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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 Funct Integr Genomics 20, 151-162, (2020) DOI: 10.1007/s10142-019-00665-4

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.
Publikationen in Druck

Grunewald, S.; Marillonnet, S.; Hause, G.; Haferkamp, I.; Neuhaus, H. E.; Veß, A.; Hollemann, T.; Vogt, T. The Tapetal Major Facilitator NPF2.8 is Required for Accumulation of Flavonol Glycosides on the Pollen Surface in Arabidopsis thaliana Plant Cell (2020) DOI: 10.1105/tpc.19.00801

The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Whereas the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear, how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that AtNPF2.8, a member of the nitrate/peptide NTR/PTR family of transporters is required for accumulation and transport of pollen-specific flavonol 3-O-sophorosides, characterized by a glycosidic β-1,2-linkage, to the pollen surface of Arabidopsis. Ectopic, transient expression of this flavonol sophoroside transporter, termed AtFST1, fused to green fluorescent protein (GFP) demonstrated localization of AtFST1 at the plasmalemma in epidermal leaf cells of Nicotiana benthamiana whereas the tapetum-specific AtFST1-expression was confirmed by promAtFST1:GFP-reporter lines. In vitro characterization of AtFST1-activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1-line by complementation with a genomic fragment of the AtFST1 gene restored flavonol glycoside accumulation of pollen grains to wild-type levels corroborating the requirement of AtFST1 for transport of flavonol-3-O-sophorosides from the tapetum to the pollen surface.

Marillonnet, S.; Grützner, R. Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline Curr Protoc Mol Biol 130, e115, (2020) DOI: 10.1002/cpmb.115

Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one‐pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline.

Schubert, R.; Dobritzsch, S.; Gruber, C.; Hause, G.; Athmer, B.; Schreiber, T.; Marillonnet, S.; Okabe, Y.; Ezura, H.; Acosta, I. F.; Tarkowska, D.; Hause, B. Tomato MYB21 Acts in Ovules to Mediate Jasmonate-Regulated Fertility Plant Cell 31, 1043-1062, (2019) DOI: 10.1105/tpc.18.00978

The function of the plant hormone jasmonic acid (JA) in the development of tomato (Solanum lycopersicum) flowers was analyzed with a mutant defective in JA perception (jasmonate-insensitive1-1, jai1-1). In contrast with Arabidopsis (Arabidopsis thaliana) JA-insensitive plants, which are male sterile, the tomato jai1-1 mutant is female sterile, with major defects in female development. To identify putative JA-dependent regulatory components, we performed transcriptomics on ovules from flowers at three developmental stages from wild type and jai1-1 mutants. One of the strongly downregulated genes in jai1-1 encodes the MYB transcription factor SlMYB21. Its Arabidopsis ortholog plays a crucial role in JA-regulated stamen development. SlMYB21 was shown here to exhibit transcription factor activity in yeast, to interact with SlJAZ9 in yeast and in planta, and to complement Arabidopsis myb21-5. To analyze SlMYB21 function, we generated clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9 (Cas9) mutants and identified a mutant by Targeting Induced Local Lesions in Genomes (TILLING). These mutants showed female sterility, corroborating a function of MYB21 in tomato ovule development. Transcriptomics analysis of wild type, jai1-1, and myb21-2 carpels revealed processes that might be controlled by SlMYB21. The data suggest positive regulation of JA biosynthesis by SlMYB21, but negative regulation of auxin and gibberellins. The results demonstrate that SlMYB21 mediates at least partially the action of JA and might control the flower-to-fruit transition.
Bücher und Buchkapitel

Marillonnet, S.; Werner, S. Assembly of Complex Pathways Using Type IIs Restriction Enzymes (Eds. Santos C N S, Ajikumar P K). Meth Mol Biol 1927, 93-109, (2019) ISBN: 978-1-4939-9142-6 DOI: 10.1007/978-1-4939-9142-6_7

Efficient DNA assembly methods are essential tools for synthetic biology and metabolic engineering. Among several recently developed methods that allow assembly of multiple DNA fragments in a single step, DNA assembly using type IIS enzymes provides many advantages for complex pathway engineering. In particular, it provides the ability for the user to quickly assemble multigene constructs using a series of simple one-pot assembly steps starting from libraries of cloned and sequenced parts. 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. Basic parts that are not yet available need to be made. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Hausner, J.; Jordan, M.; Otten, C.; Marillonnet, S.; Büttner, D. Modular Cloning of the Type III Secretion Gene Cluster from the Plant-Pathogenic Bacterium Xanthomonas euvesicatoria ACS Synth Biol 8, 532-547, (2019) DOI: 10.1021/acssynbio.8b00434

Type III secretion (T3S) systems are essential pathogenicity factors of most Gram-negative bacteria and translocate effector proteins into plant or animal cells. T3S systems can, therefore, be used as tools for protein delivery into eukaryotic cells, for instance after transfer of the T3S gene cluster into nonpathogenic recipient strains. Here, we report the modular cloning of the T3S gene cluster from the plant-pathogenic bacterium Xanthomonas euvesicatoria. The resulting multigene construct encoded a functional T3S system and delivered effector proteins into plant cells. The modular design of the T3S gene cluster allowed the efficient replacement and rearrangement of single genes or operons and the insertion of reporter genes for functional studies. In the present study, we used the modular T3S system to analyze the assembly of a fluorescent fusion of the predicted cytoplasmic ring protein HrcQ. Our studies demonstrate the use of the modular T3S gene cluster for functional analyses and mutant approaches in X. euvesicatoria. A potential application of the modular T3S system as protein delivery tool is discussed.

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 Sci Rep 9, 10932, (2019) DOI: 10.1038/s41598-019-47376-1

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 Mutagenesis 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.

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.

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.

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.
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