Banana bunchy top virus (BBTV) is a circular, single-stranded (css) DNA virus that belongs to the genus Babuvirus in the family Nanoviridae. BBTV is responsible for the most devastating virus disease of banana known as "bunchy top", for which conventional control measures are generally ineffective. Genetically engineered resistance appears to be the most promising strategy to generate BBTV-resistant bananas but the success of this strategy is largely dependent upon the molecular characterisation of the target virus and knowledge of the virus life cycle, particularly the replication strategy. This PhD study was aimed at the molecular characterisation of the intergenic regions of BBTV, in order to complement the molecular information currently available and to potentially contribute to the development of transgenic resistance strategies against BBTV in banana. Three putative iterative sequences (iterons; GGGAC) previously identified in the BBTV intergenic regions were initially characterised. In order to determine their role in the binding of the master BBTV replication initiation protein (M-Rep), the putative iterons (F1 and F2 in the virion sense, and R in the complementary sense) were independently mutated in a BBTV DNA-6 greater-than-genome-length clone (1.1 mer). The DNA-6 1.1 mers (native and mutants) and the M-Rep-encoding component (DNA-1) were co-bombarded into banana (Musa spp. cv."Lady finger") embryogenic suspension cells and transient replication was evaluated by Southern hybridisation. Analysis of the DNA-6 replicative forms showed a significant decrease of approximately 41% for the F1 iteron mutant and 61% for the R iteron mutant in comparison with native levels. However, the mutation in the F2 iteron caused the most dramatic effect, decreasing replication to levels barely detectable by Southern hybridisation. These results suggest that the three iterons all play a role in BBTV replication, most likely as recognition and binding sites for the M-Rep, but that the F2 iteron appears to be the most important in replication. Following the observation that all BBTV isolates sequenced to date have identical iteron sequences, the extent to which the M-Rep would recognise, bind and initiate replication of heterologous components from geographically diverse BBTV isolates (the South Pacific and the Asian groups) was evaluated. Cross replication assays revealed that heterologous M-Reps from Fiji, Hawaii (South Pacific group) and Vietnam (Asian group) were able to initiate replication of the coat protein-encoding component (DNA-3) from the Australian BBTV isolate (South Pacific group). However, replication of DNA-3 from the Vietnamese isolate was not initiated by heterologous M-Reps from the two South Pacific isolates tested (Australia and Hawaii). These results suggest that a broad-range transgenic resistance strategy based on replication using Australian BBTV intergenic regions may be successful as this region will be recognised by the M-Reps from both Asian and South Pacific BBTV isolates. However, a Rep protein-mediated resistance strategy will more likely be specific to geographical isolates and, therefore, less suitable as a broad-range control strategy. To further characterise the BBTV intergenic regions and to gain a better understanding of the BBTV transcription process, the 5' untranslated regions (UTRs) of the major open reading frames (ORFs) associated with each of the six BBTV DNA components were mapped. In all cases, the transcription start sites were located 3' of a putative TATA box and the 5' UTRs varied in length from 23 nucleotides (DNA-6) to 5 nucleotides (DNA-3). Two potential transcription start sites (nt 84 and 87) were mapped for DNA-1, but whether these represent the transcription start sites of the two genes associated with DNA-1 remains to be determined. Two start sites were also associated with DNA-2 which is thought to be monocistronic. Whether one of these start sites is an artefact or whether they are due to natural sequence variability of BBTV is unknown. These results now enable us to define the transcribed regions of each BBTV DNA component and accurately predict their promoter regions in an attempt to gain a fundamental understanding of BBTV gene expression patterns.
| Identifer | oai:union.ndltd.org:ADTP/265208 |
| Date | January 2006 |
| Creators | Herrera Valencia, Virginia Aurora |
| Publisher | Queensland University of Technology |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
| Rights | Copyright Virginia Aurora Herrera Valencia |
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