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.

Soil was collected from a compost heap in a Mowbray suburban garden and a compost heap in a Plumstead suburban garden. The soil and ‘worm tea’ of a vermiculture farm from the same Mowbray suburban garden were also sampled. Using four different types of media (7H9, CZ, ISP2 and GOT) 135 isolates were putatively identified as actinobacteria based on colony morphology. These isolates were screened for antimycobacterial activity against the test bacterium Mycobacterium aurum A+. A Kribbella strain, isolated and identified by an intern in the lab, and a Micromonospora strain, isolated and identified during the authors Honours project, were also screened for antimycobacterial activity. Sixty-four (64) actinobacterial isolates displayed moderate antibiotic activity or higher (ZOI >1001 mm2 ) based on the standard overlay method. Kribbella strain SK5 displayed very strong antimycobacterial activity (3309 mm2 ). Forty (40) of the actinobacterial strains that exhibited moderate/strong/very strong antimycobacterial activity and/or had interesting morphological features were selected for genus identification via a standard nucleotide-nucleotide blastn analysis of their 16S rRNA gene sequences. Thirty-one (31) strains were identified as Streptomyces species, six strains were identified as Micromonospora species, one strain was identified as a Nocardia species, one strain was identified as a Kitasatospora species, and one strain was identified as a member of the genus Tsukamurella. These isolates were subjected to phylogenetic analysis using the partial 16S rRNA gene sequences. Based on analysis of the 16S rRNA gene sequences, Streptomyces strain PR10 was found to be the most interesting of the Streptomyces isolates and should be pursued as a novel species (99.7% sequence similarity to the top blastn hit and less than 98.8% sequence similarity from the third blastn hit onwards). Further analysis of the gyrase subunit B (gyrB) gene sequence of the Kitasatospora isolate (strain PR3) revealed that the isolate is more closely related to members of the genus Streptomyces. Further evidence to support the assignment of strain PR3 to the genus Streptomyces (rather than Kitasatospora) is that it has two Streptomyces-specific gyrB gene indels signatures. Tsukamurella strain G4 was noted for characterisation as a novel species. The potential for seven isolates to produce ansamycin, glycopeptide, non-ribosomal peptide, and/or TypeII polyketide antibiotics was determined by detection of antibiotic biosynthetic gene clusters using PCR. Strain M27 demonstrated the potential to produce all the aforementioned antibiotics. Strain Y10 demonstrated the potential to produce a non-ribosomal peptide antibiotic. Strains PR10, PR28, PR47 and UK1 demonstrated the potential to produce Type-II polyketide and non-ribosomal peptide antibiotics. The PCR products were sequenced and analysed via blastn to compare them to the known antibiotic biosynthetic gene sequences in the GenBank database. The non-ribosomal peptide synthetase (NRPS) A domain sequences were analysed using the NRPSpredictor2 software to identify the A domain substrate specificity Solvent extraction was done on the broth cultures of Streptomyces strains PR3, UK1 and Y30 and Kribbella strain SK5 to isolate the antimycobacterial compounds. It was found that the cell mass extract of the three Streptomyces isolates had active compounds against M. aurum A+. The culture broth extract of the Kribbella isolate was found to have an active compound against M. aurum A+ and Staphylococcus aureus ATCC 25923. One-dimensional and two-dimensional TLC of the culture broth extract from strain SK5 revealed that a single compound was active against M. aurum A+ and S. aureus ATCC 25923. Nocardamine was purified from the culture broth extract of strain SK5 by Mr Kojo Acquah (PhD student, Department of Chemistry, University of Cape Town). In a side-by-side spot bioautography analysis of the purified nocardamine and the strain SK5 culture broth extract, it was found that the active compound in the culture broth extract was not nocardamine, because nocardamine only had activity against M. aurum A+ while the culture broth extract had activity against M. aurum A+ and S. aureus ATCC 25923. Using the polyphasic taxonomic approach, Kribbella strain SK5 was tentatively characterised as a novel species, for which the name Kribbella stellenboschensis sp. nov. is proposed. The closest phylogenetic relatives were identified as the type strains of Kribbella aluminosa, Kribbella karoonensis, Kribbella pittospori, Kribbella shriazensis, ‘Kribbella sindirgiensis’ and ‘Kribbella soli’. Genetic distances of 0.030 and 0.016 were calculated for ‘K. soli’ and ‘Kribbella sindirgiensis’, respectively, for the concatenated gene sequence of five housekeeping genes (gyrB, rpoB, recA, relA, and atpD). Thus, DNA-DNA hybridisation (DDH) will need to be carried out to confirm that strain SK5 is a separate species. Phenotypic differences were observed between strain SK5 and all the type strains of the most closely related species. Chemotaxonomically, strain SK5 possessed the key characters definitive of the genus Kribbella: i) MK9(H4) as the major menaquinone; ii) LL-diaminopimelic acid as the diagnostic diamino acid; iii) anteiso-C15:0 and iso-C16:0 as the major fatty acids (>10%); and iv) phosphatidylcholine in the polar lipid profile.

The evolution of a strikingly elongated and webbed FL in bats, which contrasts with a small, free-toed HL, has seen extensive research into bat wing development in an effort to determine the molecular mechanism driving limb development. A recent RNA-seq and ChIP-seq study carried out on M. natalensis showed differences in FL and HL activity for several genetic pathways known to be involved in bone formation during key bat development stages CS15-CS17. In this project the prediction made from the literature and the RNA-seq results was that the observed decreased Wnt/β-catenin signalling and increased BMP signalling in the bat FL may lead to elevated levels of Sox9 expression, and larger fields of mesenchymal condensations. This was tested by annotating Sox9 in the M. natalensis genome to further analyse the expression levels and associated ChIP-seq data. In addition the behaviour of condensing mesenchymal cells during bat and mouse limb development was observed by visualising the various stages of chondrogenesis, using H&E and PNA stains. In addition the RNA-seq study found 3000 genes to be differentially expressed. Thus, the project also set out to create an immortalised bat autopod cell line to facilitate future testing and predictions. The Sox9 gene was successfully annotated and revealed to not be differentially expressed between FL and HL as predicted. However downstream targets of Sox9 were further inspected as potential ideas for further investigation. The histological stains provided a set of data characterising mesenchymal condensation in both mouse and bat stages, revealing many interesting features such as the non-specific binding behaviours of PNA prior to digit formation. In addition, quantitative results demonstrated the bat FL digits are already longer than the HL digits at CS16. Cell line work established a working protocol for the storage, dissociation and plating of bat primary cells that retain their bat limb expression identity. Mouse cells were successfully immortalised and a cell line was established from a HL digit cell. This project has facilitated further studies in understanding extreme digit elongation in the bat FL autopod during development.

Molecular farming is gaining traction as a cost-effective platform to produce recombinant proteins. Further improvements can be made, however, to increase overall yield especially for difficult to express proteins. In this study virus-derived silencing suppressors and replication elements were used with the aim of increasing expression and yield of enhanced green fluorescent protein (EGFP) and the Zika PrME polyprotein in Nicotiana benthamiana. A comparison of four viral silencing suppressor proteins was performed: these were tomato spotted wilt virus non-structural protein, NSs, tomato aspermy virus (TAV) 2b, tomato bushy stunt virus P19 and begomovirus alphasatellite Rep. Differences in EGFP expression in N. benthamiana due to the silencing suppression were determined using immunoblotting and fluorescence of EGFP. In addition, replication elements from three viruses (bean yellow dwarf virus [BeYDV], beak and feather disease virus [BFDV] and begomovirus alphasatellite) were assembled into novel plant expression vectors using GoldenBraid (GB) cloning technology and assessed using EGFP. Finally, the two approaches were combined in an attempt to express the Zika PrME polyprotein, which was assessed using immunoblotting. EGFP expression was found to be greatest in the presence of the TAV 2b protein and no difference in fluorescence intensity between the original BeYDV replicating plant expression vector and that constructed using GB could be detected; however, the GB assembly of the BFDV and alphasatellite plant expression vectors was unsuccessful. The TAV 2b combined with the BeYDV replicating elements were used for the expression of Zika PrME. The gene was successfully cloned into the replicating BeYDV vector and a vector that does not replicate (negative control). The PrME was not detected using anti-His tag immunoblotting despite optimisation for Agrobacterium infiltration density, harvest day post infiltration, signal peptides and buffers during extraction. In this study I demonstrated the following: that the TAV 2b protein out-performed all other silencing suppressors; that the GB cloning technology can be successfully applied in the development of novel plant expression vectors, although further optimisation is required for these and for Zika PrME expression. Further work in characterising the effect of silencing suppression on recombinant protein expression can be assessed using RT-qPCR to measure the effect on mRNA levels. In summary, these improvements in plant recombinant protein expression can be readily applied to large scale production of novel therapeutics and vaccines.

Common to orthodox seeds, desiccation tolerance (DT) is exceedingly rare in the vegetative tissues of modern angiosperms, being limited to a small number of "resurrection plants". While the molecular mechanisms of DT, as well as the transcription factors regulating the seed and vegetative DT programmes, have been identified, very little is known with regards to the role of regulatory noncoding RNAs (ncRNAs). To investigate the presence and roles of possible ncRNA players, RNA-Seq was performed on desiccating Xerophyta humilis leaves and a bioinformatic pipeline assembled to identify the potential decoy lncRNAs and miRNAs present. Interaction mapping was performed, identifying a number of small regulatory networks each regulating a small subset of the desiccation transcriptome. Predicted networks were screened for function related to DT and expression consistent with functional regulatory interactions. Of the predicted networks, two appear highly promising as potential regulators of key DT response genes. The results indicate that differentially expressed (DE) desiccation response ncRNAs are present in the vegetative tissues of X. humilis and likely play a key role in the regulation of DT. This suggests that ncRNAs appear to play a more important role in DT than previously thought, and may have facilitated the evolution of vegetative DT through reprogramming of seed DT programs in vegetative tissues.