Cdc25-Mediated Activation of the Small GTPase RasB Is Essential for Hyphal Fusion and Symbiotic Infection of Epichloë festucae

Epichloë festucae is a filamentous endophytic fungus that symbiotically colonizes the intercellular spaces of aerial tissues in perennial ryegrass ( Lolium perenne ) without causing disease symptoms. This mutualistic association enhances host resistance to both biotic and abiotic stresses. Balanced and coordinated growth of E. festucae with its host is essential for the establishment and long-term maintenance of the symbiotic relationship. Various E. festucae mutants defective in symbiosis with host plants have been isolated, and notably, many of these symbiosis-defective mutants also lack hyphal fusion ability under culture conditions, supporting a close functional connection between signal transduction required for hyphal fusion and symbiosis establishment. Using a plasmid insertional mutagenesis approach, we identified Cdc25 as an essential regulator of hyphal fusion in E. festucae . Cdc25 encodes a guanine nucleotide exchange factor (GEF) that activates the small GTPase Ras. The Δcdc25 strain lost both hyphal fusion ability and the capacity to infect host plants. Furthermore, yeast two-hybrid assays revealed that Cdc25 specifically interacts with RasB, one of the five Ras proteins in E. festucae . Expression of constitutively active (CA) RasB in the Δcdc25 strain restored both hyphal fusion and host infection, whereas expression of CA-RasB in the ΔmpkB deletion strain failed to rescue its defect in hyphal fusion, suggesting that the Cdc25-RasB signaling module acts upstream of the MAPK cascade. In pathogenic fungi, this signaling module is known to regulate infection-related morphogenesis. These findings indicate that E. festucae has evolutionarily repurposed the conserved Cdc25-RasB module to coordinate hyphal fusion and maintain a stable mutualistic interaction with its host.