Project description:DNA oligonucleotide microarrays were designed with 307 probes for 96 internal transcribed spacer (ITS1, located between 18S and 26S rRNA genes) sequences of known species and strains from the genus Pseudo-nitzschia (Bacillariophyceae). In addition, microarrays also carried 1893 probes targeting ITS1 aequences of marine Crenarchaeota and Alphaproteobacteria of SAR11 clade. In order to assign microarray profiles to Pseudo-nitzschia ribotypes and species and to 'train' the data analysis system, we grew cultures of Pseudo-nitzschia in the laboratory with identities confirmed through rDNA sequence analysis. In total, 9 cultures and 35 environmental water samples were hybridized to microarrays, in some cases, in duplicate or triplicate. Analysis of microarray data allowed us to identify and map Pseudo-nitzschia spp. in the coastal waters along Washington and Oregon coast of the Eastern Pacific Ocean, and to observe seasonal changes in diatom community composition.
Project description:Pseudo-nitzschia multiseries (Ps-n) is a toxigenic marine diatom that produces the neurotoxin, domoic acid. We screened for candidate genes that may be involved in domoic acid production by determining changes in transcript profiles in Ps-n cultures that were in late exponential (low-domoic-acid-producing) vs. stationary (high-domoic-acid-producing) growth states. We also identified a number of candidate reference genes for future RT-qPCR studies, based on their stability in this study.
Project description:Background: Marine phytoplankton are responsible for 50% of the CO2 that is fixed annually worldwide and contribute massively to other biogeochemical cycles in the oceans. Diatoms and coccolithophores play a significant role as the base of the marine food web and they sequester carbon due to their ability to form blooms and to biomineralise. To discover the presence and regulation of short non-coding RNAs (sRNAs) in these two important phytoplankton groups, we sequenced short RNA transcriptomes of two diatom species (Thalassiosira pseudonana, Fragilariopsis cylindrus) and validated them by Northern blots along with the coccolithophore Emiliania huxleyi. Results: Despite an exhaustive search, we did not find canonical miRNAs in diatoms. The most prominent classes of sRNAs in diatoms were repeat-associated sRNAs and tRNA-derived sRNAs. The latter were also present in E. huxleyi. tRNA-derived sRNAs in diatoms were induced under important environmental stress conditions (iron and silicate limitation, oxidative stress, alkaline pH), and they were very abundant especially in the polar diatom F. cylindrus (20.7% of all sRNAs) even under optimal growth conditions. Conclusions: This study provides first experimental evidence for the existence of short non-coding RNAs in marine microalgae. Our data suggest that canonical miRNAs are absent from diatoms. However, the group of tRNA-derived sRNAs seems to be very prominent in diatoms and coccolithophores and may be used for acclimation to environmental conditions.
Project description:DNA oligonucleotide microarrays were designed with 307 probes for 96 internal transcribed spacer (ITS1, located between 18S and 26S rRNA genes) sequences of known species and strains from the genus Pseudo-nitzschia (Bacillariophyceae). In addition, microarrays also carried 1893 probes targeting ITS1 aequences of marine Crenarchaeota and Alphaproteobacteria of SAR11 clade. In order to assign microarray profiles to Pseudo-nitzschia ribotypes and species and to 'train' the data analysis system, we grew cultures of Pseudo-nitzschia in the laboratory with identities confirmed through rDNA sequence analysis. In total, 9 cultures and 35 environmental water samples were hybridized to microarrays, in some cases, in duplicate or triplicate. Analysis of microarray data allowed us to identify and map Pseudo-nitzschia spp. in the coastal waters along Washington and Oregon coast of the Eastern Pacific Ocean, and to observe seasonal changes in diatom community composition. Total DNA was isolated from 9 Pseudo-nitzschia laboratory cultures and 35 environmental water samples collected during 7 field campaigns in 2007-2009. The environmental samples were collected at distances of 5 to 55 km from the coast, along the following transects in the Pacific Ocean covering over 300 km of the coastline: La Push (LP), Grays Harbor (GH), Columbia River (CR), and Newport Hydroline (NH). The DNA samples were subjected to PCR amplification with the primers specific for ITS1 sequences. The resultant biotin-labeled target samples were analyzed using microarray hybridization with the CombiMatrix ElectraSense 4X2K format. Out of 44 analyzed samples, 40, 2, and 2 were used for single, duplicate and triplicate hybridizations, respectively.
Project description:Background: Marine phytoplankton are responsible for 50% of the CO2 that is fixed annually worldwide and contribute massively to other biogeochemical cycles in the oceans. Diatoms and coccolithophores play a significant role as the base of the marine food web and they sequester carbon due to their ability to form blooms and to biomineralise. To discover the presence and regulation of short non-coding RNAs (sRNAs) in these two important phytoplankton groups, we sequenced short RNA transcriptomes of two diatom species (Thalassiosira pseudonana, Fragilariopsis cylindrus) and validated them by Northern blots along with the coccolithophore Emiliania huxleyi. Results: Despite an exhaustive search, we did not find canonical miRNAs in diatoms. The most prominent classes of sRNAs in diatoms were repeat-associated sRNAs and tRNA-derived sRNAs. The latter were also present in E. huxleyi. tRNA-derived sRNAs in diatoms were induced under important environmental stress conditions (iron and silicate limitation, oxidative stress, alkaline pH), and they were very abundant especially in the polar diatom F. cylindrus (20.7% of all sRNAs) even under optimal growth conditions. Conclusions: This study provides first experimental evidence for the existence of short non-coding RNAs in marine microalgae. Our data suggest that canonical miRNAs are absent from diatoms. However, the group of tRNA-derived sRNAs seems to be very prominent in diatoms and coccolithophores and may be used for acclimation to environmental conditions. RNA-seq study of sRNA populations in two species of diatoms using Illumina GAII high-throughput sequencing
Project description:Diatoms, which are responsible for up to 40% of the 45 to 50 billion metric tons of organic carbon production each year in the sea, are particularly sensitive to Fe stress. Here we describe the transcriptional response of the pennate diatom Phaeodactylum tricornutum to Fe limitation using a partial genome microarray based on EST and genome sequence data. Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport and nitrate assimilation are down-regulated to cope with the reduced cellular iron quota. This retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and storage carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electron s by mitochondrial alternative oxidase (AOX), augmented Fe-independent oxidative stress responses, and sensitized iron capture mechanisms. Keywords: Marine phytoplankton, pinnate diatom
Project description:In this work, we have used deep sequencing to study the viral small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species and have different type of genome (dsRNA, (+)ssRNA, and (-)ssRNA). In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5' end, and are unevenly distributed along the viral genome showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking concomitances with vsiRNAs produced by plant viruses suggesting similar pathways of viral targeting in plants and fungi
Project description:Background: Ocean temperatures are projected to increase over the coming century, with dramatic consequences for the marine biosphere. Diatoms are important contributors to marine primary production and the ocean carbon cycle, yet the molecular mechanisms that regulate their acclimation and adaptation to temperature are poorly understood. Method: Here we use a transcriptomic approach to identify the molecular mechanisms associated with temperature acclimation and adaptation in closely related colder- and warmer-adapted diatom species. Results: We find contrasting patterns of differential expression at sub- and supra-optimal temperatures across the two species, which may be due to adaptive changes in baseline expression. Frontloaded and divested pathways indicate protein processing machinery, membrane structure, and the balance between temperature-independent photosynthesis and temperature-dependent metabolism are key elements of adaptation to temperature changes. Conclusions: Our findings suggest that transcriptional frontloading and divestment may provide a framework to interpret diatom acclimation and adaptation to temperature and success under future warming.
Project description:Diatoms, which are responsible for up to 40% of the 45 to 50 billion metric tons of organic carbon production each year in the sea, are particularly sensitive to Fe stress. Here we describe the transcriptional response of the pennate diatom Phaeodactylum tricornutum to Fe limitation using a partial genome microarray based on EST and genome sequence data. Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport and nitrate assimilation are down-regulated to cope with the reduced cellular iron quota. This retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and storage carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electron s by mitochondrial alternative oxidase (AOX), augmented Fe-independent oxidative stress responses, and sensitized iron capture mechanisms. Keywords: Marine phytoplankton, pinnate diatom Wild-type Phaeodactylum tricornutum was grown under Fe replete (10,000 nM) and Fe limiting (5nM) conditions. Partial genome gene expression analysis of iron-inducible genes was conducted using a two-color competitive hybridization microarray.
Project description:The oceanic diatom Pseudo-nitzschia granii was cultured in the laboratory under steady-state iron-replete and iron-limited conditions. Transcriptomic and proteomic analyses were performed to determine how this organism reorganizes major metabolic processes as a function of iron supply.