<i>Fusarium graminearum</i> Ste2 and Ste3 receptors undergo peroxidase-induced heterodimerization when expressed heterologously in <i>Saccharomyces cerevisiae</i>

<i>Fusarium graminearum Fg</i>Ste2 and <i>Fg</i>Ste3 are G-protein-coupled receptors (GPCRs) shown to play roles in hyphal chemotropism and fungal plant pathogenesis in response to activity arising from host-secreted peroxidases. Here, we follow up on the observation that chemotropism is dependent on both <i>Fg</i>Ste2 and <i>Fg</i>Ste3 being present; testing the possibility that this might be due to formation of an <i>Fg</i>Ste2-<i>Fg</i>Ste3 heterodimer. Bioluminescence resonance energy transfer (BRET) analyses were conducted in <i>Saccharomyces cerevisiae</i>, where the addition of horse radish peroxidase (HRP) was found to increase the transfer of energy from the inducibly expressed <i>Fg</i>Ste3-Nano luciferase donor, to the constitutively expressed <i>Fg</i>Ste2-yellow fluorescent protein (YFP) acceptor, compared to controls. A partial response was also detected when an HRP-derived ligand-containing extract was enriched from <i>F. graminearum</i> spores and applied instead of HRP. In contrast, substitution with pheromones or an unrelated bovine GPCR, rhodopsin-YFP used as acceptor, eliminated all BRET responses. Interaction results were validated by affinity pulldown and receptor expression was validated by confocal immunofluorescence microscopy. Taken together these findings demonstrate the formation of HRP and HRP-derived ligand stimulated heterodimers between <i>Fg</i>Ste2 and <i>Fg</i>Ste3. Outcomes are discussed from the context of the roles of ligands and reactive oxygen species in GPCR dimerization.