Researchers at the National Institute of Chemistry coordinated a research project explaining microbe cytolysin specifics and binding mechanism

Plant pathogenic microorganisms have a vast selection of effector molecules that are used for infecting and spreading throughout tissues. Researchers from two departments of the Institute of Chemistry, in cooperation with the Biotechnical Faculty of the University of Ljubljana, have determined a receptor on the plant cell surface and explained why certain microbe toxins are dicotyledon specific.

Plant pathogenic organisms usually have a vast selection of molecules for infecting plants, allowing them to spread. A family of protein molecules named NLP (abbreviation for Nep1-like proteins) has been known to exist in bacteria, fungi and oomycetes for some time now. These proteins are involved in many diseases of plants that are important for humans, such as various types of vegetables, the cocoa tree, grapevine etc. The most common symptoms include rotting, rust, blackened crops, plant withering and seed dying, all quickly spreading through the crops. One of the most obvious plant reactions to the NLP proteins is the death of plant tissue. Cell death only occurs in dicotyledonous plants, such as potato, tobacco and the cocoa tree, while it does not occur in monocotyledonous plants, such as corn, wheat or rice. In our research, published in the prestigious science periodical “Science”, we have explained which molecule is the first NLP protein contact with a plant and why monocotyledons are unaffected by the NLP proteins. The plant cell membranes contain lipids with different numbers of sugar units attached that extend beyond the outer membrane layer. However, these lipids are different in monocotyledons and dicotyledons, and because of the different lipid structure, monocotyledons are not susceptible to the NLP proteins. The structure of these microbe toxins is such that a specific pocket on a toxin is able to bind to a sugar molecule, which is a terminal and thus exposed construction element of the lipid receptors in the membranes of dicotyledons. Unlike dicotyledons, the lipids in monocotyledons have an additional sugar bound at this location, which - after the toxin has been bound on this lipid receptor - prevents the toxin being incorporated into the cell membrane due to too great a distance – thereby monocotyledons remain unaffected. We have also shown that by binding the NLP protein to the sugar unit of the lipid in the plant membrane, a change of the protein structure occurs, which is likely to facilitate incorporation into the plant cell membrane.

We will continue to research the molecular basis of plant cell damage caused by the NLP proteins. We would like to understand how these toxins destroy the properties of plant cell membranes. On the other hand, our discovery is already enabling us to develop substances that prevent the action of the NLP proteins. The NLP proteins are mainly found in plant pathogens and as such represent an ideal target for fighting those organisms. With a targeted attack on NLP proteins, the toxic effects of pathogens could be eliminated and their effect on other proteins or organisms reduced.

25 scientists from 14 institutions from six countries have been involved in the research. From Slovenia, eight scientists from the Institute of Chemistry and the Biotechnical Faculty of the University of Ljubljana have participated. Tea Lenarčič and dr. Vesna Hodnik share primary authorship with two colleagues from Germany. From Department of Molecular Biology and Nanobiotechnology of the Institute of Chemistry, the following scientists also participated: dr. Katja Pirc, dr. Polona Bedina, dr. Marjetka Podobnik, and prof. dr. Gregor Anderluh, who was also one of the two leading authors of the study. Dr. David Pahovnik and dr. Ema Žagar from the Department of Polymer Chemistry and Technology of the Institute of Chemistry have also participated. In addition to scientists from the Institute of Chemistry, researchers of the Eberhard-Karls University from Tübingen have designed and coordinated the study.

Publication: Tea Lenarčič, Isabell Albert, Hannah Böhm, Vesna Hodnik, Katja Pirc, Apolonija B. Zavec, Marjetka Podobnik, David Pahovnik, Ema Žagar, Rory Pruitt, Peter Greimel, Akiko Yamaji-Hasegawa, Toshihide Kobayashi, Agnieszka Zienkiewicz, Jasmin Gömann, Jenny C. Mortimer, Lin Fang, Adiilah Mamode-Cassim, Magali Deleu, Laurence Lins, Claudia Oecking, Ivo Feussner, Sébastien Mongrand, Gregor Anderluh, Thorsten Nürnberger. Science (2017) Eudicot plant-specific sphingolipids determine the host selectivity of microbial NLP cytolysins.

Associated publication:
Ottmann C, Luberacki B, Küfner I, Koch W, Brunner F, Weyand M, Mattinen L, Pirhonen M, Anderluh G, Seitz HU, Nürnberger T, Oecking C. Proc Natl Acad Sci U S A. (2009) A common toxin fold mediates microbial attack and plant defence. 106:10359-10364.



Cytotoxic NLPs are causing necrosis on dicotyledon plants. The pictures above show damage to tobacco leaves caused by injected NLP proteins (NLPPp and NLPPya). Similar proteins affecting vertebrate cells do not cause such damage (e.g. proteins on the left side, EqtII, FraC in Brioporin).

A detailed view of the binding location for glucosamine. The NLP protein without a sugar bound to it is marked with grey, while the toxic NLP proteins in the complex with glucosamine are marked with orange. Little bars mark the glucosamine (green) and amino acids involved in sugar binding, the coordination of the magnesium ion Mg2+ (violet ball) and the stabilisation of membrane interaction. The magnesium ion can have different positions in the protein (marked with numbers), depending on the sugar bound.

Schematic model of the NLP protein interactions with the plant cell membrane. The NLP protein is shown in grey, the protein in the complex with the sugar part of lipid receptor is shown in yellow. The plasma membrane is shown in blue, target lipids in dicotyledons are shown in green, and lipids containing several sugar units, usually present in other plants (monocotyledons) are shown in red. The magnesium ion at the active location of the toxin is shown as a violet ball.


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