Browsing by Author "Bradley, Ellie Lynn"

Now showing 1 - 2 of 2

Identification and functional characterisation of glycoside hydrolases from the kauri dieback pathogen, Phytophthora agathidicida : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science at Massey University, Manawatū, New Zealand
(Massey University, 2022) Bradley, Ellie Lynn
The survival of kauri, an ancient conifer species endemic to New Zealand, is currently threatened by kauri dieback disease, caused by the oomycete plant pathogen Phytophthora agathidicida. As P. agathidicida continues to spread throughout kauri forests in the northern North Island of New Zealand, encouraging research has indicated there may be natural tolerance to the disease within the kauri population. This resistance is likely governed, in part, by the plant immune system, which is activated upon recognition of pathogen invasion patterns such as microbe-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs), and effectors (virulence factors required for host colonisation), which are recognised at the plant cell surface by plant immune receptors. To better understand how P. agathidicida interacts with its plant host on a molecular level, pathogenproduced proteinaceous invasion patterns need to be identified and characterized to aid in the identification of cognate immune receptors in the plant host, which may be involved in activation of the plant immune system. As the role of glycoside hydrolase (GH) proteins in virulence and pathogenicity of fungal and oomycete plant pathogens is well established (Chapter two), an effectoromics approach was used to identify six P. agathidicida GH12 proteins that appear to act as MAMPs in both Nicotiana benthamiana and Nicotiana tabacum (Chapter three). Furthermore, nuclear magnetic resonance was used to identify considerable changes in kauri leaf apoplastic wash fluid of approximately 17 metabolites, including sucrose and glucose, in response to P. agathidicida inoculation (Chapter four), thus suggesting a role for GH proteins in the hydrolysis of some of these metabolites. Finally, proteomic analysis of P. agathidicida culture filtrates via liquid chromatography-mass spectrometry (LC–MS) was used to validate the expression of predicted P. agathidicida proteins and to investigate the capacity of this method to identify candidate invasion patterns for future analysis. Chapter five established that LC–MS analysis of Phytophthora culture filtrate was an effective method for the identification of putative apoplastic invasion pattern candidates and confirmed the production of all six P. agathidicida GH12 cell death elicitors in culture. Collectively, this thesis has advanced our understanding of the molecular mechanisms underpinning the interaction of P. agathidicida with its host and has contributed to the identification of candidate apoplastic effectors.
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
(Massey University, 2018) Bradley, Ellie Lynn
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.