Identification of regulatory elements in the cutinase promoter from Fusarium solani f. sp. pisi (Nectria haematococca)

Abstract

The cutinase gene from Fusarium solani f. sp. pisi (Nectria haematococca) is induced upon contact with the plant cuticular polymer, cutin, by the unique hydroxy fatty acid monomers released by cutinase carried by virulent strains of the fungus, and this gene is also catabolite-repressed by glucose. Functional elements of the cutinase promoter were studied in vivo by transforming F. solani pisi with fusions of 5'-flanking regions of the cutinase gene and the gene encoding chloramphenicol acetyltransferase (cat). DNA-binding proteins from F. solani pisi were analyzed in vitro by gel shift experiments, methylation interference analysis, and UV-cross-linking experiments. Thus, we identified four promotor elements involved in cutinase gene regulation: a silencer, positive-acting G-rich element, an element that binds a basal transcription factor, and a palindrome necessary for induction by cutin monomer. A silencer between -287 and -249 keeps basal gene expression low but also influences the inducibility of the gene. To restore high levels of induction, a G-rich positive-acting element with sequence similarities to other fungal elements acts as an antagonist to the silencer. Basal transcription is mediated by the first 141 base pairs of the cutinase promoter. The binding site of transcription factor CTF2 was identified between the TATA box and the transcription initiation sites. Gene induction by cutin monomers is regulated by CTF1, most probably a dimeric DNA-binding protein of 49 kDa with a palindromic recognition site at -170.