3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Transcriptome analysis of cassava (Manihot esculenta Crantz) in response to mealybug (Phenacoccus manihoti) infestation(2018) Rauwane, Molemi EvelynCassava mealybug (Phenacoccus manihoti) is one of the most damaging pests of cassava (Manihot esculenta Crantz) globally. While biological control of mealybugs through the use of natural predators has been successfully practiced, breeding for resistant cultivars remains an important means of control. Understanding plant responses to insect herbivory, by determining and identifying differentially expressed genes (DEGs), is a vital step towards the understanding of molecular mechanisms of defence responses in plants and the development of resistant cultivars by gene editing. Mealybug isolates were collected from different cassava growing regions of South Africa and a detailed morphological and molecular characterization of the pests was undertaken. Morphological and molecular analysis confirmed the mealybug identity as Phenacoccus manihoti (Matile-Ferrero). The positively identified mealybug isolates were used to artificially infest three cassava genotypes with contrasting response to mealybug. The response of cassava genotypes AR23.1 (which has resistance to multiple diseases and to green mites), P40/1 (which has no known resistances) and 98/0581 (which has multiple pest resistances and resistance to cassava mosaic disease) was further investigated by analysing their transcriptome after mealybug infestation at 24 and 72 hours in comparison with non-infested mock tissues. A total of 301, 206 and 269 transcripts from AR23.1, P40/1 and 98/0581 were differentially expressed (log2 fold and P ≤ 0.05) per time point following mealybug infestation. In two genotypes (AR23.1 and 98/0581), mealybug infestation resulted in a higher number of down-regulated than up-regulated genes between mealybug-infested and mock-infested tissues, while in P40/1 between infested leaves at 24 hpi and 72 hpi, more genes were up-regulated than were down-regulated. There was an increase in the number of DEGs from 24 hpi to 72 hpi suggesting an early induction of defence responses during the mealybug infestation. Gene expression was further compared between AR23.1, P40/1 and 98/0581 to determine whether there were any genotype-specific expression patterns. Within the Gene Ontology (GO) functional classification, DEGs in the class Secondary Metabolic Processes were significantly enriched in AR23.1 in comparison with P40/1 and 98/0581. Additionally, DEGs in the class Regulation of Molecular Function were significantly enriched in P40/1, while DEGs in the classes Reproduction and Reproductive Processes, and Nutrient Reservoir, were enriched in the multi-pest resistant 98/0581 genotype compared to P40/1 and AR23.1. In the metabolic pathway analysis performed using the KEGG database, DEGs in the classes ABC Transporters and MAPK signalling pathways were present in AR23.1 and 98/0581; and not in P40/1. There were both up- and down-regulated genes that were mapped to the classes Plant-Pathogen Interactions, and Plant Hormone Signal Transduction pathways in genotypes AR23.1 and 98/0581; while in P40/1, there were only down-regulated genes classified under Plant-Pathogen Interactions pathway. The up-regulated DEGs associated with these pathways are suggested to be involved in defence in response to P. manihoti feeding since these stress-associated genes, including those related to transcription factors, phytohormones and secondary metabolism were significantly induced in the AR23.1 (resistant) and 98/0581 (tolerant) genotypes, and not in the P40/1 (susceptible) genotype. Stress-associated genes such as 2-oxogluterate / Fe (II)-dependent oxygenase superfamily protein, terpene synthase 21, heat shock family proteins and cytochrome P450 superfamily proteins were induced in AR23.1 and 98/0581 genotypes. The results revealed a significantly different response to mealybug infestation in the three genotypes studied, with genotypes AR23.1 and 98/0581 showing a higher proportion of differentially expressed transcripts compared with the susceptible genotype P40/1 during infestation with mealybugs. Putative candidate defence-related genes that were overexpressed in the AR23.1 genotype post-infestation will be useful in future functional studies towards the control of mealybugs. These results will further form the basis for more detailed future studies on the specific role of all the differentially expressed transcripts identified. The transcripts identified in this study will also contribute significantly to the cassava EST database, and can be used to improve the annotation of the cassava genome.Item Elucidation of the role of NOA1 and myosins in host response to infection by SACMV(2017) Mwaba, Imanu Msifu ImmaculeeDifferent host genes playing a role in replication, transcription and movement of geminiviruses have been identified, allowing a better understanding of host response during infection. The cytoskeletal protein myosin has been shown to associate with RNA viruses movement protein and mediate its movement, however no geminivirus association with myosin has been established. Arabidopsis thaliana nitric oxide associated protein 1 (AtNOA1), once thought to be an enzyme involved in a nitric oxide (NO) production, has been reported to be differentially regulated in response to biotic and abiotic stress. In this study we sought to identify the role that myosins and NOA1 play in the development of disease by south african cassava mosaic virus (SACMV). Using a bioinformatics approach, 24 myosin transcripts were identified in Nicotiana benthamiana, and phylogeny analysis revealed that seven were class VIII myosins and 17 class XI. Five myosins silencing constructs M15.1 (transcript Niben101Scf11288g00015.1), MYOSIN XI-F (M11.F), MYOSIN XI-K (M11.K), MYOSIN XI-2 (M11.2) and MYOSIN VIII.B were selected for silencing using a virus induced gene silencing (VIGS) approach with SACMV and TRV-VIGS vectors. At 14 days post inoculation (dpi), both SACMV and TRV-VIGS vectors successfully silenced myosins with SACMV-VIGS silencing all five and TRV-VIGS silencing all but M11. F. At 28 dpi, SACMV-VIGS induced silencing of myosin of only two myosins and TRV-VIGS three. TRV-VIGS was found to be more efficient at silencing as the suppression of myosin induced by TRV-VIGS was stronger than that of SACMV-VIGS. To assess the effect of myosin silencing on SACMV infectivity in a separate experiment, 7 dpi of silencing, N. benthamiana plants were challenged with SACMV and reduction of myosin expression was assessed as well as viral accumulation. TRV-VIGS did not induce any silencing of myosin at 14 dpi, and at 28 dpi, the expression of M11.K and M11.F were silenced. SACMV-VIGS induced silencing of M11.F at both 14 and 28 dpi. In TRV-VIGS silenced M11.K, viral load at 28 dpi was not lower than the control, however the fold increase in viral load at 28 dpi compared to 14 dpi was 3-fold (p value 0.03) for M11.K silenced TRV-VIGS plants and 86-fold for the control 6-fold for the M11.K suggesting that silencing of M11.K decreases the spread of SACMV. In TRV-VIGS silenced M11.K, viral load at 28 dpi was lower than the control (9-fold p value 0.03) and the increase in viral load at 28 dpi compared to 14 dpi was insignificant, suggesting that spreading of SACMV was also hampered. The reduction in myosin M11.F expression induced iv by SACMV-VIGS resulted in an increase in viral load compared to the control. We hypothesise that the increase in viral load observed in M11.F silenced plants induced by SACMV-VIGS is due to the perceived resistance of SACMV-VIGS control (SACMV-challenged no silencing construct) to SACMV-challenge, and therefore results from the SACMV-VIGS study were inconclusive. From the TRV-VIGS study however, we have identified two candidate myosins in N. benthamiana myosin XI-K and myosin XI-F as potential interactor of SACMV during infectivity. Further research into their role in the development of SACMV disease is warranted. Nitric oxide associated 1 (NOA1) in plants is a cyclic GTPase involved in protein translation in the chloroplast and has been indirectly linked to nitric oxide (NO) accumulation. To understand the role played by NOA1 in response to (SACMV) infection, a bioinformatics approach was used to identify NOA1 homologues in cassava T200. Using the cassava genome data on Phytozome, a putative NOA1 namely cassava 4.1_007735m, was identified. Based on its protein sequence, cassava4.1_007735m shared a 69.6% similarity to Arabidopsis NOA1 (AtNOA1). The expression of cassava4.1_007735.m (MeNOA1) and N. benthamiana NOA1 (NbNOA1) and the accumulation of NO in leaf samples was compared between SACMV-infected and non-infected at early infection stage (14 dpi for N. benthamiana and 28 dpi for cassava T200) and full systemic stage (28 dpi for N. benthamiana and 56 dpi for cassava T200). Real-time PCR was used to measure SACMV viral load which increased significantly by 2-fold (p value 0.05) from 14 to 28 dpi for N. benthamiana and 8-fold from 28 to 56 dpi in cassava T200 (p value 0.04) as chlorosis and symptom severity concomitantly progressed. At 14 and 28 dpi, NbNOA1 expression was significantly lower than mock inoculated plants (2-fold lower at 14 dpi, p value 0.01 and 4 fold lower at 28, (p value 0.00) and the abundance of NO in infected N. benthamiana leaf tissue was 10% lower at 14 dpi and 40% lower at 28 dpi when compared to mock inoculated. In cassava T200, MeNOA1 expression was unchanged at 28 dpi and NO levels were decreased by 40% and at 56 dpi, MeNOA1 expression was 4-fold lower and NO accumulation was 37 % higher than that of mock inoculated leaf tissue. At 28 dpi for N. benthamiana and 56 for cassava T200, the decrease in NOA1 expression was accompanied by chloroplast dysfunction, evident from the significant reduction in chlorophylls a and b and carotenoids in SACMV-infected leaf samples. Furthermore, the expression of v chloroplast translation factors (chloroplast RNA binding, chloroplast elongation factor G, translation initiation factor 3-2, plastid-specific ribosomal protein 6 and) were found to be repressed in infected N. benthamiana and infected cassava T200 relative to mock inoculated plants. GC-MS analysis showed a decrease in fumarate and an increase in glucose in SACMV-infected N. benthamiana in comparison to mock samples suggesting a decrease in carbon stores. Collectively, these results provide evidence that in response to SACMV infection in N. benthamiana, decrease in photopigment and carbon stores, accompanied by an increase in glucose and decrease in fumarate, lead to a decline in NbNOA1 and NO levels. This is manifested by suppressed translation factors, and disruption of the chloroplast, resulting in chlorotic disease symptoms. In cassava T200 however, the link could not be established as the level of glucose was not significantly decreased and fumaric acid was not detected and although the concomitant decrease in the expression of MeNOA1 and chloroplast translation factors indicate dysfunction of the chloroplast, the link between MeNOA1 expression, carbon store, NO and chloroplast activity could not be established.Item Gene expression studies towards the elucidation of host responses to South African cassava mosaic virus(2014-04-22) Allie, FarhahnaUnable to load abstract.