Project description:Genome sequence data results are reported from experimental and bioinfomatic work using the technique 'Bulk Segregant Analysis' to determine the genetic basis of observed resistance to the azole antifungal compound itraconazole in the opportunistic fungal pathogen Aspergillus fumigatus.
Project description:The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4 sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveals new insights into SREBPM-bM-^@M-^Ys complex role in infection site adaptation and fungal virulence. 4 hour and 12 hour ChIP experiments were completed. Input control samples for each set were collected.
Project description:The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from grapevine wood infected by a fungal pathogen in the presence of a root biological control agent. One of the goals was to obtain molecular data about the fungus pathogen (Phaeomoniella chlamydospora) during grapevine wood infection. Grapevine pathogen-infected wood mRNA profiles of 2-month-old plantlets (14 days post infection) were generated by deep sequencing, in triplicate, using Illumina Hiseq2500. The sequence reads that passed quality filters were analyzed by TopHat followed by Cufflinks. qRTaPCR validation was performed using SYBR Green assays. Using an optimized data analysis workflow, we mapped sequence reads to the grapevine genome (build IGGP 12x) and identified pathogen transcripts. RNAseq analyses, using a ribosomal RNA depletion technology for library preparation, provided identification of genes expressed by P. chlamydospora during infection: as for genes related to effector biosynthesis enzymes, carbohydrate-active enzymes and transcription regulators involved in known regulation pathways in fungi. Insights about P. oligandrum modulation of grapevine infection by this pathogen were also found. Our study represents the first detailed analysis of grapevine wood infection by a fungal pathogen generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive evaluation of mRNA content within grapevine wood tissue. We conclude that RNA-seq based transcriptome characterization would permit the dissection of complex biologic interactions.
Project description:We have focused our investigation on the characterization of the role of the fungal specific SWI/SNF subunit, Snf6. Our data show that, although the C. albicans subunit has only limited sequence similarity to other fungal orthologs, Snf6 was copurified with SWI/SNF complex subunits including the catalytic ATPase subunit, Snf2. We show that Snf6 plays a critical role in biological processes that are essential for fungal pathogenesis including carbon metabolic flexibility, stress response and morphogenesis. The Snf6 regulon was determined by combining both genome-wide location (ChIP-chip) and transcriptional profiling (microarrays) to identify targets of the SWI/SNF complex under both yeast- and hyphal-promoting conditions.