Objectives: Host toxicity and the development of fungal resistance threaten the longevity of currently used clinical antifungals. The need for the development of new antifungal molecules to add to the repertoire for control of fungal pathogens is urgent. Naturally occurring antifungal peptides from plants are an attractive option for the development of next generation antifungals. Plants lack the adaptive immune system found in mammals and are thus fully dependent on their arsenal of innate immunity molecules. These antifungals are the result of millions of years of an evolutionary tug-of-war between hosts and fungal pathogens. One of the largest families of plant antifungal peptides are the plant defensins. The plant-defensin fold is extremely stable to extremes in pH and temperature as well as proteolytic degradation. Interactions with lipids and polysaccharides are key components of the mechanisms of many plant defensins. One of the major hurdles in the development of plant defensins for treatment of systemic fungal infections is that the majority of plant defensins are rendered inactive in physiological salt concentrations. Understanding why so many of these molecules lose activity in the presence of salt and identifying members of the plant defensin family that do not lose activity in the presence of salt is crucial for the development of plant defensins as antifungal molecules. Plant defensins bind lipids in the presence of physiological salt leading to the hypothesis that the fungal cell wall caused the loss of activity in the presence of salt.
Methods: The biochemical interaction between plant defensins and fungal cell wall polysaccharides were assessed by generating binding isotherms. Chemical and genetic modification of cell wall polysaccharide levels via treatment with chemical inhibitors or deletion of genes encoding key synthases were used to assess the effect of varying cell wall polysaccharide levels on defensin activity. Salt tolerance of defensins was assessed using micro broth dilution assays with increasing salt concentrations. Defensin activity on spheroplasts in the presence and absence of salt was also assessed.
Results: By investigating the interactions of plant defensins with fungal cell wall polysaccharides we have revealed that binding to β-glucan and chitin in the fungal cell wall is a major contributor to the loss of activity of plant defensins in high salt. We have also identified a defensin that does not bind to either β-glucan or chitin and found that it retains activity in high salt medium. We also discovered that defensins with very strong positive charges retain their activity at physiological salt concentrations. 
Conclusion: Interaction between plant defensins and fungal cell wall carbohydrates is the mechanism for the loss of antifungal activity in elevated salt concentrations. Future work will focus on further assessing the potential for these molecules to be developed as antifungals by looking at their effects on various mammalian cell types and efficacy at clearing fungal infections in animal models. The differences in the mechanisms of salt-tolerant and intolerant defensins will also be investigated.

2 / Interactions with fungal cell wall polysaccharides determines the salt tolerance of antifungal plant defensins.