9 research outputs found
A Versatile High Throughput Screening Platform for Plant Metabolic Engineering Highlights the Major Role of ABI3 in Lipid Metabolism Regulation
Traditional functional genetic studies in crops are time consuming, complicated andcannot be readily scaled up. The reason is that mutant or transformed crops need tobe generated to study the effect of gene modifications on specific traits of interest.However, many crop species have a complex genome and a long generation time. Asa result, it usually takes several months to over a year to obtain desired mutants ortransgenic plants, which represents a significant bottleneck in the development of newcrop varieties. To overcome this major issue, we are currently establishing a versatileplant genetic screening platform, amenable to high throughput screening in almost anycrop species, with a unique workflow. This platform combines protoplast transformationand fluorescence activated cell sorting. Here we show that tobacco protoplasts canaccumulate high levels of lipid if transiently transformed with genes involved in lipidbiosynthesis and can be sorted based on lipid content. Hence, protoplasts can be usedas a predictive tool for plant lipid engineering. Using this newly established strategy, wedemonstrate the major role ofABI3in plant lipid accumulation. We anticipate that thisworkflow can be applied to numerous highly valuable metabolic traits other than storagelipid accumulation. This new strategy represents a significant step toward screeningcomplex genetic libraries, in a single experiment and in a matter of days, as opposed toyears by conventional means.This work was partly funded through the CSIRO Synthetic
Biology Future Science Platform and the CSIRO Research Office
CERC Postdoctoral Fellowship schem
Library screening for metabolic engineering applications
Plant based research is undermined by the lack of plant based systems that allow rapid testing of large gene libraries. Most current screening system are based on technologies that are not transferrable between species and do not allow tissue-specific or cell-type-specific screening. We are currently developing a new strategy using protoplast transformation, cell sorting based on physiological trait, and single cell sequencing, to allow screening of large gene libraries. Our group is focusing primarily on oil accumulation, but once set-up this tool could be used for screening a multitude of physiological trait. In addition, because this technology is based on protoplast, there is strong chances that this technology could be transferable to a wide diversity of plants and tissues
Rational Design of Novel Fluorescent Enzyme Biosensors for Direct Detection of Strigolactones
Strigolactones are plant hormones and rhizosphere signaling molecules with key roles in plant development, mycorrhizal fungal symbioses, and plant parasitism. Currently, sensitive, specific, and high-throughput methods of detecting strigolactones are limited. Here, we developed genetically encoded fluorescent strigolactone biosensors based on the strigolactone receptors DAD2 from Petunia hybrida, and HTL7 from Striga hermonthica. The biosensors were constructed via domain insertion of circularly permuted GFP. The biosensors exhibited loss of cpGFP fluorescence in vitro upon treatment with the strigolactones 5-deoxystrigol and orobanchol, or the strigolactone analogue rac-GR24, and the ShHTL7 biosensor also responded to a specific antagonist. To overcome biosensor sensitivity to changes in expression level and protein degradation, an additional strigolactone-insensitive fluorophore, LSSmOrange, was included as an internal normalization control. Other plant hormones and karrikins resulted in no fluorescence change, demonstrating that the biosensors report on compounds that specifically bind the SL receptors. The DAD2 biosensor likewise responded to strigolactones in an in vivo protoplast system, and retained strigolactone hydrolysis activity. These biosensors have applications in high-throughput screening for agrochemical compounds, and may also have utility in understanding strigolactone mediated signaling in plants. </p
Rational Design of Novel Fluorescent Enzyme Biosensors for Direct Detection of Strigolactones
Strigolactones are plant hormones and rhizosphere signaling molecules with key roles in plant development, mycorrhizal fungal symbioses, and plant parasitism. Currently, sensitive, specific, and high-throughput methods of detecting strigolactones are limited. Here, we developed genetically encoded fluorescent strigolactone biosensors based on the strigolactone receptors DAD2 from Petunia hybrida, and HTL7 from Striga hermonthica. The biosensors were constructed via domain insertion of circularly permuted GFP. The biosensors exhibited loss of cpGFP fluorescence in vitro upon treatment with the strigolactones 5-deoxystrigol and orobanchol, or the strigolactone analogue rac-GR24, and the ShHTL7 biosensor also responded to a specific antagonist. To overcome biosensor sensitivity to changes in expression level and protein degradation, an additional strigolactone-insensitive fluorophore, LSSmOrange, was included as an internal normalization control. Other plant hormones and karrikins resulted in no fluorescence change, demonstrating that the biosensors report on compounds that specifically bind the SL receptors. The DAD2 biosensor likewise responded to strigolactones in an in vivo protoplast system, and retained strigolactone hydrolysis activity. These biosensors have applications in high-throughput screening for agrochemical compounds, and may also have utility in understanding strigolactone mediated signaling in plants. </p
Duplicate maize wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis
L'article original est publié par The American Society of Plant BiologistsWRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs
Transcriptome database resource and gene expression atlas for the rose
International audienceBackground: For centuries roses have been selected based on a number of traits. Little information exists on the genetic and molecular basis that contributes to these traits, mainly because information on expressed genes for this economically important ornamental plant is scarce. Results: Here, we used a combination of Illumina and 454 sequencing technologies to generate information on Rosa sp. transcripts using RNA from various tissues and in response to biotic and abiotic stresses. A total of 80714 transcript clusters were identified and 76611 peptides have been predicted among which 20997 have been clustered into 13900 protein families. BLASTp hits in closely related Rosaceae species revealed that about half of the predicted peptides in the strawberry and peach genomes have orthologs in Rosa dataset. Digital expression was obtained using RNA samples from organs at different development stages and under different stress conditions. qPCR validated the digital expression data for a selection of 23 genes with high or low expression levels. Comparative gene expression analyses between the different tissues and organs allowed the identification of clusters that are highly enriched in given tissues or under particular conditions, demonstrating the usefulness of the digital gene expression analysis. A web interface ROSAseq was created that allows data interrogation by BLAST, subsequent analysis of DNA clusters and access to thorough transcript annotation including best BLAST matches on Fragaria vesca, Prunus persica and Arabidopsis. The rose peptides dataset was used to create the ROSAcyc resource pathway database that allows access to the putative genes and enzymatic pathways. Conclusions: The study provides useful information on Rosa expressed genes, with thorough annotation and an overview of expression patterns for transcripts with good accuracy
Bilan 2015 du dispositif national de surveillance de la santé des mollusques marins
Depuis 1992, la surveillance de la santĂ© des mollusques marins du littoral français est assurĂ©e par le rĂ©seau de Pathologie des Mollusques (Repamo). Ses activitĂ©s sâinscrivent dans le cadre de la Directive EuropĂ©enne 2006/88/CE. Depuis son Ă©valuation par la plateforme nationale dâĂ©pidĂ©miosurveillance en santĂ© animale en 2012, lâobjectif de surveillance est la dĂ©tection prĂ©coce des infections dues Ă des organismes pathogĂšnes exotiques et Ă©mergents affectant les mollusques marins sauvages et dâĂ©levage. LâannĂ©e 2015 est la premiĂšre annĂ©e de transition pour laquelle un dĂ©but dâĂ©volution des modalitĂ©s de surveillance de la santĂ© des mollusques marins animĂ©es par lâIfremer a Ă©tĂ© amorcĂ©. Un dispositif hybride de surveillance a Ă©tĂ© mis en place, sâappuyant sur lâexistant et intĂ©grant des dĂ©buts dâĂ©volution. La surveillance Ă©vĂ©nementielle a constituĂ© lâactivitĂ© principale du dispositif en 2015 et sâest appuyĂ©e sur des rĂ©seaux existants : (1) la surveillance des mortalitĂ©s observĂ©es sur des animaux sentinelles dĂ©ployĂ©s sur les sites ateliers des rĂ©seaux Ifremer RESCO 2 (12 sites) pour lâhuĂźtre creuse Crassostrea gigas et MYTILOBS 2 (8 sites) pour la moule bleue Mytilus edulis. Pour lâhuĂźtre creuse Crassostrea gigas, la mortalitĂ© cumulĂ©e moyenne Ă©tait de 50,3% (Ă©cart-type 10,9%) pour le naissain standardisĂ© Ifremer (NSI), de 11,0% (Ă©cart-type 9,1%) pour les huĂźtres de 18 mois et de 7,3% (Ă©cart-type 5,6%) pour les huĂźtres de 30 mois. Les mortalitĂ©s ont Ă©tĂ© observĂ©es principalement entre le dĂ©but du mois de mai et la mi-juillet. Lors de ces Ă©pisodes de mortalitĂ©, des prĂ©lĂšvements dâanimaux ont Ă©tĂ© rĂ©alisĂ©s en vue dâanalyses diagnostiques : 7 prĂ©lĂšvements pour le NSI, 2 pour les huĂźtres de 18 mois et 1 pour les huĂźtres de 30 mois. Aucun agent rĂ©glementĂ© nâa Ă©tĂ© dĂ©tectĂ© dans les Ă©chantillons dâhuĂźtres creuses prĂ©levĂ©s et analysĂ©s. Le virus OsHV-1 a Ă©tĂ© dĂ©tectĂ© dans les 7 Ă©chantillons analysĂ©s de NSI, dans 2 Ă©chantillons analysĂ©s dâhuĂźtres de 18 mois et dans 1 Ă©chantillon analysĂ© dâhuĂźtres de 30 mois. La bactĂ©rie Vibrio aestuarianus a Ă©tĂ© dĂ©tectĂ©e dans 5 Ă©chantillons analysĂ©s de NSI, dans 1 Ă©chantillon dâhuĂźtres de 18 mois et dans 1âĂ©chantillon dâhuĂźtres de 30 mois. Pour la moule bleue Mytilus edulis, des mortalitĂ©s cumulĂ©es variant de 9% sur le site du Vivier Ă 51% sur le site des filiĂšres du Pertuis Breton ont Ă©tĂ© estimĂ©es. Les mortalitĂ©s ont Ă©tĂ© observĂ©es au printemps sur des moules ĂągĂ©es dâune annĂ©e et en automne sur des moules plus jeunes. Lors de ces Ă©pisodes de mortalitĂ©s, des prĂ©lĂšvements dâanimaux ont Ă©tĂ© rĂ©alisĂ©s en vue dâanalyses diagnostiques : 2 prĂ©lĂšvements pour les moules dâune annĂ©e et 1 pour les jeunes moules. Ces prĂ©lĂšvements ont eu lieu dans le Pertuis Breton. Aucun agent rĂ©glementĂ© nâa Ă©tĂ© dĂ©tectĂ© dans les Ă©chantillons de moules prĂ©levĂ©s et analysĂ©s. Des bactĂ©ries du groupe Splendidus ont Ă©tĂ© dĂ©tectĂ©es dans les 3 Ă©chantillons de moules analysĂ©s. (2) la surveillance sâappuyant sur les dĂ©clarations de mortalitĂ©s de mollusques par les conchyliculteurs et pĂȘcheurs Ă pied professionnels auprĂšs des Directions dĂ©partementales des territoires et de la mer (DDTM). Cette modalitĂ© sâapplique aux huĂźtres creuses et aux moules bleues lorsquâil nâexiste pas de site atelier RESCO 2 ou MYTILOBS 2 dans la zone oĂč des mortalitĂ©s sont dĂ©clarĂ©es par les conchyliculteurs ou pĂȘcheurs Ă pied. Le rĂ©seau REPAMO 2 a rĂ©alisĂ© 22 interventions, dont 15 pour les moules Mytilus edulis, 4 pour les coques Cerastoderma edule, 2 pour les palourdes Ruditapes sp. et 1pour les coquilles saint Jacques Pecten maximus. La recherche dâagents infectieux dans ces espĂšces de mollusques prĂ©levĂ©s lors de hausse de mortalitĂ© a permis de mettre en Ă©vidence les parasites rĂ©glementĂ©s Perkinsus olseni dans 1 lot de palourdes, et Marteilia refringens dans 4 lots de moules, ainsi que le virus OsHV-1 dans 1 lot de palourdes et 1 lot de coques, la bactĂ©rie Vibrio aestuarianus dans 3 lots de coques, et des bactĂ©ries du groupe Splendidus dans 3 lots de coques et dans 13 lots de moules. LâannĂ©e 2015 a Ă©galement permis la dĂ©monstration sur un site atelier dâun exercice de surveillance programmĂ©e, ciblĂ©e et fondĂ©e sur les risques dâintroduction et dâinstallation dâun organisme pathogĂšne exotique. Elle a concernĂ© le parasite Mikrocytos mackini de lâhuĂźtre creuse Crassostrea gigas, sur un site atelier de la Charente-Maritime, suivi par le rĂ©seau RESCO 2. Le parasite Mikrocytos mackini nâa pas Ă©tĂ© dĂ©tectĂ©. En revanche, le parasite Marteilia refringens a Ă©tĂ© dĂ©tectĂ© dans Ÿ des prĂ©lĂšvements dâhuĂźtres rĂ©alisĂ©s. Dans le cadre du soutien scientifique et technique de lâĂ©volution de la surveillance Ă©vĂ©nementielle, lâannĂ©e 2015 a Ă©galement permis de poursuivre la dĂ©marche relative aux dĂ©veloppements mĂ©thodologiques en lien avec la surveillance Ă©vĂ©nementielle des mortalitĂ©s de mollusques marins. Une Ă©tude de faisabilitĂ© de la recherche prospective de regroupements spatio-temporels dâĂ©vĂ©nements de mortalitĂ©s dâhuĂźtres creuses a Ă©tĂ© prĂ©parĂ©e en collaboration avec tous les acteurs de la santĂ© des mollusques marins en Normandie. Un outil de collecte et dâanalyse des donnĂ©es de signalements des mortalitĂ©s, automatisĂ©, simple dâutilisation et flexible, a Ă©tĂ© Ă©laborĂ©