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Publications - Stress and Develop Biology

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Publications

Widjaja, I.; Naumann, K.; Roth, U.; Wolf, N.; Mackey, D.; Dangl, J. L.; Scheel, D.; Lee, J.; Combining subproteome enrichment and Rubisco depletion enables identification of low abundance proteins differentially regulated during plant defense Proteomics 9, 138-147, (2009) DOI: 10.1002/pmic.200800293

Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone‐responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2‐DE facilitated the discovery of low abundance proteins – enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA‐binding protein, and a C2‐domain‐containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA‐binding protein, involvement of PTM and post‐transcriptional regulation are implicated, respectively.
Publications

Vadassery, J.; Ranf, S.; Drzewiecki, C.; Mithöfer, A.; Mazars, C.; Scheel, D.; Lee, J.; Oelmüller, R.; A cell wall extract from the endophytic fungus Piriformospora indica promotes growth of Arabidopsis seedlings and induces intracellular calcium elevation in roots Plant J. 59, 193-206, (2009) DOI: 10.1111/j.1365-313X.2009.03867.x

Calcium (Ca2+), as a second messenger, is crucial for signal transduction processes during many biotic interactions. We demonstrate that cellular [Ca2+] elevations are early events in the interaction between the plant growth‐promoting fungus Piriformospora indica and Arabidopsis thaliana . A cell wall extract (CWE) from the fungus promotes the growth of wild‐type seedlings but not of seedlings from P. indica ‐insensitive mutants. The extract and the fungus also induce a similar set of genes in Arabidopsis roots, among them genes with Ca2+ signalling‐related functions. The CWE induces a transient cytosolic Ca2+ ([Ca2+]cyt) elevation in the roots of Arabidopsis and tobacco (Nicotiana tabacum ) plants, as well as in BY‐2 suspension cultures expressing the Ca2+ bioluminescent indicator aequorin. Nuclear Ca2+ transients were also observed in tobacco BY‐2 cells. The Ca2+ response was more pronounced in roots than in shoots and involved Ca2+ uptake from the extracellular space as revealed by inhibitor studies. Inhibition of the Ca2+ response by staurosporine and the refractory nature of the Ca2+ elevation suggest that a receptor may be involved. The CWE does not stimulate H2O2 production and the activation of defence gene expression, although it led to phosphorylation of mitogen‐activated protein kinases (MAPKs) in a Ca2+‐dependent manner. The involvement of MAPK6 in the mutualistic interaction was shown for an mpk6 line, which did not respond to P. indica . Thus, Ca2+ is likely to be an early signalling component in the mutualistic interaction between P. indica and Arabidopsis or tobacco.
Publications

Tennstedt, P.; Peisker, D.; Böttcher, C.; Trampczynska, A.; Clemens, S.; Phytochelatin Synthesis Is Essential for the Detoxification of Excess Zinc and Contributes Significantly to the Accumulation of Zinc Plant Physiol. 149, 938-948, (2009) DOI: 10.1104/pp.108.127472

The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn2+ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn2+ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd2+ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn2+-elicited PC2 accumulation in roots reached about 30% of the level of Cd2+-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn2+ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms.
Publications

Lin, Y.-F.; Liang, H.-M.; Yang, S.-Y.; Boch, A.; Clemens, S.; Chen, C.-C.; Wu, J.-F.; Huang, J.-L.; Yeh, K.-C.; Arabidopsis IRT3 is a zinc-regulated and plasma membrane localized zinc/iron transporter New Phytol. 182, 392-404, (2009) DOI: 10.1111/j.1469-8137.2009.02766.x

ZIP transporters (ZRT, IRT‐like proteins) are involved in the transport of iron (Fe), zinc (Zn) and other divalent metal cations. The expression of IRT3, a ZIP transporter, is higher in the Zn/cadmium (Cd) hyperaccumulator Arabidopsis halleri than is that of its ortholog in Arabidopsis thaliana , which implies a positive association of its expression with Zn accumulation in A. halleri. IRT3 genes from both A. halleri and A. thaliana functionally complemented the Zn uptake mutant Spzrt1 in Schizosaccharomyces pombe ; and Zn uptake double mutant zrt1zrt2 , Fe‐uptake mutant fet3fet4 and conferred Zn and Fe uptake activity in Saccharomyces cerevisiae . By contrast, the manganese (Mn) uptake mutant smf1 phenotypes were not rescued. Insufficient Cd uptake for toxicity was found.Expression of IRT3‐green fluorescent protein (GFP) fusion proteins in Arabidopsis root protoplasts indicated localization of both IRT3 proteins in the plasma membrane.Overexpressing AtIRT3 in A. thaliana led to increased accumulation of Zn in the shoot and Fe in the root of transgenic lines. Therefore, IRT3 functions as a Zn and Fe‐uptake transporter in Arabidopsis.
Publications

Halim, V. A.; Altmann, S.; Ellinger, D.; Eschen-Lippold, L.; Miersch, O.; Scheel, D.; Rosahl, S.; PAMP-induced defense responses in potato require both salicylic acid and jasmonic acid Plant J. 57, 230-242, (2009) DOI: 10.1111/j.1365-313X.2008.03688.x

To elucidate the molecular mechanisms underlying pathogen‐associated molecular pattern (PAMP)‐induced defense responses in potato (Solanum tuberosum ), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep‐13, a PAMP from Phytophthora , induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive‐like cell death. We have previously shown that SA is required for Pep‐13‐induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12‐oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F‐box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep‐13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep‐13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA‐deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans .
Publications

Eschen-Lippold, L.; Draeger, T.; Teichert, A.; Wessjohann, L.; Westermann, B.; Rosahl, S.; Arnold, N.; Antioomycete Activity of γ-Oxocrotonate Fatty Acids against P. infestans J. Agr. Food Chem. 57, 9607-9612, (2009) DOI: 10.1021/jf902067k

Infections with Phytophthora infestans, the causal agent of potato and tomato late blight disease, are difficult to control and can lead to considerable agricultural losses. Thus, the development of new effective agents against the pathogen is of great interest. In previous work, (E)-4-oxohexadec-2-enoic acid (3) was isolated from Hygrophorus eburneus, which exhibited fungicidal activity against Cladosporium cucumerinum. Here, the inhibitory effect of 3 on P. infestans spore germination and mycelium growth in vitro is demonstrated. The in vivo effect on infections of whole potato plants was investigated by spraying plants with the sodium salt of 3, sodium (2E)-4-oxohexadec-2-enoic acid (4), prior to P. infestans inoculation. Additionally, the influence of 3 on mycelium growth of Colletotrichum coccodes, the causal agent of potato black dot disease, was analyzed. In all approaches, a significant inhibition of pathogen development was achieved. Importantly, the unsaturated fatty acid exerted no toxic effect when sprayed on plants, a prerequisite for its commercial use.
Publications

Engelhardt, S.; Lee, J.; Gäbler, Y.; Kemmerling, B.; Haapalainen, M.-L.; Li, C.-M.; Wei, Z.; Keller, H.; Joosten, M.; Taira, S.; Nürnberger, T.; Separable roles of the Pseudomonas syringae pv. phaseolicola accessory protein HrpZ1 in ion-conducting pore formation and activation of plant immunity Plant J. 57, 706-717, (2009) DOI: 10.1111/j.1365-313X.2008.03723.x

The HrpZ1 gene product from phytopathogenic Pseudomonas syringae is secreted in a type‐III secretion system‐dependent manner during plant infection. The ability of HrpZ1 to form ion‐conducting pores is proposed to contribute to bacterial effector delivery into host cells, or may facilitate the nutrition of bacteria in the apoplast. Furthermore, HrpZ1 is reminiscent of a pathogen‐associated molecular pattern (PAMP) that triggers immunity‐associated responses in a variety of plants. Here, we provide evidence that the ion pore formation and immune activation activities of HrpZ1 have different structure requirements. All HrpZ1 orthologous proteins tested possess pore formation activities, but some of these proteins fail to trigger plant defense‐associated responses. In addition, a C‐terminal fragment of HrpZ1 retains the ability to activate plant immunity, whereas ion pore formation requires intact HrpZ1. Random insertion mutagenesis of HrpZ1 further revealed the C terminus to be important for the PAMP activity of the protein. HrpZ1 binds to plant membranes with high affinity and specificity, suggesting that the activation of plant immunity‐associated responses by HrpZ1 is receptor‐mediated. Our data are consistent with dual roles of HrpZ1 as a virulence factor affecting host membrane integrity, and as a microbial pattern governing the activation of plant immunity during infection.
Publications

Cheng, Y. T.; Germain, H.; Wiermer, M.; Bi, D.; Xu, F.; García, A. V.; Wirthmueller, L.; Després, C.; Parker, J. E.; Zhang, Y.; Li, X.; Nuclear Pore Complex Component MOS7/Nup88 Is Required for Innate Immunity and Nuclear Accumulation of Defense Regulators in Arabidopsis Plant Cell 21, 2503-2516, (2009) DOI: 10.1105/tpc.108.064519

Plant immune responses depend on dynamic signaling events across the nuclear envelope through nuclear pores. Nuclear accumulation of certain resistance (R) proteins and downstream signal transducers are critical for their functions, but it is not understood how these processes are controlled. Here, we report the identification, cloning, and analysis of Arabidopsis thalianamodifier of snc1,7 (mos7-1), a partial loss-of-function mutation that suppresses immune responses conditioned by the autoactivated R protein snc1 (for suppressor of npr1-1, constitutive 1). mos7-1 single mutant plants exhibit defects in basal and R protein–mediated immunity and in systemic acquired resistance but do not display obvious pleiotropic defects in development, salt tolerance, or plant hormone responses. MOS7 is homologous to human and Drosophila melanogaster nucleoporin Nup88 and resides at the nuclear envelope. In animals, Nup88 attenuates nuclear export of activated NF-κB transcription factors, resulting in nuclear accumulation of NF-κB. Our analysis shows that nuclear accumulation of snc1 and the defense signaling components Enhanced Disease Susceptibility 1 and Nonexpresser of PR genes 1 is significantly reduced in mos7-1 plants, while nuclear retention of other tested proteins is unaffected. The data suggest that specifically modulating the nuclear concentrations of certain defense proteins regulates defense outputs.
Publications

Böttcher, C.; Westphal, L.; Schmotz, C.; Prade, E.; Scheel, D.; Glawischnig, E.; The Multifunctional Enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) Converts Cysteine-Indole-3-Acetonitrile to Camalexin in the Indole-3-Acetonitrile Metabolic Network of Arabidopsis thaliana Plant Cell 21, 1830-1845, (2009) DOI: 10.1105/tpc.109.066670

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, γ-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate–treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.
Publications

Bethke, G.; Scheel, D.; Lee, J.; Sometimes new results raise new questions: the question marks between mitogen-activated protein kinase and ethylene signaling Plant Signal Behav. 4, 672-674, (2009) DOI: 10.4161/psb.4.7.9039

In Arabidopsis thaliana, mitogen activated protein kinase (MAPK) signaling cascades that contain MPK3, MPK4 and MPK6 have been implicated in various aspects of developmental processes and stress responses. We identified an ethylene response factor (ERF104), which controls innate immunity, to be a specific substrate of MPK6 and showed that ethylene signaling regulates the release of the ERF104 substrate from its kinase. Implications and questions that arise from our findings are addressed. To promote discussions, previously unpublished data, that are rather confounding, are presented and possible explanation provided on how these may fit into our current model.
  • Die Leibniz-Gemeinschaft
  • Digitalization in agriculture
  • Universität Halle
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