Plant-associated microbial pathogens secrete effector proteins to aid to infection and support parasitic lifestyles. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are widely-distributed effectors, produced by phytopathogens enrolled in diseases of most important crops. Many NLPs are cytolytic, causing cell death and tissue necrosis by plant plasma membrane disruption. Recently, a major plant membrane constituent, sphingolipids glycosylinositol phosphorylceramides (GIPCs), were identified as the target for NLP attachment to plant plasma membranes. However, the molecular mechanism by which NLPs damage plant membranes remained unknown.
Identification of NLP’s membrane receptor enabled exploitation of various GIPCs containing lipid model systems. We prepared a series of model plant membranes and examined membrane interaction of NLPPya from phytopathogenic oomycete Pythium aphanidermatum. Liposome sedimentation assay, surface plasmon resonance (SPR), quartz crystal microbalance (QCM) and neutron reflectometry (NR) revealed electrostatically-driven and shallow membrane binding. The presence of plant sterols strengthened the NLP-membrane interaction. High salt dependence of the NLP-GIPC association correlates with the low salt environment in the extracellular compartment where the toxin binds. The results indicate that cytotoxicity does not result from membrane reorganization or large-scale membrane defects but rather from small membrane ruptures. The data provide important insights into molecular mechanism of membrane damage induced by NLP toxins and narrow the gap in knowledge between initial NLP-receptor binding and final membrane disruption.