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.
Highlights
PromAontearlysis oCf tuhteinase Gene fFroumsarium standard media as described in Sambrook et al [14]
Stocks (15%glycerol) at -80 OC; spores from frozen stocks were plated As mutagenesis primers, the following oligonucleotidepairs were used: on potato dextrose agar (Difco) supplemented with 0.5 g/liter ground for pCAT433AInd: oligo 12: GAA GCG GAA GAG CTT GCA TCT AGA
Lyophilized pea stems (PPDA-Medium)and incubated for 4-7 days at TAT CTA TGG TGG 'RT CCT GAA GCG and oligo 13:CGC TTC AGG
Summary
Confer induction and repression [16]. In a first set of experiments, we used different 5' deletions of the promoter fragment of plasmid pCAT433 generated by digestion with exonuclease. Promoter fragments 209 bp andlonger conferred inducibility by cutin hydrolysate and glucose repression, quantitative expression vaned between the different fragments. CAT expression was conferred by a 209-bp promoter fragment, while a 141-bp fragment did not allow regulated gene expression; the elements responsible for induction by cutin hydrolysate have tobe located between -209 and -141. -360 and -310 functions as an antagonist to thenegative element and restoreisnducibility, without affecting the level of constitutive gene expression.In a previous study [16], transformants harboring a 225-bp promoter fragment linked to the CAT gene did not show significant inducibility, possibly becausethetransformantsharboring different deletion constructs used in that studygave only very low CAT activity in the range of 2% conversion
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
