The Fungal Cell-Wall Integrity MAPK Cascade Is Crucial for Hyphal Network Formation and Maintenance of Restrictive Growth of Epichloë festucae in Symbiosis With Lolium perenne.

Epichloë festucae is a mutualistic symbiont that systemically colonizes the intercellular spaces of Lolium perenne leaves to form a highly structured and interconnected hyphal network. In an Agrobacterium tumefaciens T-DNA forward genetic screen, we identified a mutant TM1066 that had a severe host interaction phenotype, causing stunting and premature senescence of the host. Molecular analysis revealed that the mutation responsible for this phenotype was in the cell-wall integrity (CWI) mitogen-activated protein kinase kinase (MAPKK), mkkA. Mutants generated by targeted deletion of the mkkA or the downstream mpkA kinase recapitulated the phenotypes observed for TM1066. Both mutants were defective in hyphal cell­cell fusion, formed intrahyphal hyphae, had enhanced conidiation, and showed microcyclic conidiation. Transmission electron microscopy and confocal microscopy analysis of leaf tissue showed that mutant hyphae were more abundant than the wild type in the intercellular spaces and colonized the vascular bundles. Hyphal branches failed to fuse but, instead, grew past one another to form bundles of convoluted hyphae. Mutant hyphae showed increased fluorescence with AF488-WGA, indicative of increased accessibility of chitin, a hypothesis supported by changes in the cell-wall ultrastructure. These results show that the CWI MAPK pathway is a key signaling pathway for controlling the mutualistic symbiotic interaction between E. festucae and L. perenne.