The role of the G protein and cAMP/PKA signalling pathway in establishment and maintenance of the mutualistic Epichloë festucae - ryegrass association : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

Abstract

Growth of the fungal endophyte 𝘌𝘱𝘪𝘤𝘩𝘭𝘰𝘦̈ 𝘧𝘦𝘴𝘵𝘶𝘤𝘢𝘦 in mutualistic symbiotic association with 𝘓𝘰𝘭𝘪𝘶𝘮 𝘱𝘦𝘳𝘦𝘯𝘯𝘦 (perennial ryegrass) is highly regulated and synchronised with the growth of the host plant leaf. To maintain this pattern of fungal growth 𝘪𝘯 𝘱𝘭𝘢𝘯𝘵𝘢, specific signalling between symbiont and its host grass is required. To sense the extracellular environment and respond to changes, filamentous fungi rely on G protein-coupled receptors (GPCRs), which transmit signals predominantly via heterotrimeric G proteins to downstream pathways such as the cAMP/Protein Kinase A (PKA) and MAP kinase signalling pathways. In phytopathogenic fungi, G protein signalling and the associated cAMP/PKA pathways are often essential for a normal host interaction. Signal transduction using the second messenger cAMP to activate the PKA activity is finely balanced through a regulatory feedback loop for signal attenuation regulated by 3’-5’-cyclic nucleotide phosphodiesterases (PDE).

Using a blast-based bioinformatic approach, a total of 40 genes encoding putative GPCRs were identified in the genome of 𝘌. 𝘧𝘦𝘴𝘵𝘶𝘤𝘢𝘦, grouping into 13 of 14 classes of the recent classification system for fungal GPCRs. Among genes encoding components of the G protein signalling cascade, nine of these GPCRs including the two cAMP receptor-like GPCRs, Gpr1b and Gpr2, show significant transcriptional up-regulation in association with the host compared to the respective expression level when grown in axenic culture. A reverse genetics approach was used to functionally characterise the identified cAMP receptor-like GPCRs (Gpr1a, Gpr1b and Gpr2). While deletion of 𝘨𝘱𝘳1𝘢 was unsuccessful, plants infected with an 𝘌. 𝘧𝘦𝘴𝘵𝘶𝘤𝘢𝘦 Δ𝘨𝘱𝘳1𝘣 mutant showed a severe breakdown of the 𝘌. 𝘧𝘦𝘴𝘵𝘶𝘤𝘢𝘦–ryegrass association, whilst no effects were observed for Δ𝘨𝘱𝘳2-inoculated plants. Among numerous other genes putatively involved in G-protein and cAMP/PKA signalling, two putative PDEs involved in regulation of the cAMP-mediated signal were also identified. Deletion of 𝘱𝘥𝘦𝘏 (Δ𝘱𝘥𝘦𝘏), a gene encoding a PDE with high affinity towards cAMP, had a dramatic effect on the endophyte-plant association. In contrast, deletion of pdeL (ΔpdeL), a gene encoding a PDE with low affinity towards cAMP, had no effect on the host interaction phenotype, while primarily modulating the intracellular cAMP level during nutrient-induced activation of the cAMP/PKA signalling pathway in axenic culture. Finely balanced cAMP levels are crucial for various cellular processes including hyphal growth, cellular differentiation of asexual development and conidiogenesis.

This research identified Gpr1b as an important receptor involved in maintenance of the highly restricted endophytic growth pattern of 𝘌. 𝘧𝘦𝘴𝘵𝘶𝘤𝘢𝘦 in association with the host, potentially regulated by plant-derived molecules. These signals are mediated by the cAMP/PKA signalling pathway for an appropriate alteration in gene expression and subsequent cellular adaptation. Intracellular cAMP levels triggering these concentration-dependent processes are regulated by PdeH, which specifically acts 𝘪𝘯 𝘱𝘭𝘢𝘯𝘵𝘢. This study shows that G-protein and cAMP/PKA signalling play an important role in regulating environmental signalling for establishment and maintenance of the mutualistic association between 𝘌. 𝘧𝘦𝘴𝘵𝘶𝘤𝘢𝘦 and 𝘓. 𝘱𝘦𝘳𝘦𝘯𝘯𝘦.