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Publications - Stress and Develop Biology

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Publications

Haapalainen, M.; Engelhardt, S.; Küfner, I.; Li, C.-M.; Nürnberger, T.; Lee, J.; Romantschuk, M.; Taira, S.; Functional mapping of harpin HrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction Mol. Plant Pathol. 12, 151-166, (2011) DOI: 10.1111/j.1364-3703.2010.00655.x

Harpin HrpZ is one of the most abundant proteins secreted through the pathogenesis‐associated type III secretion system of the plant pathogen Pseudomonas syringae. HrpZ shows membrane‐binding and pore‐forming activities in vitro, suggesting that it could be targeted to the host cell plasma membrane. We studied the native molecular forms of HrpZ and found that it forms dimers and higher order oligomers. Lipid binding by HrpZ was tested with 15 different membrane lipids, with HrpZ interacting only with phosphatidic acid. Pore formation by HrpZ in artificial lipid vesicles was found to be dependent on the presence of phosphatidic acid. In addition, HrpZ was able to form pores in vesicles prepared from Arabidopsis thaliana plasma membrane, providing evidence for the suggested target of HrpZ in the host. To map the functions associated with HrpZ, we constructed a comprehensive series of deletions in the hrpZ gene derived from P. syringae pv. phaseolicola, and studied the mutant proteins. We found that oligomerization is mainly mediated by a region near the C‐terminus of the protein, and that the same region is also essential for membrane pore formation. Phosphatidic acid binding seems to be mediated by two regions separate in the primary structure. Tobacco, a nonhost plant, recognizes, as a defence elicitor, a 24‐amino‐acid HrpZ fragment which resides in the region indispensable for the oligomerization and pore formation functions of HrpZ.
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

Qutob, D.; Kemmerling, B.; Brunner, F.; Küfner, I.; Engelhardt, S.; Gust, A. A.; Luberacki, B.; Seitz, H. U.; Stahl, D.; Rauhut, T.; Glawischnig, E.; Schween, G.; Lacombe, B.; Watanabe, N.; Lam, E.; Schlichting, R.; Scheel, D.; Nau, K.; Dodt, G.; Hubert, D.; Gijzen, M.; Nürnberger, T.; Phytotoxicity and Innate Immune Responses Induced by Nep1-Like Proteins Plant Cell 18, 3721-3744, (2006) DOI: 10.1105/tpc.106.044180

We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)–like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses.
Publications

Racapé, J.; Belbahri, L.; Engelhardt, S.; Lacombe, B.; Lee, J.; Lochman, J.; Marais, A.; Nicole, M.; Nürnberger, T.; Parlange, F.; Puverel, S.; Keller, H.; Ca2+-dependent lipid binding and membrane integration of PopA, a harpin-like elicitor of the hypersensitive response in tobacco Mol. Microbiol. 58, 1406-1420, (2005) DOI: 10.1111/j.1365-2958.2004.04910.x

PopA is released by type III secretion from the bacterial plant pathogen Ralstonia solanacearum and triggers the hypersensitive response (HR) in tobacco. The function of PopA remains obscure, mainly because mutants lacking this protein are not altered in their ability to interact with plants. In an attempt to identify the site of PopA activity in plant cells, we generated transgenic tobacco plants expressing the popA gene under the control of an inducible promoter. Immunocytologic analysis revealed that the HR phenotype of these plants correlated with the presence of PopA at the plant plasma membrane. Membrane localization was observed irrespective of whether the protein was designed to accumulate in the cytoplasm or to be secreted by the plant cell, suggesting a general lipid‐binding ability. We found that the protein had a high affinity for sterols and sphingolipids in vitro and that it required Ca2+ for both lipid binding and oligomerization. In addition, the protein was integrated into liposomes and membranes from Xenopus laevis oocytes where it formed ion‐conducting pores. These characteristics suggest that PopA is part of a system that aims to attach the host cell plasma membrane and to allow molecules cross this barrier.
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