RNA-seq analysis of Aspergillus fumigatus response to low and supra-MIC concentrations of voriconazole
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ABSTRACT: The purpose of this experiment was to detect Aspergillus fumigatus genes that are up- and downregulated in the presence of low concentrations, and also in supra-MIC concentrations of voriconazole, which is persister growth.
Project description:Expression profiling of Drosophila melanogaster halo[AJ] snail[Df(2L)TE116GW11] and halo[AJ] sna[V2] homozygous mutant embryos at two timepoints of embryogenesis. Embryos were manually sorted and assayed at cellularisation stage (stage 5 to early stage 6) and at gastrulation stage (late stage 6 to early stage 8). Homozygous mutant embryos were sorted based on their reduced transparency caused by the recessive mutation of halo[AJ]. Stage-matched halo[AJ] embryos served as wildtype control. Total RNA was extracted, amplified and hybridized to Affymetrix GeneChip Drosophila Genome array version 2. Three biological replicates at each condition were generated.
Project description:The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the RIB40 reference strain. We now report the existence of a complimentary MAT1-2 gene and sequencing of an idiomorph region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of MAT1-1 and MAT1-2 genotype among 180 strains assayed including industrial tane-koji isolates. Strains used for sake and miso production showed a near 1:1 ratio of MAT1-1 and MAT1-2 mating-types, whereas strains used for soy sauce production showed a significant bias towards the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and comparisons were made of gene expression by DNA microarray and RT-PCR methodologies under conditions when MAT genes were expressed. 33 genes were found to be up-regulated greater than 10-fold in either the MAT1-1 host or MAT1-2 gene replacement strains relative to each other, showing that both MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating-type dependent manner, the first such report from a supposedly asexual fungus. MAT1-1 expression specifically up-regulated an a-pheromone precursor gene, but most genes affected were of unknown function. Results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed. On a condition that induces mating-type genes descrived in growth protocol section, each cogenic MAT1-1 and MAT1-2 mating-type strains were processed into RNA extraction and hybridization on Affymetrix microarrays. In a speculation that genes involved in putative mating process were reguated in mating-type dependent manner, we designed the cogenic strains that differs only in mating-type gene locus to analyze differential gene expression of MAT1-1 vs MAT1-2 strains.
Project description:The development of affinity purification technologies together with mass spectrometric analyses of the purified protein mixtures (AP-MS) has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we have investigated whether ectopic expression of an affinity tagged transcription factor as bait in AP-MS experiments perturbs gene expression in cells resulting in false positive identification of bait associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA-Seq, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then copurify non-specifically and be misidentified as bait associated proteins. Therefore typical controls should be sufficient and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFêB1, NFêB2, Rel, RelB, IêBá, IêBâ and IêBå). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFêB family transcription factors. The work here therefore provides a conceptual and experimental framework for analyzing transcription faction protein interactions. Gene expression profiles were assayed in triplicate from HEK293 cells expressing either Halo-RelA, Halo-NFkB1, or Halo tag alone.
Project description:This experiment analyzes the set of RNAs copurifying with the protein TNIP2 (amino acids 196-346) HEK293 cells were transfected with constructs expressing either Halo tag (controls) or Halo-TNIP2 196-346. Total RNA was purified from an aliquot of the whole cell extract (Input samples). Halo-tagged proteins were purified from the remainder of the whole cell extract, and RNA subsequently purified from the Halo purified samples (Pulldown samples).
Project description:Cryptococcus neoformans lab strain H99 was spread on YPD plate supplemented with 0.25ug/ml tunicamycin. Randomly 30 adaptors were chosen. 10 of them were used to obtain revertatants which lost aneuploidy step by step. These adaptors, and the revertants, as well as the parent, were sequenced.
Project description:Mycoparasitism is a key feature of Trichoderma biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase, in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study we identified the most robustly regulated targets governed by Tmk1 during mycoparasitism by whole transcriptome gene expression profiling using a custom microarray.
Project description:We carried out an RNA-seq based transcriptome study on two rice varieties, Cocodrie (CCDR; rice sheath blight susceptible) and MCR10277 (MCR; rice sheath blight resistant), to profile the time-series wide genome-scale transcriptional differences in response to sheath blight (SB), an infection caused by R. solanii (LR172) . Our approach is cross-referencing differentially expressed genes with significant variants of two phenotypically different varieties to validate known and discover novel variants and to further understand rice's physiological response to SB.
Project description:Plant diseases are a major cause for yield losses and new strategies to control them without harming the environment are urgently needed. Plant-associated bacteria contribute to their host’s health in diverse ways, among which the emission of disease-inhibiting volatile organic compounds (VOCs). We have previously reported that VOCs emitted by potato-associated bacteria caused strong in vitro growth inhibition of the late blight causing agent Phytophthora infestans. This work focuses on sulfur-containing VOCs (sVOCs) and demonstrates the high in planta protective potential of S-methyl methane thiosulfonate (MMTS), which fully prevented late blight disease in potato leaves and plantlets without phytotoxic effects, in contrast to other sVOCs. Short exposure times were sufficient to protect plants against infection and MMTS also displayed curative effects. We further show that MMTS’s protective activity was not mediated by the plant immune system but lied in its anti-oomycete activity. Using quantitative proteomics, we determined that different sVOCs caused specific proteome changes in P. infestans, highlighting sulfur metabolism, protein translation and redox balance as main targets. This work brings new perspectives for sustainable protection against the devastating Irish Famine pathogen, while opening new research avenues on the role of sVOCs in the interaction between plants and their microbiome.
Project description:Dissection of catalytic and non-catalytic functions of Tet1. DNA methylation (RRBS) profiling of wild type, Tet1 knockout and catalytic mutants during EpiLC differentiation.
Project description:BRAF targeted drug vemurafenib have shown very good clinical benefit in melanoma patient containing BRAF V600E mutation. However, resistance always occurs in patient. Early stage of the resistance development require the tumor cell adapted to the targeted drug. We are trying to study the kinetic of melanoma cell adaptation towards vemurafenib. 10 melanoma cell lines with BRAF mutation are treated with targeted therapy vemurafenib. RNA-seq samples are collected after drug treatment for different time (day3 and day21) to compare with DMSO-treated control samples for each cell line. Except innate resistant cell line M381, all other cell lines shows inhibition of proliferation. However, a small cluster of cell lines (M397, M229 and M263) shows some other unique transcriptomic change. For cell line M397, M229 and M263, we also collected the RNA-seq data for long-term (73day/90day) drug treatment condition where the cells developed resistance to vemurafenib treatment. Dedifferentiation is enriched in these unique transcriptomic change within these 3 cell lines. Similar cell state dedifferentiation phenomenon is also reported to cause resistance towards immunotherpay where the resistant de-differentiated melanoma cells are induced by TNF which is secreted by tumor-infiltrating T cells. We also treat our cultured melanoma cells with TNF and collected the treated sample for RNA-seq experiment.