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

## TL;DR

A Cdc25-RasB signaling pathway is crucial for hyphal fusion and infection in the symbiotic fungus Epichloë festucae.

## Contribution

The study identifies Cdc25 as a key regulator of hyphal fusion and symbiosis in Epichloë festucae through a conserved signaling module.

## Key findings

- The Δcdc25 strain loses hyphal fusion and host infection abilities.
- Cdc25 interacts specifically with RasB, and CA-RasB restores fusion and infection in Δcdc25.
- The Cdc25-RasB module acts upstream of the MAPK cascade in symbiosis.

## Abstract

Epichloë festucae is a filamentous endophytic fungus that symbiotically colonises the intercellular spaces of aerial tissues in perennial ryegrass 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. Yeast two‐hybrid assays revealed that Cdc25 specifically interacts with RasB, one of 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 strain failed to rescue its defect in hyphal fusion, suggesting that the Cdc25‐RasB signalling module acts upstream of the MAPK cascade. In pathogenic fungi, this signalling 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.

A conserved Cdc25–RasB signalling pathway, essential for hyphal fusion and host infection, is repurposed in Epichloë festucae to maintain a stable mutualistic symbiosis with grass hosts.