Student Number : 9610284H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Cassava Mosaic Disease is the most devastating disease affecting cassava (Manihot esculenta Crantz) crops worldwide. This disease is associated with eight species of geminiviruses, all belonging to the genus Begomovirus of the family Geminiviridae. In South Africa, in particular, CMD is caused by South African cassava mosaic virus (SACMV). Currently, there are no adequate methods for control of this disease as mechanisms within virus-host interactions are poorly understood. This brings about the need for development of virus-disease control strategies. This study was therefore conducted to identify the host’s response to an invading virus. The model plant, Arabidopsis was chosen as it is a well-characterized plant system, with expression databases readily available as its entire genome has been sequenced. This study was conducted, firstly, to phenotypically determine if Arabidopsis was resistant or susceptible to SACMV infection, and secondly, to identify the host’s response to pathogen infection on a molecular level through gene expression studies utilizing microarrays. Results from the symptomatology study revealed that Arabidopsis plants were fully symptomatic 28 days post-inoculation, displaying characteristic disease symptoms such as stunting, yellowing, and leaf deformation. This indicated that Arabidopsis was susceptible to SACMV infection. Microarray analyses revealed 86 differentially expressed genes, of which 48 showed up-regulation and 38 down-regulation. Relative quantification real-time PCR was performed on selected genes to confirm these results. Many up-regulated genes were shown to be primarily involved in a general stress response induced by the host, whereas those genes that were downregulated seemed to be involved in more specific responses to viral invasion, probably a consequence of suppression of host genes by SACMV to enhance its own replication. The majority of genes identified fell under the predominant functional categories involved in metabolism, transcription, and transport. To our knowledge, this is the first study in which a DNA geminivirus has been used in a host-pathogen interaction utilizing microarrays.

Cassava (Manihot esculenta Crantz) is a basic staple food crop in Tanzania. Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) constitutes a major limiting factor to cassava production in the country. This study was undertaken to characterize the CMGs occurring in Tanzania using molecular techniques and to map their geographical distribution to generate information on which the formulation of control measures can be based. Using Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) for analysis of CMGs DNA-A genomes, different CMGs were found to be associated with CMD. Higher molecular diversity was observed among East African cassava mosaic viruses (EACMVs) than African cassava mosaic viruses (ACMVs), which was confirmed later by complete nucleotide sequence analysis. In addition to EACMV and ACMV isolates, two isolates of EACMV Cameroon virus (EACMCV) were found in Tanzania. These were confirmed to be strains of EACMCV Cameroon, originally described in Cameroon, West Africa and here named EACMCV- [TZ1] and EACMCV-[TZ7]. They had high (92%) overall DNA-A nucleotide sequence identity and EACMCV-[TZ1] was widespread in the southern part of the country. A subgenomic DNA form of CMG that appeared to be truncated was identified in a CMD-infected cassava plant. It was confirmed upon sequence analysis to be a defect of EACMV DNA-A and had a capacity of attenuating symptoms when coinoculated with wild-type EACMV. In addition, this study revealed for the first time the presence of two novel non-geminivirus single-stranded DNA (ssDNA) sub-genomic molecules associated with CMG infection. They were shown to be dependent on CMG for replication and movement within the plants, confirming their status as satellite molecules named here as satDNA-II and satDNA-III. When present in coinfection with CMGs, they enhance symptoms and can break high levels of resistance in a cassava landrace. Finally a simple, inexpensive technique is described of archiving, transporting and recovering plant DNA for downstream geminivirus characterisation. / Thesis (PhD)--University of Pretoria, 2007. / Microbiology and Plant Pathology / Unrestricted