Purification, crystallization and preliminary X-ray diffraction analysis of an oomycete-derived Nep1-like protein

Luberacki, Borries; Weyand, Michael; Seitz, Ulrich; Koch, Wolfgang; Oecking, Claudia; Ottmann, C.

doi:10.1107/s1744309108037640

Cited by 4 publications

(5 citation statements)

References 14 publications

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“…For crystallization of NLPPya and mutants D93A and H101A, recombinant proteins were produced without signal peptides as described in ref. 15 Leaf Infiltration and Electrolyte Leakage Assay. Purified NLPs (0,5 M, 10 l) or mock solution were infiltrated abaxialy into leaves of N. tabacum (4 -6 weeks old) or A. thaliana (3-4 weeks old).…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant NLP Pya (lacking the N-terminal signal peptide for secretion) [supporting information (SI) Fig. S1] was produced in Escherichia coli, purified under denaturing conditions and reconstituted into its biologically active form (15). The refolded protein crystallized in orthorhombic crystals whose structure was solved at a resolution of 1.35 Å.…”

Section: The Nlp Fold Is Highly Conserved and Is Distantly Related Tomentioning

confidence: 99%

See 1 more Smart Citation

A common toxin fold mediates microbial attack and plant defense

Ottmann

Luberacki

Küfner

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Many plant pathogens secrete toxins that enhance microbial virulence by killing host cells. Usually, these toxins are produced by particular microbial taxa, such as bacteria or fungi. In contrast, many bacterial, fungal and oomycete species produce necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) that trigger leaf necrosis and immunity-associated responses in various plants. We have determined the crystal structure of an NLP from the phytopathogenic oomycete Pythium aphanidermatum to 1.35Å resolution. The protein fold exhibits structural similarities to cytolytic toxins produced by marine organisms (actinoporins). Computational modeling of the 3-dimensional structure of NLPs from another oomycete, Phytophthora parasitica, and from the phytopathogenic bacterium, Pectobacterium carotovorum, revealed a high extent of fold conservation. Expression of the 2 oomycete NLPs in an nlp-deficient P. carotovorum strain restored bacterial virulence, suggesting that NLPs of prokaryotic and eukaryotic origins are orthologous proteins. NLP mutant protein analyses revealed that identical structural properties were required to cause plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. In sum, NLPs are conserved virulence factors whose taxonomic distribution is exceptional for microbial phytotoxins, and that contribute to host infection by plasma membrane destruction and cytolysis. We further show that NLP-mediated phytotoxicity and plant defense gene expression share identical fold requirements, suggesting that toxin-mediated interference with host integrity triggers plant immunity-associated responses. Phytotoxin-induced cellular damage-associated activation of plant defenses is reminiscent of microbial toxin-induced inflammasome activation in vertebrates and may thus constitute another conserved element in animal and plant innate immunity.crystal structure ͉ immunity ͉ pathogen ͉ plant immunity ͉ phytotoxin

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Section: Methodsmentioning

confidence: 99%

Section: The Nlp Fold Is Highly Conserved and Is Distantly Related Tomentioning

confidence: 99%

A common toxin fold mediates microbial attack and plant defense

Ottmann

Luberacki

Küfner

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

223

280

View full text Add to dashboard Cite

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“…The NLP Pya protein was expressed and purified as described previously ( 7 , 9 , 35 ). The production of deuterated d-NLP Pya for NR experiments followed the same protocol, with the following modifications.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were cultivated at 26°C and 180 rpm for 15 hours. The protein was purified with ion-exchange chromatography on a gravity flow nickel affinity column (Ni-NTA Superflow, Qiagen) ( 35 ). Protein-containing fractions were pooled and dialyzed against D 2 O.…”

Section: Methodsmentioning

confidence: 99%

An oomycete NLP cytolysin forms transient small pores in lipid membranes

et al. 2022

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Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1–like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

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“…Protein expression was induced with 0.5 mM IPTG for 20 h at 20 °C. NLPPya was purified by affinity chromatography (Ni–NTA agarose, Qiagen) ( 82 ), followed by gel filtration (GE Healthcare) ( 36 ).…”

Section: Methodsmentioning

confidence: 99%