Doctoral Degrees (Genetics)

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Browsing Doctoral Degrees (Genetics) by browse.metadata.advisor "Burger, Johan"

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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.