An antifungal protein was isolated from the intercellular washing fluid (IWF) of leaves of sugar beet (Beta vulgaris L., cv. Monova) and purified to homogeneity. The protein, IWF6, comprising 37 amino acids with six cysteines, was able to inhibit the growth of the pathogen Cercospora beticola (Sacc.) in vitro, by 75% after 120 h of growth at a concentration of 20 mu g ml (-1). The amino acid sequence data were used to generate a polymerase chain reaction (PCR) clone, employed for the isolation of a corresponding cDNA clone. The cDNA encodes a precursor protein with an N-terminal putative signal sequence of 45 amino acids, followed by the mature protein of 37 amino acids. Antibodies raised against a synthetic peptide covering the complete sequence of IWF6 were used in immunolocalization studies. The protein was recognized by the antibody in nearly all leaf cell types except epidermal cells. In necrotic tissue, the protein was mainly recognized on C, beticola hyphae growing in a 'pellet' (ball-like) structure. The hyphal 'pellets' are primarily located beneath the stomata. IWF6 shows less than 26% identity to any previously described protein. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.