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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.

Genome-editing in plants

The understanding of TAL-DNA binding [8] and the development of TALENs [9] caused an enormous genome editing hype, as it became for the first time conceivable to generate targeted mutations at virtually any genome position with reasonable effort. However, it was not until the discovery of the CRISPR/Cas system and its potential use in generating double strand breaks [10] that genome editing started to be considered as a tool for everybody, since the assembly of TALENs necessitated availability of a repeat library and some experience for complex DNA assemblies. However, although CRISPR/Cas assumedly made genome editing easy, no tools were actually available when we developed an interest in using this technique in plants. This definitely changed by early 2015, but in the meantime, we developed a user friendly modular toolkit for the PCR-free assembly of genome editing constructs. Different vectors containing Cas9 under control of a 2x35S or a Ubi promoter (PcUbi4-2, Petroselinum crispum) and a plant selectable marker (BASTA or Kanamycin resistance) were generated either for the expression of simple nucleases or for multiplex genome editing. To generate simple nucleases, vectors additionally contain a ccdB cassette flanked by an AtU6 Promoter and the sgRNA scaffold. The ccdB cassette can be excised by BpiI digestion, and is replaced by a guide sequence loaded into the vectors as two hybridized oligonucleotides in a single step. For multiplex genome editing, a set of shuttle vectors each containing a ccdB cassette flanked by an AtU6 promoter and the sgRNA scaffold were generated. Guide sequences are equally loaded into these shuttle vectors as hybridized oligos by BpiI cut/ligation. The sgRNA transcription units are then mobilized into the final genome editing vectors by BsaI cut/ligation. Depending on shuttle vectors used, two, four or eight different sgRNA transcription units can be combined in an sgRNA array in the final genome editing vectors in a single step. Given the modular design, e.g. HDR repair templates for knock-in approaches are also easily implemented in the cloning scheme. All constructs were validated in Agrobacterium-based transient expression assays, and were also successfully used for stable transformation of Nicotiana benthamiana and Arabidopsis. We will use these tools to generate different mutants valuable for other projects and to gain experience in targeted genome modification. A similar vector set for genome editing in monocots employing the ZmUbi (Zea mays) promoter for Cas9 expression, hygromycin as plant selectable marker, and the OsU6 promoter for sgRNA expression was also generated, but is yet awaiting functional validation. We will be happy to share material for future experiments, and additional information will be made available here soon.

People: Johannes Stuttmann, Jana Ordon

References:

8. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, et al. (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326: 1509-1512.

9. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, et al. (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186: 757-761.

10. 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.

Figure 3: Overview of our genome editing toolbox and cloning procedures


Publications

Stephan Wagner, Johannes Stuttmann, Steffen Rietz, Raphael Guerois, Elena Brunstein, Jaqueline Bautor, Karsten Niefind, Jane E. Parker (2013). Structural Basis for Signaling by Exclusive EDS1 Heteromeric Complexes with SAG101 or PAD4 in Plant Innate Immunity. Cell Host Microbe 14(6):619-30. doi: 10.1016/j.chom.2013.11.006.

Equal contributions as first authors

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Hoser R, Zurczak M, Lichocka M, Zuzga S, Dadlez M, Samuel MA, Ellis BE, Stuttmann J, Parker JE, Hennig J, Krzymowska M. (2013). Nucleocytoplasmic partitioning of tobacco N receptor is modulated by SGT1. New Phytol. 200(1):158-71. doi: 10.1111/nph.12347

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Stuttmann J, Hubberten HM, Rietz S, Kaur J, Muskett P, Guerois R, Bednarek P, Hoefgen R, Parker JE (2011). Perturbation of Arabidopsis amino acid metabolism causes incompatibility with the adapted biotrophic pathogen Hyaloperonospora arabidopsidis. Plant Cell. 23(7):2788-803

This article was highlighted in “In Brief”:
Interconnected Metabolism of Host Plants and Obligate Biotrophic Pathogens.
Eckhardt NA
Plant Cell. 2011 Jul;23(7):2788-803

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Stuttmann J, Parker JE, Noël LD (2009). Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants. Plant Signal Behav. 4(9):896-8

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Stuttmann J, Parker JE, Noël LD (2008). Staying in the fold: The SGT1/chaperone machinery in maintenance and evolution of leucine-rich repeat proteins. Plant Signal Behav. 3(5):283-5

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Stuttmann J, Lechner E, Guérois R, Parker JE, Nussaume L, Genschik P, Noël LD (2009). COP9 signalosome- and 26S proteasome-dependent regulation of SCFTIR1 accumulation in Arabidopsis. J Biol Chem. 284(12):7920-30

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Lorenz C, Kirchner O, Egler M, Stuttmann J, Bonas U, Büttner D (2008). HpaA from Xanthomonas is a regulator of type III secretion. Mol Microbiol. 69(2):344-60

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Noël LD, Cagna G, Stuttmann J, Wirthmüller L, Betsuyaku S, Witte CP, Bhat R, Pochon N, Colby T, Parker JE (2007). Interaction between SGT1 and cytosolic/nuclear HSC70 chaperones regulates Arabidopsis immune responses. Plant Cell. 19(12):4061-76

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Büttner D, Noël L, Stuttmann J, Bonas U (2007). Characterization of the nonconserved hpaB-hrpF region in the hrp pathogenicity island from Xanthomonas campestris pv. vesicatoria. Mol Plant Microbe Interact. 20(9):1063-74

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