P-191 - NEP1-LIKE PROTEINS FROM PHYLOGENETICALLY DISTINCT PLANT PATHOGENS BIND AND PERMEABILIZE MODEL MEMBRANES CONTAINING EUDICOT AND MONOCOT PLANT-DERIVED GLYCOSYLINOSITOL PHOSPHORYLCERAMIDES

Plant-pathogen interplay affect plant health. Nep1-like proteins (NLPs) are microbial effectors secreted by oomycetes, fungi and bacteria. It was established that NLP_Pya from oomycete Phytium aphanidermatum binds to glycosylinositol phosphoryceramides (GIPCs), major sphingolipids in plant plasma membrane. However, this was not yet shown for other NLPs and the interaction of NLPs with plant plasma membrane remains poorly characterized. For long, NLPs were considered eudicot-specific plant effectors, a hypothesis that has been challenged in the recent years.

We investigate binding and pore-forming activity of two cytotoxic NLPs, NLP_Pya and MpNEP2 from fungi Moniliopthora perniciosa, on model membranes containing GIPCs from dicot (tobacco) and monocot (leek) plants. Using multilamellar vesicles (MLVs) and giant unilamellar vesicles (GUVs), we demonstrate both NLPs selectively bind GIPC-containing membranes. Real-time imaging of NLPs-treated GUVs in a microfluidic chamber, revealed binding and fluorescent probe leakage, confirming pore formation. We observed differences between the two NLPs - NLP_Pya binding and pore-formation led to vesicle collapse and membrane permeabilization occurred more rapidly in dicot than in monocot GIPCs-containing membranes. The differences in lipid composition and protein characteristics may lead to distinct mechanisms of interactions and steps in pore-formation. These findings provide insights into the molecular basis of NLP cytotoxicity.