Fungal cells are encased by a cell wall, which is unique to fungi. The precise composition of the cell wall varies between species but three layers predominate: a chitin layer adjacent to the plasma membrane, a β-1,3-glucan layer and an outer glycoprotein layer. For a molecule to enter into the fungal cell it must cross the thick cell wall, and pass through the plasma membrane. Antifungal plant defensins, like NaD1, require passage across both these barriers to exert their cell killing activity.
Plant defensins are highly stable peptides that often have potent antifungal activity and have the potential for use in agricultural and pharmaceutical applications. NaD1, a plant defensin from the ornamental tobacco, Nicotiana alata, is active against both plant and animal fungal pathogens. It acts via a three-step process, beginning with interaction with the fungal cell wall, followed by permeabilisation of the plasma membrane, and entry into the cytoplasm. Unlike other antifungal molecules, NaD1 requires an intact cell wall for its antifungal activity NaD1 binds to β-1,3-glucan and chitin, the main structural components of the cell wall. Furthermore, NaD1 binds with a higher affinity to chitin than β-1,3-glucan leading to the hypothesis that NaD1 might be directed through the wall and onto the plasma membrane by an affinity gradient. Once through the wall, NaD1 must pass through the membrane to enter the cytoplasm. NaD1 interaction with membranes of varying lipid composition was studied in detail using liposome pull-down assays, calcein-loaded liposomes and dual polarisation interferometry. This revealed that NaD1 interacts specifically with membranes containing phosphatidylinositol 4,5-bisphosphate, a low abundance phospholipid important in many cellular processes.