Plant defensins interact with phospholipids in bilayers as part of their cytotoxic activity. Solanaceous class II defensins with the loop 5 sequence pattern "S-[KR]-[ILVQ]-[ILVQ]-[KR]-[KR]" interact with PI(4,5)P2. Here, the prototypical defensin of this class, NaD1, is used to characterise the biophysical interactions between these defensins and phospholipid bilayers. Binding of NaD1 to bilayers containing PI(4,5)P2 occurs rapidly and the interaction is very strong. Dual polarisation interferometry revealed that NaD1 does not dissociate from bilayers containing PI(4,5)P2. Binding of NaD1 to bilayers with or without PI(4,5)P2 induced disorder in the bilayer. However, permeabilisation assays revealed that NaD1 only permeabilised liposomes with PI(4,5)P2 in the bilayer, suggesting a role for this protein-lipid interaction in the plasma membrane permeabilising activity of this defensin. No defensins in the available databases have the PI(4,5)P2 binding sequence outside the solanaceous class II defensins, leading to the hypothesis that PI(4,5)P2 binding co-evolved with the C-terminal propeptide to protect the host cell against the effects of the tight binding of these defensins to their cognate lipid as they travel along the secretory pathway. This data has allowed us to develop a new model to explain how this class of defensins permeabilises plasma membranes to kill target cells.