A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

Background: Leishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor. Results: IFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 mu L - 100 mu L). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter. Conclusions: Using IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources

Plant Transcription Factors @ uni-potsdam.de

We present the Plant Transcription Factor Database (PlnTFDB), and the putative complete set of TFs in the algae _Chlamydomonas reinhardtii_, _Ostreococcus tauri_ and the vascular plants _Oryza sativa_ and _Arabidopsis thaliana_

A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors

<p>Abstract</p> <p>Background</p> <p>Quantitative reverse transcription – polymerase chain reaction (qRT-PCR) has been demonstrated to be particularly suitable for the analysis of weakly expressed genes, such as those encoding transcription factors. Rice (<it>Oryza sativa </it>L.) is an important crop and the most advanced model for monocotyledonous species; its nuclear genome has been sequenced and molecular tools are being developed for functional analyses. However, high-throughput methods for rice research are still limited and a large-scale qRT-PCR platform for gene expression analyses has not been reported.</p> <p>Results</p> <p>We established a qRT-PCR platform enabling the multi-parallel determination of the expression levels of more than 2500 rice transcription factor genes. Additionally, using different rice cultivars, tissues and physiological conditions, we evaluated the expression stability of seven reference genes. We demonstrate this resource allows specific and reliable detection of the expression of transcription factor genes in rice.</p> <p>Conclusion</p> <p>Multi-parallel qRT-PCR allows the versatile and sensitive transcriptome profiling of large numbers of rice transcription factor genes. The new platform complements existing microarray-based expression profiling techniques, by allowing the analysis of lowly expressed transcription factor genes to determine their involvement in developmental or physiological processes. We expect that this resource will be of broad utility to the scientific community in the further development of rice as an important model for plant science.</p

EBE, an AP2/ERF Transcription Factor Highly Expressed in Proliferating Cells, Affects Shoot Architecture in Arabidopsis

We report about ERF BUD ENHANCER (EBE; At5g61890), a transcription factor that affects cell proliferation as well as axillary bud outgrowth and shoot branching in Arabidopsis (Arabidopsis thaliana). EBE encodes a member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor superfamily; the gene is strongly expressed in proliferating cells and is rapidly and transiently up-regulated in axillary meristems upon main stem decapitation. Overexpression of EBE promotes cell proliferation in growing calli, while the opposite is observed in EBE-RNAi lines. EBE overexpression also stimulates axillary bud formation and outgrowth, while repressing it results in inhibition of bud growth. Global transcriptome analysis of estradiolinducible EBE overexpression lines revealed 48 EBE early-responsive genes, of which 14 were up-regulated and 34 were downregulated. EBE activates several genes involved in cell cycle regulation and dormancy breaking, including D-type cyclin CYCD3;3, transcription regulator DPa, and BRCA1-ASSOCIATED RING DOMAIN1. Among the down-regulated genes were DORMANCYASSOCIATED PROTEIN1 (AtDRM1), AtDRM1 homolog, MEDIATOR OF ABA-REGULATED DORMANCY1, and ZINC FINGER HOMEODOMAIN5. Our data indicate that the effect of EBE on shoot branching likely results from an activation of genes involved in cell cycle regulation and dormancy breaking.Fil: Mehrnia, Mohammad. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Balazadeh, Salma. Institut Max Planck Fur Molekulare Physiologie; Alemania. University of Potsdam. Institute of Biochemistry and Biology; AlemaniaFil: Zanor, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Mueller Roeber, Bernd. Institut Max Planck Fur Molekulare Physiologie; Alemania. University of Potsdam, Institute of Biochemistry and Biology; Alemani

A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors

<p>Abstract</p> <p>Background</p> <p>Quantitative reverse transcription – polymerase chain reaction (qRT-PCR) has been demonstrated to be particularly suitable for the analysis of weakly expressed genes, such as those encoding transcription factors. Rice (<it>Oryza sativa </it>L.) is an important crop and the most advanced model for monocotyledonous species; its nuclear genome has been sequenced and molecular tools are being developed for functional analyses. However, high-throughput methods for rice research are still limited and a large-scale qRT-PCR platform for gene expression analyses has not been reported.</p> <p>Results</p> <p>We established a qRT-PCR platform enabling the multi-parallel determination of the expression levels of more than 2500 rice transcription factor genes. Additionally, using different rice cultivars, tissues and physiological conditions, we evaluated the expression stability of seven reference genes. We demonstrate this resource allows specific and reliable detection of the expression of transcription factor genes in rice.</p> <p>Conclusion</p> <p>Multi-parallel qRT-PCR allows the versatile and sensitive transcriptome profiling of large numbers of rice transcription factor genes. The new platform complements existing microarray-based expression profiling techniques, by allowing the analysis of lowly expressed transcription factor genes to determine their involvement in developmental or physiological processes. We expect that this resource will be of broad utility to the scientific community in the further development of rice as an important model for plant science.</p

QuantPrime – a flexible tool for reliable high-throughput primer design for quantitative PCR

<p>Abstract</p> <p>Background</p> <p>Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays.</p> <p>Results</p> <p>Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses. QuantPrime can be used online through the internet <url>http://www.quantprime.de/</url> or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant <it>Arabidopsis thaliana</it>, the crop <it>Hordeum vulgare </it>and the model green alga <it>Chlamydomonas reinhardtii </it>show success rates of designed primer pairs exceeding 96%.</p> <p>Conclusion</p> <p>QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative <it>in situ </it>hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of RNA expression assays.</p

PlnTFDB: an integrative plant transcription factor database

BACKGROUND: Transcription factors (TFs) are key regulatory proteins that enhance or repress the transcriptional rate of their target genes by binding to specific promoter regions (i.e. cis-acting elements) upon activation or de-activation of upstream signaling cascades. TFs thus constitute master control elements of dynamic transcriptional networks. TFs have fundamental roles in almost all biological processes (development, growth and response to environmental factors) and it is assumed that they play immensely important functions in the evolution of species. In plants, TFs have been employed to manipulate various types of metabolic, developmental and stress response pathways. Cross-species comparison and identification of regulatory modules and hence TFs is thought to become increasingly important for the rational design of new plant biomass. Up to now, however, no computational repository is available that provides access to the largely complete sets of transcription factors of sequenced plant genomes. DESCRIPTION: PlnTFDB is an integrative plant transcription factor database that provides a web interface to access large (close to complete) sets of transcription factors of several plant species, currently encompassing Arabidopsis thaliana (thale cress), Populus trichocarpa (poplar), Oryza sativa (rice), Chlamydomonas reinhardtii and Ostreococcus tauri. It also provides an access point to its daughter databases of a species-centered representation of transcription factors (OstreoTFDB, ChlamyTFDB, ArabTFDB, PoplarTFDB and RiceTFDB). Information including protein sequences, coding regions, genomic sequences, expressed sequence tags (ESTs), domain architecture and scientific literature is provided for each family. CONCLUSION: We have created lists of putatively complete sets of transcription factors and other transcriptional regulators for five plant genomes. They are publicly available through . Further data will be included in the future when the sequences of other plant genomes become available

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here

JUNGBRUNNEN1 confers drought tolerance downstream of the HD-Zip I Transcription factor AtHB13

Low water availability is the major environmental factor limiting growth and productivity of plants and crops and is therefore considered of high importance for agriculture affected by climate change. Identifying regulatory components controlling the response and tolerance to drought stress is thus of major importance. The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) from Arabidopsis thaliana extends leaf longevity under non-stress growth conditions, lowers cellular hydrogen peroxide (H2O2) level, and enhances tolerance against heat stress and salinity. Here, we additionally find that JUB1 strongly increases tolerance to drought stress in Arabidopsis when expressed from both, a constitutive (CaMV 35S) and an abiotic stress-induced (RD29A) promoter. Employing a yeast one-hybrid screen we identified HD-Zip class I TF AtHB13 as an upstream regulator of JUB1. AtHB13 has previously been reported to act as a positive regulator of drought tolerance. AtHB13 and JUB1 thereby establish a joint drought stress control module.Fil: Ebrahimian Motlagh, Saghar. University of Potsdam; Alemania. Max Planck Institute of Molecular Plant Physiology; AlemaniaFil: Ribone, Pamela Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Thirumalaikumar, Venkatesh P.. Max Planck Institute of Molecular Plant Physiology; Alemania. University of Potsdam; AlemaniaFil: Allu, Annapurna D.. Max Planck Institute of Molecular Plant Physiology; Alemania. University of Potsdam; AlemaniaFil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Mueller Roeber, Bernd. University of Potsdam; Alemania. Max Planck Institute of Molecular Plant Physiology; AlemaniaFil: Balazadeh, Salma. University of Potsdam; Alemania. Max Planck Institute of Molecular Plant Physiology; Alemani

Genome-wide analysis of ABA-responsive elements ABRE and CE3 reveals divergent patterns in Arabidopsis and rice

Gomez-Porras J, Riano-Pachon DM, Dreyer I, Mayer JE, Mueller-Roeber B. Genome-wide analysis of ABA-responsive elements ABRE and CE3 reveals divergent patterns in Arabidopsis and rice. BMC GENOMICS. 2007;8(1): 260.Background: In plants, complex regulatory mechanisms are at the core of physiological and developmental processes. The phytohormone abscisic acid ( ABA) is involved in the regulation of various such processes, including stomatal closure, seed and bud dormancy, and physiological responses to cold, drought and salinity stress. The underlying tissue or plant-wide control circuits often include combinatorial gene regulatory mechanisms and networks that we are only beginning to unravel with the help of new molecular tools. The increasing availability of genomic sequences and gene expression data enables us to dissect ABA regulatory mechanisms at the individual gene expression level. In this paper we used an insilico-based approach directed towards genome-wide prediction and identification of specific features of ABA-responsive elements. In particular we analysed the genome-wide occurrence and positional arrangements of two well-described ABA-responsive cis-regulatory elements ( CREs), ABRE and CE3, in thale cress ( Arabidopsis thaliana) and rice ( Oryza sativa). Results: Our results show that Arabidopsis and rice use the ABA-responsive elements ABRE and CE3 distinctively. Earlier reports for various monocots have identified CE3 as a coupling element ( CE) associated with ABRE. Surprisingly, we found that while ABRE is equally abundant in both species, CE3 is practically absent in Arabidopsis. ABRE-ABRE pairs are common in both genomes, suggesting that these can form functional ABA-responsive complexes ( ABRCs) in Arabidopsis and rice. Furthermore, we detected distinct combinations, orientation patterns and DNA strand preferences of ABRE and CE3 motifs in rice gene promoters. Conclusion: Our computational analyses revealed distinct recruitment patterns of ABA-responsive CREs in upstream sequences of Arabidopsis and rice. The apparent absence of CE3s in Arabidopsis suggests that another CE pairs with ABRE to establish a functional ABRC capable of interacting with transcription factors. Further studies will be needed to test whether the observed differences are extrapolatable to monocots and dicots in general, and to understand how they contribute to the fine-tuning of the hormonal response. The outcome of our investigation can now be used to direct future experimentation designed to further dissect the ABA-dependent regulatory networks