Characterization and localization of new antifungal cysteine-rich proteins from Beta vulgaris

Two novel antifungal proteins, AX1 and AX2, were isolated from leaves of sugar beet infected with Cercospora beticola. AX1 (MW = 5078 +/- 3D) and AX2 (MW = 5193 +/- 3D) were N-terminally sequenced and identified as monomeric, basic proteins consisting of 46 amino acid residues, of which eight are cysteines. Both AX proteins strongly inhibit growth of C. beticola and other filamentous fungi, but have little or no effect against bacteria. Based on primary sequence homology (24 to 46% identity), they are related to the superfamily of gamma-thionins, which have been isolated recently from seeds of monocotyledons and Brassicaceae. Specific antibodies were raised against the AX proteins after conjugation to diphtheria toxoid. Using immunoblotting and immunohistology, we detected high concentrations of AX proteins extracellularly in cell walls and in globular bodies around necrotic lesions in sugar beet leaves infected with C. beticola, suggesting that AX proteins are involved in antifungal defense. Furthermore, AX proteins or serologically related proteins were detected in xylem, stomata, and stomatal cells as well as in sugar beet styles.