Abstract
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 beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.
Highlights
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
In order to investigate which amino acids are essential for the antifungal activity of Rs-AFP2, we have undertaken a mutational analysis of this peptide
Conception and Production of Rs-AFP2 Variants—Proteins belonging to the family of plant defensins have been purified and sequenced from a range of taxonomically divergent plant species, while others have been identified via cDNA sequencing [9]
Summary
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. Well known examples of peptides with antimicrobial properties are the cecropins of invertebrates (reviewed in Ref. 1) and magainins of amphibians (reviewed in Ref. 2) Another class comprises cysteine-rich peptides, among which are the mammalian and insect defensins [3,4,5], both small, basic proteins with a cysteine-stabilized three-dimensional folding pattern involving antiparallel -sheets. Plant defensins are small cysteine-rich peptides consisting of 45–54 amino acids with four intramolecular disulfide bridges They are encountered in different plant species and various tissues such as seed, flowers, and pathogen-stressed leaves. In this study we have performed a structure-function analysis of Rs-AFP2, a plant defensin isolated from radish seed and member of the plant defensin group causing hyperbranching of fungal hyphae. Information on amino acid substitutions resulting in either a decreased or enhanced antifungal activity, taken together with preliminary data on the three-dimensional configuration of Rs-AFP2, allows prediction of the sites which possibly interact with the yet unknown target site on the fungal hyphae
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