Identification of a fungal cutinase promoter that is inducible by a plant signal via a phosphorylated trans-acting factor.

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Abstract
Plant cutin monomers trigger, and glucose suppresses, the expression of the cutinase gene of pathogenic fungi. To identify the cutinase promoter region responsible for induction by the unique plant components, a promoter analysis was done with transformants. Plasmids were constructed that contained (i) the 5' flanking region of the cutinase gene or its deletion mutants from Fusarium solani pisi fused with a chloramphenicol acetyltransferase (CAT) reporter gene and (ii) a constitutive promoter fused with a hygromycin phosphotransferase gene. Hygromycin-resistant transformants of F. solani pisi generated by electroporation were assayed for CAT activity inducible by cutin hydrolysate and for glucose repression of this induction. CAT was induced in a glucose-repressible manner when fused with a 360-base-pair (bp), or longer, segment of the 5' flanking region of the cutinase gene, and deletion of the next 135 bp abolished this induction. Gel retardation assays showed that a protein(s) in nuclear extract from the fungus bound to the 5' flanking region of cutinase gene, and this binding was also abolished when the same 135-bp segment was deleted. These results show that the -225 to -360 segment of the cutinase gene contains a cis-acting regulatory element that binds trans-acting factor(s) in the nuclei. Treatment of the nuclear extract with immobilized phosphatase abolished binding to the promoter, suggesting that binding required a phosphorylated form of the protein. With isolated nuclei, phosphorylation of a protein occurred only in the presence of both cutin monomer and the fungal protein factor. The presence of protein kinase inhibitor H7 during the preincubation of nuclei with the monomer and protein factor inhibited cutinase gene transcription. These results suggest that cutin monomer causes phosphorylation of a transcription factor that binds to the -225 to -360 segment of the cutinase gene and enhances transcription of this gene.