Evidence of cellular adaptations in a fungal cultivar promoting resource exchange with leafcutter ant farmers.

Leafcutter farming systems are ant-fungus mutualisms whose ecological success hinges on differentiation of fungal hyphae into swollen cells called gongylidia that ants consume. While gongylidium cells are unique signatures of coevolved crop domestication, their cell biology is poorly understood. Each gongylidium cell contains a large vacuole that is thought to protectively store plant degradation enzymes that ants ingest and vector unharmed in faecal droplets back to the fungus. We hypothesized that enzyme storage requires gongylidium vacuoles to have distinct levels of pH and reactive oxygen species (ROS) compared to the vacuoles of undifferentiated hyphae that likely degrade cellular waste. We used live-cell fluorescence microscopy of fungal isolates with targeted probes to first show that both cell types had vacuoles with lower pH than the surrounding cytosol. In contrast, while hyphal vacuoles stored ROS, gongylidium vacuoles excluded these potentially harmful molecules. These findings suggest derived cellular adaptations in a mutualistic fungus where gongylidia protect ant-vectored enzymes through specialized subcellular ROS compartmentalization.