Browsing by Author "Mostert, Ilani"

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    Genetic diversity and identification of putative recombination events in Grapevine rupestris stem pitting-associated virus
    (Stellenbosch : Stellenbosch University, 2017-12) Mostert, Ilani; Maree, H. J.; Burger, J. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Phylogenetic analysis for variant classification plays a key role in the characterisation of the aetiological role of viruses. The genomic regions selected to identify viral variants and the occurrence of recombination has the potential to influence tree topologies. To investigate the impact of these factors on variant classification, and to evaluate the success of certification schemes in eliminating virus infection, a diversity study was performed on Grapevine rupestris stem pitting-associated virus (GRSPaV), a ubiquitous virus commonly detected in cultivated vines and reportedly associated with Rupestris Stem Pitting disorder. Three surveys were conducted to characterise and compare the genetic diversity of GRSPaV on a global and local level, using a phylogenetic approach. The first constituted a collection of accessions from various countries to represent global virus diversity. A second survey was carried out on local mother blocks that previously conformed to certification requirements for South Africa. Finally, GRSPaV diversity in South African vineyards established prior to the implementation of current sanitary protocols was investigated. Two genomic areas, the coat protein and replicase domains, were selected for this study as these were used to characterise the sequence diversity of GRSPaV in previous studies. Mixed infections were found to occur within single vines, the genetic diversity of GRSPaV was confirmed with the clustering of sequences into five of the six distinct, currently recognised lineages, and a seventh, previously unclassified lineage was detected. Furthermore, the ability of the two domains to detect and classify variants was compared. Additional evidence for recombination in GRSPaV was provided and a correlation between recombinant sequences and inconsistencies between topologies generated by the two genome regions, was observed. Results indicate that disease control methods were moderately successful, but less effective at eliminating non-symptomatic variants. The study illustrates the effect of recombination on phylogenetic trees, and emphasises the importance of accounting for such factors in the characterisation of virus diversity. Increased knowledge of the recombination events within the GRSPaV genome could promote the development of a standardised method for variant classification and the clarification of the aetiological role of the virus.
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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.