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Item Characterising CG5846 (Peep) in Drosophila melanogaster neural function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2024) Wilson, Sarah JeanHistone deacetylase 4 (HDAC4) is a transcriptional regulator that has been implicated in a number of neurodevelopmental and neurodegenerative diseases that are associated with intellectual disability, cognitive defects, and/or memory loss. Both the accumulation of nuclear HDAC4 and its loss-of-function have been linked to these conditions, therefore exploring HDAC4’s role in neuronal function is essential to understand the molecular mechanisms underlying these diseases. In Drosophila, overexpression of HDAC4 results in defects in morphogenesis of axons in the mushroom body, a structure essential for memory formation, as well as long-term memory defects and disruption to the development of the compound eye. The molecular mechanisms underlying these HDAC4-induced phenotypes are currently unknown. RNA-sequencing on fly heads in which HDAC4 was overexpressed has previously been performed and showed few genes were transcriptionally regulated by HDAC4. In addition, an enhancer/suppressor rough eye phenotype screen has also been performed which identified a number of genes that interact genetically in the same molecular pathway as HDAC4. To further investigate the molecular mechanisms underlying HDAC4 dysfunction, an RNA interference (RNAi) based candidate screen for potential HDAC4-interactors was performed, which involved quantification of developmental defects in the mushroom body and eye following RNAi knockdown of each candidate. It was hypothesised that if a phenotype resulting from RNAi knockdown was similar to that induced by HDAC4 overexpression, that candidate may function in similar molecular pathways. A single candidate-interactor was selected (CG5846, named Peep) for further investigation. On overexpression, Peep and HDAC4 co- distribute in nuclei of mushroom body neurons, however no physical interaction was detected. Furthermore, overexpression of Peep did not rescue the HDAC4-induced mushroom body or eye defects. Due to the uncharacterised nature of Peep, a thorough investigation was performed to assess the importance of Peep in survival, longevity, motor function, brain development, courtship learning and memory, and wing development. Peep was observed to be essential for survival of glial cells and for normal mushroom body development, which warrants further investigation. Reduced expression of Peep also resulted in a unique severe necrotic eye phenotype, and through this, Peep was shown to play a potential role in processes involved in regulating mitochondrial and proteasomal function, apoptosis and oxidative stress. These data provide the first documented characterisation of the functional role of Peep in Drosophila development and provide the basis for further investigation into the underlying molecular mechanisms involved in mushroom body and eye development.Item Investigating HDAC4 aggregation in a Drosophila model of neuronal development : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2024-06-21) Hawley, Hannah RoseHistone deacetylase four (HDAC4) is essential in neuronal development and function, and dysregulation of HDAC4 has been observed in a number of neurodevelopmental and neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. In particular, its aberrant nuclear accumulation is a common feature among these diseases, and it has been observed that upon upregulation or accumulation in the nucleus, HDAC4 forms punctate foci in neuronal nuclei. Previous research in a Drosophila model determined that overexpression of HDAC4 disrupted both neuronal development and long-term memory, and this was largely mediated by the nuclear pool of HDAC4. Based on these data, it was hypothesised that aggregates of HDAC4 are responsible for the neurotoxicity that leads to disrupted neurodevelopment and memory. Therefore, this study aimed to determine whether the presence of HDAC4 nuclear aggregates correlated with neurodevelopmental deficits in a Drosophila model of neurodevelopment, and if so, how they mediate their toxic effects. The N-terminus of HDAC4 forms homo-oligomers in solution, and it was hypothesised that full-length HDAC4 similarly oligomerises, and that this is required for its aggregation in neuronal nuclei. Mutations predicted to prevent oligomerisation were introduced into the N- terminus of HDAC4 and were shown to significantly reduce aggregation of HDAC4 in Drosophila neurons. Furthermore, their presence also reduced the severity of HDAC4 overexpression-induced impairments in neurodevelopment. Conversely, stabilisation of oligomerisation increased aggregation and the severity of neurodevelopmental phenotypes, together indicating that aggregation positively correlates with the severity of neurodevelopmental deficits. HDAC4 aggregates have been previously shown to sequester the transcription factor MEF2, and further investigation revealed that the presence of MEF2 stabilised aggregation and increased the severity of defects in neuronal development. Importantly, targeting the interaction between HDAC4 and MEF2 reduced the severity of these defects. Other than MEF2, the composition of HDAC4 aggregates is unknown, and therefore immunoprecipitation-coupled mass spectrometry was performed on nuclear HDAC4 to identify candidate interactors of aggregates. This revealed a number of proteins with roles in neuronal development and function, as well as those involved in splicing and protein homeostasis, suggesting that aggregates may be impairing these processes to mediate toxicity. Together these data indicate that nuclear aggregation of HDAC4 impairs neurodevelopment, and may constitute a novel biomarker of disease or therapeutic target. Given the overlap in aetiology between neurodevelopmental and neurodegenerative diseases, further investigation of whether HDAC4 aggregation contributes to the severity and/or progression of neurodegenerative disorders is warranted.Item Identification and characterisation of novel virulence factors from Dothideomycete pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand(Massey University, 2023) McCarthy, HannahThe Dothideomycetes class of fungi contains some of the most important plant pathogens, including Dothistroma septosporum and Fulvia fulva. Dothistroma needle blight, caused by D. septosporum, is a devasting disease of pines that has been increasing in severity and incidence worldwide. Tomato leaf mould, caused by F. fulva, has recently risen in importance after overcoming resistance breeding efforts. There is now an urgent need to identify novel genes from these pathogens that encode virulence factors, such as effector proteins. These proteins contribute to pathogen virulence, so the study of virulence factors not only enables a deeper understanding of how pathogens and their hosts interact at the molecular level, but also provides information that may lead to the development of new methods for disease control. As such, the aim of this thesis was to identify and characterise new candidate virulence factors from D. septosporum and F. fulva. Candidate virulence factor CfEcp11-1 from F. fulva, previously identified to trigger plant defences in wild tomato cultivars, was a particular focus in Chapter 2. Two new homologues of CfEcp11-1 were identified from Fusarium oxysporum and suggested to be part of the same Leptosphaeria AviRulence and Suppressing (LARS) effector family. Recognition of CfEcp11-1 by the tomato receptor was also examined through the design of chimeric and mutant protein sequences. CRISPR/Cas9 was used for the first time in F. fulva to disrupt CfEcp11-1 and generate single- and double-copy mutants; to the best of our knowledge this was the first report of multiple gene copy disruption by CRISPR/Cas9 in a fungal pathogen. In Chapter 3, candidate virulence factors from D. septosporum and F. fulva were identified through a prediction pipeline, which selected proteins with predicted roles as effectors that manipulate plant defences, or as transcription factors which regulate the expression of other genes. Because D. septosporum and F. fulva are hemibiotrophic pathogens, they transition from colonising living plant tissue to killing and feeding on dead tissue, a transition termed the necrotrophic switch. It is currently unknown what mechanisms govern this important disease process, so a key focus was on candidate virulence factors with a possible role in the necrotrophic switch. In Chapter 4, some of these candidates were further characterised, and new candidates identified, from proteomic analysis of the culture filtrates of D. septosporum and F. fulva grown in different conditions. Existing transcriptomic data were used to assess which of these proteins were likely to be functional in planta. Among those identified were several characterised effectors, such as Cf/DsEcp2-1, Cf/DsEcp20-1, Cf/DsEcp20-3, and CfAvr4E, which were secreted in culture despite having known functions in planta. Novel candidate virulence factors from D. septosporum and F. fulva were also identified in this analysis, including a Nis1 domain-containing protein from D. septosporum with a possible role in the necrotrophic switch. This analysis illustrates that in culture proteomic analysis can be a useful tool for the identification of candidate effector proteins. In Chapter 5, two candidate virulence factor genes of D. septosporum with predicted roles in the necrotrophic switch were disrupted through CRISPR/Cas9 gene editing; this was the first use of this method for gene disruption in D. septosporum. The disruption mutants were tested for virulence on the P. radiata host. One of the mutants was disrupted in DsCE3, which was suggested to be a virulence factor, with a possible role in the necrotrophic switch. Overall, the results presented in this thesis have provided new research methodologies as well as valuable knowledge about the molecular tools these two pathogens use to invade their hosts. Whilst further work is required, these developments will ultimately aid future disease control strategies in pine and tomato.Item Investigating the role of HDAC4 in Drosophila neuronal function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand(Massey University, 2022) Tan, Wei JunHDAC4 plays an essential role in brain functions including neurodevelopment and memory formation, and increased levels of HDAC4 have also been associated with neurodegenerative disorders including Alzheimer’s disease. Histone deacetylases are enzymes that are traditionally known to regulate gene expression in the nucleus, however in neurons, HDAC4 shuttles between the nucleus and cytoplasm with a predominant distribution in the cytoplasm. Although studies have identified potential differences in subcellular function in which accumulation of nuclear HDAC4 has been shown to promote neurodegeneration, while cytoplasmic HDAC4 is neuroprotective, the mechanistic pathways through which it acts are still unknown. Therefore, this project aimed to determine the importance of nuclear and cytoplasmic pools of HDAC4 to the neurological functions of Drosophila melanogaster, as well as to determine the domains within the protein that are required for its function(s). This was carried out by expressing HDAC4 with mutations that resulted in altered subcellular distribution or carrying mutations in binding domain/motifs that have previously been shown to be important for HDAC4 function. Increased expression of wild-type HDAC4 disrupted development of the retina and the mushroom body (MB, a brain structure derived from Kenyon cells which are crucial for learning and memory), and expression of each mutant revealed the importance of specific domains/motifs to HDAC4 function in these tissues. Of interest, impairments to MB formation were exacerbated by mutation of the ankyrin-binding site and by mutation of serine residues that promote nuclear exit when phosphorylated (i.e. resulting in restriction to the nucleus). Mutation of the MEF2-binding site ameliorated these phenotypes, suggesting that HDAC4 acts through MEF2 to regulate MB development. However, while deacetylase activity was found to be dispensable in the MB, an active deacetylase domain was required in order for the phenotype to manifest in the retina, and mutation of the MEF2-binding site had no impact on the deficits caused by nuclear restriction of HDAC4 and mutation of the ankyrin-binding domain. Together these data indicate that HDAC4 acts through varying mechanism(s) depending on the cell type. Transcriptional changes in the Drosophila brain resulting from the expression of HDAC4 or its mutant variants was also explored using RNA-Seq. However only wild-type HDAC4 resulted in a large number of differentially expressed genes and the low level of differential gene expression in HDAC4 variants suggests that non-transcriptional processes may be involved in the induction of phenotypes caused by expression of these mutants. Additionally, further analysis of genes that were differentially regulated revealed a number of processes related to mitochondrial energy production. These findings have provided new insights into the role of HDAC4 in Drosophila neurodevelopment which opens up additional research avenues to focus on in the future.Item Identification of novel avirulence effectors in the Dothideomycete plant pathogens, Venturia inaequalis and Cladosporium fulvum : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Sciences at Massey University, Manawatū, New Zealand(Massey University, 2022) de la Rosa, SilviaVenturia inaequalis and Cladosporium fulvum are important fungal pathogens of crop species, causing scab and leaf mould disease of apple and tomato, respectively. Resistance to these pathogens is governed by Rvi (apple) and Cf (tomato) resistance (R) genes. These R genes encode immune receptors that recognize specific pathogen virulence factors, termed avirulence (Avr) effectors, to activate plant defenses. Notably, isolates or strains of V. inaequalis and C. fulvum have emerged that can overcome resistance mediated by specific R genes in their respective hosts. To better understand how these pathogens cause disease or overcome resistance, and to monitor the occurrence of resistance-breaking isolates or strains in the field, Avr effectors from V. inaequalis and C. fulvum must be identified and functionally characterized. Using a combined comparative genomics and phenotyping approach based on progeny from a sexual cross between V. inaequalis isolates that differ in their ability to overcome Rvi4 resistance in apple, a strong candidate for the corresponding AvrRvi4 effector gene was identified (Chapter 2). Similarly, using a comparative genomics approach based on in planta-expressed effector candidates from C. fulvum strains that differ in their ability to overcome Cf-9B resistance in tomato, combined with functional assays, the corresponding Avr9B effector gene was identified (Chapter 4). In the resistance-breaking isolates or strains studied, the candidate AvrRvi4 gene was disrupted, while the Avr9B gene had been deleted. Consistent with most fungal Avr effectors and their genes, both the AvrRvi4 candidate and Avr9B are highly expressed in planta, and encode small, secreted cysteine-rich proteins. The AvrRvi4 candidate forms part of an expanded protein family in V. inaequalis, with members predicted to adopt a β sandwich fold similar to structurally characterized fungal effectors. Avr9B, however, is predicted to adopt a novel protein fold. Finally, using a heterologous expression approach, three in planta-expressed candidate effectors from V. inaequalis were found to trigger defense responses in non-host plants (Nicotiana spp.), suggesting they are recognized by R proteins in these species (Chapter 3). Taken together, this thesis has increased our understanding of the molecular mechanisms responsible for the activation and circumvention of resistance by V. inaequalis and C. fulvum, which will in turn direct host cultivar deployment and disease control strategies in the field.Item Molecular and immunological analysis of New Zealand isolates of Mycoplasma ovipneumoniae : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand(Massey University, 2021) Bridgeman, BenjaminMycoplasma ovipneumoniae is a pathogen of caprinae expected to infect the majority of domestic sheep in early life. M. ovipneumoniae is suspected to be the primary cause of chronic non-progressive pneumoniae (CNP) in lambs. Currently there is no effective vaccine against M. ovipneumoniae, and many commonly used antibiotics have limited effect. Diagnosis of M. ovipneumoniae infections can be difficult, due in part to the limited overt symptoms seen in cases of CNP. Despite the significance of M. ovipneumoniae infections there is limited information on the molecular and immunological data of M. ovipneumoniae. Thus, the major focus of this thesis was to identify potential immunogenic proteins for a vaccine or diagnostic purposes and construct genomes from New Zealand isolates of M. ovipneumoniae. Draft genomes were produced for three New Zealand isolates of M. ovipneumoniae, (isolates 16, 90, and 103), and annotated. Using antisera produced against these isolates, immunogenic proteins were identified from hydrophobic membrane protein fractions of M. ovipneumoniae using Western blotting. Those protein bands were subjected to mass spectrometry analysis, and by comparing mass spectrometry data to the genomes, six novel proteins, GAPDH, P146, MATE, hypothetical protein 1, hypothetical protein 2, hypothetical protein 3, and two previously described immunogenic proteins, EF-Tu and HSP70, were identified. Five of the proteins were produced as recombinant proteins for further study. The nucleotide sequence of EF-Tu was edited using an overlap polymerase chain reaction (PCR) to convert the sequence to Escherichia coli codon preference. This sequence was then cloned into the pET22b (+) vector for expression of histidine-tagged fusion protein. Additionally, EF-Tu, GAPDH, P146, hypothetical protein 3 and an epitope of hypothetical protein 1 were produced as recombinant proteins by GenScript. Antisera were produced in sheep against each of the recombinant proteins for further studies. Enzyme-linked immunosorbent assay (ELISA) confirmed that each protein was immunogenic and elicited antibody responses in sheep. Immunofluorescence microscopy confirmed the localisation of native EF-Tu and GAPDH proteins on the surface of M. ovipneumoniae cells. Antisera produced against whole cell antigens from M. ovipneumoniae cross-reacted with P146 and hypothetical protein 3 suggesting a potential role for these proteins to be used to detect animals infected with M. ovipneumoniae. Sheep vaccinated with M. ovipneumoniae produced weak IFN-γ responses but stronger IL-17 responses in whole blood cultures stimulated with whole cell antigens. Of the five proteins tested as recall antigens in the IL-17A assay, recombinant GAPDH produced a strong IL-17A response in sheep vaccinated with M. ovipneumoniae suggesting a potential role for GAPDH in a diagnostic assay for measuring IL-17A responses in M. ovipneumoniae infected sheep. Mycoplasmas are known to adhere to host epithelial cells as part of the pathogenesis process. A bovine endometrial epithelial cell line was established as a model to study adherence of M. ovipneumoniae to epithelial cells and determine if antibodies against the surface proteins could interfere with adherence. M. ovipneumoniae cells adhered to the endometrial epithelial cells. Adherence was inhibited by antibodies directed against whole cells of M. ovipneumoniae but not against the recombinant proteins.Item Establishing systems to characterise MH pathogenic RyR1 variants : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry(Massey University, 2021) Stephens, JeremyMalignant hyperthermia (MH) is a potentially fatal, autosomal dominant, metabolic disorder triggered in susceptible individuals upon exposure to volatile anaesthetics. Following the onset of an MH episode, a patient will enter a hypermetabolic state, displaying the symptoms of intense muscle contraction, metabolic acidosis, increased oxygen consumption. Prolonged episodes can result in rhabdomyolysis. If left untreated, MH can manifest as an increase in body temperature and death by cardiac arrest. MH is diagnosed by the invasive in vitro contracture test, which requires a muscle tissue biopsy. DNA screening has been implemented and is commonly used to diagnose a genetic predisposition to MH; however, the test is currently limited to fifty variants confirmed to be pathogenic out of approximately 350 variants linked to the disorder. DNA-based tests are limited because of the technical difficulties associated with functional analysis. Thus, additional variants must be functionally characterised. The structural implications of MH-linked variants potentially leading to the onset of MH are not yet well defined. Potential structural changes induced by pathogenic variants have been modelled in silico, where variants were mapped to the rabbit RyR1 structure characterised by cryo electron microscopy. However, this does not confirm the role the variants play in the structural and functional alteration of the channel. To address, this a functionally significant region of RyR1 was cloned for recombinant expression in E. coli. The RyR1 region was shown to be soluble and efforts were made to purify the protein. However, the protein could not be purified to an extent acceptable for either biochemical analysis or crystallisation trials or for subsequent X-ray crystallography. A number of pathogenic variants were instead modelled in silico to provide some insights into their potential pathogenic functional role. The viability of a new cell-based system for the functional characterisation of variants was also tested. Patient derived myoblasts were immortalised using lentivirus transduction with the cDNA for human telomerase and cyclin dependent protein kinase 4. The genome editing tool CRISPR Cas 9 was then used to successfully introduce the pathogenic variant c.14497C>T p.his 4833 tyr into the genome of MH negative myoblasts. Functional characterisation of the introduced variant has yet to be performed.Item Recovery of α-lactalbumin from whey protein isolate and osteopontin from milk by anion exchangers : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Chemistry at Massey University, New Zealand(Massey University, 1998) Chen, YishanA series of amines, DMEA, DMH, DMO, DMD, DMDo, and Do, were coupled to Sepharose, which was activated by epichlorohydrin first, to prepare amino anion exchangers DMEA-Seph, DMH-Seph, DMO-Seph, DMD-Seph, DMDo-Seph, and Do-Seph. The batch binding of α-lactalbumin and ß-lactoglobulin in 25 mM NaCl at around proteins' IEP to these exchangers and a commercial anion exchanger Q-Seph-ff were tested. Q-Seph-ff, DMEA-Seph, DMH-Seph, and DMO-Seph bound both of proteins at pH > IEP. Q-Seph-ff, DMEA-Seph, DMH-Seph did not bind either of the proteins at pH < IEP. DMO-Seph, DMD-Seph, DMDo-Seph, and Do-Seph bound both of the proteins, especially α-lactalbumin, by hydrophobic interaction at and below the proteins' IEP. The proteins bound by these exchangers except DMO-Seph could not eluted by HCl at low pH. HCI at pH 2.5 could be used to elute these proteins from DMO-Seph. Recovery of α-lactalbumin from WPI in 25 mM NaCl at pH 3.8-5 by DMO-Seph, DMD-Seph, DMDo-Seph, and Do-Seph were tested. These exchangers were able to bind α-lactalbumin in preference to ß-lactoglobulin at and below the proteins' IEPs. Thus DMD-Seph gave an α-lactalbumin yield and purity of 70 and 80% at pH 4.3, DMO-Seph 77 and 81% at pH 4.4. However DMD-Seph had difficulty in eluting all of the α-lactalbumin unless using ethanol. The batch binding of α-lactalbumin and ß-lactoglobulin in high concentration of NaCl at low pH (2.5) by DMO-Seph was tested. The exchanger showed strong hydrophobic affinity for a-lactalbumin but not ß-lactoglobulin in 200-500 mM NaCl. Recovery of native α-Iactalbumin from WPI in 400 mM NaCl at pH 2.5 by DMO-Seph was tested. This gave an α-lactalbumin yield and purity of 79 and 73% and a capacity of DMO-Seph 0.73 g/g in 400 mM NaCl at pH 2.5, compared to 67%, 84% and 16 mg/g of DMO-Cell. DMO-Cell was prepared. Cellulose was modified by propyl oxide and epichlorohydrin and then activated by ECH. The activated cellulose was then coupled with DMO. The effects of cellulose particle size, cellulose type and substitution level of DMO-Cell on binding of whey proteins were investigated. DMO-Cell, activated by 1,4-butanediol diglycidyl ether, with substitution level 0.55 meq/g was prepared. It showed a better binding capacity than DMO-Cell activated by epichlorohydrin. Recovery of native α-lactalbumin from different WPI in 400 mM NaCl at pH 2.5 by DMO-Cell was tested. DMO-Cell showed good selectivity for α-lactalbumin from all of three WPI. This gave an α-lactalbumin yield and purity of 13.5 mg/g from WPI (PT8253). From a low α-lactalbumin content WPI this gave an α-lactalbumin yield and purity of 70 and 91%.Item Identification of mechanisms defining resistance and susceptibility of Camellia plants to necrotrophic petal blight disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand(Massey University, 2019) Kondratev, NikolaiSpecies in the genus Camellia, which includes the tea crops, oil-producers and valuable ornamental plants, have economic and cultural significance for many countries. The fungus Ciborinia camelliae causes petal blight disease of Camellia plants, which has a short initial asymptomatic phase and results in rapid necrosis and fall of blooms. Ciborinia camelliae is a necrotrophic pathogen of the family Sclerotiniaceae, which also includes two broad-host range necrotrophic pathogens, Botrytis cinerea and Sclerotinia sclerotiorum. Previously it was shown that some Camellia plants, such as Camellia lutchuensis, are naturally resistant to petal blight. In order to find molecular mechanisms underpinning this resistance, a genome-wide analysis of gene expression in C. lutchuensis petals was conducted. The analysis revealed a fast modulation of host transcriptional activity in response to C. camelliae ascospores. Interaction network analysis of fungus-responsive genes showed that petal blight resistance includes increased expression of important plant defence pathways, such as WRKY33-MPK3, phenylpropanoid and jasmonate biosynthesis. A much-delayed activation of the same pathways was observed in the susceptible Camellia cultivar, Camellia ‘Nicky Crisp’ (Camellia japonica x Camellia pitardii var. pitardii), suggesting that failure to activate early defence enables C. camelliae to invade and cause tissue necrosis. Early artificial induction of defence pathways using methyl jasmonate reduced the rate of petal blight in susceptible ‘Nicky Crisp’ plants, further verifying the role of a rapid defence activation in petal-blight resistance. Overall, transcriptomic and functional analysis of the Camellia spp.- C. camelliae interaction demonstrated that the same plant defence pathways contribute to both resistance and susceptibility against this necrotrophic pathogen, depending on the timing of their activation. To further understand the molecular mechanisms of petal blight resistance, the role of the phenylpropanoid pathway, identified as a key feature in the transcriptome study above, was investigated in more detail. This pathway produces various metabolites, including phenolic acids, aldehydes, and alcohols, which have numerous physiological functions and also participate in the production of flavonoids and lignin. Resistant C. lutchuensis was shown to rapidly activate the expression of core phenylpropanoid genes after treatment with C. camelliae ascospores. LC-MS-based quantification of phenylpropanoid compounds demonstrated that within the first 6 h of the infection, resistant plants had already accumulated coumaric, ferulic and sinapic acids, while at 24 hpi, concentrations of coumaraldehyde, sinapaldehyde, and caffeyalcohol were significantly increased. Thus, I further hypothesized that the compounds produced by the phenylpropanoid pathway may have fungistatic activity. Indeed, all tested phenylpropanoids inhibited the growth of C. camelliae in agar plates with different efficacy. Moreover, the application of phenylpropanoid compounds, including ferulic and coumaric acids, fully prevented the formation of petal blight lesions on susceptible Camellia ‘Nicky Crisp’ petals. Taken together, it can be concluded that the phenylpropanoid pathway may contribute to the early defence against the petal blight via the rapid production of fungistatic compounds. Ultimately, these compounds could be used to develop natural antifungal sprays to protect susceptible Camellia flowers. The analysis of the C. camelliae secretome using LC-MS/MS detection of proteins showed that the pathogen produces a large number of carbohydrate-active enzymes in liquid culture and plant petals. Injection of these proteins induced necrosis not only in susceptible Camellia petals but also in petals of the resistant species and leaves of non-host Nicotiana benthamiana. It was proposed that these enzymes can contribute to the virulence of the pathogen by inducing cell death and facilitating necrosis propagation. Thus, the early defence responses of resistant Camellia plants may possibly stop the development of C. camelliae before it starts releasing carbohydrate-active enzymes during the necrotrophic step of the infection. Overall, the results of this research further expand our understanding of plant- necrotroph interactions, suggesting that the timing of plant immune responses may be a crucial factor defining the outcome of the necrotrophic infection.Item Investigating the role of HDAC4 subcellular distribution in Drosophila development and memory : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2019) Main, Patrick JamesThe class IIa histone deacetylase HDAC4 has been previously demonstrated to play an essential role in brain development, learning and memory. However, the molecular pathways through which it acts are unknown. HDAC4 undergoes activity-dependent nucleocytoplasmic shuttling, disruption of the balance of nuclear and cytoplasmic HDAC4 has been identified as a factor in developmental and neurodegenerative disorders. This project used Drosophila melanogaster as a model to investigate the effects of altered subcellular distribution of HDAC4 on neural development and memory formation through the overexpression of Drosophila HDAC4 and wild-type human HDAC4 (hHDAC4), as well as nuclear- and cytoplasm-localising mutants of hHDAC4 named 3SA and L175A, respectively. The nuclear or cytoplasmic abundance of HDAC4 was adjusted by expressing the mutants during development or in adult flies. It was established that increased nuclear abundance of hHDAC4 in the brain impaired long-term memory and development, whereas increasing the cytoplasmic abundance did not. Further investigation showed that, contrary to vertebrate models, HDAC4 does not appear to repress memory in Drosophila through inactivation of MEF2 or CREB. Investigation of the transcriptomic changes induced by nuclear and cytoplasmic HDAC4 via RNASeq on RNA isolated from fly heads showed that L175A unexpectedly up-regulates the expression of genes in transcription and DNA synthesis. The relatively low number of transcriptional changes induced by 3SA suggested that it may be acting through largely transcriptionally independent means to impair memory and development in Drosophila. The localisation of HDAC4 to punctate foci in nuclei, potentially forming protein aggregates similar to Marinesco bodies seen in Parkinson’s Disease warrants further investigation. This project has shown that nuclear but not cytoplasmic HDAC4 impairs development and memory in Drosophila. Furthermore, cytoplasmic HDAC4 may play a role in transcriptional regulation of neurons, possibly regulation metabolic activity, suggesting that the activity-dependent nucleocytoplasmic shuttling of HDAC4 may not be primarily to remove HDAC4 from the nucleus and but instead to return HDAC4 to the cytoplasm.