Small cysteine-rich antifungal proteins from radish: their role in host defense.

Radish seeds have previously been shown to contain two homologous, 5-kD cysteine-rich proteins designated Raphanus sativus-antifungal protein 1 (RsAFPl) and RsAFP2, both of which exhibit potent antifungal activity 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, RsAFPs are preferentially released during seed germination after disruption of the seed coat. The amount of 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 RsAFP1 cDNA-derlved probe were present in near-mature and mature seeds. Such transcripts as well as the corresponding proteins were barely detectable in healthy uninfected leaves but accumulated systemically at high levels after localized fungal infection. The induced leaf proteins (designated RsdFP3 and RsdFP4) were purified and shown to be homologous to seed RsAFPs and to exert similar antifungal activity in vitro. A chimeric RsAFP2 gene under the control of the constitutive cauliflower mosaic virus 35s promoter conferred enhanced resistance to the foliar pathogen Alternaria longipes in transgenic tobacco. The term "plant defensins" is proposed to denote these defense-related proteins.