Thesis (Ph. D)--Ohio State University, 2002. / Title from first page of PDF file. Document formatted into pages; contains xxi, 201 p.: ill. Includes abstract and vita. Advisor: Richard Sayre, Dept. of Plant Biology. Includes bibliographical references (p. 184-201).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1975. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 154-166).

Title from title page of PDF (University of Missouri--St. Louis, viewed March 2, 2010). Includes bibliographical references (p. 58-62).

Thesis (M.Inst.Agrar.(Agricultural Economics))--University of Pretoria, 1999. / Summary in English. Includes bibliographical references.

Thesis (Ph.D.) - University of KwaZulu-Natal, Pietermaritzburg, 2009. / Submitted to the African Centre for Crop Improvement. Full text also available online. Scroll down for electronic link.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy in Science at the School of Molecular and Cell Biology, 2017 / Different 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. / XL2017

Thesis (M.S.)--University of Wisconsin--Madison, 1969. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

Geminiviruses causes diseases to many staple food and cash crops of great economic importance worldwide. Currently eight species of Begomoviruses belonging to the Geminivirus family exist, of which South African cassava mosaic virus SACMV-[ZA:99] is a member, and is known to cause cassava mosaic disease (CMD). Cassava (Manihot esculenta, Crantz) is considered to be an important food crop consumed in many tropical, sub-tropical and African countries, and is increasingly becoming well-known for its ethanol production on a global a scale. Various strategies to control CMD are currently being implemented, one of which is to elucidate mechanisms involved in host-virus interactions with the aim of identifying defence-related genes involved in the disease process. Many defence genes within the plant kingdom are evolutionary conserved, potentially providing methods of control not only to CMD but to other diseases as well. The research outlined in this thesis aimed to identify networks and pathways involved in disease susceptibility between the model plant host system, Arabidopsis thaliana and cassava T200 upon SACMV-[ZA:99] infection. Conclusions were also drawn from within host comparisons between susceptible cassava T200 and resistant cassava TME3 cultivars in order to explore if similarities, differences or common patterns of expression existed between genes governing resistance and susceptibility. Before transcriptomic profiling studies were carried out, it was important to improve South African cassava mosaic virus (SACMV-[ZA:99]) and African cassava mosaic virus (ACMV-[NG:Ogo:90]) infection efficiencies in recalcitrant crop systems such as cassava. Susceptible cassava cultivars T200, TMS60444, and SM14334 were tested for these purposes following infection with three different Agrobacterium strains (C58C1; AGL1; LBA4404). Results demonstrated that an overall increase in infection efficiency was achieved for each genotype and virus tested, although with varying infectivity levels, suggesting that although an improved method was established, basal levels of susceptibility differed between genotypes and therefore it was not possible to achieve 100% infection efficiencies for agroinfection methods. A 4 x 44k microarray whole genome study was then conducted to identify susceptible host genes involved in the interaction between the model plant system Arabidopsis thaliana and SACMV-[ZA:99]. An infectivity assay was carried out across three time points (14, 24, and 36 dpi), confirming that disease symptoms and virus infectivity levels correlated with an increase in differentially expressed transcripts across time points, with SACMV-[ZA:99] predominantly causing host-gene suppression. Many complex genes and pathways were disrupted and were shown to be involved in categories pertaining to stress and defence responses, phytohormone signalling pathways, cellular transport, metabolism and cell-cycle regulation strongly suggesting an attempt made by SACMV-[ZA:99] to affect homeostasis and antagonize host defence responses. This was the first geminivirus study identifying differentially expressed transcripts across 3 time points. Next generation sequencing (NGS) using the ABI Solid platform was then carried out on SACMV-[ZA:99] – infected susceptible cassava T200 cultivar at 3 time points (12, 32, and 67 dpi), comparing infection responses to mock-inoculated healthy controls. Similarly to the Arabidopsis microarray study, findings from this analysis also revealed a shift from up-regulated to down-regulated genes across time points, once again reflecting virus-specific suppression on host genes suggesting SACMV-[ZA:99] specific alterations were induced in the host, regardless of the host (Arabidopsis and cassava T200) or platform (microarray and NGS) used. Genes identified pertaining particularly to the susceptible cassava T200 - SACMV-[ZA:99] interaction such as the disease resistance protein families (TIR-NBS-LRR), RPP1, RPM1, and NHO1 were showing down-regulation demonstrating that SACMV-[ZA:99] pathogenicity proteins may be causing this suppression leading to inactivation of basal immunity. Comparisons between tolerant cassava TME3 and susceptible T200 showed similarities and differences in responses between the cultivars. Many similarities such as cell wall precursor proteins and glutathione-S-transferases were up-regulated in both cultivars, which may be due to the host attempting to mount appropriate defences. Opposite patterns of expression was observed for genes in categories involved in transcription and phytohormone signalling such as WRKY‘s, NAC, JAZ, and ERF where suppression was evident in susceptible cassava T200, confirming the suppressive nature of SACMV-[ZA:99] to establish a replication-competent environment. Findings in this study contributed to the little that is known about geminivirus disease progression within a previously uncharacterised susceptible host such as cassava.

A thesis presented to The Faculty of Science, University of the Witwatersrand, Johannesburg in fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology 2015 / Cassava brown streak disease (CBSD) ranks among the top seven biological threats to global food security and is considered to be a major risk to food security in tropical Africa. In Uganda, overall CBSD incidence has increased by c. 20% since 2004, and persistently reduces cassava yields and storage root quality. Presently the disease negatively impacts the livelihoods of over 80% of the farming families who rely on cassava as a staple food and source of income. Two distinct ipomoviruses, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) cause CBSD. The viruses systemically infect primary host plants and accumulate, and cause severe disease symptoms as the plant matures, reducing yields through the induction of necrotic lesions in the storage roots and suppressing utility of cassava stems for subsequent vegetative propagation. Effective control strategies require screening of available germplasm for sources of natural resistance in combination with improved understanding of host-virus interaction to facilitate targeted breeding. Due to a lack of known sources of resistance to CBSD in the cassava germplasm, incorporating new virus resistance into existing cassava genotypes through transgenic RNA interference (RNAi) approaches offers an additional, relevant avenue to reduce the increasing impact of CBSD. The research presented in this thesis provides insights into the complex mechanisms of virus-host interactions linking genotype to phenotype in CBSV- and UCBSV-cassava pathosystems and provides proof of principle for CBSD control by RNAi-mediated technology. Both are contributions to progress towards potential control of the CBSD epidemic in East Africa. To correlate CBSD symptoms with virus titer, within-host CBSV and UCBSV accumulation was studied in leaf, stem and storage root samples collected from 10 genotypes of field-grown cassava with varied levels of resistance to CBSD. CBSV was found to be present in 100% of CBSD samples collected from symptomatic plants. Presence of both CBSV and UCBSV was seen in 45.3% of the samples. Quantitative PCR (RT-qPCR) analysis showed that tolerant genotypes were infected with CBSV alone and accumulated lower virus titer compared to susceptible genotypes, which were co-infected with CBSV and UCBSV. To further comprehend the molecular interaction between CBSD viruses and cassava, deep sequencing was performed to compare profiles of virus-derived small RNAs (vsRNAs) in CBSV- and UCBSV-infected cassava genotypes of NASE 3 (CBSD tolerant), TME 204 and 60444 (CBSD susceptible). The results showed an abundance of 21-24 nt sized vsRNAs which when mapped were shown to cover the entire CBSV and UCBSV genomes. The 21- and 22-nt sizes were predominant compared to the 23- and 24-nt size classes. CBSV-infected plants accumulated higher populations of vsRNAs across the genotypes compared to UCBSV-infected plants, which accumulated moderate amounts of UCBSV-derived sRNAs in TME 204 and 60444, and insignificant amounts in UCBSV-challenged NASE 3, respectively. Quantitative RT-PCR analysis was performed to determine transcript levels of cassava homologues of Dicer (DCL) proteins, particularly DCL4 and DCL2, which are involved in the biogenesis of 21- and 22-nt small RNAs, and to correlate to the abundance of 21- and 22-nt vsRNAs in CBSV- and UCBSV-infected cassava. Similarly, RT-qPCR was performed to determine the expression of Argonaute (AGO) proteins, specifically AGO2 which preferentially sort and bind sRNAs with 5’ adenine (A) or uracil (U) to effector complexes to target mRNAs repression or cleavage, since in this study a major proportion of the vsRNAs were found to have A or U at the first 5’-end. Expression levels of cassava homologues of AGO2, DCL2 and DCL4, which are core components of the gene-silencing pathway, were found to be affected in virus-infected plants across all three genotypes. The levels of viral RNA and vsRNAs correlated with disease phenotype in infected plants. CBSV-infected plants showed more severe CBSD symptoms compared with UCBSV-infected plants of the same genetic background. These results showed that CBSV is more aggressive compared to UCBSV and supports the hypothesis of occurrence of genotype-specific resistance to CBSD viruses. The abundance of 21- and 22-nt vsRNAs in CBSV- and UCBSV-infected plants signifies the viruses activated the RNA-silencing mechanism, referred to as transcriptional or post-transcriptional gene silencing (TGS or PTGS). To test efficacy of RNAi-mediated resistance to control CBSD under field conditions, 14 lines of cassava plants transgenically modified to express, as inverted repeats, two RNAi constructs p718 and p719 targeting near full-length (894 bp) and N-terminal (402 bp) portions of UCBSV coat protein sequence were tested under confined field trial conditions at Namulonge, Uganda. Transgenic plants expressing p718 showed a 3-month delay in CBSD symptom development, while 100% of non-transgenic plants (n = 60) developed CBSD shoot symptoms. Over the 11-month trial duration, 98% of clonal replicates within line 718-001 were found to remain free of CBSD symptoms. RT-PCR analysis detected UCBSV within leaves of 57% of non-transgenic plants compared to only 0.5% across the 14 transgenic lines. Presence of the non-homologous CBSV was detected in all transgenic plants that developed CBSD symptoms. However, 93% of plants of line 718-001 were free of CBSV and UCBSV. At harvest, 90% of storage roots of non-transgenic plants showed severe necrosis, whereas plants of lines 718-001 and 718-005 showed significant suppression of CBSD. Line 718-001 had 95% of roots free from necrosis and was RT-PCR negative for presence of both viral pathogens. To determine durability of RNAi-mediated resistance to CBSD, stem cuttings were obtained from mature plants of lines p718-001, p718-002 and p718-005, replanted and monitored for 11 more months. CBSV but not UCBSV was detected in tissues of plants of lines p718-002 and p718-005, whereas all leaves and roots of p718-001 plants were free of CBSV and UCBSV. Thus, RNAi constructs conferred durable CBSD resistance across the vegetative cropping cycle, providing proof of concept for application of RNAi technology to control CBSD in farmers’ fields. The findings presented in this thesis contribute to understanding the complex interconnected mechanisms involved in CBSV- and UCBSV-host interactions and will contribute to the long-term goals of devising new methods of CBSD control.