The Experts below are selected from a list of 6369 Experts worldwide ranked by ideXlab platform
Chris Bowler - One of the best experts on this subject based on the ideXlab platform.
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genome wide analysis of the diatom cell cycle unveils a novel type of cyclins involved in environmental signaling
Genome Biology, 2010Co-Authors: Cindy Martens, Marie J J Huysman, Klaas Vandepoele, Jeroen Gillard, Edda Rayko, Marc Heijde, Chris BowlerAbstract:Background Despite the enormous importance of diatoms in aquatic ecosystems and their broad industrial potential, little is known about their life cycle control. Diatoms typically inhabit rapidly changing and unstable environments, suggesting that cell cycle regulation in diatoms must have evolved to adequately integrate various environmental signals. The recent genome sequencing of Thalassiosira pseudonana and Phaeodactylum tricornutum allows us to explore the molecular conservation of cell cycle regulation in diatoms.
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gene silencing in the marine diatom Phaeodactylum tricornutum
Nucleic Acids Research, 2009Co-Authors: Valentina De Riso, Florian Maumus, Chris Bowler, Raffaella Raniello, Alessandra Rogato, Angela FalciatoreAbstract:Diatoms are a major but poorly understood phytoplankton group. The recent completion of two whole genome sequences has revealed that they contain unique combinations of genes, likely recruited during their history as secondary endosymbionts, as well as by horizontal gene transfer from bacteria. A major limitation for the study of diatom biology and gene function is the lack of tools to generate targeted gene knockout or knockdown mutants. In this work, we have assessed the possibility of triggering gene silencing in Phaeodactylum tricornutum using constructs containing either anti-sense or inverted repeat sequences of selected target genes. We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes. To highlight the utility of the approach we also report the first phenotypic characterization of a diatom mutant (cpf1). Our data open the way for reverse genetics in diatoms and represent a major advance for understanding their biology and ecology. Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms. Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.
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the Phaeodactylum genome reveals the evolutionary history of diatom genomes
Nature, 2008Co-Authors: Chris Bowler, Kamel Jabbari, Cindy Martens, Florian Maumus, Uma Maheswari, Andrew E Allen, Jonathan H Badger, Jane Grimwood, Robert OtillarAbstract:Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one- fifth of the primary productivity on Earth(1,2). The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology(3-5). Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes (similar to 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.
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molecular toolbox for studying diatom biology in Phaeodactylum tricornutum
Gene, 2007Co-Authors: Magali Siaut, Andrew E Allen, Angela Falciatore, Chris Bowler, Marc Heijde, Anton Montsant, Manuela Mangogna, Sacha Coesel, Alessandro ManfredoniaAbstract:Research into diatom biology has now entered the post-genomics era, following the recent completion of the Thalassiosira pseudonana and Phaeodactylum tricornutum whole genome sequences and the establishment of Expressed Sequence Tag (EST) databases. The thorough exploitation of these resources will require the development of molecular tools to analyze and modulate the function of diatom genes in vivo. Towards this objective, we report here the identification of several reference genes that can be used as internal standards for gene expression studies by quantitative real-time PCR (qRT-PCR) in P. tricornutum cells grown over a diel cycle. In addition, we describe a series of diatom expression vectors based on Invitrogen Gateway technology for high-throughput protein tagging and overexpression studies in P. tricornutum. We demonstrate the utility of the diatom Destination vectors for determining the subcellular localization of a protein of interest and for immunodetection. The availability of these new resources significantly enriches the molecular toolbox for P. tricornutum and provides the diatom research community with well defined high-throughput methods for the analysis of diatom genes and proteins in vivo.
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genetic and phenotypic characterization of Phaeodactylum tricornutum bacillariophyceae accessions1
Journal of Phycology, 2007Co-Authors: Alessandra De Martino, Kehou Pan, Agnes Meichenin, Juan Shi, Chris BowlerAbstract:In the last few years, genome-based studies in diatoms have received a major boost following the genome sequencing of the centric species Thalassiosira pseudonana Hasle et Heimdal and the pleiomorphic raphid pennate diatom Phaeodactylum tricornutum Bohlin. In addition, molecular tools, such as genetic transformation, have been developed for both species. Despite these molecular advances, relatively little is known regarding the genetic diversity of the available strains of these diatoms. In this study, we have compiled a historical summary of the known P. tricornutum species resources and have provided a genetic and phenotypic overview of 10 different axenic strains. Examination of intraspecies genetic diversity based on internal transcribed spacer 2 (ITS2) sequence and amplified fragment length polymorphism (AFLP) analyses indicate four different genotypes. Seven strains are predominantly fusiform, whereas one strain is predominantly oval, and another is predominantly triradiate. Another is defined as a tropical strain because it appears better acclimated to growth at higher temperatures. Observations in the natural environment indicate that P. tricornutum is a coastal marine diatom that is able to adapt to unstable environments, such as estuaries and rock pools. Because it has rarely been noted in nature, we have developed specific primers to amplify ITS2 sequences and have successfully identified it in environmental samples. These resources should become useful tools for the diatom community when combined with the whole genome sequence and will open up a range of new possibilities for experimental investigations that can exploit the genotypic and phenotypic characteristics described.
Andrew E Allen - One of the best experts on this subject based on the ideXlab platform.
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multiplexed knockouts in the model diatom Phaeodactylum by episomal delivery of a selectable cas9
Frontiers in Microbiology, 2020Co-Authors: Mark Moosburner, Andrew E Allen, Pardis Gholami, James K Mccarthy, Maxine Tan, Vincent A BielinskiAbstract:Marine diatoms are eukaryotic microalgae that play significant ecological and biogeochemical roles in oceans. They also have significant potential as organismal platforms for exploitation to address biotechnological and industrial goals. In order to address both modes of research, sophisticated molecular and genetic tools are required. We presented here new and improved methodologies for introducing CRISPR-Cas9 to the model diatom Phaeodactylum tricornutum cells and a streamlined protocol for genotyping mutant cell lines with previously unknown phenotypes. First, bacterial-conjugation was optimized for the delivery of Cas9 by transcriptionally fusing Cas9 to a selectable marker by the 2A peptide. An episome cloning strategy using both negative and positive selection was developed to streamline CRISPR-episome assembly. Next, cell line picking and genotyping strategies, that utilize manual sequencing curation, TIDE sequencing analysis, and a T7 endonuclease assay, were developed to shorten the time required to generate mutants. Following this new experimental pipeline, both single-gene and two-gene knockout cell lines were generated at mutagenesis efficiencies of 48% and 25%, respectively. Lastly, a protocol for precise gene insertions via CRISPR-Cas9 targeting was developed using particle-bombardment transformation methods. Overall, the novel Cas9 episome design and improved genotyping methods presented here allow for quick and easy genotyping and isolation of Phaeodactylum mutant cell lines (less than 3 weeks) without relying on a known phenotype to screen for mutants.
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integrated regulatory and metabolic networks of the marine diatom Phaeodactylum tricornutum predict the response to rising co2 levels
mSystems, 2017Co-Authors: Jennifer Levering, Andrew E Allen, Christopher L Dupont, Bernhard O Palsson, Karsten ZenglerAbstract:Diatoms are eukaryotic microalgae that are responsible for up to 40% of the ocean's primary productivity. How diatoms respond to environmental perturbations such as elevated carbon concentrations in the atmosphere is currently poorly understood. We developed a transcriptional regulatory network based on various transcriptome sequencing expression libraries for different environmental responses to gain insight into the marine diatom's metabolic and regulatory interactions and provide a comprehensive framework of responses to increasing atmospheric carbon levels. This transcriptional regulatory network was integrated with a recently published genome-scale metabolic model of Phaeodactylum tricornutum to explore the connectivity of the regulatory network and shared metabolites. The integrated regulatory and metabolic model revealed highly connected modules within carbon and nitrogen metabolism. P. tricornutum's response to rising carbon levels was analyzed by using the recent genome-scale metabolic model with cross comparison to experimental manipulations of carbon dioxide. IMPORTANCE Using a systems biology approach, we studied the response of the marine diatom Phaeodactylum tricornutum to changing atmospheric carbon concentrations on an ocean-wide scale. By integrating an available genome-scale metabolic model and a newly developed transcriptional regulatory network inferred from transcriptome sequencing expression data, we demonstrate that carbon metabolism and nitrogen metabolism are strongly connected and the genes involved are coregulated in this model diatom. These tight regulatory constraints could play a major role during the adaptation of P. tricornutum to increasing carbon levels. The transcriptional regulatory network developed can be further used to study the effects of different environmental perturbations on P. tricornutum's metabolism.
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integrated regulatory and metabolic networks of the marine diatom Phaeodactylum tricornutum predict the response to rising co 2 levels
mSystems, 2017Co-Authors: Jennifer Levering, Andrew E Allen, Christopher L Dupont, Bernhard O Palsson, Karsten ZenglerAbstract:Diatoms are eukaryotic microalgae that are responsible for up to 40% of the ocean's primary productivity. How diatoms respond to environmental perturbations such as elevated carbon concentrations in the atmosphere is currently poorly understood. We developed a transcriptional regulatory network based on various transcriptome sequencing expression libraries for different environmental responses to gain insight into the marine diatom's metabolic and regulatory interactions and provide a comprehensive framework of responses to increasing atmospheric carbon levels. This transcriptional regulatory network was integrated with a recently published genome-scale metabolic model of Phaeodactylum tricornutum to explore the connectivity of the regulatory network and shared metabolites. The integrated regulatory and metabolic model revealed highly connected modules within carbon and nitrogen metabolism. P. tricornutum's response to rising carbon levels was analyzed by using the recent genome-scale metabolic model with cross comparison to experimental manipulations of carbon dioxide. IMPORTANCE Using a systems biology approach, we studied the response of the marine diatom Phaeodactylum tricornutum to changing atmospheric carbon concentrations on an ocean-wide scale. By integrating an available genome-scale metabolic model and a newly developed transcriptional regulatory network inferred from transcriptome sequencing expression data, we demonstrate that carbon metabolism and nitrogen metabolism are strongly connected and the genes involved are coregulated in this model diatom. These tight regulatory constraints could play a major role during the adaptation of P. tricornutum to increasing carbon levels. The transcriptional regulatory network developed can be further used to study the effects of different environmental perturbations on P. tricornutum's metabolism.
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the Phaeodactylum genome reveals the evolutionary history of diatom genomes
Nature, 2008Co-Authors: Chris Bowler, Kamel Jabbari, Cindy Martens, Florian Maumus, Uma Maheswari, Andrew E Allen, Jonathan H Badger, Jane Grimwood, Robert OtillarAbstract:Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one- fifth of the primary productivity on Earth(1,2). The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology(3-5). Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes (similar to 40%) are not shared by these representatives of the two lineages. Analysis of molecular divergence compared with yeasts and metazoans reveals rapid rates of gene diversification in diatoms. Contributing factors include selective gene family expansions, differential losses and gains of genes and introns, and differential mobilization of transposable elements. Most significantly, we document the presence of hundreds of genes from bacteria. More than 300 of these gene transfers are found in both diatoms, attesting to their ancient origins, and many are likely to provide novel possibilities for metabolite management and for perception of environmental signals. These findings go a long way towards explaining the incredible diversity and success of the diatoms in contemporary oceans.
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molecular toolbox for studying diatom biology in Phaeodactylum tricornutum
Gene, 2007Co-Authors: Magali Siaut, Andrew E Allen, Angela Falciatore, Chris Bowler, Marc Heijde, Anton Montsant, Manuela Mangogna, Sacha Coesel, Alessandro ManfredoniaAbstract:Research into diatom biology has now entered the post-genomics era, following the recent completion of the Thalassiosira pseudonana and Phaeodactylum tricornutum whole genome sequences and the establishment of Expressed Sequence Tag (EST) databases. The thorough exploitation of these resources will require the development of molecular tools to analyze and modulate the function of diatom genes in vivo. Towards this objective, we report here the identification of several reference genes that can be used as internal standards for gene expression studies by quantitative real-time PCR (qRT-PCR) in P. tricornutum cells grown over a diel cycle. In addition, we describe a series of diatom expression vectors based on Invitrogen Gateway technology for high-throughput protein tagging and overexpression studies in P. tricornutum. We demonstrate the utility of the diatom Destination vectors for determining the subcellular localization of a protein of interest and for immunodetection. The availability of these new resources significantly enriches the molecular toolbox for P. tricornutum and provides the diatom research community with well defined high-throughput methods for the analysis of diatom genes and proteins in vivo.
Guangce Wang - One of the best experts on this subject based on the ideXlab platform.
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elevated co 2 improves both lipid accumulation and growth rate in the glucose 6 phosphate dehydrogenase engineered Phaeodactylum tricornutum
Microbial Cell Factories, 2019Co-Authors: Aiyou Huang, Shan Gao, Manoj Kumar, Phaikeem Lim, Li Huan, Guangce WangAbstract:Background Numerous studies have shown that stress induction and genetic engineering can effectively increase lipid accumulation, but lead to a decrease of growth in the majority of microalgae. We previously found that elevated CO2 concentration increased lipid productivity as well as growth in Phaeodactylum tricornutum, along with an enhancement of the oxidative pentose phosphate pathway (OPPP) activity. The purpose of this work directed toward the verification of the critical role of glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme in the OPPP, in lipid accumulation in P. tricornutum and its simultaneous rapid growth rate under high-CO2 (0.15%) cultivation.
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metabolic flux ratio analysis and cell staining suggest the existence of c4 photosynthesis in Phaeodactylum tricornutum
Journal of Applied Microbiology, 2016Co-Authors: Aiyou Huang, Lixia Liu, Peipei Zhao, Chen Yang, Guangce WangAbstract:AimsMechanisms for carbon fixation via photosynthesis in the diatom Phaeodactylum tricornutum Bohlin were studied recently but there remains a long-standing debate concerning the occurrence of C-4 photosynthesis in this species. A thorough investigation of carbon metabolism and the evidence for C-4 photosynthesis based on organelle partitioning was needed.
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silicon enhances the growth of Phaeodactylum tricornutum bohlin under green light and low temperature
Scientific Reports, 2015Co-Authors: Peipei Zhao, Aiyou Huang, Xiujun Xie, Shan Gao, Baoyu Zhang, Jianfeng Niu, Peng A Lin, Guangce WangAbstract:Phaeodactylum tricornutum Bohlin is an ideal model diatom; its complete genome is known, and it is an important economic microalgae. Although silicon is not required in laboratory and factory culture of this species, previous studies have shown that silicon starvation can lead to differential expression of miRNAs. The role that silicon plays in P. tricornutum growth in nature is poorly understood. In this study, we compared the growth rate of silicon starved P. tricornutum with that of normal cultured cells under different culture conditions. Pigment analysis, photosynthesis measurement, lipid analysis, and proteomic analysis showed that silicon plays an important role in P. tricornutum growth and that its presence allows the organism to grow well under green light and low temperature.
Imogen Foubert - One of the best experts on this subject based on the ideXlab platform.
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alkaline flocculation of Phaeodactylum tricornutum induced by brucite and calcite
Bioresource Technology, 2015Co-Authors: Dries Vandamme, Imogen Foubert, Phillip I Pohl, Annelies Beuckels, Patrick V Brady, John C Hewson, Koenraad MuylaertAbstract:Alkaline flocculation holds great potential as a low-cost harvesting method for marine microalgae biomass production. Alkaline flocculation is induced by an increase in pH and is related to precipitation of calcium and magnesium salts. In this study, we used the diatom Phaeodactylum tricornutum as model organism to study alkaline flocculation of marine microalgae cultured in seawater medium. Flocculation started when pH was increased to 10 and flocculation efficiency reached 90% when pH was 10.5, which was consistent with precipitation modeling for brucite or Mg(OH)2. Compared to freshwater species, more magnesium is needed to achieve flocculation (>7.5mM). Zeta potential measurements suggest that brucite precipitation caused flocculation by charge neutralization. When calcium concentration was 12.5mM, flocculation was also observed at a pH of 10. Zeta potential remained negative up to pH 11.5, suggesting that precipitated calcite caused flocculation by a sweeping coagulation mechanism.
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influence of extraction solvent system on extractability of lipid components from different microalgae species
Algal Research-Biomass Biofuels and Bioproducts, 2014Co-Authors: Eline Ryckebosch, Charlotte Bruneel, Koenraad Muylaert, Romina Termoteverhalle, Imogen FoubertAbstract:Abstract The purpose of this work was to evaluate two food grade solvent systems, hexane/isopropanol (3:2; HI) and hexane (H), for the extraction of lipids from different omega-3 LC-PUFA rich microalgae: Isochrysis galbana, Nannochloropis gaditana, Nannochloropsis sp. and Phaeodactylum tricornutum. We not only focused on differences in lipid yield, but also lipid class, omega-3 LC-PUFA, carotenoid and sterol yields. Furthermore, an estimation of the feasibility of these microalgae oils as an alternative for fish oil was made. For all tested microalgae species, the highest food grade lipid, lipid class, omega-3 PUFA, carotenoid and sterol yield were obtained with HI, with a general recovery highest from Isochrysis, lowest from both Nannochloropsis species, and intermediate from Phaeodactylum. Total lipid recovery values between 14 and 76% depending on solvent and species were obtained. It was also shown that the omega-3 fatty acid content of all oils was quite similar, while only the H extract was enriched in neutral lipids. Carotenoids were co-extracted in a significant amount, although the content in the various oils was quite different.
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impact of feed supplementation with different omega 3 rich microalgae species on enrichment of eggs of laying hens
Food Chemistry, 2013Co-Authors: Charlotte Lemahieu, Charlotte Bruneel, Koenraad Muylaert, Johan Buyse, Romina Termoteverhalle, Imogen FoubertAbstract:Abstract Four different omega-3 rich autotrophic microalgae, Phaeodactylum tricornutum , Nannochloropsis oculata , Isochrysis galbana and Chlorella fusca , were supplemented to the diet of laying hens in order to increase the level of omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) in egg yolk. The microalgae were supplemented in two doses: 125 mg and 250 mg extra n-3 PUFA per 100 g feed. Supplementing these microalgae resulted in increased but different n-3 LC-PUFA levels in egg yolk, mainly docosahexaenoic acid enrichment. Only supplementation of Chlorella gave rise to mainly α-linolenic acid enrichment. The highest efficiency of n-3 LC-PUFA enrichment was obtained by supplementation of Phaeodactylum and Isochrysis . Furthermore, yolk colour shifted from yellow to a more intense red colour with supplementation of Phaeodactylum , Nannochloropsis and Isochrysis , due to transfer of carotenoids from microalgae to eggs. This study shows that besides Nannochloropsis other microalgae offer an alternative to current sources for enrichment of hen eggs.
Angela Falciatore - One of the best experts on this subject based on the ideXlab platform.
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lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum
Nature Communications, 2019Co-Authors: Jochen M Buck, Angela Falciatore, Johann Lavaud, Jonathan Sherman, Carolina Bártulos, Manuel Serif, Marc Halder, Jan Henkel, Maxim Y GorbunovAbstract:Diatoms possess an impressive capacity for rapidly inducible thermal dissipation of excess absorbed energy (qE), provided by the xanthophyll diatoxanthin and Lhcx proteins. By knocking out the Lhcx1 and Lhcx2 genes individually in Phaeodactylum tricornutum strain 4 and complementing the knockout lines with different Lhcx proteins, multiple mutants with varying qE capacities are obtained, ranging from zero to high values. We demonstrate that qE is entirely dependent on the concerted action of diatoxanthin and Lhcx proteins, with Lhcx1, Lhcx2 and Lhcx3 having similar functions. Moreover, we establish a clear link between Lhcx1/2/3 mediated inducible thermal energy dissipation and a reduction in the functional absorption cross-section of photosystem II. This regulation of the functional absorption cross-section can be tuned by altered Lhcx protein expression in response to environmental conditions. Our results provide a holistic understanding of the rapidly inducible thermal energy dissipation process and its mechanistic implications in diatoms. Photosynthetic organisms can dissipate excess absorbed light energy as heat to avoid photodamage. Here the authors show that induced thermal dissipation in the diatom Phaeodactylum tricornutum Pt4 is Lhcx protein-dependent and correlates with a reduced functional absorption cross-section of photosystem II.
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Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum
Philosophical Transactions of the Royal Society B: Biological Sciences, 2017Co-Authors: Valeria Villanova, Angela Falciatore, Eric Maréchal, Antonio Emidio Fortunato, Dipali Singh, Melissa Conte, Toshihiro Obata, Juliette Jouhet, Alisdair R. Fernie, Julien PagliardiniAbstract:Diatoms are prominent marine microalgae, interesting not only from an ecological point of view, but also for their possible use in biotechnology applications. They can be cultivated in phototrophic conditions, using sunlight as the sole energy source. Some diatoms, however, can also grow in a mixotrophic mode, wherein both light and external reduced carbon contribute to biomass accumulation. In this study, we investigated the consequences of mixotrophy on the growth and metabolism of the pennate diatom Phaeodactylum tricornutum, using glycerol as the source of reduced carbon. Transcriptomics, metabolomics, metabolic modelling and physiological data combine to indicate that glycerol affects the central-carbon, carbon-storage and lipid metabolism of the diatom. In particular, provision of glycerol mimics typical responses of nitrogen limitation on lipid metabolism at the level of triacylglycerol accumulation and fatty acid composition. The presence of glycerol, despite provoking features reminiscent of nutrient limitation, neither diminishes photosynthetic activity nor cell growth, revealing essential aspects of the metabolic flexibility of these microalgae and suggesting possible biotechnological applications of mixotrophy. This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.
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genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology
Nature Communications, 2014Co-Authors: Fayza Daboussi, Sophie Leduc, Alan Marechal, Valerie Guyot, Christophe Perezmichaut, Gwendoline Dubois, Alberto Amato, Angela Falciatore, Alexandre JuilleratAbstract:Diatoms, a major group of photosynthetic microalgae, have a high biotechnological potential that has not been fully exploited because of the paucity of available genetic tools. Here we demonstrate targeted and stable modifications of the genome of the marine diatom Phaeodactylum tricornutum, using both meganucleases and TALE nucleases. When nuclease-encoding constructs are co-transformed with a selectable marker, high frequencies of genome modifications are readily attained with 56 and 27% of the colonies exhibiting targeted mutagenesis or targeted gene insertion, respectively. The generation of an enhanced lipid-producing strain (45-fold increase in triacylglycerol accumulation) through the disruption of the UDP-glucose pyrophosphorylase gene exemplifies the power of genome engineering to harness diatoms for biofuel production. Diatoms are photosynthetic microalgae with underutilized biotechnological potential. Here, the authors carry out targeted gene modifications of lipid metabolism genes in the diatom, Phaeodactylum tricornutum, resulting in a strain that exhibits a 45-fold increase in triacylglycerol accumulation.
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gene silencing in the marine diatom Phaeodactylum tricornutum
Nucleic Acids Research, 2009Co-Authors: Valentina De Riso, Florian Maumus, Chris Bowler, Raffaella Raniello, Alessandra Rogato, Angela FalciatoreAbstract:Diatoms are a major but poorly understood phytoplankton group. The recent completion of two whole genome sequences has revealed that they contain unique combinations of genes, likely recruited during their history as secondary endosymbionts, as well as by horizontal gene transfer from bacteria. A major limitation for the study of diatom biology and gene function is the lack of tools to generate targeted gene knockout or knockdown mutants. In this work, we have assessed the possibility of triggering gene silencing in Phaeodactylum tricornutum using constructs containing either anti-sense or inverted repeat sequences of selected target genes. We report the successful silencing of a GUS reporter gene expressed in transgenic lines, as well as the knockdown of endogenous phytochrome (DPH1) and cryptochrome (CPF1) genes. To highlight the utility of the approach we also report the first phenotypic characterization of a diatom mutant (cpf1). Our data open the way for reverse genetics in diatoms and represent a major advance for understanding their biology and ecology. Initial molecular analyses reveal that targeted downregulation likely occurs through transcriptional and post-transcriptional gene silencing mechanisms. Interestingly, molecular players involved in RNA silencing in other eukaryotes are only poorly conserved in diatoms.
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molecular toolbox for studying diatom biology in Phaeodactylum tricornutum
Gene, 2007Co-Authors: Magali Siaut, Andrew E Allen, Angela Falciatore, Chris Bowler, Marc Heijde, Anton Montsant, Manuela Mangogna, Sacha Coesel, Alessandro ManfredoniaAbstract:Research into diatom biology has now entered the post-genomics era, following the recent completion of the Thalassiosira pseudonana and Phaeodactylum tricornutum whole genome sequences and the establishment of Expressed Sequence Tag (EST) databases. The thorough exploitation of these resources will require the development of molecular tools to analyze and modulate the function of diatom genes in vivo. Towards this objective, we report here the identification of several reference genes that can be used as internal standards for gene expression studies by quantitative real-time PCR (qRT-PCR) in P. tricornutum cells grown over a diel cycle. In addition, we describe a series of diatom expression vectors based on Invitrogen Gateway technology for high-throughput protein tagging and overexpression studies in P. tricornutum. We demonstrate the utility of the diatom Destination vectors for determining the subcellular localization of a protein of interest and for immunodetection. The availability of these new resources significantly enriches the molecular toolbox for P. tricornutum and provides the diatom research community with well defined high-throughput methods for the analysis of diatom genes and proteins in vivo.
