Quantitative Phosphoproteome analyses of Ophiocordyceps sinensis Reveal Phosphoproteins Modulating Primordium Formation

Ophiocordyceps sinensis is a highly prized medicinal and edible fungus. However, its commercial cultivation is challenging because the mechanisms underlying the development of its fruiting bodies are poorly understood. Primordium (PR) formation is the initial and critical stage of fruiting body development. Here, we employed 4D label-free quantitative phosphoproteomics to elucidate phosphorylation dynamics during the fungal sclerotium (ST)-to-PR transition. In total, 400 expressed phosphorylated proteins (DPPs) (266 upregulated, 134 downregulated) and 642 differentially phosphorylated peptides (477 upregulated, 165 downregulated) were screened. Most DPPs displayed markedly increased phosphorylation level during the transition, demonstrating the essential involvement of protein phosphorylation in governing PR formation in O. sinensis. Functional enrichment analysis identified the mitogen-activated protein kinase (MAPK) signaling pathway, exocytosis, glycolysis, glycerophospholipid metabolism, and oxidative stress as critical pathways involved in PR development. Moreover, totals of 25 core DPPs were identified, including the exocyst component Sec8 (T5AQW1), the SNARE protein Sec9 (T5A976), a Sec7 domain-containing protein (T4ZY17), lysophospholipase NTE1 (T5ACX5), MAPK (T5AIB8) and MAPKK Ste7 (T5ADZ7). Combined with protein protein interaction (PPI) analysis, we demonstrated that MAPKK Ste7 serves as the core component of the MAPK signaling cascade, coordinating the processes of cell wall remodeling and oxidative stress responses to facilitate PR initiation. Bioinformatic analysis revealed that Ste7 shows high evolutionary conservation across multiple fungal species and the potential function of the Ste7–Fus3 module in PR formation in O. sinensis. The deletion of Ste7 homology completely abolished PR formation in C. militaris, indicating that Ste7 is essential for PR development in this fungus. Transcriptomic analysis further revealed that Ste7 activates genes associated with cell wall remodeling，and the oxidative stress response, while repressing genes associated with vegetative growth. These expression patterns were further validated using quantitative RT-PCR. Given the high conservation of Ste7, it was implied that Ste7 regulates PR formation in O. sinensis through a similar mechanism. Furthermore, yeast two-hybrid assays combined with AlphaFold 3.0 structural prediction suggested that Ste7 physically interacts with Fus3 in O. sinensis, with Ser362 and Thr388 acting as crucial phosphorylation sites. These findings provide novel insights into how phosphorylation modification dynamically modulates PR development in O. sinensis.