3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item 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.Item The recombinant expression and structural characterization of movement protein BC1 from South African cassava mosaic virus(2018) Nankoo, NikitaSouth African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises of DNA A (encodes six genes) and DNA B (encodes BC1 (cell-to-cell movement protein) and BV1 (nuclear shuttle protein)). Begomoviruses cause cassava mosaic disease (CMD) resulting in substantial root yield losses impacting farmers and potential starch yields for industrial purposes. The structural and physiochemical characteristics of begomoviruses have not been elucidated to date. Additionally, it is crucial to identify protein-protein interactions between SACMV BC1 and cassava host proteins that facilitate SACMV movement. In this study, in silico characterization studies for BC1 were undertaken. The FASTA amino acid sequence of BC1 was uploaded onto a webserver (Predict Protein (https://www.predictprotein.org)) and resulted in the predicted secondary structure of BC1 as well as predicted protein binding sites. BC1 was cloned into a pET-28a(+) expression vector and transformed into host cells E.coli BL21 (DE3). The optimal conditions for the expression of BC1 protein were found to be induction with 0.25 mM IPTG at an OD600 of ~0.6 expressed at 37 °C for four hours. The protein was refolded using stepwise dialysis. The molecular weight of BC1 was 35 kDa (SDS-PAGE). The secondary structure of BC1 was confirmed to be predominantly β-strands (CD). An ANS (1-anilino-8-naphthalene sulphonate) binding assay confirmed that BC1 possesses hydrophobic pockets with the ability to bind ligands such as ATP. A 2’ (3’)-N-methylanthraniloyl-ATP (MANT-ATP) assay showed binding of MANTATP to BC1. Intrinsic tryptophan fluorescence studies indicated significant conformational change in the denatured form of BC1 in the presence of ATP compared to in the absence of ATP. Literature and the PredictProtein webserver were employed to in silico predict suitable cassava host prey proteins for the yeast-two-hybrid (Y2H) system. Cloning of host proteins (HSP70 and Histone H3) into pDEST™22 plasmids was done at GenScript USA. The in silico study and structural characterization of BC1 provide insights to the structural characteristics of BC1 and further explains its functionality. Completion of Y2H assays will confirm or refute that cassava host proteins Histone H3 and HSP70 interact with the cell-to-cell movement protein of SACMVItem Screening of cassava improved germplasm for potential resistance against cassava mosaic disease(2017) Mvududu, DonTafadzwa KudzanaiWith growing populations and climate change associated drought predicted for the future, cassava can provide one solution for food security and a source of starch for industrial use and biofuels in South Africa, and other countries in the SADC region. One of the severe constraints on cassava production is cassava mosaic disease (CMD) caused by cassava infecting begomoviruse species, including African cassava mosaic virus (ACMV), South African cassava mosaic virus (SACMV) and East African cassava mosaic virus (EACMV). Cassava begomoviruses (CBVs) are responsible for significant yield loss of the starchy tubers. Since no chemical control of virus diseases of plants is possible, one approach to develop virus resistance is via biotechnology, through genetic engineering (GE) of cassava with hairpin RNA (hpRNA) silencing constructs that express small interfering RNAs targeting CBVs and preventing severe disease development. The aim of this project was to subject previously transformed five CMM6 cassava lines (cv. 60444 transformed with a non-mismatched Africa cassava mosaic virus-[Nigeria:Ogorocco;1990] (ACMV-[NG:Ogo:90])-derived hpRNA construct, six AMM2 (cv. 60444 transformed with a mismatched ACMV-[NG:Ogo:90]-derived hpRNA construct), six CMM8 cassava lines (cv.60444 transformed with a non-mismatched SACMV BC1-derived hpRNA construct) and seven AMM4 cassava lines (cv.604444 transformed with a mismatched SACMV BC1-derived hpRNA construct) to reproducible trials, and evaluate for response to virus challenge. The ACMV-[NG:Ogo:90] hpRNAi constructs target 4 overlapping virus open reading frames (ORFs) (AC1 replication associated protein/AC4 and AC2 transcriptional/AC3 replication enhancer), while the SACMV hpRNAi constructs target the cell-to cell movement BC1 ORF. Non mismatched constructs consist of a transformation cassette that has an intron separating the sense and antisense arms of the viral transgene whilst mismatched constructs have the sense arm of the viral transgene treated with bisulfite to induce base mutation. This mutated sense arm is then separated from the non mutated antisense arm by a small spacer. Furthermore, a 229 bp inverted repeat hpRNA construct (DM-AES) was designed to target ACMV-[NG:Ogo:90] 117 nt putative promoter region (2714-49 nt), a 91 nt overlapping sequence (1530-1620 nt) between ACMV-[NG:Ogo:90] AC1 3’ end and AC2 5’ end (AC1 3’/AC2 5’-ter) as well as being efficient against SACMV and EACMV due to the inclusion of a 21 nt conserved sequence (1970-1990) of AC1/Rep shared between ACMV, EACMV and SACMV. Cassava landrace T200 friable embryogenic callus (FEC) were transformed with this construct. The selected transgenic lines were infected with either ACMV-[NG:Ogo:90] (CMM6 and AMM2 transgenic lines) or SACMV (CMM8 and AMM4 transgenic lines) by agro-inoculation and monitored at 14, 36 and 56, 180 and 365 days post infection (dpi) for symptom development, plant growth and viral load. From the ACMV trials 3 lines (CMM6-2, CMM6-6 and line AMM2-52) showed significantly lower symptom scores and lower viral load at 36, 56 and 365 dpi, compared with viral challenged untransgenic cv.60444. This phenotype is described as tolerance, not resistance, as despite ameleriorated symptoms virus replication persists at lower levels. From the SACMV infectivity trials even though all CMM8 and AMM4 transgenic lines had lower symptom severities and viral loads compared with infected untransformed cv.60444, the results were not highly significant (p˃ 0.05). From this study, tolerance or reduction of viral load and symptoms was attributed to the accumulation of transgene-derived siRNAs prior to infection. However there was no observable correlation between levels (semi-qauntitative northern blots) of siRNAs and tolerance or susceptible phenotypes. Tuber yield evaluation of the three tolerant lines (CMM6-2, CMM6-6 and line AMM2-52) showed that the tuber fresh and dry weight at 365 dpi was not affected by the viral presence. These are promising lines for larger greenhouse and field trials. A comparison between the two different constructs showed that the two tolerant CMM6 lines-2 and 6 appeared to perform better (viral load) compared with AMM2 tolerant line-52 with regards to levels of viral amplification. The mismatched construct in AMM4 lines and the nonmismatched construct in CMM8 lines induced the same viral and symptom severity score (sss) reduction. Transformation of T200 FECs with the DM-AES construct was unsuccessful due to the age (more than six months old) of the FECs. FECs are more likely to lose their regeneration and totipotent nature with age. We therefore propose the use of fresh T200 FECs in future transformation studies to test the DM-AES construct.