cAMP signalling regulates synchronised growth of symbiotic Epichloë fungi with the host grass Lolium perenne

The seed-transmitted fungal symbiont, Epichloë festucae colonises grasses by infecting host tissues as they form on the shoot apical meristem (SAM) of the seedling. How this fungus accommodates the complexities of plant developmental to successfully colonise the leaves and inflorescences is unclear. Since adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent signalling is often essential for host colonisation by fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the E. festucae adenylate cyclase gene (acyA). Consistent other fungi, disruption mutants had a slow radial growth rate in culture and hyphae were convoluted and hyper-branched suggesting that fungal apical dominance had been disrupted. Nitro blue tetrazolium staining of hyphae showed that cAMP disruption mutants were impaired in their ability to synthesise superoxide, indicating that cAMP signalling regulates accumulation of ROS. Despite significant defects in hyphal growth and ROS production E. festucae ΔacyA mutants were infectious and capable of forming symbiotic associations with grasses, albeit at a lesser infection frequency than wild-type. Plants infected with E. festucae ΔacyA were indistinguishable from controls, however microscopic evidence showed that hyphae were hyper-branched, and leaf tissues heavily colonised, indicating that the tight regulation of hyphal growth normally observed in maturing leaves requires a functional cAMP signalling cascade.