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.

An antimicrobial protein of about 1 O kD, called AceAMPl, was isolated from onion (Allium cepa L.) seeds. Based on the nearcomplete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. l h e mature protein is homologous t o plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. AceAMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 pg mL-'. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. AceAMP1 is also active on two Cram-positive bacteria but is apparently not toxic for Cram-negative bacteria and cultured human cells. In contrast t o nsLTPs such as those isolated from radish or maize seeds, AceAMPI was unable t o transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. l h e relevance of these findings with regard to the function of nsLTPs is discussed.Although plant seeds are usually sown on a substrate that is extremely rich in microorganisms, infection of seeds or seedling tissues normally occurs at relatively low frequency. It is believed that seed proteins that exhibit antimicrobial activity may participate in the protection of seeds against potential microbial invaders. Different types of antimicrobial proteins have been purified from plant seeds '

Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI ␤-turn connecting ␤-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting ␤-strand 1 and the ␣-helix and contiguous residues on the ␣-helix and ␤-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca 2؉ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca 2؉ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca 2؉ uptake.