The role of Epichloë festucae boiA and boiB in symbiotic maintenance of endophyte-grass associations : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand

Abstract

The filamentous fungal endophyte Epichloë festucae and its plant host, perennial ryegrass, form a highly regulated mutualistic symbiosis. A key component in the regulation and maintenance of this symbiosis is the production of reactive oxygen species (ROS) by the NADPH oxidase (Nox) complex. Mutations in fungal Nox complex components lead to a disruption in the mutualistic association. In these mutants’ hyphae are often defective in cell-cell fusion and undergo proliferative growth, while the infected plant host is severely stunted. The fungal NoxA complex consists of membrane bound NoxA and NoxD components and cytosolic subunits NoxR, RacA, BemA and Cdc24, which are recruited to the membrane in response to some as yet unidentified signal. This study investigates the hypothesis that BemA is recruited to the membrane via interaction with a BOI1-like protein, known to interact with Bem1 in yeast. E. festucae has two BOI1-like proteins, designated BoiA and BoiB. Phylogenetic analysis suggested the ancestral Boi gene was duplicated in Letiomyceta after the whole genome duplication (WGD) in yeast which resulted in the presence of BOI1 and BOI2. Previous transcriptomic studies have shown boiA is highly expressed in planta compared to in culture and is differentially expressed in mutants that disrupt the mutualistic interaction, suggesting an important role for boiA in symbiotic maintenance. Microscopy analysis showed that in comparison to wild-type, ΔboiA deletion mutants displayed loss of hyphal cell-cell fusion in culture, while in planta ΔboiA mutants exhibited multiple hyphae per intercellular space, intrahyphal hyphae, and hyphal breakage. The resulting ΔboiA infected plants were stunted compared to wild-type infected plants. In contrast, no phenotype was observed for ΔboiB mutants in culture, though in planta intrahyphal hyphae and hyphal breakage was observed. These results suggest that BoiA is required for hyphal cell fusion and regulation of the mutualistic association between E. festucae and perennial ryegrass. While hyphal breakage suggests a role in intercalary growth, the novel mechanism by which E. festucae keeps up with rapid host growth in planta. This study provides an in-depth analysis on the origin and function of E. festucae BoiA and BoiB in culture and in planta.