MYCOPARASITIC INTERACTION RELIEVES BINDING OF THE CRE1 CARBON CATABOLITE REPRESSOR PROTEIN TO PROMOTER SEQUENCES OF THE ECH42 (ENDOCHITINASE-ENCODING) GENE IN TRICHODERMA-HARZIANUM

The fungus Trichoderma harzianum is a potent mycoparasite of various p lant pathogenic fungi. We have studied the molecular regulation of myc oparasitism in the host/mycoparasite system Botrytis cinerea/T. harzia num. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EM-SAs) with tao promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expressio n is triggered during mycoparasitic interaction [Carsolio, C., Gutierr ez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Pro c. Natl. Acad. Sci. USA 91, 10903-10907]. All cell-free extracts forme d high-molecular Height protein-DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with grea ter mobility, Competition experiments, using oligonucleotides containi ng functional and nonfunctional consensus sites for binding of the car bon catabolite repressor Cre1. provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragmen ts of the ech-42 promoter, The presence of two and three consensus sit es for binding of Cre1 in the two ech-42 promoter fragments used is co nsistent with these findings, In contrast, the formation of the protei n-DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Ad dition of equal amounts of protein of cell-free ext nets from nonmycop arasitic mycelia converted the mycoparasitic DNA-protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::gluta thione S-transferase protein to mycoparasitic cell-free extracts produ ced the same effect, These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites i n the ech-42 promoter, (ii) binding of a ''mycoparasitic'' protein-pro tein complex to the ech-42 promoter in vicinity of the Cre1 binding si tes, and (iii) functional inactivation of Cre1 upon mycoparasitic inte raction to enable the formation of the mycoparasitic protein-DNA compl ex.