Fruiting body-associated Pseudomonas contact triggers ROS-mediated perylenequinone biosynthesis in Shiraia mycelium culture.

Perylenequinones (PQs) from Shiraia fruiting bodies serve as potent photosensitizers for anticancer and antimicrobial photodynamic therapy (PDT). Although these fruiting bodies harbor diverse endophytic bacteria, their interactions with the host fungus remain poorly understood. In this study, we used an in vitro confrontation bioassay to investigate the interaction between Shiraia sp. S9 and dominant Pseudomonas isolates, analyzing fungal transcriptional responses and PQ biosynthesis. Comparative assessment of co-cultures with freely suspended live P. fulva SB1 versus dialysis membrane-separated bacteria revealed that direct physical contact is essential for eliciting fungal PQ production, particularly extracellular secretion of hypocrellin A (HA), HC, and elsinochrome A-C. Bacterial elicitation with P. fulva SB1 at 400 cells/mL stimulated both intracellular PQ biosynthesis and extracellular secretion, resulting in a total PQ yield of 362.2 mg/L, a 2.4-fold increase over axenic cultures. RNA-seq analysis after 24 h of co-culture identified 646 differentially expressed genes (DEGs), with 445 upregulated and 201 downregulated, showing significant enrichment in oxidative stress defense, carbohydrate metabolism, and membrane transport functions. Bacterial contact induced reactive oxygen species (ROS) generation, specifically O