Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by a variety of plant-associated pathogens that infect a plethora of important crops, causing enormous economic losses worldwide. Many NLPs trigger cell death and tissue necrosis in dicot plants. Based on their structural similarity to well-known pore-forming toxins actinoporins, and ability to induce leakage from plasma membrane vesicles, membrane disruption was proposed to be the mechanism of this observed cytotoxicity. Recently, a breakthrough discovery exposed glycosylinositol phosphorylceramides (GIPCs), a major class of plant sphingolipids, as targets for NLPs binding to plant plasma membranes. A model of early steps of NLP membrane interaction was further presented, but the exact mechanism of disruption remains unclear.
By confocal microscopy, we are exploiting giant unilamellar vesicles (GUVs) as model systems to observe NLP action on membranes, composed of palmitoyloleoylphosphatidylcholine and plant-isolated GIPCs. Phytosterols are added to mimic the composition of plant plasma membrane. By optimizing electroformation protocol, GUVs from such complex lipid mixtures with lipids purified from natural sources, are successfully prepared. Visual information after NLP – membrane interaction about localized toxin binding, changes in the morphology of the vesicles, and differential leakage of different-sized probes allow making speculations about membrane-damaging mechanism. NLPPya, a model NLP protein from oomycete Pythium aphanidermatum, forms small pores on GUVs, which become permeable to 4 kDa fluorescent dextran (2.8 nm in diameter), but not 10 kDa and 70 kDa dextrans (4.6 and 12 nm in diameter). On the other hand, does NLPPya not open discreet pores in planar lipid bilayers with GIPCs, but increases the noise of the ionic current and triggers gradual openings that subsequently end with the rupture of the membrane. Our study will eventually solve the mystery of the unique NLP cytolytic mechanism, be it either via pore formation or some other membrane destabilizing process.