Genetics - Theses
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In vivo functional characterization of nicotinic acetylcholine receptors in Drosophila melanogaster
Nicotinic acetylcholine receptors (nAChRs) are responsible for fast excitatory synaptic transmission in insect central nervous system. Their role as targets for commercial insecticides have resulted in extensive studies on their structure and pharmacological properties. However, many other aspects of their fundamental biology remain less understood. For example, what behaviours are underpinned by the activity of nicotinic acetylcholine receptors? Here, we used reverse genetics to address this question. The precise genome editing power of CRISPR/Cas9 technology was used to generate a collection of Drosophila melanogaster lines harbouring precise genomic deletions of the genes of interest, including the subunits for the nicotinic acetylcholine receptors as well as a couple of their accessory proteins. The overall strategy was to remove as much as of the genomic locus as possible by having two sgRNAs directing Cas9 to cut at the 5’ and 3’ ends of the gene’s coding sequence and relying on non-homologous end joining repair to ligate the termini together creating a deletion. In total, nine knockout strains were generated for four genes, successfully removing genomic sequences ranging from 4 to 83kb in length. For three genes, Dα4, Dα6 and DmRIC3, the same allele was recapitulated for three backgrounds. The role of nAChRs in regulating sleep behaviour in vinegar flies was investigated using null alleles of the receptor subunits. For seven of the ten subunits, flies harbouring null alleles were viable as adults for behavioural assays. All mutants showed changes in total sleep amount compared to their controls, which most strongly correlated with changes in sleep episode duration. Additionally, genotypes carrying partial deletions or point mutations displayed different sleep changes, suggesting that allelic variation within subunits can yield different phenotypes. These data confirmed a role in sleep regulation for most nAChR subunits. Furthermore, the role of the nAchR accessory proteins were considered. Lines with a deletion of the nAChR-specific chaperone DmRIC3 responded to two commercial insecticides in similar manner to loss of the subunit Dα1. Those lines also phenocopied sleep behaviour of flies lacking receptor subunits. This is the first in vivo evidence of the functional significance of DmRIC3 to nAChRs in D. melanogaster. Altogether, these results show that significant behavioural changes might be considerable fitness costs beyond viability for resistant alleles of genes with important functions in the central nervous system such as nAChRs. However, resistance could still arise from disruption to other proteins interacting and regulating nAChRs with less severe costs.
Population genomics and transcriptomics in the cotton bollworm, Helicoverpa armigera
Helicoverpa armigera is an agricultural pest that causes billions of dollars' worth of damage each year. As H. armigera has evolved resistance to insecticides, an understanding of resistance genes will provide useful insights into managing this pest. One approach to identify candidate genes is to scan the genome for signs of strong and recent selective sweeps. This extends the search beyond typical candidate genes (detoxifying enzymes and molecular targets) although a limitation of the approach is that the selective agent causing a sweep may not be an insecticide. Another approach is to compare the differences between lab-selected and unselected cohorts. Genes that are differentially expressed are good candidates for further investigation. Here, I present estimates of some baseline parameters such as nucleotide diversity and the extent of linkage disequilibrium to lay a foundation for detecting selective sweeps in H. armigera, and I identify a gene exhibiting the hallmarks of strong and recent selection. I also present some preliminary findings from an analysis of differentially-expressed genes between selected and unselected cohorts of H. armigera in response to a pyrethroid insecticide.
Understanding aphids: transcriptomics, molecular evolution and pest control
Aphids exhibit fascinating biological features including parthenogenesis, symbiosis, altruism and host-plant preference; all of which would be better understood if genetic tools and molecular biological techniques were applied to them. Aphids are also agricultural pests that vector plant viruses and new approaches to control them are required. This thesis addresses questions motivated by an interest in the biology of aphids and a desire to improve the agricultural impact of aphids. It does so through transcriptomic analyses and RNA interference (RNAi) technology. I examined the ways in which the transcriptome of aphid changes with host-plant, between tissues, within species and between species. The three-aphid species studied (the green peach aphid: Myzus persicae, the mustard aphid: Lipaphis erysimi, and the cabbage aphid: Brevicoryne brassicae) are all pests of economically important brassica crops (such as cabbage, cauliflower, mustard and canola). These data may provide insights into the way different aphid species deal with plant secondary compounds such as glucosinolates. These data also allowed me to examine the structure, function and evolution of myrosinase enzymes that have allowed some aphid species to develop an anti-predator ‘mustard bomb’. RNAi has been suggested as a way to specifically target pest that would be more ‘environmentally friendly’ than conventional insecticides. I experimentally assessed the feasibility of orally-delivered RNAi to control aphids and the potential of this technology to be developed as a functional genomic tool. RNAi was fed to aphids via artificial diets at various concentrations and with various delivery agents and via transgenic Arabidopsis thaliana plants that I created that produced dsRNA’s corresponding to aphid genes. These studies lead me to suggest that more work needs to be done to limit the effects of RNase enzymes of the aphid gut digesting orally delivered RNAi and to more carefully characterize factors that may affect within-species variation in RNAi efficacy.
Identifying downstream targets of FILAMENTOUS FLOWER, a YABBY transcription factor that promotes organ polarity and lateral growth in Arabidopsis
Angiosperm leaves are typically polar structures with a distinct arrangement of cell types along the adaxial-abaxial (upper-lower) axis. Studies of leaf development in the model dicot plant Arabidopsis thaliana have shown that adaxial-abaxial patterning is not only associated with the formation of distinct cell types, but also triggers growth along the lateral axis leading to the formation of the leaf blade or lamina. Adaxial-abaxial patterning factors identified in a variety of plants including Arabidopsis, Antirrhinum and maize are either small regulatory RNAs or transcription factors. Although first categorized as being involved in adaxial-abaxial patterning, the YABBY (YAB) family of transcription factors is now thought to play a pivotal role in coordinating various developmental programs involved in leaf blade formation. As part of an approach to identify targets of the Arabidopsis YAB gene FILAMENTOUS FLOWER (FIL), this study generated transgenic lines with inducible FIL activity. Constitutive activation of FIL resulted in the partial abaxialisation of leaves and reduced blade growth, phenotypes that have previously been attributed to ectopic YAB expression. Further analysis showed that constitutive FIL activity increased the rate of cell cycle progression in specific regions of the developing leaf, as well as increasing sensitivity to exogenously applied auxin. The latter phenotype was inferred from increased activity of an auxin-signalling reporter, suggesting that FIL modulates auxin responses in Arabidopsis. Transcriptional profiling with microarrays was subsequently used to monitor genome-wide changes in gene expression following FIL activation. This analysis identified groups of genes that were either positively or negatively regulated by FIL and extensive testing of a subset of these genes showed that some were direct targets. On the basis of these results it is proposed that FIL functions as both a transcriptional activator as well as repressor during leaf development. Among the positively regulated genes identified as FIL targets, two are well-known abaxial patterning factors, KANADI1 (KAN1) and AUXIN RESPONSE FACTOR4 (ARF4). Given the well-defined role these factors play during leaf development, this study focused on their regulation. Analysis of mutant lines lacking activity of the leaf-expressed YABs revealed a significant reduction in KAN1 expression, but not ARF4 expression. These results confirm that FIL is a regulator of KAN1, but presumably regulates ARF4 in combination with other factors. In conclusion, this study identified direct targets of the bifunctional plant transcription factor FIL. Finding that two of these targets promote abaxial cell identity during leaf development supports the case for YABs being important polarity regulators. Given the expression pattern of the YABs, it is argued that YABs are unlikely to function upstream of KAN1/ARF4. Instead, a model is proposed in which YABs promote adaxial/abaxial patterning through a system of positive feedback loops that ultimately maintain the activity of these early abaxial patterning genes during leaf blade formation.
The genetic basis of resistance to the Ryanodine Receptor modulator chlorantraniliprole in Drosophila melanogaster
The development of synthetic insecticides in the mid 20th century lead to a revolution in pest control. However, issues with environmental toxicity, adverse human exposure and insecticide resistance have meant new safer alternative pest control methods are required. Chlorantraniliprole belongs to a promising new class of insecticides that exert control by targeting the Ryanodine Receptor. As this class, the group 28 synthetic diamides, has a unique chemistry and a mode of action that is distinct from most insecticides, its market share has rapidly increased since it was first introduced in 2007. Here, I use genomic, transcriptomic and phenotypic analyses of the Drosophila Genetic Reference Panel (DGRP) to examine the way chlorantraniliprole interacts with an insect’s biology. This research reveals that a novel muscle- associated gene, Stretchin Myosin Light Kinase, is strongly associated with resistance in the DGRP. In addition, a co-expressed set of detoxification enzymes, under control of the Cap ‘n Collar/Keap1 pathway were found to be constitutively up-regulated in a subset of the DGRP and that their transcriptional abundance was correlated with survivorship on chlorantraniliprole. Transgenic ‘knock up’ of one of these putative detoxification enzymes, Cyp12d1, confers increased resistance to chlorantraniliprole and resistance to the related compound cyantraniliprole. Furthermore, a lab selection experiment based on a large Australian population of D. melanogaster also confirms an association between Cyp12d1 and resistance. This contributes to a growing body of evidence suggesting that cytochrome P450 enzymes play a role in chlorantraniliprole resistance. Through the quantitative genetic approaches employed, it is possible to demonstrate that the genetic architecture underpinning resistance changes with dose. Furthermore, as the DGRP was established before the introduction of chlorantraniliprole this study demonstrates that alleles of large effect are pre- existing in naïve populations and such alleles may increase in frequency as this class of insecticides become more widespread. Finally, this study illustrates the systems genetic approach offers unprecedented power to understand the biology perturbed by insecticides.
The significance of low level mosaicism in Fragile X Syndrome.
Fragile X Syndrome (FXS) is the most common single disorder associated with intellectual disability (ID) and autism spectrum disorder (ASD). FXS is fundamentally caused by a trinucleotide CGG sequence repeat expansion within the 5’ untranslated portion of the FMR1 gene, to more than 200 repeats which is called Full Mutation (FM). This is associated with abnormal methylation of this gene’s promoter and silencing of FMR1 expression. To date, the type and severity of the neurocognitive phenotype has been related more closely with the methylation status than with the number of CGG repeats in the FM range. Smaller CGG expansions are called premutation (PM) (55 and 200 CGGs), grey zone (GZ) (45-54 CGGs) and normal size (<44 CGGs) alleles, and are usually associated with presence of an unmethylated FMR1 promoter, and normal FMR1 expression. There is a proportion of FXS individuals in which two or more population of cells can be found, and who are called mosaic or mosaicism. These individuals possess cells with FM and smaller CGG sizes (e.g. Normal, GZ or PM), which lead to milder forms of the FXS phenotype. This PhD explores the clinical and diagnostic significance of low level mosaicism (LLM) and DNA methylation variation amongst different CpG sites within FMR1. The first aim of this study was to define the lower limit of detection LLM of six FMR1 tests currently used in diagnostic laboratories. This was achieved using three FMR1 PCR commercial kits (AmplideXTM, X-Sense and FastFraXTM) targeting the CGG region and three methylation tests (Methylation Specific- Quantitative Melt Analysis [MS-QMA], Sequenom®EpiTYPER system [MALDI-TOF MS] and mSouthern blot) targeting methylation of Exon1/Intron1 boundary region and specific CpG sites within FMR1 promoter. The second aim was to screen a large number of males with ID and ASD of unknown etiology referred for FXS testing to determine the prevalence of mosaicism containing FM together with a normal or GZ allele (cryptic FMR1-FM) who are not being identified as a part of standard FXS testing. This was achieved using MS-QMA and the positive results were confirmed with the FMR1 tests assessed in Aim 1. The third aim of this study was to examine the association between DNA methylation of FMR1 and mRNA levels and severity of intellectual functioning impairment in individuals with a cryptic FMR1-FM identified in Aim 2 and males and females with typical FXS (only FM and PM/FM alleles). Three significant contributions to the FXS field resulting from this PhD are: i) implementation of a new highly sensitive assay for detection LLM missed by standard testing; ii) determination of the prevalence of cryptic FM-FMR1 in males with idiopathic ID/ASD referred for FXS testing; iii) clinical and molecular characterization of LLM and its methylation characterization amongst tissues and CpG sites; and demonstration that LLM levels are significantly correlated with severity of cognitive impairment and ASD as well as FMR1 mRNA levels.
A genome-wide analysis of carbon catabolite repression in Schizosaccharomyces pombe
The process by which eukaryotic microorganisms preferentially utilise glucose as a carbon source is coordinated by a network of sensory and signalling pathways, which converge at the transcriptional level to control the function of a conserved regulatory mechanism known as carbon catabolite repression (CCR). In the fission yeast, Schizosaccharomyces pombe, CCR is mediated by Scr1, a C2H2 zinc finger transcriptional repressor orthologous to Saccharomyces cerevisiae Mig1, and Aspergillus nidulans CreA. In addition, a conserved co-regulatory complex, comprised of Tup11, Tup12 and Ssn6 proteins (hereafter Tup/Ssn6), is required for maximal transcriptional repression. Also implicated in this process is the transcriptional activator, Rst2, which in the absence of glucose, induces the expression of gluconeogenesis and sexual differentiation genes. To date, the molecular mechanism of CCR in S.ipombe has not been characterised in depth, and so there is limited knowledge of the range of genes that are subject to transcriptional repression, or of the functional relationship between Scr1, Tup/Ssn6, Rst2, or other factors that influence the establishment and/or maintenance of CCR. This study combined genetic techniques with a suite of high-throughput sequencing approaches to investigate the process of CCR in S. pombe. RNA-seq and ChIP-seq approaches showed that Scr1 represses approximately 2% of the S. pombe genome in the presence of glucose including hexose uptake, glycolysis, TCA cycle, pentose phosphate pathway, and gluconeogenesis genes. In addition, unexpected roles for Scr1 in the regulation of iron homeostasis and stress-induced meiosis were discovered, integrating Scr1 and CCR more broadly into the regulation of general metabolism and stress responses in S.ipombe. Biochemical pulldown approaches showed that Scr1 physically interacts with the Tup/Ssn6 complex in vitro and further ChIP-seq showed co-localisation of Tup11 with Scr1 at gene promoters in glucose-sufficient conditions. Interestingly, Tup11 was shown to remain at the promoter of certain target genes that were activated in the absence of glucose, suggesting roles for the Tup/Ssn6 complex in gene activation. Additional ChIP-seq analysis of Rst2 in the absence of glucose revealed localisation to gene promoters formerly repressed by Scr1 in glucose-sufficient conditions. Surprisingly, Rst2 was also found to co-localise with Scr1 and Tup11 at certain genes in the presence of glucose suggesting unforeseen regulatory roles for this factor in glucose-sufficient conditions and hinting at a potential competitive or co-operative relationship between Scr1 and Rst2 at these genes. In addition to increasing knowledge of CCR, these findings also have important biotechnological implications. S. pombe is industrially utilised to produce bioethanol, a renewable biofuel of significant environmental and economic importance. In a concurrent approach, an S. pombe isolate used for industrial scale bioethanol production from sugarcane molasses was analysed for modifications to carbon metabolism, CCR or other processes. Whole genome sequencing identified structural variation, including a 100kb duplication of a subtelomeric region in chromosome III, and potential evidence for horizontal gene transfer of coding sequences from Schizosaccharomyces octosporus. Further transcriptomic analysis identified a distinct transcriptional signature of this industrial isolate in both laboratory media and the molasses feedstock. Importantly, regulatory rewiring of central carbon metabolism and stress response pathways was evident. Finally, direct examination of CCR within this strain via Scr1 ChIP-seq, revealed significant plasticity with respect to the number of Scr1 targets, particularly in the molasses feedstock where Scr1 was associated with multiple actively transcribed genes, suggesting alteration of the CCR pathway within the industrial strain genetic background. Overall, this study has shown that CCR in S. pombe forms a core regulatory network that responds to glucose primarily at the transcriptional level to facilitate the regulation of a range of metabolic processes in both laboratory and industrial contexts. Thus, this work significantly improves our understanding of the CCR process in S. pombe and forms an important resource for the study of carbon regulation in eukaryotes. These findings will also be useful for the development of fission yeast strains that possess improved bioethanol production characteristics.
The evolution of pathogenicity and isolate variation in Talaromyces marneffei
The opportunistic fungal pathogen of humans, Talaromyces marneffei, is one of very few pathogens in an order of over a thousand species and the only species that has the capacity to switch between two morphologically distinct growth forms (known as dimorphism). Growth at 25°C results in a saprophytic multicellular, hyphal form while infectious growth in a host occurs as a uninucleate unicellular yeast that resides within phagocytic cells of the immune system. The intracellular niche of T. marneffei differs significantly from the niches of other Talaromycetes. The identification of the mechanisms by which T. marneffei can survive and grow in this intracellular niche is a major aim of this study. Comparisons of the genomes of three closely related non-dimorphic, non-pathogenic species with the T. marneffei genome identified unique features that contribute to niche specific growth and the ability to cause disease. Most significant of these were an overall reduction in genome size and gene number in T. marneffei with substantial gene losses in families responsible for environmental interaction. These and other findings strongly indicate that T. marneffei has adapted to an intracellular host niche distinct from its saprophytic relatives. Against this background of gene loss three gene families were identified that had been significantly expanded in T. marneffei. These expanded gene families showed putative extracellular and cell surface localisation and consisted of cell wall galactomannoproteins (mpl family), aspartyl proteases (pop family) and a family of small proteins with very little functional characterisation in any species (mib family). Genes in the pop, mpl and mib families were over-represented in subtelomeric regions, under positive selection, had copy number variation in T. marneffei isolates and many had high levels of repetitive adjacent sequences including several transposon families. In the host T. marneffei grows as an intracellular pathogen within phagocytes and as such extracellular proteins interact directly with the host. Therefore another aim of this study was to characterise these expanded gene families and their role in pathogenesis. Deletion studies in pop genes revealed roles in yeast cell formation during intracellular growth, while high variability in cell-to-cell protein production for two mib genes suggested a role in cell surface variation when interacting with the host. Understanding the type and degree of variation within the population of a fungal pathogen can reveal its population structure and potential to adapt to stressors such as antifungal compounds. Genome wide variation in the T. marneffei population had yet to be examined therefore an aim of this study was to characterise the degree and type of this variation. To this end several clinical and environmental isolates of T. marneffei were examined for variation in chromosomal structure, which is a common means of generating phenotypic variation in other fungi. While no obvious abnormalities were observed, gene copy number variation in subtelomeric regions was widespread and several strains showed specific small mutations with impacts in antifungal resistance and phenotypic instability. Overall this study has revealed the genomic and genetic changes within T. marneffei and between it and other Talaromycetes. Many of these changes help to explain its unique niche as an intracellular pathogen within an almost entirely non-pathogenic clade. This research also highlights specific genes and gene families with roles in this pathogenesis and identifies potential therapeutic targets and genes involved in host interactions for future investigation.
Probing insecticide biology using Drosophila melanogaster
Insecticides are often used to control insect pests, but resistance to these chemicals arises quickly, leading to agricultural losses and public health concerns. Understanding how insects cope with insecticides is necessary when designing rational pest management strategies, but much still remains unknown regarding the fate of insecticides once inside the body. Furthermore, the genetic variation that governs an insects ability to survive insecticide exposures has not been fully described. Here, a 3 pronged approach is applied to study insecticide biology using the model insect Drosophila melanogaster. First, an acute, sub-lethal insecticide response assay was developed, which provided information complementary to that obtained from more common toxicology assays. In particular, behavioural response observed in a hyper-resistant target site mutant suggests additional target sites for the insecticide spinosad. This bioassay was then applied in a forward genetics approach to describe the genetic basis of resistance to the insecticide imidacloprid. This approach identified a variety of neuronal genes and the previously identified drug metabolizing enzyme Cyp6g1, which was explored through genetic manipulation. Finally, a reverse genetics approach was employed in order to study the effect of an ABC transporter protein Mdr65 on insecticide resistance. Removing the gene made the insects more susceptible to a subset of the insecticides tested, and this was confirmed with genetic and chemical complementation tests. These data provide information both on the genetics and kinetics of insecticide biology. Such information will help to better understand insecticide resistance and design rational resistance management strategies.
The genomic basis of climate and host adaptation
Many species are currently threatened by the direct and indirect effects of anthropogenically driven climate change. The elevation of global temperatures and increase in variability in both temperature and precipitation pose a risk to biodiversity as species are pushed close to their thermal safety margins. Current predictions suggest a dramatic loss of species diversity and the contraction of geographical ranges of many species. Many ectothermic insects that cannot regulate their body temperature are likely to be threatened, particularly ecologically- restricted herbivorous insects that depend for on plants for food and that are often in phenological synchrony with their plant hosts. However, adaptive shifts in these species in response to host loss and climatic extremes may counter the effects of climate change to some extent. This highlights the importance of studying species-specific adaptation mechanisms including host interactions. This dissertation contributes to this overall aim by studying the genomic basis of climatic and host adaptation. I use Drosophila melanogaster as a model system at the intraspecific level, and Drosophila species from the repleta group as a model system for the comparative level. In assessing the genomic basis of host responses, I consider a much broader range of insect taxa. This dissertation begins with a study on the use of chromosome level sequencing of D. melanogaster populations from two ends of a thermal cline. I present genomic evidence for the role of the inversion 3R Payne in capturing alleles favourable to local climatic conditions in the non-inverted form, and therefore driving adaptation to climate change. The study further elucidates the impact of climatically important chromosomal inversions in driving higher linkage disequilibrium on the non-inverted form - potentially benefiting both karyotypes. In the second chapter, I develop a new pipeline, Orthonome, and tools for multi-species comparisons for prediction of orthologues and inparalogues with the highest accuracy and recall. Using Orthonome, I was able to identify a much greater level of conservation across Drosophilid lineages than earlier thought, amounting to nearly 33% better resolution than industry-accepted methods. I then use Orthonome in the third chapter to compare the genomes of 58 insect species – most of which are known to be agricultural pests. Testing across eight gene families, I present evidence for genomic patterns in only four gene families (P450s, CCEs, GSTs and ABCs) as being associated with polyphagy or particular host ranges. While three of them have been reported before, I find that ABC transporters present much stronger evidence than reflected in earlier studies, with feeding behaviour as well as host tissue displaying an effect on gene gain in more voracious pest species. Finally, in the last study, I use novel genomic data and evidence from the repleta group of drosophilids to carry out phylogenetically constrained analyses of genes potentially associated with host and thermal stress adaptation. My aim here is to find mutually exclusive evolutionary pathways to neofunctionalisation between stress tolerant cactophilic specialists and less tolerant generalists in the group. I also find a different adaptive response in the cactophilic species compared to the generalist species; these species show little lineage specific gene gain, suggesting an exception to current standing theories on neofunctionalisation for adaptation. I further discuss the applicability the species level and order level analyses for an overall detailed and systematic approach to identify the genomic basis of climatic and host adaptation.
Identification and analysis of factors influencing intracellular growth of the pathogenic fungus Talaromyces marneffei
Fungal pathogens of animals and plants are a major concern in society with huge economic and public health consequences. The prevalence of pathogenic fungi and the emergence of new, opportunistic fungal pathogens, about which we know little, compounds the current problem. Talaromyces marneffei is a dimorphic, opportunistic pathogenic fungus that infects immunocompromised individuals. At 25 ̊C T. marneffei grows in a multinucleate hyphal form that can undergo a process of asexual development to produce conidia, which are the infectious agents. Upon inhalation, conidia reach the alveoli of the lungs where they are phagocytosed by resident phagocytes such as alveolar macrophages. The transition to 37 ̊C, which is the human host body temperature, induces the dimorphic switch to a pathogenic, uninucleate, fission yeast form. The yeast form is able to utilize macrophages as a niche from within which to avoid detection by the host immune system. T marneffei yeast must then be able to withstand macrophage-killing responses and obtain nutrients in order to proliferate inside these phagocytic cells. The objectives of this study were to determine the transcriptional response of T. marneffei to the host environment, including those induced by growth at body temperature as well as to host-derived cellular signals. This would lead to the identification of genes and pathways necessary for establishing infection. For this purpose RNAseq analysis was used to create a transcriptomic profile of T. marneffei during in vitro growth at 25°C (hyphal) and 37°C (yeast) and during murine and human macrophage infection (ex vivo). To identify pathways that are important during the establishment of the pathogenic yeast cell type, expression data for hyphal growth was compared to yeast growth in vitro and during intracellular macrophage infection. Key nutritional and cell protective pathways that show common upregulation during the yeast growth phase were identified and these included carbon and nitrogen utilization, micronutrient uptake, melanin generation and oxidative stress protection. Additionally, to separate host specific responses from temperature driven expression and identify genes that are specifically regulated during infection, macrophage infection specific transcriptional data sets were compared against the expressionprofile of T. marneffei grown at 37 ̊C in vitro. Twelve genes were chosen for phenotypic characterization using gene deletion as a way of validating the output of the screen, and these genes were shown to be important for different aspects of establishment and maintenance of T. marneffei yeast growth in macrophages. The analysis was extended for a novel gene designated msgA, encoding a guanyl nucleotide exchange factor,which was found to be essential for maintaining appropriate cell morphology, which in turn is crucial for T. marneffei occupying its macrophage niche. Overall the findings of this study revealed that T. marneffei yeast and hyphal forms have adapted specific metabolic programs tailored to the diverse environmental conditions encountered by each cell type. The identification of genes which are specifically required for establishing yeast growth during macrophage infection, uncovered components of pathways that respond to host–specific rather than temperature specific signals. Together these provide valuable insights into the initiation of infection and pathogenicity establishment in T. marneffei, and may serve to broaden our understanding of the means by which to target opportunistic fungal infections.
Using insecticides to probe nicotinic acetylcholine receptors in Drosophila melanogaster
Insecticides remain the most effective means of insect control for both our personal protection and for the protection of our food and economic crops. The knowledge gained through the study of current and past insecticides can be a valuable tool in both the design of new, more effective insecticides and as a guide for integrated pest management. To best utilise a new insecticide and for it to retain field efficacy, its mode of action on its molecular target must be thoroughly understood. For this, a genetically tractable model organism can be used to enhance the understanding of insecticide:insect interactions through characterisation of resistance alleles, analysis of the insecticide target(s) and other resistance mechanisms. The advancement of genome engineering technology and the increasing availability of pest genome sequences has increased the predictive and diagnostic capacity of the Drosophila model. The Drosophila model can be extended to investigate the basic biology of the interaction between insecticides and the proteins they target. In this study, the vinegar fly, Drosophila melanogaster, has been used to identify and manipulate insecticide resistance genes. Recently an in vivo system was developed that permits the expression and study of key insecticide targets, the nicotinic acetylcholine receptors (nAChRs), in controlled genetic backgrounds. Rescue of the spinosad resistance phenotype in the Dα6 loss of function mutant, was possible with not only individual isoforms of this gene, but also with pest α6-like orthologues. It has also been found that a chimera of the Dα7 N-terminal and Dα6 C-terminal region is able to rescue the response to the insecticide spinosad. In this study, an incompletely dominant, spinosad resistance mechanism that may evolve in pest species is examined. First generated using chemical mutagenesis, the Dα6P146S mutation was recreated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 system, the first reported use of this technology to introduce a resistant mutation into a controlled genetic background. Both alleles present with the same incompletely dominant, spinosad resistance phenotype, proving the P146S replacement to be the causal mutation. The proximity of the P146S mutation to the conserved Cys-loop indicates that it may impair receptor gating. The Dα6 in vivo expression system was used here to assist in characterizing this dominant allele. The results of this study enhance our understanding of nAChR structure:function relationships, in particularly the interaction between spinosad and the Dα6 subunit. A complete in vivo rescue model was developed here for analysis of the Da1 subunit. A Dα1 deletion was generated using ends-out gene knockout technology and this knockout was found to be highly resistant to neonicotinoid insecticides. By combining this deletion with the GAL4:UAS binary expression system this study was able to rescue the phenotype of susceptibility to neonicotinoids as well as confirm the resistance potential of two nAChR subunits from the pest species, Helicoverpa armigera. This system is also used to investigate other phenotypes of the Dα1 deletion. The endogenous role of the nAChRs may enhance our understanding of why these are such effective insecticide targets, but also why they appear to be functionally redundant in terms of insect viability. Knowledge of other phenotypes present in lossof function mutants gives an insight into their function as well as hinting at fitness costs that may be associated with their loss. Targets with important roles are less likely to evolve resistance mutations if they also impact endogenous functions. If the target does have an important function, resistance modifications may lead to decreased fitness that would not be able to persist in natural populations without insecticide selection. A phenotype for the Da1 subunit was discovered whereby it appears to play a role in Drosophila courtship and mating behaviour. Although this allele can be cultivated in laboratory conditions, it would cause dramatic fitness effects under field conditions. Field evolved resistance mechanisms have been identified for both insecticides classes used in this study. Furthermore the neonicotinoid, imidacloprid, has recently been implicated in colony collapse disorder that is currently impacting honeybee populations. The results in this study provide information about the basic biology of the molecular targets of these insecticides. This information may help extend the life of these insecticides through more efficient use or perhaps provide ideas for new, more specific insecticides.