Doctoral Degrees (Genetics)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/551

Browse

Browsing Doctoral Degrees (Genetics) by browse.metadata.advisor "Bester, Rachelle"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    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.