Various mechanisms to fend off microbial invaders have been devised by a11 living organisms, including microorganisms themselves. The most sophisticated of these mechanisms relies on the synthesis of immunoglobulins directed against specific microbial targets. However, immunoglobulin-based immunity operates only in a relatively minor subset of living species, namely the higher vertebrates. A much more ancient and widespread defense strategy involves the production of small peptides that exert antimicrobial properties. As products of single genes, antimicrobial peptides can be synthesized in a swift and flexible way, and because of their small size they can be produced by the host with a minimal input of energy and biomass. Wellknown examples of antimicrobial peptides are the cecropins that accumulate in the hemolymph of many invertebrates in response to injury or infection (reviewed by Boman and Hultmark, 1987) and the magainins that are secreted by glands in the skin of amphibians (reviewed by Bevins and Zasloff, 1990). Cecropins and magainins are small (2040 residues) basic peptides displaying an amphipathic a-helical structure that can integrate in microbial membranes to form ion channels (Duclohier, 1994).Another class of antimicrobial peptides is formed by the Cys-rich peptides, which in contrast to cecropins and magainins, have a complex cystine-stabilized three-dimensional folding pattern often involving antiparallel P-sheets. Defensins are one class among the numerous types of Cys-rich antimicrobial peptides, which differ in length, number of cystine, bonds, or folding pattern (reviewed by Boman, 1995). Insect defensins (3443 residues, three disulfide bridges) are, like cecropins, produced in a pathogeninducible manner by the insect fat body and secreted in the hemolymph (reviewed by Hoffmann and Hétru, 1992).

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From seeds of Aesculus hippocastanum, Clitoria ternatea, Dahlia merckii and Heuchera sanguinea five antifungal proteins were isolated and shown to be homologous to plant defensins previously characterised from radish seeds and ~/-thionins from Poaceae seeds. Based on the spectrum of their antimicriobial activity and the morphological distortions they induce on fungi the peptides can be divided into two classes. The peptides did not inhibit any of three different a-amylases.

Treatment of hyphae of Neurospora crassa with antifungal plant defensins, i.e. Rs-AFP2 and Dm-AMP1 isolated from radish and dahlia seed, respectively, induced a rapid K+ efflux, Ca2+ uptake, and alkalinization of the incubation medium. The Rs-AFP2-induced alkalinization of the incubation medium could be inhibited with G-protein inhibitors. alpha-Hordothionin, an antifungal thionin from barley seed, caused a sustained increased Ca2+ uptake at subinhibitory concentrations but only a transient increased uptake at inhibitory concentrations. alpha-Hordothionin also caused increased K+ efflux and alkalinization of the medium, but these fluxes occurred more rapidly compared to those caused by plant defensins. Furthermore, alpha-hordothionin caused permeabilization of fungal hyphae to the non-metabolite alpha-aminoisobutyric acid and, in addition, altered the electrical properties of artificial lipid bilayers, consistently leading to rupture of the lipid bilayers. The plant defensins did not form ion-permeable pores in artificial membranes and did not exhibit substantial hyphal membrane permeabilization activity. Our results are consistent with the notion that thionins inhibit fungal growth as a result of direct protein-membrane interactions, whereas plant defensins might act via a different, possibly receptor-mediated, mechanism.

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