3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Geminivirus replication-association protein (Rep) as a target for the development of small molecule inhibitors by(2020) Kgomokaboya, Maite CodlinneReplication-association protein (Rep) is an indispensable protein for the replication of Geminivirus single-stranded DNA in its life cycle. Geminivirus, in particular African Cassava Mosaic Virus (ACMV), affects agricultural crops such as Cassava (Manihot esculenta Crantz) that have the potential to bridge food insecurity in sub-Saharan Africa where it is mostly produced. For this virus to replicate, it encode for a Rep protein to initiate the rolling cycle replication (RCR) therefore making the protein indispensable for viral life cycle. Understanding the mechanism of viral replication proteins is essential because of the involvement with replication and spread of the disease. There is an urgent need to develop novel strategies to control the virus. Therefore, the main aim of this study was to investigate ACMV Rep protein functional activity as a target for the development of small molecule inhibitors. To achieve this aim, overexpression of full length ACMV Rep recombinant protein in Escherichia coli (E.coli) BL21 (DE3) pLysS bacterial cells and purification on nickel affinity chromatography was successfully done. Biochemical and biophysical characterisation is a crucial step to follow in order to understand the role of proteins in the viral life cycle. he structural determination of ACMV Rep protein was first predicted using online bioinformatics tool ExPASy and further determined with Fourier-transform infrared (FTIR)and intrinsic fluorescence spectroscopy. The secondary structure prediction from ExPASy resulted in the prediction of helices, sheets and coils. From FTIR spectroscopy analyses, the Amide I region was detected which was reported to be common in proteins representing the secondary structure. The overlapping peaks of the Amide I region were resolved through deconvolution on OriginPro 8 and this resulted inα-helices, β-sheets, β-turns and coils which correlates with the ExPASy results. Intrinsic fluorescence spectra showed an emission maximum wavelength of 355 nm at both 280 nm and 290 nm excitation wavelengths. The emission maxima shows that the tryptophan is exposed to the solvent. Assessing the activity of proteins is another step to determine proper folding of the protein. This was determined with binding and cleavage assays using Electromobility Shift Assay (EMSA). Results showed that purified ACMV Rep was functional in both the binding and cleavage activity at increasing concentrations (≥0.4 μM). Enzyme-linked Immunosorbent Assay (ELISA), a microtiter plate assay for protein functionality, was also developed for the interaction of ACMV Rep and ACMV DNA. This assay is amenable to high throughput screening of potential inhibitors. ELISA results also confirmed the binding activity of the purified Rep protein to ACMV DNA, this assay can be adapted to screen potential small molecule inhibitors of ACMV Rep interaction with ACMV DNA. The identification of small molecules that inhibit the activity of ACMV Rep protein to viral DNA on EMSA was done. Natural phenolic compounds such as Epigallocatechin gallate (EGCG) and Chicoric acid (CA) have inhibition activity on the binding of ACMV Rep to DNA at a concentration of ≥100 μM. In addition, EGCG inhibits the cleavage activity of DNA by ACMV Rep protein with a concentration of ≥ 100 μM while CA showed no inhibition towards cleavage activity of the ACMV Rep on the DNA. From this study, more can be done to understand the crystal structure of the protein and its mechanism of interactionItem The transcription factor interacting network of tolerant TME3 and susceptible T200 cassava landraces infected with SACMV(2019) Freeborough, WarrenCassava, Manihot esculenta Crantz, is categorized as a food security crop, producing large starchy tubers that are gaining interest from both international and local agro-processing industries for products such as bioethanol, textiles, and food additives. However, cassava is currently under threat from a group of begomoviruses that cause cassava mosaic disease (CMD) in all countries in sub-Saharan Africa where cassava is cultivated. CMD can result in up to 100% crop loss. South African cassava mosaic virus (SACMV) is particularly a threat to the growing cassava industry in southern Africa. Despite extensive breeding programs over the past 70 years to develop CMD-resistant farmer-preferred cassava landraces, total resistance has not been achieved. Furthermore, the high mutational rates of begomoviruses, and mixed infections in the field, have exacerbated the problem. TME3 is a West African landrace that displays tolerance to begomoviruses, including SACMV. Infection of TME3 by SACMV leads to recovery, hallmarked by low virus loads and milder symptoms compared to a susceptible southern African landrace T200. The molecular processes that govern tolerance in crops, including cassava, are not well understood. However, systemic immune responses, which are controlled by hormoneresponsive transcription factors (TFs), are required by the plant to successfully combat an invading pathogen. Two different branches of systemic immunity have been described, namely systemic acquired resistance (SAR), facilitated by salicylic acid (SA) signalling, and induced systemic resistance (ISR), which is induced through jasmonic acid (JA) and ethylene (ET) signalling in the presence of beneficial rhizobacteria. In 2014, Allie et al. compared global transcriptomic responses occurring in TME3 and the T200 during early 12 days’ post inoculation (dpi), middle (32 dpi) and late (67 dpi) stages of SACMV infection. In order to give greater context to transcriptomic data, which is inheritably large and complex, network analysis may be implemented. By placing the differentially expressed (DE) gene homologs/orthologs identified from the cassava transcriptome datasets into protein-protein networks, functions of SACMV-responsive genes, interacting partners, and potential hubs, can be derived. Cassava gene functions are based on the model crop Arabidopsis thaliana, as despite the sequencing of the cassava genome, the annotations are incomplete. The aim of this study was to identify potential candidate TFs, and their associated hormones and other network partners, that confer either tolerance (TME3) or susceptibility (T200) to SACMV.