicola infection (STB) disease of wheat. Wheat genotypes vary in their response to this disease. Cultivar (cv.) Longbow is susceptible and cv. Flame is resistant to STB disease, with cv. Flame possessing the STB resistance locus Stb6 that confers resistance to pathogen strain IPO323. Gene expression profiling (conducted using Affymetrix wheat gene chip) identified transcripts that accumulate in leaves of both these wheat cultivars as an early response to M. graminicola strain IPO323 (at 24h post-treatment). At this initial time point, microscopic analysis verified that fungal spores had germinated, but not penetrated the leaves of both genotypes. Results showed that basal defence genes were activated in both the compatible and incompatible interactions. A subset of genes were identified that were more pathogen-responsive in the cv. Flame v. IPO323 incompatible interaction as compared to the cv. Longbow v. IPO323 compatible interaction, including defence genes such as peroxidases, beta-1,3-glucanase, annexin, chitinases, brassinosteroid-associated kinase 1 and a jasmonate-inducible protein.
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:Mycosphaerella graminicola is a major pathogen of wheat worldwide, causing Septoria leaf blotch disease. Targeted gene disruption in M. graminicola, by Agrobacterium tumefaciens-mediated transformation, has become an established functional genomics tool for M. graminicola research in recent years. However, in order to advance research into this economically important pathogen, further functional genomics tools need to be developed. Here, we report three new capabilities for M. graminicola research: (i) two selectable markers have been shown to work robustly in M. graminicola, namely G418 and the fungicide carboxin; (ii) the generation of a strain of M. graminicola in which the KU70 (MUS-51) homologue has been disrupted; in this strain, homologous recombination efficiencies increased to more than 95%, whilst maintaining wild-type growth in vitro and full pathogenicity on wheat leaves; (iii) the ability to efficiently target and generate precise mutations of specific genes in the genomic context in M. graminicola. In addition, the insertion of the E198A mutation into the beta-tubulin gene (MgTUB1), conferring resistance to the fungicide benomyl, suggests that this mutant allele may provide an additional selectable marker. The collective use of these tools will permit further advancements in our knowledge of the biology and pathogenicity of this important plant pathogen.
Project description:Pitkanen2014 - Metabolic reconstruction of Mycosphaerella graminicola using CoReCo
This model was reconstructed with the CoReCo method (Comparative ReConstruction of genome-scale metabolic networks) from protein sequence and phylogeny data.
This model is described in the article:
Comparative Genome-Scale Reconstruction of Gapless Metabolic Networks for Present and Ancestral Species
Esa Pitkänen, Paula Jouhten, Jian Hou, Muhammad Fahad Syed, Peter Blomberg, Jana Kludas, Merja Oja, Liisa Holm, Merja Penttilä, Juho Rousu, Mikko Arvas
PLoS Comput Biol. 2014 Feb 6;10(2):e1003465
Abstract:
We introduce a novel computational approach, CoReCo, for comparative metabolic reconstruction and provide genome-scale metabolic network models for 49 important fungal species. Leveraging on the exponential growth in sequenced genome availability, our method reconstructs genome-scale gapless metabolic networks simultaneously for a large number of species by integrating sequence data in a probabilistic framework. High reconstruction accuracy is demonstrated by comparisons to the well-curated Saccharomyces cerevisiae consensus model and large-scale knock-out experiments. Our comparative approach is particularly useful in scenarios where the quality of available sequence data is lacking, and when reconstructing evolutionary distant species. Moreover, the reconstructed networks are fully carbon mapped, allowing their use in 13C flux analysis. We demonstrate the functionality and usability of the reconstructed fungal models with computational steady-state biomass production experiment, as these fungi include some of the most important production organisms in industrial biotechnology. In contrast to many existing reconstruction techniques, only minimal manual effort is required before the reconstructed models are usable in flux balance experiments. CoReCo is available at http://esaskar.github.io/CoReCo/.
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Project description:The hemibiotrophic fungal pathogen Colletotrichum graminicola is the causal agent of anthracnose disease on maize stalks and leaves. After the formation of appressoria the host cell wall is penetrated by the conversion of appressorial turgor pressure into forceful ejection of a penetration peg. Subsequently, C. graminicola establishes biotrophic hyphae in the penetrated epidermis cell at around 36 hours post inoculation (hpi) until a switch of hyphal morphology and lifestyle takes place during the colonization of neighboring host cells at around 72 hpi. During the ensuing necrotrophic growth, dark necrotic lesions are formed that are visible as anthracnose symptoms. We used microarrays to detail the global programme of gene expression during the infection process of Colletotrichum graminicola in its host plant to get insight into the defense response of this compatible interaction and into the metabolic reprogramming needed to supply the fungus with nutrients.
Project description:The hemibiotrophic fungal pathogen Colletotrichum graminicola is the causal agent of anthracnose disease on maize stalks and leaves. After the formation of appressoria the host cell wall is penetrated by the conversion of appressorial turgor pressure into forceful ejection of a penetration peg. Subsequently, C. graminicola establishes biotrophic hyphae in the penetrated epidermis cell at around 36 hours post inoculation (hpi) until a switch of hyphal morphology and lifestyle takes place during the colonization of neighboring host cells at around 72 hpi. During the ensuing necrotrophic growth, dark necrotic lesions are formed that are visible as anthracnose symptoms. We used microarrays to detail the global programme of gene expression during the infection process of Colletotrichum graminicola in its host plant to get insight into the defense response of this compatible interaction and into the metabolic reprogramming needed to supply the fungus with nutrients. In three independent experiments, maize plants were infected with conidia of the Colletotrichum graminicola strain CgM2 by spray inoculation of leaves. Samples from infected leaves were taken at 36 and 96 hours post infection, corresponding to initial biotrophic and necrotrophic phase, respectively. Samples from uninfected control plants were taken at the same time points.