Cassava is a staple crop for over 500 million people worldwide. Production of cassava is affected by several constraints, including viral diseases such as cassava brown streak disease (CBSD), caused by the ipomoviruses Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). CBSD is present throughout cassava growing regions in Africa and symptoms include streaking, chlorosis, dieback and tuber necrosis, leading to crop losses of up to 70%. An increased genetic and molecular understanding of the viruses that cause CBSD is essential for management of this disease. In this thesis, five strategies were evaluated in an attempt to generate infectious clones of CBSV to facilitate further understanding of this virus: fusion PCR, cloning the viral genome in a bacterial artificial chromosome (BAC) in E. coli, the use of E. coli strains developed for unstable clones, intron insertion using homologous recombination in yeast and in vitro ligation. It was determined to be not possible to construct a full-length CBSV clone in E. coli when the cylindrical inclusion (CI) gene was intact, likely due to its toxicity in E. coli leading to instability. Use of an in vitro ligation technique successfully created a cDNA infectious clone of CBSV. In this process, two segments of the genome were generated in separate plasmids and then assembled by in vitro ligation before in vitro transcription. Typical infection symptoms were observed when Nicotiana benthamiana and N clevelandii were inoculated with the infectious transcripts. Following the construction of infectious clones, functional genomic studies were possible. The HAMI gene, unique to CBSV and UCBSV in the Jpomovirus genus, was chosen as the target for functional analysis. The HAMl gene has homology with HAM I in the yeast Saccharomyces cerevisiae which is a nucleoside triphosphate pyrophosphatase (NTPase) that hydrolyses non-canonical nucleoside triphosphates (NTPs) and prevents their incorporation into nucleic acids to avoid unfavourable mis-match mutations. A bioinformatk analysis of HAMI was conducted, a 5-Fluorouracil resistance assay was performed and knockout constructs of the HAMl gene were generated to determine whether HAM I from CBSV and UCBSV has similar functionality to the HAMl in yeast. The multiple sequence analysis revealed that the HAMI from CBSV and UCBSV contains the "signature" motif(SHR) for ITPase family could therefore be expected to have a similar function to other ITPases. However, the CBSV and UCBSV HAMI genes did not confer resistance in S. cerevisiae to 5-Fluorouracil, as shown with the S. cerevisiae HAMI homologue and the UCBSV HAMI-knockout infectious clones were unable to establish infections in N benthamiana and N clevelandii. This suggests that HAMl may be required for infection, rather than function as a NTPase. The construction of the CBSV infectious clone in this thesis, and functional analysis of this virus, will assist in further research conducted on this important pathogen to facilitate management of CBSD and maintain yield of the important root crop, cassava.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:702169 |
| Date | January 2016 |
| Creators | Bunawan, Hamidun |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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