An extra sugar provides protection

NLP proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. NLP refers to “Necrosis and ethylene-inducing peptide 1–like proteins”, describing effects of these proteins in target plants, i.e. tissue cell death (necrosis) and production of the plant hormone ethylene, produced in response to environmental stress. Many NLPs are cytotoxins that facilitate microbial infection of eudicot plants (like tomato and tobacco), but not of monocots (like cereals and leek). The molecular basis of such specificity of members of the NLP family of toxins has been revealed for the oomycete plant pathogen Pythium aphanidermatum. Namely, the crystal structures of its cytotoxin NLP_Pya in complex with two different sugar moieties, complemented with other experimental evidence suggested the authors to consider glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors, and differences in structures of GIPC receptors between monocots and eudicots to justify the differences in NLP sensitivity. In plants, GIPCs are major constituents of membranes, comprising up to 40% of plasma membrane lipids, reaching up to 60 to 80% of lipids in the outer leaflet of the plasma membrane. A GIPC molecule consists of an inositol phosphoceramide, which stabilizes its position in the membrane, and a head group consisting of glucuronic acid and a variable number and form of terminal hexoses (6-carbon sugars, such as glucosamine or mannosamine). The sugar moiety is positioned at the extracellular surface of the plant cell membrane reaching out of the lipid bilayer and is thus accessible for binding of NLP proteins.

Figure 1. Structural change in NLP protein upon sugar binding.Crystal structures of NLP_Pyabefore (blue, PDB ID 3GNZ, left) and after glucosamine binding (green, PDB ID 5NNW, right). Mg²⁺ ion is depicted as a pink sphere, glucosamine as orange sticks.

Different biochemical and biophysical assays confirmed that NLPs bind GIPCs with high affinity, but not other lipids. Because monomeric sugars glucosamine and mannosamine were found to bind to the NLP protein, they were used to determine crystal structures of protein-sugar complexes using the XRD1 beamline at Elettra: sugar binding resulted in structural changes in the NLP_Pya (Figure 1) resulting in formation of a channel harbouring the sugar, thereby suggesting that NLPs use this opening to anchor themselves to GIPC sugar head groups with a subsequent recruitment of other parts of the NLP molecule. This process proceeds efficiently in dicots, while in monocots additional sugar on GIPC head group leads to more distant placement of NLP on the surface of plant cells and resistance of plants against its activity (Figure 2).

Figure 2. Differences in GIPCs head groups between dicots (image bottom) and monocots (top) allow the toxin NLP to be able to reach the surface of plant cells and harm them (bottom) producing nectrosis or not (image top, monocots).
 

This research was conducted by the following research team:

Tea Lenarčič¹, Katja Pirc¹, Apolonija B. Zavec¹, Marjetka Podobnik¹, Gregor Anderluh¹, Vesna Hodnik², Isabell Albert³,Hannah Böhm³,Rory Pruitt³, Claudia Oecking³, Thorsten Nürnberger³,David Pahovnik⁴, Ema Žagar⁴, Peter Greimel⁵, Akiko Yamaji-Hasegawa⁶, Toshihide Kobayashi⁷, Agnieszka Zienkiewicz⁸, Jasmin Gömann⁸, Ivo Feussner⁸, Jenny C. Mortimer⁹, Lin Fang⁹, Adiilah Mamode-Cassim¹⁰, Sébastien Mongrand¹⁰, Magali Deleu¹¹ and Laurence Lins¹¹

¹ Department for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia
² Department for Molecular Biology and Nanobiotechnology, National Institute of Chemistry and Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
³ Centre of Plant Molecular Biology, Eberhard-Karls-University Tübingen, Tübingen, Germany
⁴ Department of Polymer Chemistry and Technology, National Institute of Chemistry, Ljubljana, Slovenia
⁵ Lipid Biology Laboratory andLaboratory for Cell Function Dynamics, Brain Science Institute, RIKEN Institute, Wako, Saitama, Japan
⁶ Lipid Biology Laboratory and Molecular Membrane Neuroscience, Brain Science Institute, RIKEN Institute, Wako, Saitama, Japan
⁷ Lipid Biology Laboratory, RIKEN Institute, Wako Saitama, Japan and UMR 7213 CNRS, University of Strasbourg, Illkirch, France
⁸ Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences and Göttingen Center for Molecular Biosciences, University of Göttingen, Germany.
⁹ Joint Bioenergy Institute, Emeryville and Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, USA
¹⁰ Laboratoire de Biogenèse Membranaire, UMR 5200 CNRS-Université de Bordeaux, Villenave-d’Ornon Cedex, France
¹¹ Laboratory of Molecular Biophysics at Interfaces, University of Liège, Gembloux, Belgium

Maurizio Polentarutti, email: