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

Abstract

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 cell–cell 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.

An Epichloë festucae homologue of MOB3, a component of the STRIPAK complex, is required for the establishment of a mutualistic symbiotic interaction with Lolium perenne

“…Two bands were detected in WT, corresponding to phosphorylated MpkA at 47 kDa and phosphorylated MpkB, the Fus3 homologue, at 41 kDa. Although MpkA phosphorylation was not detected in the Δ mpkA and Δ mkkA CWI kinase mutants (Becker et al ., ), both MpkA and MpkB phosphorylation still occurred in the Δ mobC #21 and Δ mobC #37 mutants, with bands of similar intensity to that of WT.…”

“…Given that IHH formation and hyperconidiation are phenotypes shared among many E. festucae symbiosis mutants (Becker et al ., ; Tanaka et al ., ), we next tested whether mobC is required for the symbiotic interaction of E. festucae with L. perenne . Plants infected with Δ mobC were severely stunted compared with WT‐infected plants (Fig.…”

“…In culture, E. festucae hyphal strands adhere to one another to form cables, which extend outwards from the colony centre. Cell–cell fusion within these cables occurs by tip‐to‐side fusion events that are easy to observe and quantify using light microscopy (Becker et al ., ). To date, we have been unable to test whether cell–cell fusion mutants of E. festucae are also defective in fruiting body development, as the sexual cycle of E. festucae is highly complex and only occurs on the plant host and requires a third symbiont, a Botanophila fly, to transfer spermatia from a stroma of one mating type to a stroma of the opposite mating type (Bultman & Leuchtmann, ).…”

“…Epichloë festucae NoxA, NoxR, So, ProA, MpkA and MkkA are required for cell–cell fusion in culture and hyphal network formation in planta . These findings gave rise to the hypothesis that cell–cell fusion regulates and restricts hyphal growth in planta and is required for the establishment of a mutualistic association (Becker et al ., ; Charlton et al ., ; Takemoto et al ., ; Tanaka et al ., ). Here, we show that E. festucae MobC, the homologue of the S. macrospora STRIPAK complex component MOB3, is essential for the maintenance of a mutualistic symbiotic interaction between E. festucae and L. perenne , and plays an accessory role in regulating cell–cell fusion and expressoria formation.…”

Signal exchange and integration during self-fusion in filamentous fungi

“…The MAK-1 cell wall integrity pathway is a second MAP kinase cascade essential for germling and hyphal fusion in filamentous fungi [42,[51][52][53].…”