Control of two fungal pathogenicity traits in response to a host-specific isoflavonoid

Many plants produce a variety of chemical compounds that discourage potential pathogens. Pathogens have adapted to recognize these defense compounds as host recognition signals. Pathogens express various pathogenicity traits in response to these signals. These traits include developmental changes and specific gene expression. The ability to initiate these traits according to the perceived state of the host contributes to the success of the infection. The identity of host signals that trigger pathogenicity traits is often unknown. The components of the regulatory pathways that convey the perception of the signal compound towards the expression of the pathogenic traits are even less clear. The ability to genetically manipulate the pathogenic fungus Fusarium solani f. sp. pisi and previous characterization of leguminous secondary metabolites make the interaction between this fungus and its preferred host legume garden pea a model system in which to study pathogenic responses to host plant signals. An HPLC method was developed to identify flavonoids in pea root exudates that had germination-stimulating activity based upon previous indications that F. solani spores germinated in response to flavonoids. The pea isoflavonoid pisatin was isolated in a fraction with high stimulating activity. In addition to this developmental response, a specific gene response was investigated. Pisatin, an ordinarily fungitoxic compound, induces the fungal expression of PDA1, which encodes a cytochrome P450 with detoxifying pisatin demethylase activity. A yeast one-hybrid system was used to clone a transcription regulator that mediated the pisatin-induced expression of PDA1. The yeast one-hybrid system isolated a pisatin-responsive factor (PRF) that bound a 40 by regulatory element in the promoter region of PDA1. PRF had sequence similarity to a family of transcription regulators unique to fungi, proteins with the C6 binuclear cluster DNA-binding motif. Gel shift experiments were performed to establish binding determinants of PRF to its DNA target-binding site. Site-specific mutation within the 40 by sequence was performed to test adherence to the consensus DNA binding site of C6 zinc cluster proteins, inverted CGGs separated by a variable spacer sequence. A CGGN14CCG site within the 40bp pisatin-responsive sequence proved to be a minimal consensus DNA target sequence for PRF.