Browsing by Author "Shepherd, Dionne"

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    Adaptive evolution by recombination is not associated with increased mutation rates in Maize streak virus
    (BioMed Central Ltd, 2012) Monjane, Aderito; Pande, Daniel; Lakay, Francisco; Shepherd, Dionne; van der Walt, Eric; Lefeuvre, Pierre; Lett, Jean-Michel; Varsani, Arvind; Rybicki, Edward; Martin, Darren
    BACKGROUND: Single-stranded (ss) DNA viruses in the family Geminiviridae are proving to be very useful in real-time evolution studies. The high mutation rate of geminiviruses and other ssDNA viruses is somewhat mysterious in that their DNA genomes are replicated in host nuclei by high fidelity host polymerases. Although strand specific mutation biases observed in virus species from the geminivirus genus Mastrevirus indicate that the high mutation rates in viruses in this genus may be due to mutational processes that operate specifically on ssDNA, it is currently unknown whether viruses from other genera display similar strand specific mutation biases. Also, geminivirus genomes frequently recombine with one another and an alternative cause of their high mutation rates could be that the recombination process is either directly mutagenic or produces a selective environment in which the survival of mutants is favoured. To investigate whether there is an association between recombination and increased basal mutation rates or increased degrees of selection favoring the survival of mutations, we compared the mutation dynamics of the MSV-MatA and MSV-VW field isolates of Maize streak virus (MSV; Mastrevirus), with both a laboratory constructed MSV recombinant, and MSV recombinants closely resembling MSV-MatA. To determine whether strand specific mutation biases are a general characteristic of geminivirus evolution we compared mutation spectra arising during these MSV experiments with those arising during similar experiments involving the geminivirus Tomato yellow leaf curl virus (Begomovirus genus). RESULTS: Although both the genomic distribution of mutations and the occurrence of various convergent mutations at specific genomic sites indicated that either mutation hotspots or selection for adaptive mutations might elevate observed mutation rates in MSV, we found no association between recombination and mutation rates. Importantly, when comparing the mutation spectra of MSV and TYLCV we observed similar strand specific mutation biases arising predominantly from imbalances in the complementary mutations G->T: C->A. CONCLUSIONS: While our results suggest that recombination does not strongly influence mutation rates in MSV, they indicate that high geminivirus mutation rates are at least partially attributable to increased susceptibility of all geminivirus genomes to oxidative damage while in a single stranded state.
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    Experimental evidence indicating that mastreviruses probably did not co-diverge with their hosts
    (BioMed Central Ltd, 2009) Harkins, Gordon; Delport, Wayne; Duffy, Siobain; Wood, Natasha; Monjane, Aderito; Owor, Betty; Donaldson, Lara; Saumtally, Salem; Triton, Guy; Briddon, Rob; Shepherd, Dionne; Rybicki, Edward; Martin, Darren; Varsani, Arvind
    BACKGROUND:Despite the demonstration that geminiviruses, like many other single stranded DNA viruses, are evolving at rates similar to those of RNA viruses, a recent study has suggested that grass-infecting species in the genus Mastrevirus may have co-diverged with their hosts over millions of years. This "co-divergence hypothesis" requires that long-term mastrevirus substitution rates be at least 100,000-fold lower than their basal mutation rates and 10,000-fold lower than their observable short-term substitution rates. The credibility of this hypothesis, therefore, hinges on the testable claim that negative selection during mastrevirus evolution is so potent that it effectively purges 99.999% of all mutations that occur. RESULTS: We have conducted long-term evolution experiments lasting between 6 and 32 years, where we have determined substitution rates of between 2 and 3 x 10-4 substitutions/site/year for the mastreviruses Maize streak virus (MSV) and Sugarcane streak Reunion virus (SSRV). We further show that mutation biases are similar for different geminivirus genera, suggesting that mutational processes that drive high basal mutation rates are conserved across the family. Rather than displaying signs of extremely severe negative selection as implied by the co-divergence hypothesis, our evolution experiments indicate that MSV and SSRV are predominantly evolving under neutral genetic drift. CONCLUSION: The absence of strong negative selection signals within our evolution experiments and the uniformly high geminivirus substitution rates that we and others have reported suggest that mastreviruses cannot have co-diverged with their hosts.
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    A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family
    (BioMed Central Ltd, 2009) Varsani, Arvind; Shepherd, Dionne; Dent, Kyle; Monjane, Aderito; Rybicki, Edward; Martin, Darren
    BACKGROUND:We have characterised a new highly divergent geminivirus species, Eragrostis curvula streak virus (ECSV), found infecting a hardy perennial South African wild grass. ECSV represents a new genus-level geminivirus lineage, and has a mixture of features normally associated with other specific geminivirus genera. RESULTS: Whereas the ECSV genome is predicted to express a replication associated protein (Rep) from an unspliced complementary strand transcript that is most similar to those of begomoviruses, curtoviruses and topocuviruses, its Rep also contains what is apparently a canonical retinoblastoma related protein interaction motif such as that found in mastreviruses. Similarly, while ECSV has the same unusual TAAGATTCC virion strand replication origin nonanucleotide found in another recently described divergent geminivirus, Beet curly top Iran virus (BCTIV), the rest of the transcription and replication origin is structurally more similar to those found in begomoviruses and curtoviruses than it is to those found in BCTIV and mastreviruses. ECSV also has what might be a homologue of the begomovirus transcription activator protein gene found in begomoviruses, a mastrevirus-like coat protein gene and two intergenic regions. CONCLUSION: Although it superficially resembles a chimaera of geminiviruses from different genera, the ECSV genome is not obviously recombinant, implying that the features it shares with other geminiviruses are those that were probably present within the last common ancestor of these viruses. In addition to inferring how the ancestral geminivirus genome may have looked, we use the discovery of ECSV to refine various hypotheses regarding the recombinant origins of the major geminivirus lineages.
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    Maize streak virus (MSV) diversity in Uganda and the assessment of gene silencing as a tool for development of resistance to MSV
    (2008) Owor, Betty Elizabeth; Thomson, Jennifer Ann; Shepherd, Dionne
    Maize streak virus (MSV: Family Geminiviridae, Genus Mastrevirus) is the causal agent of maize streak disease (MSD) that contributes significantly to low maize yields in Africa, thereby threatening food security of sub-Saharan Africa’s poorest people. In Uganda, MSD has been identified as one of the most important constraints to maize production. In order to have a better understanding of the disease in that country, this thesis set out to establish MSD levels in farmers’ fields; develop a new sampling and virus isolation method; assess the diversity of MSVs throughout Uganda; and, through the cloning of sampled virus genomes, to determine the genetic characteristics of different isolates. In addition, this study also included an assessment of RNA silencing as a resistance strategy against MSV.
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    The use of maize streak virus (MSV) replication-associated protein mutants in the development of MSV-resistant plants
    (2003) Shepherd, Dionne; Thomson, Jennifer Ann; Rybicki, Edward P
    Maize streak virus (MSV) is the type member of the Mastrevirus genus of the Geminiviridae. As the causal agent of maize streak disease (MSD), MSV is the most significant pathogen of maize in Africa, resulting in crop yield losses of up to 100%. Transmitted by leafhoppers (Cicadulina spp.), MSV is indigenous to Africa and neighbouring Indian Ocean Islands. Despite maize being a crucial staple food crop in Africa, the average maize yield per hectare in Africa is the lowest in the world, leading to food shortages and famine. A major contributing factor to these low yields is MSD. To genetically engineer MSV-resistant maize using the pathogen-derived resistance (PDR) strategy, the viral replication-associated (Rep) protein gene was targeted, whose multifunctional products Rep and RepA are the only viral proteins essential for replication. Rep constructs had previously been made containing deleterious mutations in several conserved amino acid motifs. In this study, these mutants and the wild type Rep gene were truncated to remove key motifs involved in viral replication. A quantitative PCR assay was developed to determine the effects of the mutant and truncated Reps on viral replication in black Mexican sweetcorn (BMS) suspension cells. The MSVsensitive grass Digitaria sanguinalis was then transformed with Rep constructs that inhibited MSV replication in BMS, and transgenic lines were tested for virus resistance. Several plants of a D. sanguinalis line transgenic for a mutated full-length Rep gene showed excellent resistance (immunity) to MSV, but the transgene had negative effects on aspects of plant growth and development. Transformation with a mutated/truncated Rep gene resulted in healthy fertile transgenic D. sanguinalis plants, many of which showed good MSV resistance. Fertile maize (Hi-II) T 1 transgenic plants expressing the truncated/mutated Rep gene have been obtained, the offspring of which will be tested for resistance to MSV. Considering the success in achieving MSV-resistant D. sanguinalis, there is good reason to believe that the transgenic maize will too be resistant to MSV.