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Publikation

Brückner, K.; Schäfer, P.; Weber, E.; Grützner, R.; Marillonnet, S.; Tissier, A.; A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal gene expression in plants Plant J. 82, 707-716, (2015) DOI: 10.1111/tpj.12843

A library of synthetic promoters containing the binding site of a single designer transcription activator‐like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18‐base long dTALE‐binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty‐three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.
Publikation

Heinze, M.; Brandt, W.; Marillonnet, S.; Roos, W.; “Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints Plant Cell 27, 448-462, (2015) DOI: 10.1105/tpc.114.135343

The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins.
Publikation

Walter, M. H.; Stauder, R.; Tissier, A.; Evolution of root-specific carotenoid precursor pathways for apocarotenoid signal biogenesis Plant Sci. 233, 1-10, (2015) DOI: 10.1016/j.plantsci.2014.12.017

Various cleavage products of C40 carotenoid substrates are formed preferentially or exclusively in roots. Such apocarotenoid signaling or regulatory compounds differentially induced in roots during environmental stress responses including root colonization by arbuscular mycorrhizal fungi include ABA, strigolactones and C13 α-ionol/C14 mycorradicin derivatives. The low carotenoid levels in roots raise the question of whether there is a regulated precursor supply channeled into apocarotenoid formation distinct from default carotenoid pathways. This review describes root-specific isogene components of carotenoid pathways toward apocarotenoid formation, highlighting a new PSY3 class of phytoene synthase genes in dicots. It is clearly distinct from the monocot PSY3 class co-regulated with ABA formation. At least two members of the exclusive dicot PSY3s are regulated by nutrient stress and mycorrhization. This newly recognized dicot PSY3 (dPSY3 vs. mPSY3 from monocots) class probably represents an ancestral branch in the evolution of the plant phytoene synthase family. The evolutionary history of PSY genes is compared with the evolution of MEP pathway isogenes encoding 1-deoxy-d-xylulose 5-phosphate synthases (DXS), particularly DXS2, which is co-regulated with dPSY3s in mycorrhizal roots. Such stress-inducible isoforms for rate-limiting steps in root carotenogenesis might be components of multi-enzyme complexes committed to apocarotenoid rather than to carotenoid formation.
Publikation

Patron, N. J.; Orzaez, D.; Marillonnet, S.; Warzecha, H.; Matthewman, C.; Youles, M.; Raitskin, O.; Leveau, A.; Farré, G.; Rogers, C.; Smith, A.; Hibberd, J.; Webb, A. A. R.; Locke, J.; Schornack, S.; Ajioka, J.; Baulcombe, D. C.; Zipfel, C.; Kamoun, S.; Jones, J. D. G.; Kuhn, H.; Robatzek, S.; Van Esse, H. P.; Sanders, D.; Oldroyd, G.; Martin, C.; Field, R.; O'Connor, S.; Fox, S.; Wulff, B.; Miller, B.; Breakspear, A.; Radhakrishnan, G.; Delaux, P.-M.; Loqué, D.; Granell, A.; Tissier, A.; Shih, P.; Brutnell, T. P.; Quick, W. P.; Rischer, H.; Fraser, P. D.; Aharoni, A.; Raines, C.; South, P. F.; Ané, J.-M.; Hamberger, B. R.; Langdale, J.; Stougaard, J.; Bouwmeester, H.; Udvardi, M.; Murray, J. A. H.; Ntoukakis, V.; Schäfer, P.; Denby, K.; Edwards, K. J.; Osbourn, A.; Haseloff, J.; Standards for plant synthetic biology: a common syntax for exchange of DNA parts New Phytol. 208, 13-19, (2015) DOI: 10.1111/nph.13532

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease‐mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.
Bücher und Buchkapitel

Marillonnet, S.; Werner, S.; Assembly of Multigene Constructs Using Golden Gate Cloning (Castilho, A., ed.). Methods Mol. Biol. 1321, 269-284, (2015) ISBN: 978-1-4939-2760-9 DOI: 10.1007/978-1-4939-2760-9_19

Efficient DNA assembly methods are required for synthetic biology. Standardization of DNA parts is an essential element that not only facilitates reuse of the same parts for various constructs but also allows standardization of the assembly strategy. We provide here a protocol for assembly of multigene constructs from standard biological parts using the modular cloning system MoClo. Making constructs using this system requires to first define the structure of the final construct and to identify all basic parts and vectors required for the construction strategy. The cloning strategy is in large part determined by the structure of the final construct, which is then made using a series of one-pot Golden Gate cloning reactions.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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