DNA-protein interactions on the osmotin gene promoter and functional analysis of osmotin

Dong Liu, Purdue University

Abstract

The osmotin gene is regulated by many hormonal and environmental signals. Previous results indicated that a sequence-specific region ($-$248/$-$108) on the osmotin promoter, designated fragment A, is required for a minimal level of gene expression and responsiveness to various external signals. We demonstrated here that fragment A is also sufficient to direct gene expression when fused to a minimal CaMV 35S promoter. Protein factors associated with fragment A were identified in salt-adapted tobacco cells, ABA treated unadapted cells, and young cultured tobacco leaves by gel mobility-shift assays. DNase I footprinting revealed that three conserved promoter elements are involved in DNA-protein interactions on fragment A. These elements are: (1) a cluster of three overlapped G-box-like sequences (G sequence); (2) an AT-1 box-like sequence, 5$\sp\prime$-GTATTTTATTAA-3$\sp\prime$ on the bottom strand (AT sequence); (3) a sequence highly conserved in ethylene-induced PR gene promoters, 5$\sp\prime$-TAAGTGCCGCC-3$\sp\prime$ (PR sequence). Among them, the AT sequence was responsible for the majority of protein binding activity of the whole fragment A, whereas the G and PR sequences had weak protein binding activity. Transient expression assays indicated that osmotin promoter activity of these elements correlated with their protein binding activity. UV cross-linking analysis showed that the protein complex bound to fragment A consisted of at least four individual proteins with approximate molecular weights of 28, 29, 40, and 42 kD. Of these, the 28 and 29 kD proteins were associated with the G sequence, and the 40 and 42 kD proteins were associated with the AT and PR sequences. One component of this protein complex, which is associated with G sequence, is a 14-3-3-like protein. Synthesis of osmotin and osmotin-like proteins are induced by fungal infection in tobacco and potato plants. Purified osmotin exhibits antifungal activity against a variety of fungal species in vitro. Transgenic potato but not tobacco plants which overexpress osmotin gene display enhanced resistance to fungal attack. Furthermore, a 20-amino acid C-terminal peptide on osmotin was identified to be responsible for vacuole targeting. Osmotin which secreted into extracellular matrix in transgenic tobacco plants retains its antifungal activity.

Degree

Ph.D.

Advisors

Bressan, Purdue University.

Subject Area

Botany|Molecular biology

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