Interaction of an Oomycete Nep1-like Cytolysin with Natural and Plant Cell-Mimicking Membranes

Plants are attacked by various pathogens that secrete a variety of effectors to damage host cells and facilitate infection. One of the largest and so far understudied microbial protein families of effectors is necrosis- and ethylene-inducing peptide-1-like proteins (NLPs), which are involved in important plant diseases. Many NLPs act as cytolytic toxins that cause cell death and tissue necrosis by disrupting the plant's plasma membrane. Their mechanism of action is unique and leads to the formation of small, transient membrane ruptures. Here, we capture the interaction of the cytotoxic model NLP from the oomycete Pythium aphanidermatum, NLP_Pya, with plant cell-mimicking membranes of giant unilamellar vesicles (GUVs) and tobacco protoplasts using confocal fluorescence microscopy. We show that the permeabilization of GUVs by NLP_Pya is concentration- and time-dependent, confirm the small size of the pores by observing the inability of NLP_Pya monomers to pass through them, image the morphological changes of GUVs at higher concentrations of NLP_Pya and confirm its oligomerization on the membrane of GUVs. In addition, NLP_Pya bound to plasma membranes of protoplasts, which showed varying responses. Our results provide new insights into the interaction of NLP_Pya with model lipid membranes containing plant-derived sphingolipids.