@article{DortayMuellerRoeber2010,
  author    = {Dortay, Hakan and M{\"u}ller-R{\"o}ber, Bernd},
  title     = {A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker},
  issn      = {1475-2859},
  doi       = {10.1186/1475-2859-9-29},
  year      = {2010},
  abstract  = {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},
  language  = {en}
}
@article{NguyenSchippersGoniRamosetal.2013,
  author    = {Nguyen, Hung M. and Schippers, Jos H. M. and Goni-Ramos, Oscar and Christoph, Mathias P. and Dortay, Hakan and van der Hoorn, Renier A. L. and M{\"u}ller-R{\"o}ber, Bernd},
  title     = {An upstream regulator of the 26S proteasome modulates organ size in Arabidopsis thaliana},
  series = {The plant journal},
  volume    = {74},
  journal   = {The plant journal},
  number    = {1},
  publisher = {Wiley-Blackwell},
  address   = {Hoboken},
  issn      = {0960-7412},
  doi       = {10.1111/tpj.12097},
  pages     = {25 -- 36},
  year      = {2013},
  abstract  = {In both animal and plant kingdoms, body size is a fundamental but still poorly understood attribute of biological systems. Here we report that the Arabidopsis NAC transcription factor Regulator of Proteasomal Gene Expression' (RPX) controls leaf size by positively modulating proteasome activity. We further show that the cis-element recognized by RPX is evolutionarily conserved between higher plant species. Upon over-expression of RPX, plants exhibit reduced growth, which may be reversed by a low concentration of the pharmacological proteasome inhibitor MG132. These data suggest that the rate of protein turnover during growth is a critical parameter for determining final organ size.},
  language  = {en}
}
@article{DortaySchmoeckelFettkeetal.2011,
  author    = {Dortay, Hakan and Schm{\"o}ckel, Sandra M. and Fettke, J{\"o}rg and M{\"u}ller-R{\"o}ber, Bernd},
  title     = {Expression of human c-reactive protein in different systems and its purification from Leishmania tarentolae},
  series = {Protein expression and purification},
  volume    = {78},
  journal   = {Protein expression and purification},
  number    = {1},
  publisher = {Elsevier},
  address   = {San Diego},
  issn      = {1046-5928},
  doi       = {10.1016/j.pep.2011.03.010},
  pages     = {55 -- 60},
  year      = {2011},
  abstract  = {With its homo-pentameric structure and calcium-dependent specificity for phosphocholine (PCh), human c-reactive protein (CRP) is produced by the liver and secreted in elevated quantities in response to inflammation. CRP is widely accepted as a cardiac marker, e.g. in point-of-care diagnostics, however, its heterologous expression has proven difficult. Here, we demonstrate the expression of CRP in different Escherichia coli strains as well as by in vitro transcription/translation. Although expression in these systems was straightforward, most of the protein that accumulated was insoluble. We therefore expanded our study to include the expression of CRP in two eukaryotic hosts, namely the yeast Kluyveromyces lactis and the protozoon Leishmania tarentolae. Both expression systems are optimized for secretion of recombinant proteins and here allowed successful expression of soluble CRP. We also demonstrate the purification of recombinant CRP from Leishmania growth medium; the purification of protein expressed from K. lactis was not successful. Functional and intact CRP pentamer is known to interact with PCh in Ca(2+)-dependent manner. In this report we verify the binding specificity of recombinant CRP from L tarentolae (2 mu g/mL culture medium) for PCh.},
  language  = {en}
}
@article{DortayAkulaWestphaletal.2011,
  author    = {Dortay, Hakan and Akula, Usha Madhuri and Westphal, Christin and Sittig, Marie and M{\"u}ller-R{\"o}ber, Bernd},
  title     = {High-throughput protein expression using a combination of ligation-independent cloning (LIC) and infrared fluorescent protein (IFP) detection},
  series = {PLoS one},
  volume    = {6},
  journal   = {PLoS one},
  number    = {4},
  publisher = {PLoS},
  address   = {San Fransisco},
  issn      = {1932-6203},
  doi       = {10.1371/journal.pone.0018900},
  pages     = {19},
  year      = {2011},
  abstract  = {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.},
  language  = {en}
}
@article{WuAlluGarapatietal.2012,
  author    = {Wu, Anhui and Allu, Annapurna Devi and Garapati, Prashanth and Siddiqui, Hamad and Dortay, Hakan and Zanor, Maria-Ines and Asensi-Fabado, Maria Amparo and Munne-Bosch, Sergi and Antonio, Carla and Tohge, Takayuki and Fernie, Alisdair R. and Kaufmann, Kerstin and Xue, Gang-Ping and M{\"u}ller-R{\"o}ber, Bernd and Balazadeh, Salma},
  title     = {Jungbrunnen1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in arabidopsis},
  series = {The plant cell},
  volume    = {24},
  journal   = {The plant cell},
  number    = {2},
  publisher = {American Society of Plant Physiologists},
  address   = {Rockville},
  issn      = {1040-4651},
  doi       = {10.1105/tpc.111.090894},
  pages     = {482 -- 506},
  year      = {2012},
  abstract  = {The transition from juvenility through maturation to senescence is a complex process that involves the regulation of longevity. Here, we identify JUNGBRUNNEN1 (JUB1), a hydrogen peroxide (H2O2)-induced NAC transcription factor, as a central longevity regulator in Arabidopsis thaliana. JUB1 overexpression strongly delays senescence, dampens intracellular H2O2 levels, and enhances tolerance to various abiotic stresses, whereas in jub1-1 knockdown plants, precocious senescence and lowered abiotic stress tolerance are observed. A JUB1 binding site containing a RRYGCCGT core sequence is present in the promoter of DREB2A, which plays an important role in abiotic stress responses. JUB1 transactivates DREB2A expression in mesophyll cell protoplasts and transgenic plants and binds directly to the DREB2A promoter. Transcriptome profiling of JUB1 overexpressors revealed elevated expression of several reactive oxygen species-responsive genes, including heat shock protein and glutathione S-transferase genes, whose expression is further induced by H2O2 treatment. Metabolite profiling identified elevated Pro and trehalose levels in JUB1 overexpressors, in accordance with their enhanced abiotic stress tolerance. We suggest that JUB1 constitutes a central regulator of a finely tuned control system that modulates cellular H2O2 level and primes the plants for upcoming stress through a gene regulatory network that involves DREB2A.},
  language  = {en}
}
@article{MatallanaRamirezRaufFarageBarhometal.2013,
  author    = {Matallana-Ramirez, Lilian P. and Rauf, Mamoona and Farage-Barhom, Sarit and Dortay, Hakan and Xue, Gang-Ping and Droege-Laser, Wolfgang and Lers, Amnon and Balazadeh, Salma and M{\"u}ller-R{\"o}ber, Bernd},
  title     = {NAC Transcription Factor ORE1 and Senescence-Induced BIFUNCTIONAL NUCLEASE1 (BFN1) Constitute a Regulatory Cascade in Arabidopsis},
  series = {Molecular plant},
  volume    = {6},
  journal   = {Molecular plant},
  number    = {5},
  publisher = {Oxford Univ. Press},
  address   = {Oxford},
  issn      = {1674-2052},
  doi       = {10.1093/mp/sst012},
  pages     = {1438 -- 1452},
  year      = {2013},
  abstract  = {The NAC transcription factor ORE1 is a key regulator of senescence in Arabidopsis thaliana. Here, we demonstrate that senescence-induced and cell death-associated BIFUNCTIONAL NUCLEASE1 (BFN1) is a direct downstream target of ORE1, revealing a previously unknown regulatory cascade.Senescence is a highly regulated process that involves the action of a large number of transcription factors. The NAC transcription factor ORE1 (ANAC092) has recently been shown to play a critical role in positively controlling senescence in Arabidopsis thaliana; however, no direct target gene through which it exerts its molecular function has been identified previously. Here, we report that BIFUNCTIONAL NUCLEASE1 (BFN1), a well-known senescence-enhanced gene, is directly regulated by ORE1. We detected elevated expression of BFN1 already 2 h after induction of ORE1 in estradiol-inducible ORE1 overexpression lines and 6 h after transfection of Arabidopsis mesophyll cell protoplasts with a 35S:ORE1 construct. ORE1 and BFN1 expression patterns largely overlap, as shown by promoterreporter gene (GUS) fusions, while BFN1 expression in senescent leaves and the abscission zones of maturing flower organs was virtually absent in ore1 mutant background. In vitro binding site assays revealed a bipartite ORE1 binding site, similar to that of ORS1, a paralog of ORE1. A bipartite ORE1 binding site was identified in the BFN1 promoter; mutating the cis-element within the context of the full-length BFN1 promoter drastically reduced ORE1-mediated transactivation capacity in transiently transfected Arabidopsis mesophyll cell protoplasts. Furthermore, chromatin immunoprecipitation (ChIP) demonstrates in vivo binding of ORE1 to the BFN1 promoter. We also demonstrate binding of ORE1 in vivo to the promoters of two other senescence-associated genes, namely SAG29/SWEET15 and SINA1, supporting the central role of ORE1 during senescence.},
  language  = {en}
}
@article{RaufArifDortayetal.2013,
  author    = {Rauf, Mamoona and Arif, Muhammad and Dortay, Hakan and Matallana-Ramirez, Lilian P. and Waters, Mark T. and Nam, Hong Gil and Lim, Pyung-Ok and M{\"u}ller-R{\"o}ber, Bernd and Balazadeh, Salma},
  title     = {ORE1 balances leaf senescence against maintenance by antagonizing G2-like-mediated transcription},
  series = {EMBO reports},
  volume    = {14},
  journal   = {EMBO reports},
  number    = {4},
  publisher = {Nature Publ. Group},
  address   = {London},
  issn      = {1469-221X},
  doi       = {10.1038/embor.2013.24},
  pages     = {382 -- 388},
  year      = {2013},
  abstract  = {Leaf senescence is a key physiological process in all plants. Its onset is tightly controlled by transcription factors, of which NAC factor ORE1 (ANAC092) is crucial in Arabidopsis thaliana. Enhanced expression of ORE1 triggers early senescence by controlling a downstream gene network that includes various senescence-associated genes. Here, we report that unexpectedly ORE1 interacts with the G2-like transcription factors GLK1 and GLK2, which are important for chloroplast development and maintenance, and thereby for leaf maintenance. ORE1 antagonizes GLK transcriptional activity, shifting the balance from chloroplast maintenance towards deterioration. Our finding identifies a new mechanism important for the control of senescence by ORE1.},
  language  = {en}
}