OpyA Links MAPK and Calcium Signaling to Sustain Polarized Growth, Hyphal Tip Integrity, and Development in Aspergillus nidulans

The Saccharomyces cerevisiae adaptor Opy2 couples cell-surface cues to MAPK cascades, but its roles outside yeasts remain largely unexplored. We characterize OpyA, the Aspergillus nidulans Opy2 ortholog, and identify it as a major regulator of polarized growth and hyphal tip homeostasis. OpyA retains Opy2 domain organization, including the eight extracellular cysteines required for Sho1-branch signaling in yeast. ΔopyA mutants display reduced, irregular growth, hyperbranching, calcofluor white/Congo red sensitivity, and lysis of ~50% of hyphal tips, indicating compromised cell wall integrity and tip stability. Conidiation is strongly reduced, and sexual development arrests at sterile cleistothecial primordia. Signaling is selectively affected: ΔopyA mutants tolerate H2O2 and phosphorylate SakA normally in response to it, whereas SakA phosphorylation after osmotic shock is diminished. Additive conidiation defects in double mutants place OpyA parallel to the SakA and SrrA axes. Strikingly, the ΔopyA mutant phenotype is suppressed by KCl or NaCl, but not by sorbitol, and by CaCl2, but not by MgCl2, indicating ion-specific rather than purely osmotic rescue. Calcium restores colony growth and branching pattern, improving hyphal tip integrity, conidiation, and the formation of fertile cleistothecia, suggesting that OpyA loss creates a calcium-responsive defect. OpyA::GFP localizes to the plasma membrane with apical enrichment and to cytoplasmic puncta that include CMAC-positive acidic compartments, while non-lysed ΔopyA hyphae display a tubular tip vacuolar network, consistent with altered vesicle trafficking. We propose that OpyA integrates cell-surface surveillance with calcium-dependent signaling to sustain hyphal tip growth, cell wall integrity and developmental competence. Its conservation across fungi makes OpyA a potential antifungal target.