Project description:The function of the plant hormone jasmonic acid (JA) in development of tomato flowers was analyzed with a mutant defective in JA perception (jasmonate-insensitive1-1, jai1-1). In contrast to Arabidopsis JA-insensitive plants that are male sterile, the tomato mutant jai1-1 exhibits major defects in female development resulting in female sterility. To identify putative JA-dependent regulatory components, transcriptomics was performed using isolated ovules of three different stages of flower development, from both wild type and jai1-1. Among the strongly down-regulated genes in jai1-1, one encoding a MYB transcription factor (SlMYB21) was found. Its orthologue in Arabidopsis has a crucial role in JA-regulated stamen development. SlMYB21 showed transcription factor activity in yeast, interaction with SlJAZ9 in yeast and in planta, and complemented the Arabidopsis mutant myb21-5. To analyze SlMYB21 function in tomato ovule development, CRISPR/Cas9 mutants were created and a TILLING mutant was identified, all showing female sterility and therefore corroborating a function of MYB21 in tomato ovule development. Transcriptomics from wild type, jai1-1 and myb21-2 carpels revealed processes that might be controlled by SlMYB21. The data suggest a positive regulation of JA biosynthesis by SlMYB21, but a negative regulation of the action of auxin and GA. The results demonstrate that SlMYB21 mediates at least partially the action of JA and might control the flower to fruit transition.
Project description:Phosphorus, in its orthophosphate form (Pi), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole genome molecular mechanisms contributing to plant acclimation to Pi deficiency remain largely unknown. White lupin (Lupinus albus L.) has evolved unique adaptations for growth in Pi deficient soils including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to Pi supply. We de novo assembled 277,224,180 Illumina reads from 12 cDNA libraries to build the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences 50,734 were transcriptionally active (RPKM = 3) representing approximately 7.8% of the Lupinus albus genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to Pi deficiency with a = 2-fold change and a p-value = 0.05. Twelve sequences were consistently differentially expressed due to Pi deficiency stress in three species, making them ideal candidates to monitor the Pi status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in Pi deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to Pi deficiency. Examination of 2 different tissue types (roots and leaves) under phosphorus (P) -sufficient or P-deficient condition with 3 biological replications per condition in white lupin (Lupinus albus).
Project description:Anaplastic Lymphoma Kinase (ALK) is a tyrosine kinase receptor which is a clinical target of major interest in cancer, including neuroblastoma. To better understand ALK signaling, three different neuroblastoma cell lines (CLB-BAR, CLB-GE and SK-N-AS) were cultured for 1hr and 24hrs in control conditions or after treatment with the ALK inhibitors crizotinib or lorlatinib. RNA-Seq experiments were performed to determine the expression changes resulting from ALK inhibition. Together with parallel phosphoproteomic experiments, these data unveil several important conserved oncogenic pathways in neuroblastoma.
Project description:Boar taint described as an offensive odor that is released while cooking pork. Androstenone, a steroid mainly synthesized in testis and metabolized in liver is one of the major causes of boar taint. The aim of the present study was to investigate transcriptome differences in boar testis and liver tissues with divergent androstenone levels using RNA deep sequencing (RNA-Seq). For this purpose, mRNA expression from testis and liver tissue samples from 10 boars, 5 samples each for high and low androstenone levels were quantified and analyzed. The results shows that in testis samples 46 genes were differentially regulated whereas 25 genes showed differential expression in the liver. Digital gene expression analysis identified genes in keratin family, desmoplakin and Interferon-induced protein family for as candidate genes for low androstenone testis sample and genes in flavin monooxygenease family, cytochrome P450 family and hydroxysteroid dehydrogenase family as candidate genes for low androstenone liver sample. Additionally, the results revealed that mutations in genes IRG6, DSP, IFIT2 were specific for low androstenone testis tissues and mutations in genes FMO5, HIST1H4K and TSKU were specific for low androstenone liver samples. Testis and liver mRNA profile of high and low androstenone level were generated by RNA deep sequencing, in five animal for each group, Ilumina HiSeq 2000
Project description:The leaf transcriptome of the Arabidopsis thaliana aquaporin gene PIP1;2 T-DNA insertion line was compared to that of control plants. In total 730 genes were found to be differentially regulated. This regulation pattern was compared to mild drought stress and low CO2 Affymetrix data to elucidate whether loss of the aquaporin resembles transcriptomic changes of drought stress or lack of CO2 supply. Mild drought stress data were obtained from Harb A, Krishnan A, Ambavaram MMR, Pereira A (2010) Molecular and Physiological Analysis of Drought Stress in Arabidopsis Reveals Early Responses Leading to Acclimation in Plant Growth. Plant Physiology 154: 1254-1271 (GSE24177). Low CO2 data were obtained from Oliver E. Bläsing, Yves Gibon, Manuela Günther, Melanie Höhne, Rosa Morcuende, Daniel Osuna, Oliver Thimm, Björn Usadel, Wolf-Rüdiger Scheible, and Mark Stitt (2005) Sugars and Circadian Regulation Make Major Contributions to the Global Regulation of Diurnal Gene Expression in Arabidopsis. The Plant Cell, Vol. 17, 3257-3281 (GSE3423). 2 samples examined: wildtype and atpip1;2-1 mutant
Project description:To elucidate the effect of the rational ribosomal engineering on the changes in the expression of the endogenious biosynthetic gene clusters the transcriptome analysis was performed. The Streptomyces albus strains carrying mutations in rpsL gene (encode for ribosomal protein S12) and the deletion of the rsmG gene (16S rRNA methyltransferase G), as well as their combination were used for the experiment. The list of the strains with mutations is next: S. albus K88E, S. albus GI92, S. albus K88E-GI92, S. albus P91S, S. albus K88E-P91S, S. albus del rsmG, S. albus K88E-GI92 del rsmG, S. albus K88E-P91S del rsmG. Abovementioned strains along with S. albus native strain were grown in NL-19 production medium. Samples were harvested by centrifugation after 48 and 72 hours of cultivation. For total RNA isolation, S. albus cells were grown in SG medium (for ara expression) or NL19 medium (for indigenous BGC expression). Then, 2 ml of 2-day and 3-day cultures was spun down for 20 s at 14,000 × rpm, and the pellets were immediately frozen in liquid nitrogen and stored at ?80 °C. Total RNA extraction was performed using an RNeasy Kit (Qiagen, Hilden, Germany) as previously described (Huser et al. 2003). An RNase-Free DNase set (Qiagen) was used two times for on-column DNA digestion, and an additional DNase treatment was then performed with a DNase I kit (Roche Diagnostics, Mannheim, Germany) to ensure that all DNA was completely removed. To check the RNA samples for DNA contamination, PCR was performed using oligonucleotides designed to create two different products approximately 150 bp and 500 bp in size. Initially, RNA quality was checked with Trinean Xpose (Gentbrugge, Belgium) and Agilent RNA Nano 6000 kits on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A Ribo-Zero rRNA Removal Kit for bacteria was obtained from Illumina (San Diego, CA, USA) and used to remove ribosomal RNA molecules from isolated total RNA. rRNA removal was checked using an Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). A TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) was used to prepare cDNA libraries (Koepff et al. 2017). The resulting cDNAs were pair-end sequenced on an Illumina HiSeq 1500 system (San Diego, CA, USA) using a 70 bp read length. Short read alignments and differential gene expression were illustrated using ReadXplorer 2.2.0 (Hilker et al. 2014) and DEseq (Anders and Huber 2010), respectively.
Project description:Objective: We analyzed changes in A. fumigatus gene expression profile at various stages of an in vitro model of aspergillosis to study the adaptation of A. fumigatus to the blood environment. Results: Most of virulence factors described to be involved in aspergillosis were not activated during the blood phase. We found three active processes to be activated in the later phase that may help to the adaptation: Iron homeostasis, a partial secondary metabolite cluster and the formation of detoxification enzymes. Conclusions: We propose that A. fumigatus is unable to grow in blood and it requires a metabolic change that allows the organism to shut down all uptake and energy-consume mechanisms, resulting in a resting mycelial stage. We performed gene expression profile by sequencing mRNA of A. fumigatus that were growm under two conditions, Minimal Medium (M) and human blood (B), and at different times: before placing the fungus in the final medium (pre), at 30' and at 180', with 2 biological replicates per condition.
Project description:The Sordaria macrospora nox1 mutant has a block in sexual development at the stage of protoperithecia formation, and therefore is sterile. The aim of this study was to determine the transcriptome of microdissected protoperithecia from the mutant, as well as the transcriptomes of total RNA from the mutant and the wild type for comparison. The data were then compared with transcriptome data from protoperithecia of the wild type and the developemental mutant pro1; the corresponding data were generated in a previous study (Teichert et al. 2012, BMC Genomics 13: 511, GEO accession GSE33668). The samples (amplified RNA from nox1 microdissected protoperithecia and total RNA from nox1 and wild type mycelia undergoing sexual development) were sequenced as paired-end reads on an Illumina HiSeq 2000, two independent biological replicates for each nox1 sample and one replicate for the wild type.
Project description:On the example of the biosynthetically exhausted landomycin A cluster we demonstrate unbalancing of gene transcription as an efficient method for the generation of new compounds. Decoupled from the native regulators LanI and LanK, all landomycin A structural genes were set under the control of a single synthetic promoter and expressed in a heterologous host Streptomyces albus J1074. Previously being both temporarily and quantitatively regulated, these genes were transcribed as a single polycistronic mRNA leading to the production of four novel and two known compounds. No glycosylated landomycins were detected though the entire biosynthetic cluster was transcribed, showing the crucial role of the balanced gene expression for the production of landomycin A. Two new compounds, fridamycin F and G, isolated in this study were shown to originate from the interplay between the expressed biosynthetic pathway and metabolic network of the heterologous host. Structure activity studies of the isolated compounds as well as results of transcriptome sequencing are discussed in this article. Comparison of gene expression of the H2-26 cosmid (encoding landomycin A biosynthetic genes) with H2-26-act, where an additional constitutive promoter cassette was integrated to drive biosynthetic genes transcription.