African horse sickness (AHS) is a non-communicable, infectious disease that affects equids and is mainly prevalent in sub-Saharan Africa. The disease has a major impact on the economy of the equine industry as well as an emotional impact on horse owners. There are nine known serotypes of African horse sickness virus (AHSV), which is spread by Culicoides midges. Currently, a multivalent live attenuated vaccine is the only vaccine licensed for use in South Africa. However, it has the inherent risk of reverting to virulence as well as the possibility of genome segment reassortment between vaccine and outbreak strains. Additionally, it is not DIVA compliant (cannot Differentiate between Infected and Vaccinated Animals). There is therefore a need for a safer and more cost-effective alternative vaccine to protect horses against AHSV. Virus-like particles (VLPs) that display antigens on their surface may be suitable vaccine platforms. One such display particle is the phage AP205 VLP, which is comprised of AP205 coat proteins. To aid antigen display, studies have utilized the SpyTag (ST) - SpyCatcher (SC) or “plug-and-display” system, a novel conjugation system used to display several antigens fused to AP205 VLPs. This study aimed at displaying the neutralizing epitope of AHSV serotype 5, known as the VP2 domain (dom) (873bp), on phage AP205 VLP particles using the SpyTag/SpyCatcher technology. The display particle vaccine candidates were produced in Nicotiana benthamiana plants. Firstly, AHSV 5 VP2dom was expressed by being linked to either the ST or SC peptide at its C-terminus. Recombinant pEAQ-AHSV 5-VP2dom ST and pEAQ-AHSV 5- VP2domSC plasmid constructs were constructed from the full-length pEAQ-AHSV 5-VP2- SpyTag and pEAQ-AHSV 5-VP2-SpyCatcher clones using in-fusion cloning. The ST/SC constructs were transformed into Stellar™ competent E. coli cells and thereafter into Agrobacterium tumefaciens AGL-1 cells. Expression time trials were conducted on plants infiltrated with the recombinant Agrobacterial strains to examine transient AHSV 5- VP2domST/SC small-scale expression. Expression was detected for AHSV 5 VP2domSC but not AHSV 5 VP2domST using guinea pig anti-AHSV 5 and rabbit antiST-AP205 sera. Secondly, the development of a particle display vaccine candidate was investigated by coupling AHSV 5 VP2domSC to plant-expressed ST-AP205 VLPs. Three coupling techniques, namely in vitro coupling of purified products, co-infiltration and co-purification, were deployed to determine the assembly of ST-AP205_AHSV 5 VP2domSC VLPs. In vitro coupling involved carrying out separate infiltrations and purification of pEAQ-STAP205 VLPs and pEAQ-AHSV 5 VP2domSC in plants and thereafter mixing the purified products. For co-infiltration, pEAQ-ST-AP205 VLPs and VP2domSC recombinant cultures were used together to infiltrate plants and the presence of complex formations was determined. During co-purification, the presence of coupled products was analysed following separate infiltration of plants with pEAQ-ST-AP205 and VP2domSC recombinant Agrobacterial strains; the homogenates were then incubated together at different VLP: antigen leaf:weight ratios. From the three coupling techniques, copurification at a 1:1 VLP: antigen ratio was identified as the best coupling approach based on the quality and quantity of particles visualised by electron microscopy. These findings indicate the potential of producing an AHSV vaccine candidate in plants, which is ultimately a safer and cheaper alternative to the currently-produced AHSV vaccine. Moreover, this preliminary data may pave the way for developing a vaccine that provides protection against all nine serotypes of AHSV by displaying VP2domSC for other serotypes on the ST-AP205 display particles.

Plants are exposed to pathogens at specific, yet predictable times of the day-night cycle. The circadian clock, an endogenous timing mechanism, allows plants to anticipate recurring changes in pathogen abundance. A recent study showed that in Arabidopsis thaliana (hereafter: Arabidopsis), the circadian clock influences temporal differences in susceptibility to the necrotrophic pathogen, B. cinerea. Plants were less susceptible following inoculation at subjective dawn compared to subjective midnight. Based on the time required for B. cinerea spores to germinate and hyphae to penetrate host tissue and infect plants, infection takes place approximately 12 hours after inoculation. The jasmonic acid (JA) pathway regulates immune responses against B. cinerea. The paralogous basic helix-loop-helix transcription factors, MYC2, MYC3 and MYC4 are primary regulators of the JA pathway and have been implicated in immune responses against B. cinerea but it is not known whether any or all of these transcription factors are involved in regulating time-of-day variations in susceptibility. This study aimed to investigate whether MYC2, MYC3, and MYC4 are involved in the timeof-day differences in susceptibility to B. cinerea, and if they serve as molecular links to regulate interactions between the JA pathway and the circadian clock in Arabidopsis. In this work, the wild-type temporal variation in susceptibility was abolished in the myc234 triple mutant coupled with increased susceptibility following inoculation at both times of the day. The presence of MYC2, MYC3 or MYC4 alone was sufficient to maintain the wild-type time-of-day differences in susceptibility. Constitutive expression of MYC2, MYC3 or MYC4 abolished time-of-day differences in susceptibility but had different effects on overall susceptibility; constitutive expression of MYC2 and MYC4 decreased susceptibility while constitutive expression of MYC3 increased susceptibility to B. cinerea. Gene expression analyses performed on leaves revealed that the transcripts of MYC2 and MYC3 but not MYC4 are expressed rhythmically with peak transcript abundance timed to discrete times of the day under both light-dark and constant light conditions. Constitutive expression of circadian clock genes CCA1 and TOC1 abolished rhythmic MYC expression. The circadian clock modulates rhythmic leaf movement and rhythmic leaf movement was abolished in plants with constitutive MYC2 and MYC3 expression but not in plants with constitutive MYC4 expression and/or in the myc234 triple mutant. Altogether, the data suggest that the MYCs are involved in circadian-driven defence responses against B. cinerea. More specifically, MYC2, MYC3 and MYC4 function redundantly in regulating time-of-day differences in defence responses. In addition, the MYCs function as a point of convergence between the JA pathway and the circadian clock. MYC2 and MYC3 (but not MYC4) may be involved in reciprocal interactions between the JA pathway and the circadian clock.

No description available.

Nickel hyperaccumulation is a unique plant adaption that has led to roughly 390 plant taxa being able to not only withstand the toxicity associated with Ni but actively translocate it to aerial tissues. However, the underlining molecular mechanisms that drive Ni hyperaccumulation remain unclear. Senecio coronatus, a Ni hyperaccumulator, is a novel species as both hyperaccumulating and non-accumulating populations can be found on the serpentine soils of the Barberton Greenstone Belt, South Africa. A comparative RNA-seq analysis on these populations of S. coronatus revealed that ScIRT1 and ScIREG2 , putative homologues of the Arabidopsis transporters, AtIRT1 and AtIREG2 which are capable of transporting Ni, showed much higher expression in the hyperaccumulating populations compared to the non-hyperaccumulating populations, suggesting a potential role in Ni hyperaccumulation. It was thus necessary to investigate whether ScIRT1 and ScIREG2 encode functional homologues of these Arabidopsis transporters. To accomplish this, irt1 and ireg2 mutants were obtained from a T-DNA insertion seed collection and their homozygosity was then determined by PCR genotyping. Since a lack of iron induces IRT1 and IREG2 expression, loss of gene expression of homozygous irt1 and ireg2 mutants by means of reverse transcriptase PCR on plant roots grown hydroponically in the absence of Fe was then done to establish full knock-out status. From this, homozygous mutants were identified, however, absence of gene expression for irt1 and ireg2 mutants was not clear. In addition to validating homozygosity, phenotypic characterisation, with the aim of developing reliable assays to be used in complementation analysis, was done by growing homozygous mutants and Col-0 in hydroponic media deficient in Fe and supplemented with Ni. The assays revealed that under Fe-deficient and Ni-supplemented conditions, a reduction in root biomass was a more reliable phenotypic characteristic for ireg2 mutants than root length or shoot biomass. In contrast, for irt1, no observable phenotype was established under Fe-deficiency conditions. In parallel, Gateway cloning was employed to create expression clones where ScIRT1 and ScIREG2 protein coding expression was to be driven by native Arabidopsis promoters pAtIRT1 and pAtIREG2 (i.e. pAtIRT1:ScIRT1 and pAtIREG2:ScIREG2) respectively for complementation of the Arabidopsis irt1 and ireg2 mutants. The open reading frames of the S. coronatus genes and the Arabidopsis promoters were PCR amplified, cloned into appropriate pDONR221 vectors, and sequence verified. The ScIREG2 clone however, revealed point mutations and could not be used. pAtIRT1 was successfully recombined with ScIRT1 to generate a two-fragment expression clone which was verified by DNA sequencing. Thus herein, the foundations for ScIRT1 and ScIREG2 complementation experiments have been established.

Haliotis midae aquaculture within South Africa remains afflicted by infectious diseases. Understanding of how the abalone’s innate immune response functions is one of the greatest hindrances to assisting with the defence or detection of pathogenic attacks on farms. The multifunctional oxygen transporter haemocyanin was previously found to be upregulated in response to bacterial infection (Beltran 2015), indicating it may play a role in the defence response of Haliotis midae. The current knowledge regarding haemocyanin’s role in the abalone innate immune response is incomplete. A number of studies have been published that investigated haemocyanin’s potential as a broad spectrum antimicrobial peptide in many arthropod species. There has been only one study conducted in molluscs, which utilised synthetic peptides derived from a haemocyanin consensus sequence. In most organisms the haemocyanin protein is comprised of a string of eight roughly 50 kDa functional units (FU) annotated a-h. The current study determined the nucleotide sequence of the final four functional units on the C-terminal end of H. midae haemocyanin and examined the potential antimicrobial activity of the peptide product. The haliotisin coding sequence identified by Zhuang et al. (2015) was detected in FU-e of the H. midae haemocyanin and subcloned into an Escherichia coli expression vector for recombinant production of the peptide. This peptide showed some activity against Staphylococcus aureus and E. coli, suggesting it may function as an antimicrobial peptide. This study provides the first evidence that an antimicrobial peptide derived from the H. midae haemocyanin could be functioning as a component of the abalone innate immune response.

African horse sickness (AHS) is a debilitating viral disease affecting equines and has resulted in many disastrous epizootics. To date, no successful therapeutic treatment exists for AHS and the commercially used live-attenuated vaccines (LAVs) have various side effects. Insoluble particulates have been shown to increase immunogenicity when compared to soluble subunit vaccines and previous studies demonstrated protection of BALB/c mice immunised with African horse sickness virus (AHSV) VP7 against a lethal challenge of AHSV-7 (Bailey 2016; Rutkowska et al. 2011; St Clair et al. 1999; Storni et al. 2005; Wade-Evans et al. 1997). This study investigates a safer monovalent vaccine alternative based on plant-produced quasicrystals of the serogroup-specific AHSV structural protein, VP7. AHSV serotype 5 (AHSV-5) VP7 was expressed in Nicotiana benthamiana by means of Agrobacterium-mediated infiltration of plant expression vector pRIC3.0 encoding VP7 and quasi-crystals purified by means of density gradient ultracentrifugation. The presence of AHSV VP7 quasi-crystals was confirmed by western immunoblotting with anti-AHSV VLP guinea-pig serum and characterized using transmission electron microscopy. After optimizing the purification protocol and achieving satisfactory concentrations, AHSV-5 VP7 quasi-crystals were used in guinea-pig immunogenicity studies where the experimental group (n=5) was inoculated with prime- and boostinoculations of between 10 and 50 µg of purified AHSV VP7 quasi-crystals, and the control group (n=5) inoculated with a control inoculum prepared in the identical manner as the vaccine but using a pRIC3.0 expression vector lacking VP7. Western immunoblot analysis of the humoral response showed stimulation of very high titres of anti-VP7 antibodies 28 days after the boost-inoculation. In addition, RNA-seq transcriptome profiling of guinea-pig spleen derived RNA was used to investigate the global immune response to AHSV-5 VP7 quasi-crystals. Thirty genes involved in innate and adaptive immunity were found to be significantly differentially expressed (q≤0.05) in experimental transcriptome data when compared to the control. Differential expression of genes involved in T-helper (Th)1, Th2 and Th17 cell differentiation and the T-cell receptor signalling pathway suggest a possible cell-mediated immune response to AHSV-5 VP7 quasi-crystals. Upregulation of several important cytokines and cytokine receptors were noted in response to VP7 quasi-crystals e.g. TNFSF14, CX3CR1, IFNLR1 and IL17RA. TNFSF14 and CX3CR1 play a role in T-cell proliferation and cytotoxic T-cell responses respectively. And IFNLR1 and IL17RA are key cytokines in antiviral defences. Upregulation of IL17RA suggests a Th17 response which has been reported as a key component in AHSV immunity. To the best of our knowledge, this study is the first to report the expression of plantproduced AHSV VP7 quasi-crystals and the first time that the cell-mediated immune response to these particles has been assessed. While further investigation is needed, these results suggest that AHSV-5 VP7 quasi-crystals produced in N. benthamiana are immunogenic, inducing both humoral and cell-mediated responses.

Approximately 62% of the 355 cycad species in the world are classified as threatened with extinction. The African genus, Encephalartos, has a total of 65 species, approximately 70% of which are threatened. This status emphasizes the need to conserve these species; however, the recalcitrant nature of cycads seeds makes it difficult to conserve using conventional seed banking methods. Recalcitrant seeds have a short lifespan and cannot be dried or stored for prolonged periods as they become non-viable when they lose moisture. While studies on cryopreservation for conserving cycad germplasm and banking these desiccation-sensitive seeds has made some advances, cycad conservation is still limited to ex situ living plant collections. In vitro tissue culture is a promising technique for conserving cycads. While attempts have been made, there have been few reported successes and, to date, there has been no successful regeneration of Encephalartos species. As such, this study was aimed at developing an efficient and successful in vitro regeneration protocol for two Encephalartos species. Embryo regeneration of E. altensteinii and E. manikensis was assessed, testing the effects of plant growth regulators (PGRs) - 0.5 mg/L Kinetin and 0.5 mg/L 6-BAP, alone or in combination, sucrose (0, 15 and 30 g/L) and light. Within six weeks of culture, embryos of both species were able to regenerate, however, each responded differently to the tested variables. While shoot regeneration was evident for both species during this period, this was however not explained by any of the variables assessed in this study. Rooting was highest in the treatments with 0.5 mg/L Kinetin for E. altensteinii, after subculture rooting was favoured by the treatments with 0.5 mg/L Kinetin + 0.5 mg/L 6-BAP. Encephalartos manikensis rooting was significantly higher in PGR-free treatment in the first six weeks of culture. After subculture, rooting was enhanced by the treatment with the highest PGR concentration of 1.0 mg/L Kinetin + 1.0 mg/L 6-BAP. Darkness enhanced rooting while 16h photoperiod enhanced shooting for both species. However, the regeneration of both roots and shoots was more prevalent in the treatments that were initiated in complete darkness as compared to the treatments initiated under 24h of light. Both species responded to sucrose; with increase in sucrose concentration, callus induction increased for E. altensteinii while, necrosis and contamination increased for E. manikensis. In vitro-derived E. altensteinii plantlets acclimatization was unsuccessful and only 3.5% of E. manikensis were successfully acclimatized. This study suggests that although both these species belong to the same genus, in vitro culture protocol should be species specific. The overall regeneration of both species was however low, thus the second study assessed the levels of phytohormones in E. altensteinii seed tissues (embryos and megagametophytes) of the same age as those used in the in vitro regeneration. Phytohormones, as well as multiple phytohormone interactions (i.e. interplay between Abscisic acid (ABA) and Gibberellins (GAs)), play a role in the germination, growth and development of a plant. The high levels of a germination inhibiting ABA compared to the low levels of cytokinins and auxins, as well as the absence of GAs obtained in the assessed seed tissue, suggest that no real germination was taking place. Thus these results suggest that E. altensteinii seeds have a very slow developmental process with the likely chance that at this age they may be immature.

Cyanide is highly toxic as it inhibits respiration in aerobic organisms by binding to cytochrome c oxidase in the mitochondrial electron transport chain. Plants naturally produce cyanide from the hydrolysis of cyanogenic glycosides and as a by-product of ethylene biosynthesis. β-Cyanoalanine synthase prevents self-poisoning by combining endogenous cyanide with cysteine in the mitochondria to form β-cyanoalanine, which is further hydrolysed to asparagine, or aspartate and ammonia, by plant nitrilase 4 enzymes. β-Cyanoalanine synthase activity enables plants to detoxify limited concentrations of exogenous cyanide. However, phytotoxicity and death occur from exposure to relatively low concentrations of exogenous cyanide. In contrast, some microorganisms have a high capacity for cyanide detoxification due to a number of metabolic pathways including the degradation of cyanide to formate and ammonia; or formamide, by bacterial cyanidase (CynD) and fungal cyanide hydratase (CHT), respectively. Environmental contamination caused by failure to contain cyanide from anthropogenic sources is an important global problem. Hydrometallurgical gold mining utilises cyanide as a lixiviant due to the high affinity of cyanide for gold and the stability of the resulting cyanometallic complexes in aqueous solution, and thus is a significant source of cyanide contamination of soil and water. Biological treatment methods for cyanide, such as phytoremediation, could provide alternatives to the currently used chemical destruction techniques with their associated disadvantages. The use of phytoremediation would require plants to tolerate high concentrations of cyanide in soil. Two attempts have previously been made, with some success, to increase cyanide tolerance in Arabidopsis by genetic engineering: the first, by augmenting the β-cyanoalanine synthase pathway using a microbial nitrilase; and, the second, by introducing a microbial detoxification pathway targeted to the chloroplasts while overexpressing the endogenous enzyme which metabolises the product of the cyanide detoxification reaction. The aim of the current study was to determine whether Arabidopsis thaliana could co-opt the CynD and CHT genes from the cyanide-degrading Bacillus pumilus and Neurospora crassa to detoxify higher levels of cyanide using the encoded enzymes, and whether targeting CynD and CHT to the mitochondria would confer a greater enhancement of cyanide tolerance on plants compared to targeting to the cytoplasm.

High risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. The three approved prophylactic vaccines do not benefit already infected individuals; therefore, there is still an urgent need for therapeutic vaccines. The HPV oncoproteins E6 and E7 are ideal targets for the development of such vaccines, as they are expressed throughout the viral life cycle and in tumours. They could be used to elicit strong cytotoxic lymphocyte (CTL) responses which would aid in viral clearance, and could also be effective against tumours. Granadillo et al. (2011) developed an Escherichia coli-produced therapeutic vaccine candidate, consisting of the HPV-16 E7 protein and a cell membrane- penetrating and immunomodulatory peptide (LALF), whose fusion to HPV-16 E7 aided in the immunogenicity and antigen presentation of the oncoprotein. However, such vaccines need not only to be effective, but also to have a low cost. Plant expression systems represent an attractive alternative to conventional expression systems based on bacterial, yeast, mammalian and other cell cultures, and are potentially far more cost- effective. The aim of the present project was to produce LALF-E7 in Nicotiana benthamiana leaves via Agrobacterium-mediated transient expression, and to optimize its expression, extraction and purification. This was done by expressing LALF-E7 using different expression vectors, testing different subcellular localizations, and testing the effect of gene silencing suppressors. By using our group's replicating expression vector and targeting LALF-E7 to the chloroplasts, the expression of the candidate vaccine in N. benthamiana leaves was increased 26.8 fold compared to non-replicating vectors or cytoplasmic localization. Furthermore, silencing suppressors did not significantly increase the expression of LALF-E7 when expressed by the replicating vector and targeted to the chloroplasts. I showed, by fluorescence microscopy, that LALE-E7 was indeed being targeted to the plants' chloroplasts and that it possibly forms proteins bodies (PBs) that are closely associated to the chloroplast envelope. I further hypothesized a mechanism by which the PBs-like structures form. Once the expression of LALF-E7 was optimized in plant leaves, a purification strategy was developed by testing different extraction methods and using metal ion affinity chromatography. The extraction protocol developed pre-purified LALF-E7 by removing the majority of soluble proteins from the final extract. However, LALF-E7 was not fully purified by affinity chromatography, suggesting that other purification strategies should be used. Finally, I tested the partially purified plant-produced LALF-E7 candidate, and compared it to the E. coli-produced counterpart, in tumour regression experiments using mice as animal models. Due to low antigen doses and a large number tumourigenic cells used to inoculate the mice animal models, the effect of the plant-produced LALF-E7 as a therapeutic vaccine was inconclusive. However, it was suggested that it could potentially be comparable to the E. coli-produced counterpart. In summary, I report for the first time the entire chain of research involving the expression of LALF-E7 in plants, its extraction, purification and the testing of its immunogenicity in a mouse model. This research also suggests new avenues for the use of the LALF peptide as a PB-inducer which could be useful in increasing the expression of other recombinant proteins.

The accumulation of exceptionally high concentrations of heavy metals in plant tissues is an extreme phenotypic trait that has evolved independently in multiple plant taxa. The majority of research undertaken in this area has been performed on zinc/cadmium hyperaccumulators and comparatively little is known about the molecular mechanisms behind nickel accumulation. This is despite the fact that nickel hyperaccumulators constitute more than 75% of all known hyperaccumulator species. One such species is Senecio coronatus (Asteraceae), which is a useful model to study nickel hyperaccumulation - as both hyperaccumulator and non-accumulator populations have been identified on nickel-rich serpentine soils in South Africa. The nickel-transporting abilities of three proteins (ScMATE, ScVIT and ScCOP), previously shown to be constitutively over-expressed in shoot tissues of hyperaccumulating populations of S. coronatus, were investigated in order to determine if they play a role in nickel hyperaccumulation. The RNA-Seq derived nucleotide sequences of these genes were confirmed by reverse transcriptase PCR, and computational analysis suggested that the proteins encoded by these genes display identical topology to their homologues in Arabidopsis thaliana. Heterologous expression of these proteins in a metal-sensitive yeast strain was performed to determine whether they are capable of transporting nickel. Although a minor reduction in nickel sensitivity was observed in yeast expressing ScMATE, and a minor increase in ScCOP-expressing yeast, no marked changes in sensitivity to nickel were observed. C-terminal EYFP-tagged MATE and VIT fusion proteins were transiently expressed in live onion cells to determine the subcellular localization of these proteins in planta. Fluorescence microscopy indicated that MATE localises to the nucleus and VIT to the tonoplast or plasma membrane.