Doctoral Degrees (Genetics)

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    Investigating the interactome of grapevine leafroll-associated virus 3 and Vitis vinifera
    (Stellenbosch : Stellenbosch University, 2023-12) Mostert, Ilani; Maree, Hano; Bester, Rachelle; Burger, Johan; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Grapevine leafroll disease (GLD) is a globally important grapevine disease that affects the yield and fruit quality of affected vines. Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus, family Closteroviridae) has been identified as the main causal agent of GLD due to its consistent association with the symptoms of GLD. GLRaV-3 has not been successfully eliminated from mature vines, and no natural source of resistance to GLRaV-3 has been reported. Although the impact of GLRaV-3 infection on the transcriptome and metabolome of infected vines has been investigated, little is known about the exact mechanisms by which these effects occur. Furthermore, the roles of proteins encoded by GLRaV-3 open reading frames (ORFs) have largely been inferred by sequence homology or analogy to related viruses, and functional studies to determine their involvement in virion assembly and movement have not been performed. The aim of this study was to investigate the GLRaV-3 interactome to identify virus or host genes that play a key role in the proliferation and pathogenesis of GLRaV-3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were employed to screen GLRaV-3 ORFs for pairwise interactions. The majority of interactions between structural proteins suggest that GLRaV-3 shares a common mechanism of assembly with members of the genus Closterovirus, family Closteroviridae, although some unexpected interactions were also found. Interaction of p20B, a silencing suppressor, with structural proteins has not been reported for other members of the family, indicating its possible involvement in other aspects of the viral replication cycle. The GLRaV-3 transmembrane protein self-interacted; however, the mechanism by which this interaction occurs remains unknown as it lacks a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. To identify virus-host interactions, a Vitis vinifera Y2H prey library was constructed and screened against GLRaV-3 ORFs encoding proteins involved in virion assembly, intracellular movement, and suppression of host silencing. BiFC was then used to demonstrate these interactions in planta. Two interactions identified using Y2H could not be demonstrated in planta and involved GLRaV-3 p20A, a protein of unknown function proposed to play a role in suppression of host defence responses and long-distance transport. In yeast, p20A was found to interact with a V. vinifera chlorophyll a-b binding protein and a V. vinifera SMAX1-LIKE 6 protein. Y2H and BiFC assays both demonstrated the interaction of p20A with V. vinifera mitogen-activated protein kinase and a V. vinifera small heat shock protein, as well as the interaction of GLRaV-3 minor coat protein with V. vinifera 3-deoxy-D-arabino- heptulosonate 7-phosphate synthase 02. All five of these host proteins are associated with host defence responses against pathogens. Furthermore, these interactions demonstrate that the symptoms of GLD may be caused by interference with a variety of pathways. This study contributes to our knowledge on the roles of GLRaV-3-encoded proteins in its replication and spread and provides information on cellular responses by grapevine against GLRaV-3. Understanding the proteins involved in the pathogenesis of GLRaV-3 can lead to the development of novel approaches to manage GLD.
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    Characterisation of citrus tristeza virus-induced stem pitting in citrus
    (Stellenbosch : Stellenbosch University, 2023-12) Aldrich, Dirk Jacobus; Maree, Hans Jacob; Bester, Rachelle; Burger, Johan Theodorus; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Citrus tristeza virus (CTV) is the most important viral pathogen of citrus and causes several disease syndromes in different citrus hosts. CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit. The exact mechanisms of stem pitting development in CTV-infected citrus remain unclear. This study aimed to utilise CTV infectious clone mutants in a reverse genetics approach to study stem pitting induction. A panel of recombinant CTV clones was generated focussing on the open reading frames (ORFs) implicated in stem pitting induction and pathogenicity, namely p33, p18, p13 and p23. ORF replacements from severe- and mild-pitting South African CTV isolates were introduced into the mild-pitting infectious clone (CTV-fl6 - genotype T36) to determine if severe stem pitting could be induced. Stem pitting assessments were complemented with the determination of virus concentration ratios in ‘Mexican’ lime and ‘Duncan’ grapefruit to relate stem pitting outcomes to virus concentration. The various infectious clone and wild type CTV infections yielded a broad range of stem pitting outcomes and specific ORF replacements associated with enhanced stem pitting were identified. Plant responses to different stem pitting pressures were further assessed by untargeted metabolite profiling and the quantitation of the stress-responsive phytohormones, abscisic acid, jasmonic acid and salicylic acid. In both citrus hosts, the metabolite profiling yielded fourteen statistically significant compounds that differed between stem pitting groups. These compounds were mainly phenolic acids and phenolic glycosides and are known to function as antioxidant and stress signalling molecules. Significant differences in phytohormone content were also found between test groups, particularly in plants that were severely stunted. Stem pits were also characterised at the molecular anatomical level using a combination of known and novel techniques to better understand the nature of the xylem and phloem tissues impacted by severe pitting. Established methods such as biological staining and fluorescence microscopy were used to visualise CTV-induced stem pitting and virus localisation. The utility of two novel technologies that have not previously been used to study CTV-induced stem pitting, namely high-resolution CT scanning and serial block face scanning electron microscopy was also evaluated. This study contributed to our understanding of CTV-induced stem pitting in citrus. Importantly, the use of CTV infectious clones for South African CTV research was established, which can allow for targeted follow-up experiments to further characterise the local citrus-CTV pathosystem. This can also provide novel opportunities for potentially employing CTV infectious clones as a crop protection tool against other important citrus diseases on the African continent.
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    An investigation into the wheat (Triticum aestivum L.) host response to Russian wheat aphid (Diuraphis noxia Kurd.) feeding
    (Stellenbosch : Stellenbosch University, 2023-12) Fisher-Smith, Nadia; Botha-Oberholster, Anna-Maria; Van der Vyver, Christell; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjomov), is a major pest of wheat, causing damage and high yield losses worldwide. One of the undesirable effects of aphid feeding is leaf rolling, which serves as a shelter for the aphids protecting them from their natural predators and from insecticide spraying. Leaf rolling reduces the plant’s ability to photosynthesise and grow effectively, in addition, leaf rolling can also increase the aphid fitness, as it provides an ideal environment for growth. Therefore, identifying natural sources of resistance and introducing them into susceptible cultivars seems to be the most efficient strategy against RWA feeding. The use of genetic resistance is an efficient and environmentally safe method for controlling the RWA. This study aimed to ascertain if enhanced tolerance to biotic stress can be achieved by modifying plants either through genetic manipulation or chemical mutagenesis. Various studies have been done at a transcriptome level, allowing the identification of genes likely involved in RWA resistance. Utilising proteomics data in this study, allowed for the identification of differentially expressed peptides between resistant and susceptible wheat lines. Among the identified proteins were: glutathione-S-transferase (GST) and peroxidase. Literature suggests that GST forms part of the detoxification system in plants against biotic stress. This protein was uniquely expressed in the W1320-W1278 line that contains the Dn5 resistance gene. Peroxidase is associated with the oxidative burst, usually in response to stress, was identified in this study in the Gamtoos-S (Dn0) susceptible cultivar. Utilising genetic manipulation, a partial gene fragment of glutathione-S-transferase F6 (GSTF6b) was isolated from wheat and sequenced to confirm its identity. The gene fragment was cloned into a plant expression vector in the antisense orientation and bombarded into four- to six-day-old wheat immature embryos. Resulting in a putative transgenic plant, namely Gamtoos-S (Dn0)-pUBI-510:GSTF6b. Quantitative reverse- transcriptase-linked polymerase chain reaction (RT-qPCRs) were conducted to quantify the expression of the GSTF6b gene with/without RWA infestation. A reduction of nearly 50% was observed in GSTF6b expression in the respective transgenic plants when compared with the control. The T₁ was successfully hardened off, and allowed to seed and a T₂ generation was generated, which was functionally analysed through phenotypic screening, aphid fecundity, enzymatic responses and measuring oxidative burst. A decrease GST transcript level was observed post-infestation in the transgenic plants suggesting that plant susceptibility can probably be linked to a decrease in GST transcript promoting aphid growth and increasing the rate of reproduction. The last part of the study involved chemical mutagenesis, whereby drought-tolerant mutagenic M6 lines were screened for aphid resistance. A phenotyping assessment was performed on available mutant lines infested with South African (SA) biotype 1. A total of 33 mutant lines selected for drought tolerance, consisting of 21 ethyl methanesulfonate (EMS) and 12 Sodium azide (NaN₃) mutants, showed variation in aphid tolerance. Furthermore, drought-tolerant mutants were found to be more susceptible to aphid infestation, excluding the M12 (RYNOB8.012) line, shown to be intermediate to aphid feeding. The anti-oxidative enzyme GSTF6b expression was found to be significantly up-regulated in the mutagenic lines before infestation, therefore, contributing to the notion that GSTF6b is present at the basal level. A positive correlation was observed between GSTF6b gene expression and the intrinsic rate of increase (rm) in 25 mutagenic lines.
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    Characterisation of EMS mutagenic bread wheat (Triticum aestivum l.) lines to investigate their water deficit stress tolerance and adaptability
    (Stellenbosch : Stellenbosch University, 2023-03) Kayaga, Helen Ninsiima; Botha-Oberholster, Anna-Maria; Van der Vyver, Christell; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Wheat is a staple food for 2.5 billion people worldwide and is the second most important cereal grain grown in South Africa. Water deficit stress has adverse effects on wheat productivity in the world. This is exacerbated under current unpredictable climatic patterns due to global warming. In 2017, most of the wheat production areas in South Africa were lost to alternate crops due to prevailing drought conditions. This created a need to develop water deficit stress tolerant wheat lines using ethyl methanesulfonate (EMS) because currently, no officially released varieties in the country carry such a trait. This study aimed to characterise newly developed mutagenic water stress-resilient bread wheat lines. The agro-morphological traits of three wild types and ten M3 lines were determined based on wheat descriptors in a randomised experiment with three replicates in a greenhouse. The chemical properties of flour were measured using the Inframatic 9500 NIR Grain Analyser machine, and the sedimentation tests of flour were quantified using the AACC International Method 56-60.02. The M3 lines were screened for tolerance to water deficit stress in a split-plot (3x5 factorial) experimental design that induced stress at the following growth stages: stress at seedling growth (emergence), tillering (forty days after planting), anthesis (flowering), milk development stage (seed set), and control (well-watered), and 13 subplots (bread wheat lines). Results from the analysis indicated a variation in the agro- morphological traits (18 traits) of mutants from the wild types (WTs) aside from ten traits that were similar across all bread wheat lines. Baking quality analysis showed that mutant Big 8.3 had the most desirable moisture content percentage of flour, a high protein, and wet gluten. Hence its dough has good elasticity and extensibility. The mutants performed better than WTs under water deficit stress. Big 395.1 was the most tolerant at emergence and forty days after planting, while Big 8.1 and Big 8.3 were most tolerant to water deficit stress at flowering and seed set, respectively. Previously identified drought-related genes, Sal1 (an inositol polyphosphate 1- phosphatase encoding gene) and Era1 (enhanced response to Abscisic acid 1), were analysed for single nucleotide polymorphism (SNPs) variants induced by chemical mutagenesis through sequencing complementary DNA (cDNA) of the genes and aligning them to the reference wheat genome of “Chinese Spring” available on the National Centre for Biotechnology Information (NCBI) web-based platform. I could not attain results from the Era1 sequences, and alignment results showed no differences between the Sal1 sequences of the mutant lines to that of reference genome “Chinese Spring”. In conclusion, chemical mutagenesis increased the genetic diversity of the parental bread wheat lines through induction of point mutations resulting in desirable new mutant lines like Big 8.3 with a desirable agrotype, baking quality characteristics, and high tolerance to water deficit stress at seed set.
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    Analysis of interactions between glucan, water dikinase and either isoamylase or starch branching enzymes in detrmination of starch structure
    (Stellenbosch : Stellenbosch University, 2022-12) Adegbaju, Muyiwa Seyi; Lloyd, James Richard; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Department of Genetics. Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: Starch is the main form in which carbon is stored in plants and it is used in a wide array of applications, from its role as a source of calories in human diets, to livestock feed and as an industrial feedstock. It is composed of two types of glucan polymer, amylose which is com- posed mainly of α-1,4 linked chains and highly branched amylopectin which contains α1,4 chains which are connected together with α1,6 branchpoints. Synthesis of the starch polymer involves co-ordinated activities of starch synthases, starch branching enzymes (SBE) and de- branching enzymes (DBE). In addition, the enzyme glucan, water dikinase (GWD) can intro- duce phosphate into amylopectin. The critical roles these enzymes play during starch metabo- lism have been investigated by knockout or silencing of genes encoding them in some plants, mainly Arabidopsis thaliana. Interactions between some of these enzymes have also been demonstrated, but there are still many questions about how these interactions occur and how they influence starch metabolism. In this study two sets of experiments were performed. The first involved analyses of potato (Solanum tuberosum L.) plants where the expression of SBEI, SBEII and/or Glucan, Water Dikinase 1 (GWD1) were repressed using RNAi technology. Individual or joint repression of the two SBE isoforms resulted in a significant increase in starch phosphate, whereas repression of GWD1 led to synthesis of low phosphate starch. Starch phosphate decreased in SBEI/GWD1 and SBEII/GWD1 lines but not to the levels found in lines where only GWD1 was repressed. The apparent amylose content increased in starch from SBEI/GWD1 lines was greater than either the SBEI or GWD1 lines. These alterations in starch composition influenced its granule morphology, swelling power and freeze-thaw stability. Silencing of GWD1 reduced starch deg- radation in cold-stored tubers, but this was not the case in lines repressed in either starch branching enzyme. The second set of experiments examined polyglucan metabolism in potato lines repressed in isoamylase 2 (ISA2) and/or GWD1. Transgenic potato lines were produced by RNAi gene silencing and tuber starch metabolism analysed. The water-soluble glucan content in ISA2 and ISA2/GWD1 lines tuber was higher than GWD1 lines. Analysis of starch structure indicated that there was an increase in the apparent amylose content of starches from GWD1 lines whereas it was unchanged ISA2/GWD1 lines. There was a reduction in the starch phosphate in both GWD1 and ISA2/GWD1 lines, and this reduction was greatest in the GWD1 lines. The ISA2 lines contained a proliferation of small starch granules, but this was not observed in ISA2/GWD1 lines which indicates that ISA2 and GWD1 interact during granule initiation. The effect of both enzymes on cold-induced sweetening (CIS) was also investigated. As expected, CIS was reduced in lines where GWD1 was silenced. Interestingly CIS was also inhibited in the ISA2 lines indicating that the pathway of starch degradation may differ between cold-stored potato tubers and Arabidopsis leaves.