NLPs are apoplastic effectors with both immunogenic and pore-forming modes of action, yet their toxic mechanism remains poorly understood. They are secreted by microbial pathogens, such as fungi, bacteria and oomycetes. Oomycetal NLPs were reported to bind to GIPC, a sphingolipid of the plant plasma membrane. Here, we used biophysical and biochemical approaches to investigate the necrotic activity and GIPC binding of fungal MpNEP2 from Moniliophthora perniciosa, and oomycetal NLPPp from Phytophthora parasitica and NLPPya from Phytium aphanidermatum. Model membrane systems containing GIPCs extracted from tobacco (dicot) and leek (monocot) were prepared. MpNEP2 and NLPPya bound to GIPC-containing liposomes from both plant sources and exhibited pore-forming activity, demonstrating for the first time that NLPs permeabilize not only dicot but also monocot membranes. NLPPp also bound GIPC-containing liposomes, albeit more weakly, as indicated by sedimentation assay and lower response units and faster dissociation in surface plasmon resonance measurements. The crystal structure of NLPPp revealed a conserved NLP fold, but structural comparison showed a narrower, less accessible binding crevice and a more negatively charged surface relative to NLPPya and MpNEP2, consistent with its reduced binding and activity. Site-directed mutagenesis designed based on the comparison with NLPPya, targeting the binding crevice, did not significantly enhance activity, indicating that NLP function depends on the combined effects of structural organization, conformational dynamics, and electrostatic properties.
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