Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are large protein family of virulence factors that are secreted by various plant pathogens and can be found in bacteria, fungi and oomycetes. Upon membrane interactions they are able to trigger plant tissue necrosis and immunity-associated responses in various dicotyledonous plants. The molecular details of their cytolytic mechanism are not known in a great detail. Recently we have identified glycosyl inositol phospho ceramides (GIPC), plant-related sphingolipids, as a target molecule for NLPs binding to plant membranes. Surface plasmon resonance showed that NLP proteins can bind sugars glucosamine and mannosamine, which are often present as the terminal sugars in GIPCs, by millimolar affinity. Structural analysis of NLP protein from oomycete Pythium aphanidermatum in complex with the sugar have indicated structural changes in the protein induced by sugar binding and allow to elucidate the importance of the metal ion coordination for binding. Furthermore, the structure of the non-toxic representative of NLPs, HaNLP3, from oomycete Hyaloperonospora arabidopsidis indicated differences between the proteins that are important for the cytotoxicity. Collectively, we have provided important molecular insights into the mechanisms of cytolytic activity of toxic NLP proteins towards plant plasma membranes.

Financing: Slovenian Research Agency