Fusarium graminearum Ste2 and Ste3 Receptors Undergo Peroxidase Induced Heterodimerization

F. graminearumFgSte2 and FgSte3 are G-protein coupled receptors (GPCRs) recently shown to play roles in mediating fungal hyphal chemotropism and plant pathogenesis in response to activity arising from host-released peroxidases. Here, we follow up on the previous observation that chemotropism is dependent on both FgSte2 and FgSte3 being present at the same time; testing the possibility that this effect might be due to formation of an FgSte2-FgSte3 heterodimer. Initially the recombinant cell-surface expression of the F. graminearum GPCRs was validated in S. cerevisiae by confocal immunofluorescence microscopy. Bioluminescence resonance energy transfer analyses were subsequently conducted, where the addition of horse radish peroxidase (HRP) was found to increase the transfer of energy from the inducibly-expressed FgSte3-Nano luciferase (FgSte3-NLuc) donor, to the constitutively-expressed FgSte2-yellow fluorescent protein (FgSte2-YFP) acceptor, compared to controls. A partial response was also detected when an HRP-derived ligand-containing extract was enriched from F. graminearum spores and applied to the S. cerevisiae BRET system directly. The selectivity of the interaction was demonstrated by comparison to treatment with pheromones as well as an unrelated bovine GPCR, rhodopsin, fused to YFP as acceptor, that yielded no response when co-expressed with FgSte3-NLuc. Finally, the peroxidase-stimulated heterodimerization was validated by affinity pulldown. Taken together these findings demonstrate the formation of HRP and HRP-derived ligand stimulated heterodimers between FgSte2 and FgSte3. Outcomes are discussed from the context of the roles of ligands and reactive oxygen species in GPCR dimerization.