Clostridium acetobutylicum P262 is an endospore-forming Gram positive anaerobic bacterium, and for many years this organism has been used in the industrial fermentation for the production of acetone and butanol from carbohydrate substrates. The aims of this thesis included studies on small phosphorylated molecules involved in energy metabolism and cell differentiation, and an investigation into the genetics and molecular biology of C. acetobutylicum electron transport genes. To facilitate quantitation of nucleoside triphosphates in extracts of C. acetobutylicum, a chromatographic data acquisition and analysis system was constructed. Samples were prepared from C. acetobutylicum cultures by treatment with formic acid, and nucleotides contained in these extracts were separated by strong anion exchange HPLC. The developed manual integration system features the ability to collect and store chromatographic data, allowing for multiple integration using different calibration curves. Nucleoside triphosphate profiles were obtained from batch fermentations of the C. acetobutylicum P262 wild type, sporulation deficient (spo-1) and solvent deficient (ds-1) strains. The nucleoside triphosphate profiles of the wild type and spo-1 mutant were similar and were characterized by a trough in nucleotide levels which occurred just prior to the pH break point, the onset of the stationary growth phase, clostridial stage formation and the transition from the acidogenic to the solventogenic phase. The nucleoside triphosphate concentrations during the exponential growth phase were much lower than those found during the stationary phase. Exponential phase nucleotide levels in the cls-1 mutant were comparable to those observed in the wild type and spo-1 mutant. Unlike the wild type and spo-1 strains, the cls-1 mutant, which does not switch to solventogenesis, did not demonstrate an increase in nucleotide levels after the cessation of cell division. The involvement of nucleotide levels, particularly that of GTP, in the differentiation of C. acetobutylicum was indicated by the effect of inhibitors, which have been shown to decrease ribonucleotide levels in other organisms and cause an increase in sporulation. The antibacterial agent metronidazole, was used as a tool for the isolation of C. acetobutylicum electron transport genes. Since it was desired to clone these genes in Escherichia coli, and investigation into the activation of metronidazole by E. coli strains was necessary. E. coli strains with lesions in their DNA repair systems were more susceptible to metronidazole than wild type strains. However, it has been reported that DNA repair deficient strains of E. coli that also had a diminished ability to reduce chlorates and nitrates were no more susceptible to metronidazole than their wild type parents (Jackson et al., 1984; Yeung et al., 1984). To isolate a suitable E. coli cloning host for the selection of C. acetobutylicum electron transport genes which activated metronidazole, transposon mutagenesis of the recA E. coli strain CC118 with TnphoA, was used to construct the recA, metronidazole resistant E. coli strain Fl9. F19 was shown to have diminished nitroreductase activity, which was presumed to be responsible for the metronidazole resistant phenotype. However, the recA mutation renders E. coli F19 highly susceptible to the reduced toxic intermediates of metronidazole. The E. coli F19 recA, nitroreductase deficient mutant was used for the isolation of C. acetobutylicum genes on recombinant plasmids which activated metronidazole. Twenty-five E. coli F19 clones which contained different recombinant plasmids were isolated. The clones were tested for nitroreductase, pyruvate-Fd­oxidoreductase and hydrogenase activities. Nitroreductase and pyruvate-Fd­oxidoreductase activity was not demonstrated in any of the isolated clones, and only one clone tested positive for hydrogenase activity. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1 kb chromosomal fragment. It was determined that this 11.1 kb fragment contained at least two regions responsible for activating metronidazole. The one gene responsible for making E. coli F19 extremely sensitive to metronidazole was localized to a 2 kb region. The nucleotide sequence of this 2 kb region was determined and two truncated open reading frames and one complete open reading were present. The complete open reading frame was shown to be responsible for activating metronidazole. The deduced amino acid sequence of this open reading frame was determined to be 160 amino acids in length, and database searches showed good similarity to flavodoxin proteins from many organisms. Based on alignments to the amino acid sequences of these flavodoxins, as well as the fact that Chen and Blanchard (1979) reported that reduced flavodoxin can transfer electrons to metronidazole, the sequence corresponding to this C. acetobutylicum metronidazole activating gene was identified as coding for a flavodoxin gene. The role of flavodoxin in C. acetobutylicum and other organisms is presented. Possible relationships between the cloned C. acetobutylicum flavodoxin gene and metronidazole sensitivity are discussed.

In a murine model of Leishmania major infection, susceptible BALB/c mice develop a detrimental Type 2 immune response characterized by the production of interleukin (IL)-4 and IL-13, which single through a common receptor, the IL-4 receptor alpha chain (IL-4Rα). Forkhead box P3 (Foxp3⁺) Regulatory T (Treg) cells are an unique subset of CD4⁺ T cells that play important immunomodulatory roles maintaining a balance between Type 1 and Type 2 immune responses. During L. major-induced cutaneous leishmaniasis, Treg cells accumulation at the site of infection has been implicated in suppressing a detrimental Type 2 immune response by modulating early interleukin (IL)-4 production, however it remains unclear if IL- 4Rα mediated signalling on Treg cells play a significant role in this process. To investigate this further, a novel BALB/c model was utilized in which the IL-4Rα chain was conditionally knocked out on Treg cells (Foxp3ᶜʳᵉIL-4Rα⁻^/ˡᵒˣ mice). We demonstrated that the differential IL- 4Rα deletion efficiency in male (approximately 102 %) and female (approximately 32%) was maintained during L. major infection. Foxp3ᶜʳᵉIL-4Rα⁻^/ˡᵒˣ male mice, which had a greater degree of IL-4Rα deletion on Foxp3⁺ Treg cells, developed significant footpad swellings and ear swellings, increased parasitic burdens at the site of infection and within draining lymph nodes. This hypersusceptible phenotype observed in Foxp3ᶜʳᵉIL-4Rα⁻^/ˡᵒˣ BALB/c male mice was accompanied with an increased Treg cell activity and amplified Type-2 immune response with an increase in IL-4, IL-10 from L. major-infected lymph node samples and IgE antibody secretion in L. major infected serum samples. Flow cytometry analysis revealed that a L. major-induced Indoleamine 2,3 dioxygenase (IDO)-mechanism could allow for increased Leishmania replication. Collectively, these data suggest a protective role for IL-4Rα signalling on Treg cells in suppressing a detrimental Type 2 during cutaneous leishmaniasis.

A total of 56 presumptive actinobacterial strains was isolated from three different samples taken from the Silvermine Nature Reserve (Table Mountain National Park, Cape Town), namely, cave soil, the wall of the cave and sediment from the shallow waters of a reservoir. Twenty nine (29) isolates were successfully identified to the genus level by 16S-rRNA gene analysis: one Micrococcus strain, one Streptacidiphilus strain, one Micromonospora strain and 26 Streptomyces strains. The phylogenetic position of each identified strain within its genus was investigated by generating a phylogenetic tree based on its 16S-rRNA gene sequence. Further analysis of the Streptacidiphilus strain was conducted based on the gyrB gene. Metagenomic analysis was used to further analyse the actinobacterial diversity of the freshwater reservoir sediment from the Silvermine Nature Reserve. A total of 97 16S-rRNA gene clones was obtained from the reservoir sediment sample, RS1, using actinobacteriumspecific 16S-rRNA gene primers S-C-Act-0235-a-S-20-F and S-C-Act-0878-a-A-19-R and each clone was identified using the EzBioCloud database. Analysis based on unique phylotypes in the clone library revealed that 80% of the clone library was composed of actinobacterial strains belonging to the orders Acidimicrobiales, Streptomycetales, Streptosporangiales, Corynebacteriales, Sporichthyales and the family Jatrophihabitandaceae (the remaining 20% was identified as non-actinobacterial strains). The percentage composition of the actinobacterial clonal diversity for each order was as follows: Acidimicrobiales, 56%; Streptomycetales, 29%; Streptosporangiales, 9%; Corynebacteriales, 4%; Sporichthyales, 1% and family Jatrophihabitandaceae, 1%. Rarefaction analysis revealed that the total actinobacterial diversity of the sample was not represented in the clone library. Therefore, further sampling and analysis of the sample site would uncover greater actinobacterial diversity. Thirty seven (37) putative actinobacterial isolates of the 56 that were isolated from the Silvermine Nature Reserve were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard over-lay method. A total of five identified 2 actinobacterial isolates (Streptomyces strains RS6, RS7, RS9, RS13 and RS15) and an unidentified actinobacterium, strain RS4, demonstrated very strong antimycobacterial activity (zone of growth inhibition of over 3000 mm2 ). In addition, 15 of the 37 strains were active against Staphylococcus aureus ATCC 25923 and three were active against Escherichia coli ATCC 25922. Streptomyces strains CS1, CS3, CS12, CS18, CS19, CW5, RS3, RS6, RS9, RS13 and RS15, displaying varying strengths of antimycobacterial antimicrobial activity, were selected for antibiotic extraction from culture broths. The resulting crude extracts were subjected to spot bioautography to test for antibacterial activity. The organic compounds extracted from the cell mass of Streptomyces strain CS3 and the broth fraction of Streptomyces strain RS3 demonstrated strong activity against M. aurum strain A+. Furthermore, the crude extracts of 15 actinobacterial isolates (Micromonospora strain RS10 and Streptomyces strains CS1, CS3, CS12, CS18, CW2, CW5, RS3, RS6, RS7, RS9, RS13, RS15, RS18 and RS19) were additionally tested for antiplasmodial activity against Plasmodium falciparum strain NF54. Seven of these strains showed activity against Plasmodium namely, Streptomyces strains CW2, CW5, RS3, RS7, RS13, RS15 and RS19. Streptomyces strains CW2, CW5 and RS7 displayed the strongest activity against P. falciparum strain NF54 with IC50 values below the guideline threshold of 1000 ng/mL (strain CW2 culture broth crude extract: IC50 40 ng/mL, strain CW5 culture broth crude extract: IC50 128 ng/mL and strain RS7 culture broth crude extract: IC50 70 ng/mL).

Chikungunya fever is a non-fatal but highly debilitating disease that affects primates, birds and humans. The causative agent is the chikungunya virus (CHIKV), an arbovirus of the Alphavirus genus. CHIKV is responsible for the largest epidemic recorded for an Alphavirus, infecting an estimated 1.4 to 6 million patients worldwide. Furthermore, it has been recognised by the United States army as a potential biological weapon used for bioterrorism owing to the potential for infection via aerosol. CHIKV is primarily transmitted by infected Aedes aegypti mosquitos and is currently distributed in Africa, parts of Asia and South, Central and North America. As a result of the virus genetically adapting to infect the Aedes albopictus mosquito, its recent and rapid spread to non-endemic regions has occasioned increasing anxiety as well. Infection in humans presents as a sudden onset of fever, rash and severe arthralgia that persists for years. At present, there is no fast and effective diagnostic test to distinguish CHIKV from other similar viruses. This is a problem because viral infection displays the same symptoms as that of dengue, Zika, Ebola and yellow fevers while prognosis, patient care, and persistent symptoms of these viruses are very different. Usually, during the development of a diagnostic reagent, Biosafety Level 3 (BSL3) containment is required for purifying antigens from live viruses. These lab diagnostic tests are expensive to perform and, in regions facing a CHIKV epidemic, are inefficient due to their long waiting periods. This results in patients going undiagnosed or misdiagnosed and/or falling outside the window of prophylactic treatment. As such, a cheap and rapid diagnostic reagent to detect the presence of CHIKV antibodies would be most advantageous. In this study, two recombinant variants of the CHIKV E2 glycoprotein were expressed in Nicotiana benthamiana plants to assess their viability for use in a diagnostic reagent for CHIKV infection. Two versions of a tobacco sp. codon-optimised, 6xhis-tagged CHIKV E2 envelope glycoprotein gene were synthesised and cloned into the plant expression vector, pTRAkc-ERH. The E2 glycoprotein is a desirable protein candidate used for a diagnostic reagent as it is a major target for neutralizing antibody production against CHIKV during early infection. One variant contained a ~52 kDa full length E2 glycoprotein (CHIKV E2-HIS) while the other contained a ~49 kDa truncated E2 glycoprotein lacking its transmembrane domain (CHIKV E2ΔTM-HIS). Following this, an expression time trial was performed whereby the recombinant proteins were expressed in N. benthamiana plants via Agrobacterium-mediated small-scale 6 syringe-infiltration at different optical densities, OD600 = 1.0 and 0.5. To improve expression, both genes were co-infiltrated and co-expressed with a human chaperone proteins calreticulin (CRT) or calnexin (CNX), or a plant silencing-suppressor protein NSs. Expression of the recombinant protein variants alone showed low to undetectable levels of expression in plant leaves across 7 days post infiltration (dpi) for both ODs tested. CHIKV E2ΔTM-HIS yielded the highest levels of all combinations tested at an OD600 = 1.0 when co-expressed with CRT and harvested at 3 dpi. These parameters were used for subsequent scaling up and production of E2 using vacuum infiltration. Attempts at purifying CHIKV E2ΔTM-HIS proteins using Ni-NTA affinity chromatography and further investigation into the exposure of the 6xHis-tag on the native conformation of CHIKV E2ΔTM-HIS indicated that the 6xHis-tag was insufficiently exposed on E2 and thus inaccessible to facilitate purification by Ni-NTA affinity chromatography. Further attempts at purifying recombinant CHIKV E2ΔTM-HIS proteins by pH purification were also unsuccessful as large amounts of plant-protein contaminants present in all samples prevented adequate separation from CHIKV E2ΔTM-HIS. A different approach utilising ammonium sulphate precipitation facilitated separation of recombinant CHIKV E2ΔTM-HIS from some of the contaminating plant proteins in the 30 - 60% ammonium sulphate fraction; however, large amounts of recombinant CRT were co-purified with E2 in this fraction. Although expression of a candidate diagnostic reagent in plants for detecting CHIKV antibodies in the form of E2 glycoprotein was achieved, further research needs to be done to optimise a purification strategy for CHIKV E2ΔTM-HIS proteins.

Vegetative desiccation tolerance (VDT), the ability to survive loss of up to 95% of cellular water in leaves and roots, is rare amongst vascular plants. However, the trait has evolved multiple times in a small, diverse group of angiosperms collectively known as “resurrection plants”. The physiological, morphological and metabolic changes that take place during VDT have been well characterised. However, in stark contrast, the underlying regulatory mechanisms that activate the VDT programme are not well understood. A widely held view is that VDT in resurrection plants may have arisen by a genetic reprogramming of the seed maturation pathways common to the vast majority of angiosperms. In Arabidopsis thaliana, the activation of seed maturation genes is regulated by the canonical LAFL (LEC1, ABI3, FUS3 and LEC2) network of transcription factors (TFs). However, thus far there is limited evidence to indicate that the LAFL network itself regulates VDT in resurrection plants, though downstream components of this network (such as the ABI3 regulon) are active. Recently, in a transcriptomic study of the resurrection plant Xerophyta humilis, it was found that the LAFL TFs are induced during seed maturation but not during vegetative desiccation. Instead, members of the ABF family of TFs, which are associated with the vegetative abiotic stress response, were strongly upregulated, particularly XhABFA. This finding supports an alternative hypothesis: namely that the activation of VDT in X. humilis has evolved by the rewiring of the transcriptional network that controls the abiotic drought stress response in desiccation sensitive plants. The aim of this study was to investigate whether XhABFA is able to bind to the promoters of and activate the expression of three genes, XhPER1, XhECP63 and XhDSI-1VOC, which are seed-specific in desiccation sensitive plant species but are upregulated in X. humilis leaves as they desiccate. Two experimental approaches were taken in order to determine this: transient expression of XhABFA in A. thaliana protoplasts transfected with promoter:firefly luciferase reporter constructs, and a Yeast One-Hybrid analysis. A. thaliana protoplasts expressing XhABFA displayed significantly greater firefly luciferase activity than protoplasts transfected with the empty vector, indicating that XhABFA can drive transcription from the promoters of these three canonical seed genes. This is the first evidence of activation of seed-specific genes in desiccating leaves by a “vegetative” abiotic stress TF and suggests that components of the drought stress response may be important in activating VDT in X. humilis. This finding may help shed light on our understanding of the regulatory networks underpinning VDT in angiosperm resurrection plants.

Grey Leaf Spot (GLS) is a fungal disease of Zea mays (maize) that is caused by Cercospora zeina. It thrives in sub-tropical climates and causes devastating crop losses of up to 60% in southern Africa where maize is grown as a staple food source. Phytoalexins are low molecular weight anti-microbial bio-chemicals that are synthesised in planta in response to biotic stress. Related studies have characterised many phytoalexins produced in various plants against several diseases. In maize, phytoalexins fall into to two terpenoid groups: kauralexins and zealexins. To date no studies have been carried out that examine the accumulation in maize of phytoalexins in response to C. zeina. This research project found that in maize samples inoculated with C. zeina, kauralexin accumulation significantly increased with disease development stages (T0 - 0 days post inoculation, T1 - 17 dpi, T2 - 18 dpi and T3 - 24 dpi) while zealexins did not change. Gene expression of the phytoalexin biosynthesis genes TPS6 and TPS11 (both encoding the protein terpene synthase 6/11, specific for zealexins) and CPPS2 (encoding ent-copalyl diphosphate synthase 2, specific for kauralexins) increased significantly at each time point, reaching a maximum level at T2. Infiltration of maize leaves with a chitosan elicitor to mimic fungal pathogen associated molecular pattern (PAMP), and a subsequent callose assay showed positive induction of a callose defence response. However, gene expression and phytoalexin accumulation did not change following chitosan treatment, although zealexin accumulation was higher than kauralexins. Previous studies have shown that phytoalexins accumulate transiently in seedlings. Six diverse Southern African maize lines were compared for phytoalexin accumulation at seedling stage. Zealexin accumulation was generally higher than kauralexins and there were significant differences in both zealexin and kauralexin accumulation in different lines. Gene expression analysis using Genevestigator looked at microarray files and found that expression of TPS6/11 (zealexin biosynthesis) and CPPS2 (kauralexin biosynthesis) genes to be largely co-regulated and highly expressed in response to fungal pathogens, nematodes, insect pests and abiotic stresses; Ustilago maydis, Phytophthora cinnamomi, Fusarium moniliforme, Colletotrichum graminicola, Sporisorium reilianum, Meloidogyne incognita, Ostrinia nubilalis, waterlogging and drought stress. Finally promoter region analysis showed similar cis-acting regulatory elements in the 1kb region upstream of the promoter of both genes and defence specific elements. Thus kauralexin phytoalexins are produced in response to C. zeina inoculation, chitin is not likely to be the key PAMP leading to phytoalexin accumulation, phytoalexin accumulation in seedlings is genotype-dependent and phytoalexin biosynthesis genes are expressed under different conditions suggesting a wider range of action beyond repelling fungal pathogens.

The antibiotic metronidazole is used extensively in the clinical treatment of anaerobic infections, including those caused by the anaerobic pathogen Bacteroides fragilis. Metronidazole is an inert substance that requires reductive activation to become cytotoxic. In its activated form metronidazole induces DNA damage. Relatively little is known about the cytotoxic effects of this drug in vivo. The aim of the work reported in this thesis was to analyze the mode of action of metronidazole in living systems. Furthermore, the potential for bacterial cells to develop resistance mechanisms to metronidazole is largely unknown, and therefore the role played by B. fragilis genes in influencing the potency of metronidazole was investigated. Bibliography: pages 172-201.

Most higher plants cannot withstand severe water loss, except for a small group of angiosperms called resurrection plants. They can survive severe water loss without the loss of viability by employing mechanisms that aid them in desiccation tolerance. Desiccation tolerance in resurrection plants is a complex and multifaceted phenomenon and allows the plant to implement various strategies for survival. The focus of this study was a label-free proteomic analysis of Xerophyta schlechteri, a monocotyledonous and poikilochlorophyllous resurrection plant, in response to desiccation. The study investigated some of the physiological, morphological and biochemical changes of X. schlechteri leaf tissue in response to dehydration followed by proteomic analyses using a spectral counting approach. The differentially expressed proteins were identified and quantified and then subjected to gene ontological analyses to identify relevant biological processes involved in desiccation tolerance. The proteomic data was finally correlated to and validated using metabolomic analyses. X. schlechteri was subjected to a controlled dehydration stress treatment, in which changes in the relative water content (RWC) of leaf tissues, the associated changes in processes outlined above and further expanded on below, were determined. Three physiological stages were tentatively identified, namely, the early response to drying (ERD) which represents ~ 80 - 70% RWC (1.61 gH2O g ̄ˡ dwt -1.5 gH2O g ̄ˡ dwt), a mid-response to drying (MRD) represented by ~ 60 - 40% RWC (1.5 gH2O g ̄ˡ dwt -1.0 gH2O g ̄ˡ dwt) and a late response to drying (LRD), represented by ~ 40 - 10% RWC (1.0 gH2O g ̄ˡ dwt - 0.5 gH2O g ̄ˡ dwt). Morphological changes in the late stages of drying were marked by loss of green chlorophyll, increased purple anthocyanin production and leaf folding along the midrib with the abaxial surface exposed to light. Chlorophyll content analyses showed a significant decrease in chlorophyll content in the dehydrated leaf tissue as compared to the fully hydrated state. Biochemical assays to measure the activity of enzymatic antioxidants, namely, ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) were done at selected RWC points. There was a significant increase in antioxidant enzyme activity for APX, CAT, GR and SOD in the dehydrated plant tissue. The label-free proteomics approach utilized, identified a total of 3125 unique proteins in the X. schlechteri leaf tissue across the dehydration treatment of which a combined 517 proteins were significantly differentially expressed in response to drying. Amongst the differentially expressed proteins, 253 proteins were upregulated, and 264 proteins were downregulated. This was followed by functional analyses and classification of gene ontologies using bioinformatics tools such as Blast2GO, MapMan and KEGG. This allowed the identification of certain biological processes and pathways involved in the X. schlechteri desiccation response. Key biological processes and molecular processes were differentially expressed across the drying stages, these included photosynthesis, cellular respiration and antioxidant activity, respectively. The proteomic analysis was complemented and validated using metabolomics approaches based on GC MS/MS and LC/MS. The abundance of specific sugars, sugar alcohols, fatty acids, organic acids, phytohormones and amino acids of X. schlechteri during desiccation were investigated. Sugars such as raffinose and sucrose are known to play a protective role in desiccation and were found to be abundant in MRD and LRD leaf tissue while, L-histidine, an amino acid which plays a critical role in plant growth, was found to be more abundant in LRD tissue as compared to MRD. The phytohormone abscisic acid, invoked in desiccation tolerance was found to be abundant at LRD and less abundant at ERD. The metabolomic data suggested that the regulation of metabolites was towards reducing possible toxic metabolites while increasing the expression of metabolites that help and protect plant cell integrity from the negative effects of desiccation. The use of a label-free proteomics approach complemented with metabolomics allowed the identification and validation of biological processes and pathways potentially involved in establishing desiccation tolerance in X. schlechteri. As far as we are aware, this is the first label-free proteomic analysis of X. schlechteri in response to dehydration.

In order to combat the various pathogens that are constantly exposed to, plants have developed a complex multi-layered innate immune system. Arabidopsis thaliana is a widely used model organism for studying the molecular basis of the host defence response to pathogen attack. One reason for this is the phenotypic variation in resistance found across the many Arabidopsis ecotypes, which provides researchers with a rich resource to dissect the molecular basis of resistance (or susceptibility) to a given pathogen. In this study, the resistance of three Arabidopsis ecotypes (Col-0, RLD and No-0) to the necrotrophic fungal pathogen Botrytis cinerea was investigated using detached leaf infection assays, expression profiling of genes known to play a role in defence against B. cinerea, and by generating F1 hybrids from the three ecotypes. The infection assays demonstrated that Col-0 and RLD develop similar sized secondary (spreading) lesions when unable to contain the initial B. cinerea infection, but that RLD was better able to contain the primary lesion, thus preventing a secondary lesion from developing. In contrast the No-0 ecotype was highly susceptible, displaying both larger secondary lesions and no ability to prevent the initial lesion from spreading. These experiments confirmed previously published data on the susceptibility of these three ecotypes. The variation in susceptibility between the three ecotypes is not due to any modulation of circadian clock regulation ofthe host defence response, asincreased resistance to B. cinerea was observed in all three ecotypes when infected at subjective morning versus subjective night. As a first step towards understanding the molecular basis of this phenotypic variation, quantitative real-time PCR analysis of five defence genes was performed on plants infected with B. cinerea. While the expression profile of these five genes wassimilar in Col-0 and RLD, No-0 displayed a markedly different pattern of expression; no induction of ORA59 (a transcription factor which integrates jasmonic acid and ethylene signalling) or PDF1.1 (a defensin with anti-fungal activity) was observed in response to B. cinerea infection in No-0, which correlated with high constitutive expression ofthe JAZ-ZIM repressor JAZ1. Finally, to determine whether the differential susceptibility to B. cinerea exhibited by the three ecotypes falls under the control of a single genetic locus or not, the ecotypes were crossed and infection assays performed on the F1 hybrids. In all three cases the susceptibility displayed by the hybrids was intermediate between that displayed by the parental ecotypes. This pattern is not consistent with control residing in a single locus and so provides further evidence that resistance to B. cinerea is governed by multiple interacting genes in Arabidopsis.

African horse sickness is a devastating disease that causes great suffering and many fatalities amongst horses in sub-Saharan Africa. It is caused by nine different serotypes of the orbivirus African horse sickness virus (AHSV) and it is spread by Culicoid midges. The disease has significant economic consequences for the equine industry both in southern Africa and increasingly further afield as the geographic distribution of the midge vector broadens with global warming and climate change. Live attenuated vaccines (LAV) have been used with relative success for many decades, but carry the risk of reversion to virulence and/or genetic re-assortment between outbreak and vaccine strains. Furthermore, the vaccines lack DIVA capacity, the ability to distinguish between vaccine-induced immunity and that induced by natural infection. These concerns have motivated interest in the development of new, more favourable recombinant vaccines, initially focusing on the use of insect and mammalian cell expression systems. More recently, several studies have demonstrated the potential for using plant expression systems for the production of virus-like particles (VLPs), which are excellent vaccine candidates, as they do not contain virus genetic material and are DIVA compliant. A vaccine alternative to the currently used live vaccine necessarily needs to provide protection against all nine serotypes of the virus. Cross-protection has been shown to exist between certain serotypes of the virus and as capsid protein VP2 is the protein responsible for AHSV serotype specificity, the idea of a plant-produced VLP vaccine containing a representative VP2 protein from each of the different serotype groups, was conceived. Such a vaccine would potentially provideprotection against all 9 serotypes of the virus and would have DIVA capability. Furthermore, it would address local concerns regarding the use of a live vaccine and would serve as a potentially acceptable prophylactic or rapid response antidote in the wider international context. This work describes two approaches in the development of VLP vaccines in plants. In the first part of this study, the ability of 2 different serotypes of plant-produced AHSV VLPs to safely stimulate an immune response in horses, was investigated. Co-infiltration of Nicotiana benthamiana plants with Agrobacterium constructs encoding the four AHSV serotype 5 structural proteins VP2, VP3, VP5 and VP7, was shown to result in assembly of complete VLPs. Furthermore, co-infiltration with the constructs, encoding VP3 and VP7, together with constructs encoding the two outer capsid proteins VP2 and VP5 of a second serotype, AHSV 4, resulted in assembly of complete AHSV 4 VLPs. Horses vaccinated with plant-produced AHSV 4 and 5 VLPs, all seroconverted after two doses of the vaccine and the virus neutralization titres indicated that the plant-produced VLP vaccines are likely to be at least as effective as the current LAV in protecting against AHSV 4 or AHSV 5. However, they have the added advantage of being free from any of the associated risks of a live vaccine, such as reversion to virulence or genetic re-assortment with field or vaccine strains. In the second part of the study, the use of the so-called SpyTag/SpyCatcher or bacterial “superglue” technology was investigated. This technology is based on the peptide SpyTag irreversibly coupling to the SpyCatcher protein, forming an isopeptide bond when the two are mixed together. The plant-based expression system was used to produce Spy VLPs consisting of either Acinetobacter phage (AP205) VLPs or tobacco mosaic virus (TMV) VLPs displaying a SpyTag or SpyCatcher peptide. In addition, AHSV 5 VP2 displaying SpyTag was expressed in plants and several coupling strategies were tested to determine whether AP205 particles displaying AHSV 5 VP2 could be formed as a result of binding between the SpyTag/SpyCatcher moieties of the recombinant proteins. Although it was not proven that coupling occurred, this research will pave the way towards developing a multivalent vaccine platform whereby VP2 of different AHSV serotypes can be displayed on the Spy VLP surface to allow optimal presentation of these proteins to the animal's immune system. Together, the results obtained in this study show that there is great potential for the production of novel, diverse, efficacious and economically viable AHSV VLP vaccines in plants.