Partial characterization and localization of a novel type of antifungal protein (IWF6) isolated from sugar beet leaves

An antifungal protein was isolated from the intercellular washing fluid (IW F) of leaves of sugar beet (Beta vulgaris L., cv. Monova) and purified to h omogeneity. 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 cl one. The cDNA encodes a precursor protein with an N-terminal putative signa l sequence of 45 amino acids, followed by the mature protein of 37 amino ac ids. Antibodies raised against a synthetic peptide covering the complete se quence of IWF6 were used in immunolocalization studies. The protein was rec ognized by the antibody in nearly all leaf cell types except epidermal cell s. In necrotic tissue, the protein was mainly recognized on C, beticola hyp hae growing in a 'pellet' (ball-like) structure. The hyphal 'pellets' are p rimarily located beneath the stomata. IWF6 shows less than 26% identity to any previously described protein. (C) 2000 Elsevier Science Ireland Ltd. Al l rights reserved.