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    Towards better New Zealand adapted industrial hemp (Cannabis sativa L.) cultivars : a quantitative genetic analysis of key traits and evaluation of genetic diversity : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand
    (Massey Library, 2023-09-12) Komahan, Dona Harshani Shanika
    Hemp (Cannabis sativa L.) is emerging as a promising commercial crop in New Zealand. Breeding programmes developing material specifically adapted to the country’s target population of environments are at their early stages of establishment. With 20 approved hemp cultivars, 15 of which are imported, it is vital to prioritize research and development focused on the genetic improvement of hemp to generate cultivars that are well adapted to New Zealand’s crop production environments, offering both economic and agronomic benefits. The primary aim of this thesis is to lay the foundation for future hemp breeding in New Zealand by focusing on three critical initial steps: (1) conducting multi-site trials to evaluate the performance of commercial hemp cultivars based on key traits and characterize the effects of Genotype × Environment interaction (G × E interaction), (2) estimating quantitative genetic parameters and predicting genetic gain for key traits to identify breeding strategies that would improve the efficiency of maximizing genetic gain, and (3) assessing genetic diversity using molecular markers and characterizing available genetic resources. These steps are crucial for developing new cultivars that are both climate-resilient and beneficial to New Zealand’s hemp industry. Multi-site trials were conducted for two years over the 2019/2020 and 2020/2021 growing seasons using six hemp cultivars approved to be grown in New Zealand: CFX-2, CRS-1, Ferimon 12, Katani, Futura 75, and Finola. The 2019/2020 trials were conducted in Palmerston North and Wairarapa, while the 2020/2021 trials were conducted at two nearby sites in Palmerston North with different soil characteristics. Across both multi-site trials, biomass yields extend over a broad range from 1.43 to 28.41 t/ha, and seed yields ranged between 0.018 and 3.78 t/ha. The study’s findings on G × E interactions showed a complex scenario that mostly differed from prior reported research. Although significant (P < 0.05) genotypic variation was observed in most traits, only a few, specifically stand establishment in the 2019/2020 season, along with plant height and stem diameter in the 2020/2021 growing season and biomass yield across both years, showed significant (P < 0.05) G × E interactions, suggesting consistent performance across sites. The traits plant height, stem diameter, biomass yield, seed yield, and thousand seed weight have all revealed high cultivar mean broad sense heritability, indicating potential exploitable underlaying genetic variation that could be used to generate breeding pools for cultivar development. A quantitative genetic analysis was conducted using 50 half-sib families (HS families) focused on six key traits: stem diameter, plant height, number of internodes, seed yield, thousand seed weight, and biomass yield to investigate the magnitude of additive genetic variation within a breeding population. The study also aimed to predict the rate of genetic gain for these traits. Significant (P < 0.05) additive genetic variation was observed among the 50 HS families for traits: seed yield, biomass yield, stem diameter, plant height, and the number of internodes, highlighting each trait’s additive genetic variation available for breeding. Deterministic simulation of the breeding strategies, among HS family selection (AF-HS) and among- and within-family selection (AWF-HS), was conducted to predict genetic gain for these traits. AWF-HS yielded the highest genetic gains compared to AF-HS. The study estimated moderate to strong positive genetic correlations among all traits, generally higher than the phenotypic estimates. The genetic correlation coefficients suggest that genotypes with higher seed and biomass yields can be developed. There was significant (P < 0.05) differences between some HS families and the two commercial check cultivars, Fasamo and Férimon 12. HS families outperforming these commercial checks were identified. A laboratory experiment examined the genetic diversity and the degree of variability within and among six New Zealand-approved hemp cultivars using seven previously developed microsatellite (SSR) markers. STRUCTURE analysis identified two distinct genetic clusters that align with the plant’s reproductive biology (monoecious and dioecious), that further subdivided based on end-use (fibre, seed, fibre and seed, and CBD cultivars). The genetic diversity metrics revealed moderate genetic diversity among the six hemp cultivars. The average observed heterozygosity (Ho) and the expected heterozygosity (He) were 0.44 and 0.50, respectively. The Wright’s fixation index (FIS) varied from 0.26 (Futura 75) to -0.02 (Finola). The Analysis of Molecular Variance (AMOVA) revealed that a large portion of the total genetic diversity was found within individual cultivars. While only 11% of the molecular variation was attributed to differences among the cultivars, 19% was attributed to variations among individuals within each cultivar. Notably, the genetic variation within individual plants (69%) exceeded the variation observed both among individuals and among different cultivars. Additionally, considerable molecular variation was observed between male and female individuals of dioecious hemp cultivars. The genetic diversity metrics of female and male groups indicated that female individuals possess greater variation than their male counterparts. Collectively, results from this Ph.D. study lay the groundwork for future hemp breeding research and development in New Zealand. They provide an initial resource, offering insights into genetic diversity, genotypic variation, and the impact of G × E interactions on key traits evaluated among six introduced offshore cultivars. Furthermore, the estimates of quantitative genetic parameters add value to the existing knowledge base, enhancing the development of hemp cultivars suited to New Zealand crop production environments.
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    Spoilage bacteria in ewe milk and the sheep dairy farm environment : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science/Microbiology at Massey University, Manawatū, New Zealand
    (Massey University, 2023-03) Risson, Alexis
    Milk spoilage bacteria are capable of impacting the stability, processability, and overall quality of dairy products. These contaminants are highly diverse and can be found at various stages of the dairy production, but the majority originate from the dairy farm environment where they contaminate raw milk. On bovine dairy farms, research has identified several routes of transmission of spoilage microorganisms into raw milk, as well as some of the factors that modulate this process. This knowledge has aided the design, development and implementation of interventions aiming to limit the on-farm contamination of raw milk and thereby improve the microbiological quality of cow dairy products. In contrast, the microbial communities associated with the ovine dairy production have been largely overlooked. This lack of understanding of the ecology and transmission of spoilage bacteria on sheep dairy farms and in dairy products hinders attempts at reducing the farm-borne contamination of ewe milk by spoilage organisms. In this thesis, I set out to identify and characterise the microbial communities associated with the ovine dairy production, with a focus on bacteria capable of impacting ewe milk quality. In the first part, I compiled, developed, and subsequently used an extensive set of culture methods targeting the main types of bacteria implicated in dairy spoilage, namely spore-forming bacteria, and Pseudomonas species. These methods were applied to survey the microbial communities present in the farm environment of dairy ewes that were grazed or housed. For the first time, this approach provides a detailed overview of the ecology of spoilage bacteria found on sheep dairy farms and enables the identification of numerous bacterial species with spoilage potential. The diversity and principal reservoirs of spoilage bacteria were found to vary between the two farms, possibly as a result of their farming practices. In the second part, I performed source-tracking of the contaminants isolated from milking cups using phylogenetic tools such as DNA fingerprinting and whole-genome sequencing. According to this approach, sources of milking cup, and thus possibly raw milk contamination were found to vary between the two farms. In the barn environment, the silage-faeces axis was identified as the main source of spoilage bacteria in the wider farm environment and in milking cups. There was also some contribution, albeit minimal, from bedding materials and animal drinking water as potential intermediaries. In the pasture environment, the transmission of bacteria in the environment was low, but the origin of the contaminants found in milking cups was multiple, dependent on the bacteria considered, and included soil, pasture, animal faeces and drinking water. In addition, the phylogeny of Thermoactinomyces spp., which were found to be particularly abundant in both housing and grazing environments, was studied in more depth using whole-genome sequencing. Finally, I surveyed the microbiological quality of ewe raw milk samples and sheep dairy products produced in New Zealand using the culture methodology developed for the farm studies. This work provided a glimpse of the diversity and abundance of the spoilage microbiota of sheep milk and dairy products produced in New Zealand. It was found that the microbiological quality of sheep dairy products was high, however, the presence of a highly diverse range of spoilage bacteria highlights their potential to impact the quality of a variety of dairy products.
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    Phylogenomics and evolution of polyploid Azorella (Apiaceae) in New Zealand : 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, 2023) Ning, Weixuan
    Polyploid plants have more than the usual two sets of chromosomes in every cell. Analysing the macroevolutionary patterns of polyploid plants can provide further insight into the mechanisms of polyploidization or whole genome duplication (WGD) in driving species diversification. The polyploid-rich lineage, Azorella, in New Zealand (NZ) has two sections, Schizeilema and Stilbocarpa, with a total of 17 described polyploid taxa (species, subspecies, or varieties) in three known ploidy levels (4x, 6x and 10x). The divergent leaf morphologies and distinct distribution range of polyploid taxa in NZ Azorella makes this lineage an ideal system to investigate the macroevolutionary outcomes of WGD in a polyploid-rich lineage. This thesis aimed to 1) resolve the origins and species relationships of NZ Azorella using phylogenetic inference, and 2) compare the polyploidy-associated genomic, morphological, and ecological traits to understand the post-WGD diversification of Azorella polyploids. In this thesis (Chapter 1), I first reviewed the current phylogenomic approaches for resolving species relationships in groups that have complex evolutionary histories, including polyploidization and reticulation. To resolve the NZ Azorella phylogenetic relationships (Chapter 2), I applied Hyb-Seq of the Angiosperms353 bait set via Illumina sequencing to amplify 353 target-enriched single copy nuclear genes. Additionally, nrDNA and whole chloroplast DNA were recovered via genome-skimming reads to represent high copy genes/regions that are traditionally used in phylogenetics. Hyb-Seq of Angiosperms353 loci was combined with a PacBio sequencing run to improve homeologous gene extraction (Chapter 3). Finally, NZ Azorella post-polyploidization diversification patterns (Chapter 3) were assessed using the variation in genome sizes (via flow cytometry), stomatal guard cell length (using scanning electron microscopy), and ecological niches (using the R package ENMTools). Overall, from biogeographical analyses, I found two independent dispersal events of species in New Zealand Azorella sections Stilbocarpa and Schizeilema, respectively. Using the concordance factors among gene trees and single nucleotide polymorphisms from Hyb-Seq data, as well as the topological incongruence between single copy and high copy gene trees, the results indicated hybrid origins of several hexaploid (6x) species, reticulate relationships among tetraploids, and an allopolyploid origin of the 10x species A. colensoi. Furthermore, different post-polyploidization diversification patterns were compared among Azorella taxa in different ploidy levels, which showed that phylogenetic relationships (i.e., genome content), reticulate evolutionary histories, genomic modification processes (i.e., expansion or contraction), niche shifts, and the age of the polyploid species are all important factors to predict the macroevolutionary patterns of polyploid species.
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    Identification and characterization of effector proteins from pine needle 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, 2022) Massoco Tarallo, Mariana
    Collectively, Dothistroma septosporum, Cyclaneusma minus and Phytophthora pluvialis cause serious foliar diseases on Pinus radiata in New Zealand and on many other pine species worldwide. Considering the ecological and economic importance of forest trees, understanding how these pathogens interact with their hosts on a molecular level is critical as it could lead to new and durable approaches to control the diseases they cause. Pathogens have the ability to deliver proteinaceous virulence factors, termed effectors, into the apoplast and cell cytoplasm of their host plants. Effectors typically promote host colonization through suppression of the plant immune system. However, in resistant host plants, one or more of these effectors can be recognized by corresponding immune receptors to activate the plant immune system. Often, one of the main outputs of this immune system is a localised cell death reaction, termed the hypersensitive response (HR), which renders the pathogen unable to cause disease (avirulent). The general goal of this thesis was to identify shared candidate effector (CE) proteins between the three foliar pine pathogens and to characterise their virulence (or avirulence) functions. This is important because disease resistance based on core effectors that are vital for a pathogen’s ability to cause disease is more likely to be durable. Using a combination of “omics” information and bioinformatic tools, two sets of orthologous CE proteins were identified between D. septosporum, C. minus and P. pluvialis, while several other sets were identified between the two fungal pathogens. Some of these CEs had the ability to trigger cell death responses in non-host Nicotiana plants, and some were shown to activate Nicotiana benthamiana genes involved in pathogen-associated molecular pattern-triggered immunity and HR. CEs were also screened in the host, Pn. radiata, using a method developed in this thesis, where it was determined that some of these CEs also trigger cell death. Two conserved cell death elicitor families, Ecp20 and Ecp32, were identified from D. septosporum and its close relative Fulvia fulvum, and the cell death triggered by some family members in N. benthamiana was shown to require membrane-localized receptor-like proteins. Tertiary structure predictions of CEs provided insights into the possible roles and host targets of these proteins during pine infection. Moreover, a shared β-trefoil fold was found between sequence-unrelated CE proteins from the three pine pathogens, along with evidence that they are also present in many other fungal species. A CRISPR/Cas9 gene editing methodology was applied to D. septosporum for the first time, which allowed for the functional characterization of three D. septosporum CE genes, two of which are also present in C. minus and P. pluvialis. Collectively, this thesis provides a significant advance in our understanding of pine-pathogen interactions at the molecular level and provides a blueprint for similar studies in other forest pathosystems.
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    Historical biogeography of marine ray-finned fishes (Actinopterygii) of the Southwest Pacific : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Marine Evolutionary Ecology at Massey University, Auckland, New Zealand
    (Massey University, 2023) Samayoa, André Philippe
    Current environmental and anthropogenic pressures are driving significant biodiversity loss and range shifts in marine environments. Understanding how biodiversity is generated and how it responded to past environmental changes is fundamental to inform future management strategies for marine resources. As the largest ubiquitous taxonomic group among marine vertebrates, ray-finned fishes (Actinopterygii) represent the best model to understand the generation of biodiversity and the processes that shaped contemporary geographic patterns in the sea. In this sense, centers of marine endemism are of evolutionary value as they translate evolutionary and ecological mechanisms that drive biodiversity dynamics. In the Pacific Ocean, endemism centers for marine fishes are mainly located in remote oceanic islands at the periphery of the tropical West Pacific which harbors the highest levels of biodiversity. Biogeographic research suggests that marine fish endemism in the oceanic islands of the Central Pacific originated via multiple independent jump-dispersal colonization events, and that the islands have acted as sources of new unique biodiversity. However, as the evolutionary setting starts to be revealed for marine fish endemism in the Pacific, processes that generate and maintain biodiversity in other peripheral islands remain unknown. My thesis aims to fill this gap by studying the origin, evolution, and processes that have shaped endemism and biodiversity of marine fishes in the Southwest Pacific. I examined the historical biogeography of the region´s marine fish fauna using open-access molecular data to infer evolutionary histories, and geographic distribution information to assess spatial patterns of endemism and biodiversity. Data were analyzed across three research projects based on time-calibrated phylogenies, probabilistic biogeographic modeling, and statistical analysis of phylogenetic measures of endemism and biodiversity. My results confirm the role of the subtropical islands of the Southwest Pacific as sources of new unique biodiversity, identify mainland Australia as the major source of endemic lineages, highlight the significance of jump-dispersal and vicariance in shaping endemism patterns, and reveal that the processes shaping patterns of endemism and biodiversity differ at local scales. My thesis contributes to the understanding of unique contemporary biogeographic patterns in the marine fish fauna of the Southwest Pacific.
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    Molecular dynamics simulation of inter-molecular interactions : a thesis submitted to Massey University in Albany, Auckland in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Computational Biochemistry
    (Massey University, 2019) Shadfar, Shamim Zahra
    Many aspects of the operation of chemical and biological systems are based on intermolecular interactions. In this work, binding modes and interactions between molecules at a range of scales have been studied, using molecular dynamics (MD) simulations. The first chapter provides an introduction of each of the different chemical and biological systems that are studied in this work. It also introduces MD and its role in the context of this research. The second chapter corresponds to the study of host-guest interactions for cyclodextrin- bullvalene complexes. Bullvalene is a shapeshifter molecule, which interconverts between different isomers at room temperature. The goal of this chapter is to capture one favourable isomer of bullvalene (guest molecule) by binding it to cyclodextrin as a host molecule. This chapter consists of two smaller chapters (2i and 2ii). The former details the development and validation of a “host-guest binding potential energy profiling” (HGBPEP) method, which is a rotational interaction energy screening method designed for prediction of the most favourable orientation and position of bullvalene isomers with respect to cyclodextrin. The latter investigates the interaction of bullvalene isomers and cyclodextrin molecules, and finally binding free energy values of the complexes are calculated. The third chapter describes KstR, a transcriptional repressor in Mycobacteria. KstR is required for Mycobacterium tuberculosis (Mtb) pathogenesis as well as regulating the initial steps in cholesterol degradation by controlling the expression of the enzymes that carry out the early stages of cholesterol catabolism. Therefore, this protein is of great interest for development of new tuberculosis treatments. In this chapter, the stability and conformational changes of KstR in its different states – apo, DNA-bound and ligand-bound –have been studied. The main goal is to investigate the binding mechanism of KstR to DNA, as well as the effect of DNA and ligand binding on the structure and dynamics of KstR more generally, using MD simulations. In the fourth chapter, KstR2, another Mtb transcriptional repressor, is studied. KstR2 represses a 14-gene regulon involved in the later steps of cholesterol degradation. It is structurally similar to KstR, but has been proposed to act through a novel scissor-like mechanism. This chapter investigates two key questions regarding the mechanism of action of KstR2: first, the effect of mutating the key switch residue ARG170 to ALA, and second, the effect of ligand binding on its structure and motion. The focus of the fifth and final chapter is phosphatidylinositide 3-kinases (PI3Ks), which are proteins that take part in signalling pathways regulating factors like cell growth, survival and proliferation, which in turn are involved in cancer. The interaction between PI3Kα and another protein, RAS, is very important in the formation, growth and maintenance of RAS- driven tumours. A model of PI3Kα (class IA PI3K) has therefore been built, as well as of RAS associated with a model cell membrane, and MD simulations used to investigate the process by which the two proteins interact with one another and with the lipid bilayer. Altogether, this thesis uses MD simulations to provide insight into intermolecular interactions at a range of scales, with a particular focus on proteins involved in tuberculosis and in cancer.
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    Characterisation of the cell death-inducing activity of the conserved family of Ciborinia camelliae-like small secreted proteins (CCL-SSPs) of C. camelliae, Botrytis cinerea and Sclerotinia sclerotiorum : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Manawatu, New Zealand
    (Massey University, 2019) McCarthy, Hannah
    Ciborinia camelliae, the causal agent of Camellia petal blight, is a necrotrophic fungus that sequesters nutrients from dead plant cells. Candidate effector proteins have been identified from the secretome as a highly conserved clade, termed C. camelliae-like small secreted proteins (CCL-SSPs). Notably, the CCL-SSPs are not unique to C. camelliae. Indeed, a single homolog of the CCL-SSP family has shown to be encoded by the genomes of the closely related necrotrophs, Botrytis cinerea and Sclerotinia sclerotiorum, (BcSSP and SsSSP, respectively). Previous work has identified the ability of BcSSP and SsSSP to induce cell death on Camellia ‘Nicky Crisp’ petals, whereas of the ten C. camellia CCL-SSPs (CcSSPs) tested, only one induced very weak cell death. The aim of this study was to determine what specific regions of the SsSSP protein confered cell death-inducing ability, and to further characterise the cell death-inducing capability of these CCL-SSPs. In this study it was shown, through generation of chimeric regionswapped proteins and infiltration into Camellia ‘Nicky Crisp’ petals and Nicotiana benthamiana leaves, that the region encoded by Exon 2 of SsSSP is essential for cell death-inducing activity. It was also discovered that BcSSP and SsSSP may induce cell death to different extents, as a significant different was shown in quantified cell death induced on Camellia ‘Nicky Crisp’ petals. It was also found that BcSSP can induce strong cell death on Arabidopsis thaliana leaves, while SsSSP does not. This research also investigated appropriate methods for characterising cell death of CCL-SSPs, and suggested addition of a C-terminus tag for future work. The results of this study have shed further light on the CCL-SSP family as candidate effector proteins and provided several avenues for future researchers to fully elucidate the function of CCL-SSPs and their role in virulence of these three necrotrophic fungi.
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    Interactions between MCF-7 and MDA-MB-231 breast cancer cell lines and neutrophils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Anatomy and Physiology at Massey University, Albany, New Zealand
    (Massey University, 2019) Holland, Sherina
    The tumour micro-environment (TME) has an essential role in tumour development and progression. Immune cells recruited to the site of the tumour secrete soluble factors such as proteinases, growth factors, survival factors and angiogenic factors into the TME. The secretion of these factors is up-regulated via inflammatory mediators secreted by tumour cells, resulting in a pro-malignant cycle between the cancer and immune cells. A greater understanding of the molecular mechanisms underpinning these interactions is required, as this will assist towards identifying potential new drug targets for cancer and ultimately, will aid in the long-term development of targeted and effective treatments for breast cancer and MBC. The activities of certain immune cells, such as tumour associated macrophages, have been reasonably well characterised in cancer, however, until recently, less was known regarding the role of neutrophils in tumour progression. The goal of the research described in this thesis was to determine whether soluble factors secreted by breast cancer cells might alter the phenotype or lifespan of neutrophils. The latter may allow neutrophils sufficient time to participate in activities within the TME that may either help or hinder tumour progression, while soluble factors released by the neutrophils might influence the invasiveness of breast cancer cells. To investigate whether soluble factors released by breast cancer cells could delay neutrophil apoptosis, neutrophils were cultured in conditioned medium (CM) prepared from highly metastatic MDA-MB-231 or poorly metastatic MCF-7 cells. Flow cytometry experiments showed a delay in apoptosis for neutrophils cultured in MDA-MB-231 CM, but not MCF-7 CM. Quantitative RT-PCR was used to measure neutrophil mRNA expression of pro- versus anti-apoptosis peptides; neutrophils incubated in MDA-MB-231 CM, but not MCF-7 CM, demonstrated a significantly higher expression of the anti-apoptosis peptide BCL2 (A1) and significantly lower expression of the pro-apoptosis peptide BAK compared to control. Western blots showed extensive caspase-8 activation for neutrophils cultured in MCF-7 CM, consistent with apoptosis, whilst neutrophils cultured in MDA-MB-231 CM showed little activation of caspase-8, indicating low levels of apoptosis. The soluble factor contained within the MDA-MB-231 CM, responsible for the delay in neutrophil apoptosis was found to be heat stable and have a molecular weight of between 10-100kDA. Prostaglandin E2 (PGE2) was identified as a potential candidate molecule, as it is a heat stable lipid, and when bound to plasma proteins, fits the molecular weight criteria. In addition, neutrophils cultured with 10μM native or heat treated PGE2 demonstrated a delay in apoptosis, however, this was to a lesser extent compared to neutrophils cultured in MDA-MB-231 CM. Cycoloxygenase-2 (COX-2), the enzyme responsible for PGE2 synthesis, was shown to be expressed in MDA-MB-231 cells but not MCF-7 cells, which is in agreement with the results demonstrating a delay in apoptosis for neutrophils cultured in MDA-MB-231 CM but not MCF-7 CM. Freshly isolated human neutrophils, obtained from the peripheral blood of healthy volunteers, cultured in MDA-MB-231 or MCF-7 CM for 7hrs were not polarised toward a pro or anti-tumour phenotype, as determined via the expression of ICAM-1 and MMP-9. Finally, to investigate whether neutrophils could influence the process of EMT and alter the migration of breast cancer cells, neutrophils were indirectly cultured, via transwell plates, with MDA-MB-231 or MCF-7 cells. Neutrophils were not found to enhance the migration of the cancer cells, as determined via a wound scratch assay. Likewise, neutrophils were not shown to influence the process of EMT in the cancer cells, as determined by changes to cell morphology or the expression of EMT markers.
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    How does Epichloë festucae avoid the host defence response? : 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
    (Massey University, 2018) Noorifar, Nazanin
    Epichloë festucae is a filamentous fungus, which forms symbiotic associations with aerial tissues of Lolium and Festuca grass species. Chitin, a polymer of N-acetyl-Dglucosamine, is an important component of the fungal cell wall and a well-known pathogen associated molecular pattern (PAMP). Chitin promotes pathogen-triggered immunity (PTI) upon hydrolysis with plant chitinases and release of chitin oligomers. Therefore, to establish a stable and successful symbiosis, the endophyte needs to remain ‘hidden’ from the host immune system or actively suppress it. Confocal laser scanning microscopy (CLSM)-based analysis of leaf tissue infected with the E. festucae wild type strain and infiltrated with the chitin-specific molecular probe, WGA-Alexa Fluor-488, showed that only the septa of endophytic hyphae bound this probe while the entire cell wall was labelled in epiphyllous hyphae confirming previous observations that hyphal cell wall chitin is either masked or remodelled in endophytic hyphae. The aims of this project were (i) to test whether E. festucae LysM-containing proteins have a role in binding to or sequestering cell wall chitin oligomers and thereby preventing PAMPtriggered immunity and (ii) to analyse the composition of the cell wall of endophytic and epiphytic hyphae. An analysis of the E. festucae genome identified seven genes encoding proteins with LysM domains. Expression of two of these genes, lymA and lymB, increased in planta compared to in culture. Interestingly, both are divergently transcribed from chitinase encoding genes (chiA and chiB respectively), which also have increased expression in planta. Single gene deletion mutants of lymA, lymB, chiA and chiB as well as a double gene deletion ΔlymA/B were generated, and their plant interaction phenotype analysed. Plants infected with DlymA, DlymB or DchiA had the same plant-interaction phenotype as wild type whereas ΔchiB and ΔlymA/B mutants had defects in hyphal growth within the leaves. Analysis of hyphal cell wall structure using Chitin Binding Protein (CBP) and chitosan (CAP (Chitosan Affinity Protein) and OGA-488)-specific eGFP-based biosensors suggest that cell wall chitin is converted to chitosan in endophytic hyphae. This structural change is consistent with a lack of a defence response when E.festucae forms a mutualistic symbiotic association with L. perenne. Three E. festucae chitin deacetylase genes were identified (cdaA, cdaB and cdaC), and gene expression analysis showed cdaA expression is significantly increased in planta compare to in culture. Functional analysis of cdaA revealed that although plants infected with the ΔcdaA mutant had a similar whole plant interaction phenotype as wild type, they had an abnormal cellular phenotype. Patches of chitin were exposed along the endophytic hyphae confirming this mutant was unable to convert chitin to chitosan. However, hyphae in these plants still labelled with the chitosan biosensor OGA-488 demonstrating that despite the deletion of the cdaA, the hyphal cell wall of endophytic hyphae still contain chitosan suggesting that another chitin deacetylase, possibly CdaB has a redundant function in E.festucae. Collectively these results show that lymA, lymB and chiB are required for establishment of the symbiosis between E.festucae and L. perenne. In addition, this study shows that chitin is converted to chitosan in the hyphal cell wall of endophytic hyphae during the infection and colonisation of the host. The E. festucae chitin deacetylase gene cdaA is also essential for proper hyphal growth in planta and the symbiotic interaction.
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    Effector delivery and effector characterisation in Dothistroma needle blight of pines : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Sciences) in Genetics/Molecular Plant Pathology at Massey University, Manawatū, New Zealand
    (Massey University, 2018) Hunziker, Lukas
    The filamentous fungus Dothistroma septosporum causes a serious foliar disease, Dothistroma needle blight (DNB), on Pinus radiata in New Zealand and on many pine species worldwide. Potentially correlated to changes in climate, this disease has been on the rise for 20 to 30 years, and current countermeasures often struggle to contain the damage it causes. A molecular approach to combat DNB could be promising. Effectors are small proteins secreted by pathogens to promote host colonisation, and have been a major focus of plant pathologists in recent years. However, effector biology in pathogens of gymnosperms has received little research attention. Here, candidate effectors (CEs) were selected using a series of computational prediction tools, as well as RNAseq data from a compatible D. septosporum-pine interaction. A shortlist of 55 highly in planta expressed CEs, predicted to be secreted to the apoplast, was characterised in silico. While almost half of them lacked a predicted function, none were exclusive to D. septosporum. Seventeen effector candidates of particular interest were taken forward for functional characterisation. Specifically, these proteins were screened for induction of plant defences in the form of cell death in the model plants Nicotiana benthamiana and N. tabacum using an Agrobacterium transient expression assay. Five CEs induced cell death in these plants, suggesting recognition by the plant defence machinery. Of those five, three are similar to previously described proteins. Effector screening methods are not available for pine, thus various approaches to achieve this were trialled. A high-throughput method to collect each protein in the apoplastic wash fluid of N. benthamiana was developed. This fluid was applied to P. radiata shoots raised from tissue culture to screen for a response, with promising results. Along with an array of D. septosporum CEs, these shoots may ultimately be used to screen for resistant pine genotypes. Selection of genotypes at this early stage could speed up DNB resistance screening for pine breeding and the protocol could be transferred to related pathosystems. This research also contributes to the molecular understanding of forest diseases and effectors that may be common among pathogens of distantly related hosts.