9419266 Straney Fungi which enter pathogenic or symbiotic interactions with plants have developed traits which allow entry and avoidance of the plant's defenses. In addition to these traits, fungi likely developed regulatory pathways which allow these traits to be expressed at the appropriate time and level, as well as on the correct host. An appropriate signal compound for such gene expression is one which is derived from the plant host. Much excitement has arisen from the observation that several fungal genes are specifically induced during interaction with the host plant. These present systems for studying the signal exchange controlling these unique regulatory responses in fungi. The PDAl gene of Nectria haematococca (anamorph Fusarium solani) offers an excellent model for a plant-induced gene in a phytopathogenic fungus. The expression of this gene is specifically induced by pisatin, the isoflavanoid phytoalexin of its host plant. The PDA 1 gene product, pisatin demethylase, detoxifies this antimicrobial compound. Gene transfer and gene displacement experiments indicate that pisatin demethylase has a partial but significant role in fungal virulence on pea. Studies have further shown that the PDAl gene is induced in vivo, during pathogenesis of pea, concomitant with induction of a pisatin biosynthetic enzyme. The objective of this project is to elucidate the mechanism by which N. haematococca induces expression of the PDA1 gene in response to pisatin. In vitro and in vivo techniques have been developed to dissect the molecular components of the cis and trans-acting components affecting PDA promoter expression. Notably, an in vitro transcription system which reconstitutes the components needed for pisatin-induced transcription has been developed This in vitro system is the first such regulated in vitro system in filamentous fungi. The first goal is to use in vitro transcription to define the minimum cis-acting element which confers pisatininducibility upon a heterologous promoter. The sec ond goal is to test the function of the identified cis elements in vivo (in culture and during pathogenesis) through the use of promoter::GUS fusion constructs introduced into N. haematococca by transformation. The cis-elements delineated in vitro will be fused on a heterologous promoter and its response to pisatin or nutritional signals in culture will be defined. This should allow the separation of pisatin-responsiveness and nutritional responsiveness in PDAl expression. The biological importance of each regulation and their interaction will be evaluated using these transformants to infect pea plants and comparing the patterns of GUS expression in diseased tissue. The third goal of this project is to clone the gene encoding the pisatin responsive factor. Cloning of this gene would allow the investigation of questions relating to its mode of binding activation, and its appearance in other fungi which also display pisatin-induced pisatin demethylase activity. The well characterized PDAl gene and the in vitro transcription system offer an excellent system to answer these questions and provide the first comprehensive picture of regulation of a fungal virulence gene. %%% The completion of these objectives will provide an understanding of how a fungus that infects plants regulates expression of a detoxification gene in response to the host specific defense compound. The molecular components identified will serve as a tool in further detailing how this ability to respond to a plant signal may affect other genes in this and other fungi. ***
