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In ascomycetous fungi, sexual mate recognition requires interaction of the Ste2 receptor protein produced by one partner with the α-factor peptide pheromone produced by the other partner. In some fungi, Ste2 is further needed for chemotropism towards plant roots to allow for subsequent infection and colonization. Here, we investigated whether this is also true for the pine pitch canker fungus, Fusarium circinatum, which is a devastating pathogen of pine globally. Ste2 knockout mutants were generated for two opposite mating-type isolates, after which all strains were subjected to chemotropism assays involving exudates from pine seedling roots and synthetic α-factor pheromone, as well as a range of other compounds for comparison. Our data show that Ste2 is not required for chemotropism towards any of these other compounds, but, in both wild-type strains, Ste2 deletion resulted in the loss of chemotropism towards pine root exudate. Also, irrespective of mating type, both wild-type strains displayed positive chemotropism towards α-factor pheromone, which was substantially reduced in the deletion mutants and not the complementation mutants. Taken together, these findings suggest that Ste2 likely has a key role during the infection of pine roots in production nurseries. Our study also provides a strong foundation for exploring the role of self-produced and mate-produced α-factor pheromone in the growth and overall biology of the pitch canker pathogen.

In ascomycetous fungi, sexual mate recognition requires interaction of the Ste2 receptor protein produced by one partner with the α-factor peptide pheromone produced by the other partner. In some fungi, Ste2 is further needed for chemotropism towards plant roots to allow for subsequent infection and colonization. Here, we investigated whether this is also true for the pine pitch canker fungus, Fusarium circinatum , which is a devastating pathogen of pine globally. Ste2 knockout mutants were generated for two opposite mating-type isolates, after which all strains were subjected to chemotropism assays involving exudates from pine seedling roots and synthetic α-factor pheromone, as well as a range of other compounds for comparison. Our data show that Ste2 is not required for chemotropism towards any of these other compounds, but, in both wild-type strains, Ste2 deletion resulted in the loss of chemotropism towards pine root exudate. Also, irrespective of mating type, both wild-type strains displayed positive chemotropism towards α-factor pheromone, which was substantially reduced in the deletion mutants and not the complementation mutants. Taken together, these findings suggest that Ste2 likely has a key role during the infection of pine roots in production nurseries. Our study also provides a strong foundation for exploring the role of self-produced and mate-produced α-factor pheromone in the growth and overall biology of the pitch canker pathogen.

Work from our laboratories over the last 35 years that has focused on Ste2p, a G protein-coupled receptor (GPCR), and its tridecapeptide ligand α-factor is reviewed. Our work utilized the yeast Saccharomyces cerevisiae as a model system for understanding peptide-GPCR interactions. It explored the structure and function of synthetic α-factor analogs and biosynthetic receptor domains, as well as designed mutations of Ste2p. The results and conclusions are described using the nuclear magnetic resonance interrogation of synthetic Ste2p transmembrane domains (TMs), the fluorescence interrogation of agonist and antagonist binding, the biochemical crosslinking of peptide analogs to Ste2p, and the phenotypes of receptor mutants. We identified the ligand-binding domain in Ste2p, the functional assemblies of TMs, unexpected and interesting ligand analogs; gained insights into the bound α-factor structure; and unraveled the function and structures of various Ste2p domains, including the N-terminus, TMs, loops connecting the TMs, and the C-terminus. Our studies showed interactions between specific residues of Ste2p in an active state, but not resting state, and the effect of ligand activation on the dimerization of Ste2p. We show that, using a battery of different biochemical and genetic approaches, deep insight can be gained into the structure and conformational dynamics of GPCR-peptide interactions in the absence of a crystal structure.