Localisation of phosphoinositides in the grass endophyte Epichloë festucae and genetic and functional analysis of key components of their biosynthetic pathway in E. festucae symbiosis and Fusarium oxysporum pathogenesis.

• Molecular probes were used to determine the cellular localization of PI[3]P, PI[3,5]P 2 , PI[4]P and PI[4,5]P 2 in hyphae of Epichloë festucae. • Deletion of tepA allowed PI[3,4,5]P 3 to accumulate to detect and localize using a molecular probe specific to this PI to cell septa of E. festucae. • Deletion of tepA in the pathogen Fusarium oxysporum disrupted chemotropism suggesting this lipid species may be important for host signalling. Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signaling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localize the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P 2 , PI[4]P and PI[4,5]P 2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homolog of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did enable the detection of PI[3,4,5]P 3 in septa and mycelium of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P 3 in Δ tepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P 3 and that fungal PTEN homologs are functional lipid phosphatases. The F. oxysporum chemotropism defect suggests a conserved role of PTEN homologs in chemotaxis across protists, fungi and mammals. [ABSTRACT FROM AUTHOR]