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Publications - Cell and Metabolic Biology

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Books and chapters

Marillonnet, S.; Werner, S.; Golden gate cloning of multigene constructs using the modular cloning system MoClo (Schindler D.). Methods Mol. Biol. 2850, 21-39, (2025) ISBN: 978-1-0716-4094-4 DOI: 10.1007/978-1-0716-4220-7_2

Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the modular cloning system MoClo. Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.
Books and chapters

Grützner, R.; Marillonnet, S.; Golden gate cloning of MoClo standard parts (Schindler D.). Methods Mol. Biol. 2850, 1-19, (2025) ISBN: 978-1-0716-4094-4 DOI: 10.1007/978-1-0716-4220-7_1

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level-1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module.
Preprints

Mekkaoui, K.; Baral, R.; Smith, F.; Klein, M.; Feussner, I.; Hause, B.; Unraveling the role of 12-cis-oxo-phytodienoic acid in the wound response of Arabidopsis thaliana: Insights from transcriptomic and complementation studies bioRxiv (2024) DOI: 10.1101/2024.03.22.586262

In addition to jasmonoyl-isoleucine (JA-Ile), a well-established signaling molecule for plant growth and defense, its precursor, cis-12-oxo-phytodienoic acid (OPDA), is thought to possess independent signaling functions. Its perception in vascular plants is still uncharacterized. Several OPDA functions in Arabidopsis were inferred from a mutant that is affected in the function of the OPDA REDUCTASE3 (OPR3), catalyzing the conversion of OPDA within peroxisomes. Recently, opr3 plants were found to accumulate JA-Ile via a cytosolic OPR2-mediated bypass. Given the uncoupling of OPDA and JA biosynthesis in the JA-deficient mutant opr2opr3, potential OPDA signaling was investigated by a transcriptome approach comparing wild type, opr2opr3 and the JA- and OPDA-deficient mutantallene oxide synthase. Dissecting the wound response of seedlings revealed that OPDA lacked a transcriptional signature, and that previously characterized OPDA-response genes were wound-induced independently of OPDA. Exogenous application of OPDA to opr2opr3 seedlings led to JA-Ile formation and signaling even in absence of OPR2 and OPR3 and resulted in activation of sulfur assimilation. These divergent responses to endogenously synthesized and applied OPDA suggest a compartmentalization of endogenous OPDA which was investigated by a trans-organellar complementation approach. OPR3 complemented the opr2opr3 mutant in terms of fertility and wound-induced JA-Ile production irrespective of its subcellular localization. In vitro enzymatic activity of OPR3, however, showed conversion of OPDA and 4,5-didehydro-JA (4,5-ddh-JA), therefore not allowing to conclude which compound is translocated. Dissecting the conversion of either OPDA or 4,5-ddh-JA by OPR2 and OPR1 organelle variants pointed to a strong OPDA compartmentalization supporting its lacking signaling capacity.
Preprints

Balcke, G.; Saoud, M.; Grau, J.; Rennert, R.; Mueller, T.; Yousefi, M.; Davari, M. D.; Hause, B.; Csuk, R.; Rashan, L.; Grosse, I.; Tissier, A.; Wessjohann, L.; Machine learning-based metabolic pattern recognition predicts mode of action for anti-cancer drug candidates Research Square (2024) DOI: 10.21203/rs.3.rs-3494185/v1

A bottleneck in the development of new anti-cancer drugs is the recognition of their mode of action (MoA). We combined metabolomics and machine learning to predict MoAs of novel anti-proliferative drug candidates, focusing on human prostate cancer cells (PC-3). As proof of concept, we studied 38 drugs with known effects on 16 key processes of cancer metabolism, profiling low molecular weight intermediates of the central carbon and cellular energy metabolism (CCEM) by LC-MS/MS. These metabolic patterns unveiled distinct MoAs, enabling accurate MoA predictions for novel agents by machine learning. We validate the transferability of MoA predictions from PC-3 to two other cancer cell models and show that correct predictions are still possible, but at the expense of prediction quality. Furthermore, metabolic profiles of treated cells yield insights into intracellular processes, exemplified for drugs inducing different types of mitochondrial dysfunction. Specifically, we predict that pentacyclic triterpenes inhibit oxidative phosphorylation and affect phospholipid biosynthesis, as supported by respiration parameters, lipidomics, and molecular docking. Using biochemical insights from individual drug treatments, our approach offers new opportunities, including the optimization of combinatorial drug applications.
Printed publications

Dunken, N.; Widmer, H.; Balcke, G. U.; Straube, H.; Langen, G.; Charura, N. M.; Saake, P.; De Quattro, C.; Schön, J.; Rovenich, H.; Wawra, S.; Khan, M.; Djamei, A.; Zurbriggen, M. D.; Tissier, A.; Witte, C.-P.; Zuccaro, A.; A nucleoside signal generated by a fungal endophyte regulates host cell death and promotes root colonization Cell Host & Microbe 32, 1-17, (2024) DOI: 10.1016/j.chom.2024.10.020

The intracellular colonization of plant roots by the beneficial fungal endophyte Serendipita indica follows a biphasic strategy, including a host cell death phase that enables successful colonization of Arabidopsis thaliana roots. How host cell death is initiated and controlled is largely unknown. Here, we show that two fungal enzymes, the ecto-50-nucleotidase SiE5NT and the nuclease SiNucA, act synergistically in the apoplast at the onset of cell death to produce deoxyadenosine (dAdo). The uptake of extracellular dAdo but not the structurally related adenosine activates cell death via the equilibrative nucleoside transporter ENT3. We identified a previously uncharacterized Toll-like interleukin 1 receptor (TIR)-nucleotide-binding leucine-rich repeat receptor (NLR) protein, ISI (induced by S. indica), as an intracellular factor that affects host cell death, fungal colonization, and growth promotion. Our data show that the combined activity of two fungal apoplastic enzymes promotes the production of a metabolite that engages TIR-NLR-modulated pathways to induce plant cell death, providing a link to immunometabolism in plants.Graphical abstract
Publications

Wasternack, C.; Hause, B.; BFP1: One of 700 Arabidopsis F-box proteins mediates degradation of JA oxidases to promote plant immunity Mol. Plant 17, 375-376, (2024) DOI: 10.1016/j.molp.2024.02.008

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Publications

Schreiber, T.; Prange, A.; Schäfer, P.; Iwen, T.; Grützner, R.; Marillonnet, S.; Lepage, A.; Javelle, M.; Paul, W.; Tissier, A.; Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR/Cas endonucleases Mol. Plant 17, 824-837, (2024) DOI: 10.1016/j.molp.2024.03.013

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. We identified two groups of exonucleases from the Herpes Virus and the bacteriophage T7 families that conferred an up to 38-fold increase in HDR frequencies when fused to Cas9/Cas12a in a Tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrate stable and heritable knock-ins of in wheat in 1% of the primary transformants. Our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.
Publications

Launhardt, L.; Uhlenberg, J.; Stellmach, H.; Schomburg, M.; Hause, B.; Heilmann, I.; Heilmann, M.; Association of the Arabidopsis oleoyl Δ12‐desaturase FAD2 with pre‐cis‐Golgi stacks at endoplasmic reticulum‐Golgi‐exit sites Plant J. 117, 242-263, (2024) DOI: 10.1111/tpj.16492

The unsaturation of phospholipids influences the function of membranes. In Arabidopsis thaliana, the oleoyl Δ12‐desaturase FAD2 converts oleic (18:1Δ9) to linoleic acid (18:2Δ9,12) and influences phospholipid unsaturation in different cellular membranes. Despite its importance, the precise localization of Arabidopsis FAD2 has not been unambiguously described. As FAD2 is thought to modify phospholipid‐associated fatty acids at the endoplasmic reticulum (ER), from where unsaturates are distributed to other cellular sites, we hypothesized that FAD2 locates to ER subdomains enabling trafficking of lipid intermediates through the secretory pathway. Fluorescent FAD2 fusions used to test this hypothesis were first assessed for functionality by heterologous expression in yeast (Saccharomyces cerevisiae), and in planta by Arabidopsis fad2 mutant rescue upon ectopic expression from an intrinsic FAD2 promoter fragment. Light sheet fluorescence, laser scanning confocal or spinning disc microscopy of roots, leaves, or mesophyll protoplasts showed the functional fluorescence‐tagged FAD2 variants in flattened donut‐shaped structures of ~0.5–1 μm diameter, in a pattern not resembling mere ER association. High‐resolution imaging of coexpressed organellar markers showed fluorescence‐tagged FAD2 in a ring‐shaped pattern surrounding ER‐proximal Golgi particles, colocalizing with pre‐cis‐Golgi markers. This localization required the unusual C‐terminal retention signal of FAD2, and deletion or substitutions in this protein region resulted in relaxed distribution and diffuse association with the ER. The distinct association of FAD2 with pre‐cis‐Golgi stacks in Arabidopsis root and leaf tissue is consistent with a contribution of FAD2 to membrane lipid homeostasis through the secretory pathway, as verified by an increased plasma membrane liquid phase order in the fad2 mutant.
Publications

Grützner, R.; König, K.; Horn, C.; Engler, C.; Laub, A.; Vogt, T.; Marillonnet, S.; A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana Plant Biotechnol. J. 22, 1238-1250, (2024) DOI: 10.1111/pbi.14261

Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.
Publications

Frey, M.; Vahabi, K.; Cankar, K.; Lackus, N. D.; Padilla-Gonzalez, F.; Ro, D.-K.; Rieseberg, L.; Spring, O.; Tissier, A.; Sesquiterpene lactones – insights into biosynthesis, regulation and signalling roles Crit. Rev. Plant Sci. 43, 131-157, (2024) DOI: 10.1080/07352689.2024.2307240

Sesquiterpene lactones (STLs) are bitter tasting plant specialized metabolites derived from farnesyl pyrophosphate (FPP) that contain a characteristic lactone ring. STLs can be found in many plant families that are distantly related to each other and outside the plant kingdom. They are especially prevalent in the plant families Apiaceae and Asteraceae, the latter being one of the largest plant families besides the Orchidaceae. The STL diversity is especially large in the Asteraceae, which made them an ideal object for chemosystematic studies in these species. Many STLs show a high bioactivity, for example as protective compounds against herbivory. STLs are also relevant for pharmaceutical applications, such as the treatment of malaria with artemisinin. Recent findings have dramatically changed our knowledge about the biosynthesis of STLs, as well as their developmental, spatial, and environmental regulation. This review intents to update the currently achieved progress in these aspects. With the advancement of genome editing tools such as CRISPR/Cas and the rapid acceleration of the speed of genome sequencing, even deeper insights into the biosynthesis, regulation, and enzyme evolution of STL can be expected in the future. Apart from their role as protective compounds, there may be a more subtle role of STL in regulatory processes of plants that will be discussed as well.
  • Die Leibniz-Gemeinschaft
  • Digitalization in agriculture
  • Universität Halle
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