The superfamily of necrosis and ethylene-inducing peptide 1 (Nep1) like proteins (NLPs) harbors cytotoxic and non-cytotoxic, virulence-promoting members

Members of the superfamily of necrosis and ethylene-inducing peptide 1 (Nep1) like proteins (NLPs) are widely found in bacteria, fungi and oomycetes. A subset of these proteins causes leaf necrosis on dicot, but not on monocot plants. NLP cytotoxicity was shown to be crucial for microbial virulence and a necrotrophic lifestyle of the producing microbe. X-ray crystallography-based analyses of two microbial NLPs revealed substantial fold conservation of these proteins with cytolytic toxins produced by marine organisms (actinoporins). Actinoporins bind to animal host sphingomyelin prior to membrane pore formation and cytolysis. While plants do not produce sphingomyelins, we show that the target site for NLP toxins is of lipid nature and resides in the outer layer of the plasma membrane of dicot plants. Membrane binding and phytotoxicity requires the presence of a coordinately bound calcium cation within an electrophilic cavity on NLPs, suggesting that the plant docking site is negatively charged. In binding assays, NLPs preferentially bind to phosphorylated phosphatidylinositols (PIP), and incubation of NLPs with PIPs inhibits the cytotoxic activities of these proteins. Thus, NLP susceptibility of plant membranes is determined by its interaction with yet unknown PIP-like lipid structures that define a biologically significant difference in the composition of plasma membranes from monocot and dicot plants. Recently, the production by various oomycetes and fungi of non-cytotoxic members of the NLP superfamily was shown. The possible mode of action of these proteins, their biological activity as well as their contribution to microbial virulence will be discussed