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Publikationen - Stoffwechsel- und Zellbiologie

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Preprints

Püllmann, P.; Ulpinnis, C.; Marillonnet, S.; Gruetzner, R.; Neumann, S.; Weissenborn, M. J.; Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design bioRxiv (2018) DOI: 10.1101/453621

Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Muta-genesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.
Preprints

Ordon, J.; Bressan, M.; Kretschmer, C.; Dall'Osto, L.; Marillonnet, S.; Bassi, R.; Stuttmann, J.; Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation bioRxiv (2018) DOI: 10.1101/393439

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.
Publikation

van Aubel, G.; Serderidis, S.; Ivens, J.; Clinkemaillie, A.; Legrève, A.; Hause, B.; Van Cutsem, P.; Oligosaccharides successfully thwart hijacking of the salicylic acid pathway by Phytophthora infestans in potato leaves Plant Pathol. 67, 1901-1911, (2018) DOI: 10.1111/ppa.12908

Potato growing is severely threatened by the late blight agent Phytophthora infestans, which is usually controlled by massive amounts of fungicides. While variety resistance is often bypassed by the pathogen, the plant innate immunity opens the way to new biological plant protection tools e.g. the COS‐OGA elicitor. This oligosaccharide composition mimics the interaction between plants and fungal pathogens as it combines chitosan oligomers (COS) with pectin‐derived oligogalacturonides (OGA).Two different COS‐OGA elicitors were evaluated against potato late blight: FytoSave® mainly efficient against powdery mildews and FytoSol, a new composition still under development. Next to the evaluation of their protective effect, a comparative study of plant defense induction was performed focusing on the effect of repeated sprayings as well as on the stimulation of salicylic acid (SA), jasmonic acid and ethylene‐related pathways during the biotrophic and the necrotrophic growth stages of the pathogen.The FytoSave® elicitor strongly increased the SA content but failed to induce a sufficient protection against late blight while FytoSol maintained or even decreased the free SA content in presence of P. infestans and was completely efficient. Surprisingly, the necrotrophic development of P. infestans occurred along with a strong leaf accumulation of free SA and SA‐related transcripts. It may represent an attempt by P. infestans to divert plant defenses for its own benefit. Preventive sprayings with FytoSol but not FytoSave® completely impeded this hijacking. FytoSol seemed to keep the SA pathway under control, thereby preventing its diversion by P. infestans.
Publikation

Tissier, A.; Plant secretory structures: more than just reaction bags Curr. Opin. Biotech. 49, 73-79, (2018) DOI: 10.1016/j.copbio.2017.08.003

Plants have a remarkable capacity for the production of a wide range of metabolites. Much has been reported and reviewed on the diversity of these metabolites and how it is achieved, for example through the evolution of enzyme families. In comparison, relatively little is known on the extraordinary metabolic productivity of dedicated organs where many of these metabolites are synthesized and accumulate. Plant glandular trichomes are such specialized metabolite factories, for which recent omics analyses have shed new light on the adaptive metabolic strategies that support high metabolic fluxes. In photosynthetic trichomes such as those of the Solanaceae, these include CO2 refixation and possibly C4-like metabolism which contribute to the high productivity of these sink organs.
Publikation

Tannert, M.; May, A.; Ditfe, D.; Berger, S.; Balcke, G. U.; Tissier, A.; Köck, M.; Pi starvation-dependent regulation of ethanolamine metabolism by phosphoethanolamine phosphatase PECP1 in Arabidopsis roots J. Exp. Bot. 69, 467-481, (2018) DOI: 10.1093/jxb/erx408

A universal plant response to phosphorus deprivation is the up-regulation of a diverse array of phosphatases. As reported recently, the AtPECP1 gene encodes a phosphatase with in vitro substrate specificity for phosphoethanolamine and phosphocholine. The putative substrates suggested that AtPECP1 is related to phospholipid metabolism; however, the biological function of AtPECP1 is as yet not understood. In addition, whereas lipid remodelling processes as part of the phosphorus starvation response have been extensively studied, knowledge of the polar head group metabolism and its regulation is lacking. We found that AtPECP1 is expressed in the cytosol and exerts by far its strongest activity in roots of phosphate-starved plants. We established a novel LC-MS/MS-based method for the quantitative and simultaneous measurement of the head group metabolites. The analysis of Atpecp1 null mutants and overexpression lines revealed that phosphoethanolamine, but not phosphocholine is the substrate of AtPECP1 in vivo. The impact on head group metabolite levels is greatest in roots of both loss-of-function and gain-of-function transgenic lines, indicating that the biological role of AtPECP1 is mainly restricted to roots. We suggest that phosphoethanolamine hydrolysis by AtPECP1 during Pi starvation is required to down-regulate the energy-consuming biosynthesis of phosphocholine through the methylation pathway.
Publikation

Stauder, R.; Welsch, R.; Camagna, M.; Kohlen, W.; Balcke, G. U.; Tissier, A.; Walter, M. H.; Strigolactone Levels in Dicot Roots Are Determined by an Ancestral Symbiosis-Regulated Clade of the PHYTOENE SYNTHASE Gene Family Front. Plant Sci. 9, 255, (2018) DOI: 10.3389/fpls.2018.00255

Strigolactones (SLs) are apocarotenoid phytohormones synthesized from carotenoid precursors. They are produced most abundantly in roots for exudation into the rhizosphere to cope with mineral nutrient starvation through support of root symbionts. Abscisic acid (ABA) is another apocarotenoid phytohormone synthesized in roots, which is involved in responses to abiotic stress. Typically low carotenoid levels in roots raise the issue of precursor supply for the biosynthesis of these two apocarotenoids in this organ. Increased ABA levels upon abiotic stress in Poaceae roots are known to be supported by a particular isoform of phytoene synthase (PSY), catalyzing the rate-limiting step in carotenogenesis. Here we report on novel PSY3 isogenes from Medicago truncatula (MtPSY3) and Solanum lycopersicum (SlPSY3) strongly expressed exclusively upon root interaction with symbiotic arbuscular mycorrhizal (AM) fungi and moderately in response to phosphate starvation. They belong to a widespread clade of conserved PSYs restricted to dicots (dPSY3) distinct from the Poaceae-PSY3s involved in ABA formation. An ancient origin of dPSY3s and a potential co-evolution with the AM symbiosis is discussed in the context of PSY evolution. Knockdown of MtPSY3 in hairy roots of M. truncatula strongly reduced SL and AM-induced C13 α-ionol/C14 mycorradicin apocarotenoids. Inhibition of the reaction subsequent to phytoene synthesis revealed strongly elevated levels of phytoene indicating induced flux through the carotenoid pathway in roots upon mycorrhization. dPSY3 isogenes are coregulated with upstream isogenes and downstream carotenoid cleavage steps toward SLs (D27, CCD7, CCD8) suggesting a combined carotenoid/apocarotenoid pathway, which provides “just in time”-delivery of precursors for apocarotenoid formation.
Publikation

Peters, K.; Worrich, A.; Weinhold, A.; Alka, O.; Balcke, G.; Birkemeyer, C.; Bruelheide, H.; Calf, O. W.; Dietz, S.; Dührkop, K.; Gaquerel, E.; Heinig, U.; Kücklich, M.; Macel, M.; Müller, C.; Poeschl, Y.; Pohnert, G.; Ristok, C.; Rodríguez, V. M.; Ruttkies, C.; Schuman, M.; Schweiger, R.; Shahaf, N.; Steinbeck, C.; Tortosa, M.; Treutler, H.; Ueberschaar, N.; Velasco, P.; Weiß, B. M.; Widdig, A.; Neumann, S.; van Dam, N. M.; Current Challenges in Plant Eco-Metabolomics Int. J. Mol. Sci. 19, 1385, (2018) DOI: 10.3390/ijms19051385

The relatively new research discipline of Eco-Metabolomics is the application of metabolomics techniques to ecology with the aim to characterise biochemical interactions of organisms across different spatial and temporal scales. Metabolomics is an untargeted biochemical approach to measure many thousands of metabolites in different species, including plants and animals. Changes in metabolite concentrations can provide mechanistic evidence for biochemical processes that are relevant at ecological scales. These include physiological, phenotypic and morphological responses of plants and communities to environmental changes and also interactions with other organisms. Traditionally, research in biochemistry and ecology comes from two different directions and is performed at distinct spatiotemporal scales. Biochemical studies most often focus on intrinsic processes in individuals at physiological and cellular scales. Generally, they take a bottom-up approach scaling up cellular processes from spatiotemporally fine to coarser scales. Ecological studies usually focus on extrinsic processes acting upon organisms at population and community scales and typically study top-down and bottom-up processes in combination. Eco-Metabolomics is a transdisciplinary research discipline that links biochemistry and ecology and connects the distinct spatiotemporal scales. In this review, we focus on approaches to study chemical and biochemical interactions of plants at various ecological levels, mainly plant–organismal interactions, and discuss related examples from other domains. We present recent developments and highlight advancements in Eco-Metabolomics over the last decade from various angles. We further address the five key challenges: (1) complex experimental designs and large variation of metabolite profiles; (2) feature extraction; (3) metabolite identification; (4) statistical analyses; and (5) bioinformatics software tools and workflows. The presented solutions to these challenges will advance connecting the distinct spatiotemporal scales and bridging biochemistry and ecology
Publikation

Kowarschik, K.; Hoehenwarter, W.; Marillonnet, S.; Trujillo, M.; UbiGate: a synthetic biology toolbox to analyse ubiquitination New Phytol. 217, 1749-1763, (2018) DOI: 10.1111/nph.14900

Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post‐translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process.Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning.This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression.We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time‐ and cost‐effective manner.
Publikation

Gelová, Z.; ten Hoopen, P.; Novák, O.; Motyka, V.; Pernisová, M.; Dabravolski, S.; Didi, V.; Tillack, I.; Oklešťková, J.; Strnad, M.; Hause, B.; Haruštiaková, D.; Conrad, U.; Janda, L.; Hejátko, J.; Antibody-mediated modulation of cytokinins in tobacco: organ-specific changes in cytokinin homeostasis J. Exp. Bot. 69, 441-454, (2018) DOI: 10.1093/jxb/erx426

Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin
Publikation

Gantner, J.; Ordon, J.; Ilse, T.; Kretschmer, C.; Gruetzner, R.; Löfke, C.; Dagdas, Y.; Bürstenbinder, K.; Marillonnet, S.; Stuttmann, J.; Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system PLOS ONE 13, e0197185, (2018) DOI: 10.1371/journal.pone.0197185

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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