SMALL CYSTEINE-RICH ANTIFUNGAL PROTEINS FROM RADISH - THEIR ROLE IN HOST-DEFENSE

Radish seeds have previously been shown to contain two homologous, 5-k D cysteine-rich proteins designated Raphanus sativus-antifungal protei n 1 (Rs-AFP1) and Rs-AFP2, both of which exhibit potent antifungal act ivity in vitro, We now demonstrate that these proteins are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs. Moreover, Rs-AFPs are preferentially released d uring seed germination after disruption of the seed coat. The amount o f released proteins is sufficient to create a microenvironment around the seed in which fungal growth is suppressed, Both the cDNAs and the intron-containing genomic regions encoding the Rs-AFP preproteins were cloned, Transcripts (0.55 kb) hybridizing with an Rs-AFP1 cDNA-derive d probe were present in near-mature and mature seeds. Such transcripts as well as the corresponding proteins were barely detectable in healt hy uninfected leaves but accumulated systemically at high levels after localized fungal infection. The induced leaf proteins (designated Rs- AFP3 and Rs-AFP4) were purified and shown to be homologous to seed Rs- AFPs and to exert similar antifungal activity in vitro. A chimeric Rs- AFP2 gene under the control of the constitutive cauliflower mosaic vir us 35S promoter conferred enhanced resistance to the foliar pathogen A lternaria longipes in transgenic tobacco, The term ''plant defensins'' is proposed to denote these defense-related proteins.