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Dr. Johannes Stuttmann

Institute of Biology, Dept. of Genetics
Martin- Luther- University
Halle- Wittenberg
Dr. Johannes Stuttmann
Weinbergweg 10
D- 06120 Halle (Saale)
Germany

Office: +49-345-55 26345
Lab: +49-345-55 26296+49- 345- 552 6293
Fax: +49- 345- 552 7151
E-mail:

Identification of nuclear components of plant TNL signaling

Biotrophic and hemibiotrophic plant pathogenic microbes colonize and rely on obtaining nutrients from living plant tissues. To establish and maintain this intimate host-pathogen association, pathogenic microbes generally deliver so-called effector proteins directly into the host cell cytosol. Inside host cells, effectors can suppress plant defense programs or manipulate host metabolic pathways in favour of the microbial intruder, thus allowing microbial growth and colonization of host tissues. In resistant host isolates, however, effectors can become recognized by plant nucleotide-binding leucine-rich repeat-type (NLR) intracellular immune receptors. Effector recognition and receptor activation induce a rapid and rigorous defense response often associated with programmed cell death at infection sites and efficiently delimiting or fully preventing microbial spread. While molecular events leading to NLR activation are reasonably well understood, downstream signaling pathways from activated NLRs towards defense programs, including massive transcriptional reprogramming inside plant nuclei, remain largely elusive. For several NLRs, functionality depends on a nuclear receptor pool, and direct interactions of NLRs with transcription factors were also reported. However, a unified model for NLR signaling, as suggested by modular NLR architecture and highly similar defense transcriptomes and programs induced by activation of any NLR, could so far not be developed. Plant NLRs are broadly sub-divided into two major classes defined by an N-terminal coiled-coil (CC, CNLs) or Toll-Interleukin1-receptor (TIR, TNLs) domain. TNLs, but not or to lesser extent CNLs, depend on the plant-specific protein EDS1 to mediate resistance, thus suggesting EDS1 as a TNL-specific signaling component. In Arabidopsis, EDS1 can induce autoimmunity, the activation of defense programs in absence of pathogen stimulus associated with reduced plant growth or seedling necrosis, when accumulating to high levels inside nuclei by transgenic expression of an EDS1-YFPNLS fusion protein. The severe, but temperature-dependent, seedling-lethal autoimmune phenotype of an EDS1-YFPNLS-expressing line (line “NLS#A3”) was used as a read-out for a genetic suppressor screen. Approximately 50 near death experience mutants with partially or fully restored growth at ambient temperature were isolated. According to EDS1-YFPNLS nuclear localization, we expect nde loci to encode nuclear interactors of EDS1 and/or nuclear regulators of plant immunity. A first complementation group of nde mutants containing five different alleles was so far characterized. We discovered that EDS1-YFPNLS genetically interacts with RPP1Ler-R8/DM2h, a TNL encoded within a highly divergent resistance gene cluster recently also identified as a hotspot for hybrid incompatibility [1]. A small deletion within RPP1Ler-R8/DM2h within this gene cluster, discovered in the nde1-1 allele, is also sufficient to suppress autoimmunity induced by old3-1 [2] and SRF3Kas/Kond [3-5].Thus, RPP1Ler-R8 is a convergence point for autoimmunity phenotypes provoked by three different, structurally unrelated proteins (“elicitors”), localizing to different subcellular compartments. Future efforts will be made to determine a minimal module for autoimmunity induction by different elicitors, to understand how these elicitors execute receptor activation, and in which subcellular compartment resting state and activated receptors function. Also, additional nde mutations will be isolated by next generation sequencing-based mapping approaches to identify further nuclear components of the plant immune signaling network.

People: Johannes Stuttmann, Christine Wagner

References:

1. Chae E, Bomblies K, Kim ST, Karelina D,  Zaidem M, et al. (2014) Species-wide genetic incompatibility analysis  identifies immune genes as hot spots of deleterious epistasis. Cell 159:  1341-1351.

2. Tahir J, Watanabe M, Jing HC, Hunter DA, Tohge T, et al. (2013)  Activation of R-mediated innate immunity and disease susceptibility is  affected by mutations in a cytosolic O-acetylserine (thiol) lyase in  Arabidopsis. Plant J 73: 118-130.

3. Alcazar R, Garcia AV, Parker JE, Reymond M (2009) Incremental  steps toward incompatibility revealed by Arabidopsis epistatic  interactions modulating salicylic acid pathway activation. Proc Natl  Acad Sci U S A 106: 334-339.

4. Alcazar R, Garcia AV, Kronholm I, de Meaux J, Koornneef M, et al.  (2010) Natural variation at Strubbelig Receptor Kinase 3 drives  immune-triggered incompatibilities between Arabidopsis thaliana  accessions. Nat Genet 42: 1135-1139.

5. Alcazar R, von Reth M, Bautor J, Chae E, Weigel D, et al. (2014)  Analysis of a plant complex resistance gene locus underlying  immune-related hybrid incompatibility and its occurrence in nature. PLoS  Genet 10: e1004848.

Figure 1: Phenotype of the EDS1-YFPNLS-overexpressing line”NLS#A3” in comparison to Col control plants and two allelic suppressor mutants, nde1-1 and nde1-3. nde1 alleles are defective in RPP1Ler-R8, a TNL encoded within the complex RPP1-like gene cluster from accession Ler. R8, and possibly another TNL encoded within the RPP1-like gene cluster, are necessary for driving autoimmunity induced by three different elicitors, EDS1-YFPNLS, old3-1 and SRF3Kas/Kond, as summarized in the bottom scheme.

Analysis of EDS1-family protein functions in Solanaceous plants

Intracellular immune receptors of the TNL class depend on EDS1 to  initiate defense signaling in Arabidopsis and other dicot plants. EDS1  is a conserved, plant-specific protein composed of an N-terminal lipase  domain (a/b hydrolase domain) and a C-terminal EP (EDS1-PAD4) domain  [6]. Mediated by common interaction surfaces located both in the lipase  domain and the EP domain, EDS1 forms two structurally similar, but  mutually exclusive, heterodimers with the sequence-related proteins PAD4  and SAG101, which can both function in plant immunity [6]. EDS1 and  PAD4, but not SAG101, possess a hydrolase-characteristic catalytic triad  conserved in orthologous proteins. However, complementation analysis  with EDS1-PAD4 variants carrying mutations in residues critical for  hydrolase activity established that heterocomplex functions in plant  immune signaling are independent of potential substrate cleavage [6].  The availability of structural data provides an important and highly  valuable basis for functional dissection of EDS1-based heterocomplexes,  but analysis in Arabidopsis is hampered by lacking availability of  transient assays for EDS1 functionality. We made use of a previously  isolated eds1 mutant in Solanum lycopersicum (Sl, tomato) [7] to develop a system for rapid assessment of functionality of SlEDS1  variants in transient expression-based assays. As a proof of concept,  we used this system to establish that also in tomato, EDS1 functions  depend on heterocomplex formation with PAD4 and/or SAG101 orthologs,  mainly driven by hydrophobic interactions with the EDS1 aH helix. Based  on a SlEDS1-SlPAD4 heterocomplex model, SlEDS1  mutant variant analysis will be extended to conserved, surface-exposed  residues to map functionally important residues onto the EDS1 surface.  This will allow us to form new hypotheses on EDS1 functions in plant  immunity, and give way to novel, more targeted experimental approaches.  Also, we will adapt our transient complementation assay for targeted  mutagenesis of PAD4 and/or SAG101, and analyze the function of  EDS1-based heterocomplexes in the tomato system. Our final goal consists  in understanding the precise molecular function of EDS1 in the plant  immune system. Although ambitious given the wealth of analyses and  effort made in Arabidopsis, we hope that a change in system might  actually allow some critical pieces to fall into place to solve the  puzzle around this extremely important and fascinating signaling node.

People: Johannes Stuttmann, Johannes Gantner, Raphael Guerois (iBiTecS, Gif-sur-Yvette, France)

References:

6. Wagner S, Stuttmann J, Rietz S, Guerois R, Brunstein E, et al.  (2013) Structural basis for signaling by exclusive EDS1 heteromeric  complexes with SAG101 or PAD4 in plant innate immunity. Cell Host  Microbe 14: 619-630.

7. Hu G, deHart AK, Li Y, Ustach C, Handley V, et al. (2005) EDS1 in  tomato is required for resistance mediated by TIR-class R genes and the  receptor-like R gene Ve. Plant J 42: 376-391.

Figure 2: Homology model of the tomato EDS1-PAD4 heterocomlex, with amino acid conservation displayed on the surface. Agrobacterium-mediated expression of an “inducer-complementation” construct in eds1 mutant tomato tissues leads to cell death induction, which can be used as a proxy for EDS1-mediated resistances. This phenotype can be used to rapidly assess functionality of SlEDS1 variants, e.g. affected in heterocomplex formation.

Plant genome engineering: CRISPR/Cas and synthetic biology

The discovery of RNA-guided nucleases, derived from bacterial CRISPR/Cas systems, provided researchers from all fields with a useful tool for genome modification [8]. The principle is bluffingly simple: RNA-guided nucleases are used to introduce a single- or double-strand break (DSB)  at a user-defined position within the genome. Subsequently, by virtue of the cellular DNA repair mechanisms, the genome can be modified at this precise position. In its simplest version, DSBs are repaired by a repair-pathway called non-homologous end-joining, which often provokes small deletions or insertions. Thereby, a gene can be disrupted by simply "cutting and destroying" via the RNA-guided nuclease. This is a fantastic tool for fundamental plant sciences and also holds great potential for plant breeding and agriculture.

The current legal situation in which "classical" breeding methods (which include mutagenesis of plants with DNA-modifying chemicals or radiation and fusions between species that cannot normally cross) are acceptable, while genome edited plants are classified as GMOs, will not persist. It is an unacceptable waste of the potential and benefits, and is completely unfounded.

We wanted to use genome editing early on, and, therefore, had to generate our own molecular tools. Ever since, we tried to improve these systems to solve the bottlenecks we encountered. We have mainly used genome editing in Arabidopsis and wild tobacco (Nicotiana benthamiana). We have achieved tremendous improvements in the efficiency of our genome editng, and have also developed very nice markers to select non-transgenic plants. As a strong supporter of open science, all relevant material generated in the Stuttmann group is available via Addgene [https://www.addgene.org/Johannes_Stuttmann/].    Furthermore, we have a large collection of "building blocks" for genome editing systems, and can assemble custom vectors for pretty much any purpose. Please do not hesitate to contact , if you may require a vector for a special purpose.

We also generated ample ressources compatible with the Modular cloning system for hierarchical DNA assembly, or for plant pathology. Most of this material is also available via Addgene.

Find Johannes Stuttmann Lab Plasmids   

References:

8. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816-821.


Editing vectors and negative selection markers

Editing vectors and negative selection markers

Figure 3: Overview of our vector toolkit for plant genome engineering and pictures of negative selection markers (bottom). The helix is made of Arabidopsis seeds - red seeds are transgenic, green seeds not. In the other image, the same seeds were germinated on a plate containing a chemical - only the non-transgenic plants survive. We use these markers in the selection procedure for generating genome edited plants. (photos Jessica Lee Ericksson)

Publications

Ordon, Martin, Erickson, Ferik, Balcke, Bonas, Stuttmann*: Disentangling cause and consequence: Genetic dissection of the DANGEROUS MIX2 risk locus, and activation of the DM2h NLR in autoimmunity. Plant Journal (2021)    https://doi.org/10.1111/tpj.15215   

Stuttmann*, Barthel, Martin, Ordon, Erickson, Herr, Ferik, Kretschmer, Berner, Keilwagen, Marillonnet, Bonas: Highly efficient multiplex editing: One-shot generation of 8x Nicotiana benthamiana and 12x Arabidopsis mutants. Plant Journal (2021)    https://doi.org/10.1111/tpj.15197   

Gruetzner, Martin, Horn, Mortensen, Cram, Lee-Parsons, Stuttmann, Marillonnet: Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in Plants. Plant Communications (2020) https://doi.org/10.1016/j.xplc.2020.100135   

Lapin, Kovacova, Sun, Dongus, Bhandari, von Born, Bautor, Guarneri, Stuttmann, Beyer, Parker: A coevolved EDS1-SAG101-NRG1 module mediates cell death signaling by TIR domain immune receptors. Plant Cell (2019) http://www.plantcell.org/content/31/10/2430   

Gantner, Ordon, Kretschmer, Guerois, Stuttmann J*: An EDS1-SAG101 complex is essential for TNL-mediated immunity in Nicotiana benthamiana. Plant Cell (2019) http://www.plantcell.org/content/31/10/2456   

Sharma, Kretschmer, Lampe, Stuttmann, Klösgen: Determining targeting specificity of nuclear-encoded organelle proteins with the self-assembling split fluorescent protein toolkit. J Cell Science (2019) https://jcs.biologists.org/content/132/11/jcs230839   

Ordon, Bressan, Kretschmer, Dall’Osto, Marillonnet, Bassi, Stuttmann J*: Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation. Functional & Integrative Genomics (2019) https://link.springer.com/article/10.1007%2Fs10142-019-00665-4   

Gantner, Ordon, Ilse, Kretschmer, Gruetzner, Löfke, Dagdas, Bürstenbinder, Marillonnet, Stuttmann*: Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system. PLoS ONE (2018) https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197185   

Kumar, Galli, Ordon, Stuttmann, Kogel, Imani: Further analysis of barley MORC1 using a highly efficient RNA-guided Cas9 gene editing system. Plant Biotech J (2018) https://onlinelibrary.wiley.com/doi/full/10.1111/pbi.12924   

Streubel, Baum, Grau, Stuttmann, Boch: Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations. PLoS ONE (2017) https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173580   

Adlung, Prochaska, Thieme, Banik, Blüher, John, Nagel, Schulze, Gantner, Delker, Stuttmann, Bonas: Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1. Front Plant Sci (2016) https://www.frontiersin.org/articles/10.3389/fpls.2016.01796/full   

Ordon, Gantner, Kemna, Schwalgun, Reschke, Streubel, Boch, Stuttmann*: Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit. Plant Journal (2016) https://onlinelibrary.wiley.com/doi/full/10.1111/tpj.13319   

Stuttmann*, Peine, Garcia, Wagner, Choudhury, Wang, James, Griebel, Alcazar, Tsuda, Schneeberger, Parker: Arabidopsis thaliana DM2h (R8) within the Landsberg RPP1-like Resistance Locus Underlies Three Different Cases of EDS1-Conditioned Autoimmunity. PLoS Genetics (2016) https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1005990   

Wagner, Stuttmann, Rietz, Guerois, Brunstein, Bautor, Niefind, Parker: Structural Basis for Signaling by Exclusive EDS1 Heteromeric Complexes with SAG101 or PAD4 in Plant Innate Immunity. Cell Host Microbe (2013) https://pubmed.ncbi.nlm.nih.gov/24331460/   

Hoser, Zurczak, Lichocka, Zuzga, Dadlez, Samuel, Ellis, Stuttmann, Parker, Hennig, Krzymowska: Nucleocytoplasmic partitioning of tobacco N receptor is modulated by SGT1. New Phytol (2013) https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.12347   

Stuttmann, Hubberten, Rietz, Kaur, Muskett, Guerois, Bednarek, Hoefgen, Parker: Perturbation of Arabidopsis amino acid metabolism causes incompatibility with the adapted biotrophic pathogen Hyaloperonospora arabidopsidis. Plant Cell (2011) http://www.plantcell.org/content/23/7/2788   

Stuttmann, Parker, Noël: Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants. Plant Signal Behav (2009) https://www.tandfonline.com/doi/full/10.4161/psb.4.9.9526   

Stuttmann, Parker, Noël: Staying in the fold: The SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins. Plant Signal Behav (2008) https://www.tandfonline.com/doi/full/10.4161/psb.3.5.5576   

Stuttmann, Lechner, Guerois, Parker, Nussaume, Genschik, Noël: COP9 signalosome- and 26S proteasome-dependent regulation of SCFTIR1 accumulation in Arabidopsis. J Biol Chem (2009) https://www.jbc.org/content/284/12/7920.full.pdf   

Lorenz, Kirchner, Egler, Stuttmann, Bonas, Büttner: HpaA from Xanthomonas is a regulator of type III secretion. Mol Microbiol (2008) https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2958.2008.06280.x   

Noël, Cagna, Stuttmann, Wirthmüller, Betsuyaku, Witte, Bhat, Pochon, Colby, Parker: Interaction between SGT1 and cytosolic/nuclear HSC70 chaperones regulates Arabidopsis immune responses. Plant Cell (2007) http://www.plantcell.org/content/19/12/4061   

Büttner, Noël, Stuttmann, Bonas: Characterization of the nonconserved hpaB-hrpF region in the hrp pathogenicity island from Xanthomonas campestris pv. vesicatoria. Mol Plant Microbe Interact (2007) https://apsjournals.apsnet.org/doi/10.1094/MPMI-20-9-1063#   

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