Brigden, Alison2023-02-132023-02-132022https://hdl.handle.net/10539/34495A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2022Begomovirus infection causes a significant decrease in yield production in multiple economically important crops. This poses a problem to food security and livelihoods of farmers, and employers in the farming industry. Tomato curly stunt virus (ToCSV) exists as two variants, severe (ToCSV-V30) and mild (ToCSV-V22), which negatively impacts tomato production in South Africa. The non-coding intergenic region (IR) of ToCSV ssDNA is of particular interest because of its involvement in binding host factors to initiate infection and transcription of viral proteins. In addition, the IR region contains the promoter for the adjacent V2 ORF which encodes the pre-coat protein which is a known pathogenicity determinant in begomoviruses. The IR was investigated by agroinoculating Nicotiana benthamiana, an experimental host, with wild-type infectious clone constructs of ToCSV-V30 (V30), ToCSV V22 (V22) and IR mutants where nucleotides of the 3’IR in V22 were replaced with the same nucleotides as found in V30. The three V22 IR mutants constructed were a full 3’ IR swap mutant V22ΔIR-s (nt 61-137), and two shorter IR swaps on either side of the conserved TATA box; V22ΔIR-sl (nt 61-94) and V22ΔIR-sr (nt 108-138). The role of the V2 ORF was investigated by introducing single amino acid changes, creating V2 V27S and T58S substitution mutants, named V22ΔV2-V→S and V22ΔV2-T→S respectively. A mixed infection of wildtype (WT) V30 and V22 was also investigated as mixed infections are more likely to occur in the field as opposed to single viral infections. Another mixed infection of WT V30 and V30IR-s, the V30 backbone with the V22 IR, was done to further explore the role of the IR in a mixed infection. The putative C5 ORF in V22 was examined by extending the 5’ end of the ORF using site-directed mutagenesis to generate a longer, N-terminally extended protein like that encoded by the African cassava mosaic virus (ACMV) C5 ORF. V22ΔIR-s and V22ΔIR-sl resulted in a knock-out of upward leaf roll observed in wildtype V22. V22ΔV2- V→S resulted in more severe symptoms, a delayed recovery and increased viral load at 36 dpi compared to V22. V22ΔV2-T→S resulted in a delayed onset of induced symptoms compared to V22. The wildtype mixed infection resulted in an intermediary result in symptom scores and viral load compared to the wildtypes. The V30 and V30IR-s mixed infection induced more severe symptoms than both V30 and V22. Reverse transcription and PCR was performed on V22-infected leaf material at 20 dpi which verified the presence of a V22 3’ IR transcript. Furthermore, the V22 C5 mutant induced a delayed onset of symptoms compared to V22. This study concluded that a short nucleotide sequence within the 3’region of the IR plays a role in inducing upward leaf roll produced by V22 infection. Furthermore, the V22 V2 ORF was 19 concluded to play a role in symptom severity and recovery. The exploratory study of the putative V22 C5 ORF and the results observed open avenues to future studies to investigate the significance of putative ORFs and possible evolutionary links to these ORFs.enDissertation