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Edward P. Rybicki - One of the best experts on this subject based on the ideXlab platform.
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Inducible Resistance to Maize Streak Virus
2016Co-Authors: Dionne N. Shepherd, Adérito L. Monjane, Benjamin Dugdale, Jennifer A. Thomson, Darren P. Martin, Francisco Lakay, Arvind Varsani, Marion E. Bezuidenhout, James Dale, Edward P. RybickiAbstract:Maize Streak Virus (MSV), which causes Maize Streak disease (MSD), is the major viral pathogenic constraint on Maize production in Africa. Type member of the MastreVirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic Maize lines constitutively expressing ‘‘dominant negative mutant’ ’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’ ’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence o
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The Use of African Indigenous Genes in the Development of Transgenic Maize Tolerant to Drought and Resistant to Maize Streak Virus
Biotechnology in Africa, 2014Co-Authors: Jennifer A. Thomson, Sagadevan G. Mundree, Dionne M. Shepherd, Edward P. RybickiAbstract:When developing a plant with resistance to biotic and abiotic stresses and utilizing genetic engineering, why should scientists limit themselves to genes from known organisms? Why not test those from indigenous species that might have unique properties? In this chapter we describe the use of indigenous genes for the development of crops important to Africa. The first is Maize tolerant to drought, a situation which appears to be worsening on the continent, and the second is Maize resistant to the African-endemic Maize Streak Virus. The genes for drought tolerance were derived from the resurrection plant, Xerophyta viscosa, which survives even when it contains only 5 % of its relative water content. The plant can be ‘resurrected’ within 80 h of receiving moisture. Two methods were used to identify potential genes of interest. The first was complementation by functional sufficiency in Escherichia coli, resulting in the isolation of XvSap1 (which was found to code for a membrane-associated signalling protein) and XvAld, coding for aldose reducatase which converts glucose to sorbitol, an osmoprotectant. The second method was differential screening of expression libraries resulting in the isolation of XvPrx2, which codes for an antioxidant peroxiredoxin, and XvG6, which codes for a stress-responsive regulatory protein. Other genes isolated, tested, and not used further are also mentioned. For resistance to Maize Streak Virus, the approach of pathogen derived resistance was used, resulting in the isolation of dominant negative mutants of the viral replication associated protein gene, rep. In a refinement of this approach, a Virus-inducible version of the mutants was developed as well as an siRNA approach. As the development of transgenic Maize is a lengthy process, the genes were first tested in model systems. For drought tolerance the model plants were Arabidopsis and tobacco while for Virus resistance black Mexican sweetcorn in tissue culture and transgenic Digitaria sanguinalis, an MSV sensitive grass, were used. Cassettes of the genes shown to be effective, including inducible systems for both drought and Virus resistance, were introduced into Maize and results are presented. The paper concludes with a discussion on how to bring these products to the farmers’ market, in Africa.
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Replication modes of Maize Streak Virus mutants lacking RepA or the RepA-pRBR interaction motif.
Virology, 2013Co-Authors: Moritz Ruschhaupt, Holger Jeske, Marion Bezuidenhout, Darren P. Martin, Francisco Lakay, Edward P. Rybicki, Dionne N. ShepherdAbstract:Abstract The plant-infecting mastreViruses (family Geminiviridae ) express two distinct replication-initiator proteins, Rep and RepA. Although RepA is essential for systemic infectivity, little is known about its precise function. We therefore investigated its role in replication using 2D-gel electrophoresis to discriminate the replicative forms of Maize Streak Virus (MSV) mutants that either fail to express RepA (RepA − ), or express RepA that is unable to bind the plant retinoblastoma related protein, pRBR. Whereas amounts of viral DNA were reduced in two pRBR-binding deficient RepA mutants, their repertoires of replicative forms changed only slightly. While a complete lack of RepA expression was also associated with reduced viral DNA titres, the only traces of replicative intermediates of RepA − Viruses were those indicative of recombination-dependent replication. We conclude that in MSV, RepA, but not RepA–pRBR binding, is necessary for single-stranded DNA production and efficient rolling circle replication.
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Maize Streak Virus: an old and complex 'emerging' pathogen.
Molecular plant pathology, 2010Co-Authors: Dionne N. Shepherd, Darren P. Martin, Arvind Varsani, Eric Van Der Walt, Kyle C. Dent, Edward P. RybickiAbstract:SUMMARY Maize Streak Virus (MSV; Genus MastreVirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in the Genus Cicadulina, but mainly by C. mbila Naude and C. storeyi. In addition to Maize, it can infect over 80 other species in the Family Poaceae. Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant Streak disease in Maize. Severe Maize Streak disease (MSD) manifests as pronounced, continuous parallel chlorotic Streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in Maize, and/or Maize infections caused by non-Maize-adapted MSV strains, can result in narrow, interrupted Streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3–10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss. mpp_568 1..12
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Experimental observations of rapid Maize Streak Virus evolution reveal a strand-specific nucleotide substitution bias.
Virology journal, 2008Co-Authors: Eric Van Der Walt, Darren P. Martin, Arvind Varsani, Jane E. Polston, Edward P. RybickiAbstract:Background Recent reports have indicated that single-stranded DNA (ssDNA) Viruses in the taxonomic families Geminiviridae, Parvoviridae and AnelloVirus may be evolving at rates of ~10-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA Viruses and are surprisingly high given that ssDNA Virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA Virus evolution rates were first suggested in evolution experiments involving the geminiVirus Maize Streak Virus (MSV), the evolution rate of this Virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminiVirus mutation rates remain unanswered.
Dionne N. Shepherd - One of the best experts on this subject based on the ideXlab platform.
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Inducible Resistance to Maize Streak Virus
2016Co-Authors: Dionne N. Shepherd, Adérito L. Monjane, Benjamin Dugdale, Jennifer A. Thomson, Darren P. Martin, Francisco Lakay, Arvind Varsani, Marion E. Bezuidenhout, James Dale, Edward P. RybickiAbstract:Maize Streak Virus (MSV), which causes Maize Streak disease (MSD), is the major viral pathogenic constraint on Maize production in Africa. Type member of the MastreVirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic Maize lines constitutively expressing ‘‘dominant negative mutant’ ’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’ ’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence o
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Inducible Resistance to Maize Streak Virus
PLOS ONE, 2014Co-Authors: Dionne N. Shepherd, Adérito L. Monjane, Benjamin Dugdale, Jennifer A. Thomson, Marion Bezuidenhout, Darren P. Martin, Francisco Lakay, James L. Dale, Arvind Varsani, Edward Peter RybickiAbstract:Maize Streak Virus (MSV), which causes Maize Streak disease (MSD), is the major viral pathogenic constraint on Maize production in Africa. Type member of the MastreVirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic Maize lines constitutively expressing “dominant negative mutant” versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or “leaky” expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic Maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastreVirus species. The application of this new technology to MSV resistance in Maize could allow a better, more acceptable product.
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Replication modes of Maize Streak Virus mutants lacking RepA or the RepA-pRBR interaction motif.
Virology, 2013Co-Authors: Moritz Ruschhaupt, Holger Jeske, Marion Bezuidenhout, Darren P. Martin, Francisco Lakay, Edward P. Rybicki, Dionne N. ShepherdAbstract:Abstract The plant-infecting mastreViruses (family Geminiviridae ) express two distinct replication-initiator proteins, Rep and RepA. Although RepA is essential for systemic infectivity, little is known about its precise function. We therefore investigated its role in replication using 2D-gel electrophoresis to discriminate the replicative forms of Maize Streak Virus (MSV) mutants that either fail to express RepA (RepA − ), or express RepA that is unable to bind the plant retinoblastoma related protein, pRBR. Whereas amounts of viral DNA were reduced in two pRBR-binding deficient RepA mutants, their repertoires of replicative forms changed only slightly. While a complete lack of RepA expression was also associated with reduced viral DNA titres, the only traces of replicative intermediates of RepA − Viruses were those indicative of recombination-dependent replication. We conclude that in MSV, RepA, but not RepA–pRBR binding, is necessary for single-stranded DNA production and efficient rolling circle replication.
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Maize Streak Virus: an old and complex 'emerging' pathogen.
Molecular plant pathology, 2010Co-Authors: Dionne N. Shepherd, Darren P. Martin, Arvind Varsani, Eric Van Der Walt, Kyle C. Dent, Edward P. RybickiAbstract:SUMMARY Maize Streak Virus (MSV; Genus MastreVirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in the Genus Cicadulina, but mainly by C. mbila Naude and C. storeyi. In addition to Maize, it can infect over 80 other species in the Family Poaceae. Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant Streak disease in Maize. Severe Maize Streak disease (MSD) manifests as pronounced, continuous parallel chlorotic Streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in Maize, and/or Maize infections caused by non-Maize-adapted MSV strains, can result in narrow, interrupted Streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3–10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss. mpp_568 1..12
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Replicative intermediates of Maize Streak Virus found during leaf development.
The Journal of general virology, 2009Co-Authors: Julia B. Erdmann, Edward Peter Rybicki, Dionne N. Shepherd, Darren P. Martin, Arvind Varsani, Holger JeskeAbstract:GeminiViruses of the genera BegomoVirus and CurtoVirus utilize three replication modes: complementary-strand replication (CSR), rolling-circle replication (RCR) and recombination-dependent replication (RDR). Using two-dimensional gel electrophoresis, we now show for the first time that Maize Streak Virus (MSV), the type member of the most divergent geminiVirus genus, MastreVirus, does the same. Although mastreViruses have fewer regulatory genes than other geminiViruses and uniquely express their replication-associated protein (Rep) from a spliced transcript, the replicative intermediates of CSR, RCR and RDR could be detected unequivocally within infected Maize tissues. All replicative intermediates accumulated early and, to varying degrees, were already present in the shoot apex and leaves at different maturation stages. Relative to other replicative intermediates, those associated with RCR increased in prevalence during leaf maturation. Interestingly, in addition to RCR-associated DNA forms seen in other geminiViruses, MSV also apparently uses dimeric open circular DNA as a template for RCR.
Darren P. Martin - One of the best experts on this subject based on the ideXlab platform.
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Maize Streak Virus geminiviridae
2020Co-Authors: Darren P. Martin, Adérito L. MonjaneAbstract:Abstract Maize Streak Virus (MSV) is the type species of the genus MastreVirus of the family Geminiviridae and the causal agent of Maize Streak disease (MSD), one of the most important viral diseases of Maize in sub-Saharan Africa. The Virus remains largely uncontrolled in most parts of the continent and in epidemic years it contributes to widespread yield losses and famine. Eleven major MSV strains have been identified so far, of which only the MSV-A strain is known to produce economically important infections in Maize. Apart from Maize, however, these various MSV strains infect over 80 other grass species including wheat, barley, and rye. The epidemiological complexity of MSD contrasts starkly with the genomic simplicity of MSV. The MSV genome contains two intergenic regions and only three genes. Whereas the intergenic regions are involved in replication and the regulation of gene expression, the genes encode four different proteins. Two of the proteins, Rep and RepA, are expressed from a single alternatively spliced gene and are involved in genome replication and the regulation of host and Virus gene expression. The two other proteins, MP (movement protein) and CP (coat protein), are involved in systemic spread within plants and the encapsidation of viral DNA within characteristic geminate particles. Control of MSV is primarily via insecticide targeting its leafhopper vector and the use of resistant Maize genotypes that have been derived through traditional breeding approaches.
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Inducible Resistance to Maize Streak Virus
2016Co-Authors: Dionne N. Shepherd, Adérito L. Monjane, Benjamin Dugdale, Jennifer A. Thomson, Darren P. Martin, Francisco Lakay, Arvind Varsani, Marion E. Bezuidenhout, James Dale, Edward P. RybickiAbstract:Maize Streak Virus (MSV), which causes Maize Streak disease (MSD), is the major viral pathogenic constraint on Maize production in Africa. Type member of the MastreVirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic Maize lines constitutively expressing ‘‘dominant negative mutant’ ’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’ ’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence o
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Inducible Resistance to Maize Streak Virus
PLOS ONE, 2014Co-Authors: Dionne N. Shepherd, Adérito L. Monjane, Benjamin Dugdale, Jennifer A. Thomson, Marion Bezuidenhout, Darren P. Martin, Francisco Lakay, James L. Dale, Arvind Varsani, Edward Peter RybickiAbstract:Maize Streak Virus (MSV), which causes Maize Streak disease (MSD), is the major viral pathogenic constraint on Maize production in Africa. Type member of the MastreVirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic Maize lines constitutively expressing “dominant negative mutant” versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or “leaky” expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic Maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastreVirus species. The application of this new technology to MSV resistance in Maize could allow a better, more acceptable product.
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Replication modes of Maize Streak Virus mutants lacking RepA or the RepA-pRBR interaction motif.
Virology, 2013Co-Authors: Moritz Ruschhaupt, Holger Jeske, Marion Bezuidenhout, Darren P. Martin, Francisco Lakay, Edward P. Rybicki, Dionne N. ShepherdAbstract:Abstract The plant-infecting mastreViruses (family Geminiviridae ) express two distinct replication-initiator proteins, Rep and RepA. Although RepA is essential for systemic infectivity, little is known about its precise function. We therefore investigated its role in replication using 2D-gel electrophoresis to discriminate the replicative forms of Maize Streak Virus (MSV) mutants that either fail to express RepA (RepA − ), or express RepA that is unable to bind the plant retinoblastoma related protein, pRBR. Whereas amounts of viral DNA were reduced in two pRBR-binding deficient RepA mutants, their repertoires of replicative forms changed only slightly. While a complete lack of RepA expression was also associated with reduced viral DNA titres, the only traces of replicative intermediates of RepA − Viruses were those indicative of recombination-dependent replication. We conclude that in MSV, RepA, but not RepA–pRBR binding, is necessary for single-stranded DNA production and efficient rolling circle replication.
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Maize Streak Virus: an old and complex 'emerging' pathogen.
Molecular plant pathology, 2010Co-Authors: Dionne N. Shepherd, Darren P. Martin, Arvind Varsani, Eric Van Der Walt, Kyle C. Dent, Edward P. RybickiAbstract:SUMMARY Maize Streak Virus (MSV; Genus MastreVirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in the Genus Cicadulina, but mainly by C. mbila Naude and C. storeyi. In addition to Maize, it can infect over 80 other species in the Family Poaceae. Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant Streak disease in Maize. Severe Maize Streak disease (MSD) manifests as pronounced, continuous parallel chlorotic Streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in Maize, and/or Maize infections caused by non-Maize-adapted MSV strains, can result in narrow, interrupted Streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3–10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss. mpp_568 1..12
Jeffrey W. Davies - One of the best experts on this subject based on the ideXlab platform.
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Structure of the Maize Streak Virus geminate particle.
Virology, 2001Co-Authors: Wei Zhang, Timothy S. Baker, Margaret I. Boulton, Jeffrey W. Davies, Norman H. Olson, Lee Faulkner, Mavis Agbandje-mckenna, Robert MckennaAbstract:The Geminiviridae is an extensive family of plant Viruses responsible for economically devastating diseases in crops worldwide. GeminiViruses package circular, single-stranded DNA (ssDNA) genomes. The characteristic twinned or "geminate" particles, which consist of two joined, incomplete T = 1 icosahedra, are unique among Viruses. We have determined the first structure of a geminiVirus particle, the Nigerian strain of Maize Streak Virus (MSV-N), using cryo-electron microscopy and three-dimensional image reconstruction methods. The particle, of dimensions 220 x 380 A, has an overall 52-point-group symmetry, in which each half particle "head" consists of the coat protein (CP) arranged with quasi-icosahedral symmetry. We have modeled the MSV-N CP as an eight-stranded, antiparallel beta-barrel motif (a structural motif common to all known ssDNA Viruses) with an N-terminal alpha-helix. This has produced a model of the geminate particle in which 110 copies of the CP nicely fit into the reconstructed density map. The reconstructed density map and MSV-N pseudo-atomic model demonstrate that the geminate particle has a stable, defined structure.
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Cell-cycle, phase-specific activation of Maize Streak Virus promoters.
Molecular plant-microbe interactions : MPMI, 2001Co-Authors: Krisztina Nikovics, Margaret I. Boulton, Jeffrey W. Davies, Julietta Simidjieva, Adrian Peres, Ferhan Ayaydin, Taras Pasternak, Dénes Dudits, Gábor V. HorváthAbstract:It is believed that geminiviral DNA replication is coupled to the cell-cycle regulatory complex of the plant cell and that the Virus-early (complementary or C sense) gene products REP and REPA may be able to manipulate the regulation of the cycle. In this study, we examined expression from the promoters of Maize Streak Virus (MSV) in transgenic Maize plants and cells to determine whether they showed cell-cycle specificity. Histochemical staining of plant roots containing “long and short” C-sense promoter sequences upstream of the GUS (β-glucuronidase) reporter gene showed that promoter activity was restricted to the meristematic region of the roots and was enhanced by 2,4-dichlorophenoxy acetic acid (2,4-D) treatment. Analysis of reporter gene and cell-cycle-specific gene transcript levels coupled with flow cytometric data in synchronized transgenic Maize cells revealed that all of the MSV promoters showed cell-cycle specificity. The coat protein gene promoter showed highest activity in early G2, whereas ...
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Maize Streak Virus coat protein is karyophyllic and facilitates nuclear transport of viral dna
Molecular Plant-microbe Interactions, 1999Co-Authors: Huanting Liu, Margaret I. Boulton, C L Thomas, D A M Prior, Karl J Oparka, Jeffrey W. DaviesAbstract:Transport of Maize Streak Virus (MSV) DNA into the nucleus of host cells is essential for Virus replication and the presence of Virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculoVirus vector system, and also in tobacco protoplasts with a cauliflower mosaic Virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1labeled viral ss or ds DNA, was microinjected into Maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.
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Prediction of functional regions of the Maize Streak Virus replication-associated proteins by protein-protein interaction analysis
Plant Molecular Biology, 1998Co-Authors: Gábor V. Horváth, Jeffrey W. Davies, Krisztina Nikovics, Aladár Pettkó-szandtner, Metin Bilgin, Margaret Boulton, Crisanto Gutiérrez, Dénes DuditsAbstract:The replication of the geminiViruses depends on the viral encoded early (complementary-sense) proteins and on host genome encoded factors. Additionally, some of the early proteins (the AL2 protein of subgroup III, and the RepA (formerly known as C1) or Rep (C1:C2) proteins of subgroup I geminiViruses) can function as transcriptional activators of virion- (V-)sense gene expression. The yeast two-hybrid system has allowed us to predict some of the functionally important regions of the Maize Streak Virus (MSV) early proteins RepA and Rep. Defined domains of these proteins were shown to act as transactivators in yeast cells. We detected the association of the RepA and Rep proteins, and their subfragments, with the Maize retinoblastoma protein (ZmRb1) which is likely to be one of the interacting host proteins. We showed the self-association capability of the MSV proteins and suggest that homo- or hetero-oligomerization may play an important role in Virus replication. These results provide new insights into the role of different regions of the MSV proteins in relation to transcriptional activation and regulation of viral DNA replication.
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Maize Streak Virus coat protein binds single- and double- stranded DNA in vitro
Journal of General Virology, 1997Co-Authors: Huanting Liu, Margaret I. Boulton, Jeffrey W. DaviesAbstract:Maize Streak Virus (MSV) coat protein (CP) is required for Virus movement within the plant. Deletion or mutation of MSV CP does not prevent Virus replication in single cells or protoplasts but leads to a loss of infectivity in the inoculated plant. The mechanism by which MSV CP mediates the transfer of MSV DNA from cell to cell and through the vascular bundle is still unknown. Towards understanding the role of MSV CP in Virus movement, the interaction of the CP with viral DNA was investigated using the 'south-western' assay. Wild-type and truncated MSV CPs were expressed in E. coli and the expressed CPs were used to investigate interactions with single-stranded (ss) and double-stranded (ds) DNA. The results showed that MSV CP bound ss and ds viral and plasmid DNA in a sequence non-specific manner. The binding domain was mapped to within the 104 N-terminal amino acids of the MSV CP. We propose that the binding of CP to MSV DNA is involved in viral DNA nuclear transport as well as encapsidation and thus may have a role in intra- and inter-cellular movement as well as systemic infection.
Margaret I. Boulton - One of the best experts on this subject based on the ideXlab platform.
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Isolation and characterization of subgenomic DNAs encapsidated in "single" T = 1 isometric particles of Maize Streak Virus.
Virology, 2004Co-Authors: Carolina G Casado, Robert Mckenna, Mavis Agbandje-mckenna, G. Javier Ortiz, Eric Padron, Samantha Bean, Margaret I. BoultonAbstract:Abstract “Single” T = 1 isometric particles of Maize Streak Virus (MSV) have been isolated from infected Maize leaves. Biochemical and genetic characterizations show that these particles contain subgenomic (sg) MSV DNA encapsidated by the MSV coat protein. The largest sg DNA is 1.56 kb, slightly larger than half genome size, although sg DNAs as small as 0.2 kb were also cloned. The sg DNAs are not infectious, and they do not appear to play a role in the pathogenicity of MSV. This is the first report of sg DNAs for MSV and, to our knowledge, the first time that encapsidated sg DNAs have been characterized at the sequence level for any geminiVirus. These data will assist in our investigations into the role of genomic DNA in the formation of the unique geminate capsid architecture of the Geminiviridae .
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isolation and characterization of subgenomic dnas encapsidated in single t 1 isometric particles of Maize Streak Virus
Virology, 2004Co-Authors: Robert Mckenna, Carolina G Casado, Eric Padron, Samantha Bean, Javier G Ortiz, Mavis Agbandjemckenna, Margaret I. BoultonAbstract:Abstract “Single” T = 1 isometric particles of Maize Streak Virus (MSV) have been isolated from infected Maize leaves. Biochemical and genetic characterizations show that these particles contain subgenomic (sg) MSV DNA encapsidated by the MSV coat protein. The largest sg DNA is 1.56 kb, slightly larger than half genome size, although sg DNAs as small as 0.2 kb were also cloned. The sg DNAs are not infectious, and they do not appear to play a role in the pathogenicity of MSV. This is the first report of sg DNAs for MSV and, to our knowledge, the first time that encapsidated sg DNAs have been characterized at the sequence level for any geminiVirus. These data will assist in our investigations into the role of genomic DNA in the formation of the unique geminate capsid architecture of the Geminiviridae .
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Structure of the Maize Streak Virus geminate particle.
Virology, 2001Co-Authors: Wei Zhang, Timothy S. Baker, Margaret I. Boulton, Jeffrey W. Davies, Norman H. Olson, Lee Faulkner, Mavis Agbandje-mckenna, Robert MckennaAbstract:The Geminiviridae is an extensive family of plant Viruses responsible for economically devastating diseases in crops worldwide. GeminiViruses package circular, single-stranded DNA (ssDNA) genomes. The characteristic twinned or "geminate" particles, which consist of two joined, incomplete T = 1 icosahedra, are unique among Viruses. We have determined the first structure of a geminiVirus particle, the Nigerian strain of Maize Streak Virus (MSV-N), using cryo-electron microscopy and three-dimensional image reconstruction methods. The particle, of dimensions 220 x 380 A, has an overall 52-point-group symmetry, in which each half particle "head" consists of the coat protein (CP) arranged with quasi-icosahedral symmetry. We have modeled the MSV-N CP as an eight-stranded, antiparallel beta-barrel motif (a structural motif common to all known ssDNA Viruses) with an N-terminal alpha-helix. This has produced a model of the geminate particle in which 110 copies of the CP nicely fit into the reconstructed density map. The reconstructed density map and MSV-N pseudo-atomic model demonstrate that the geminate particle has a stable, defined structure.
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Cell-cycle, phase-specific activation of Maize Streak Virus promoters.
Molecular plant-microbe interactions : MPMI, 2001Co-Authors: Krisztina Nikovics, Margaret I. Boulton, Jeffrey W. Davies, Julietta Simidjieva, Adrian Peres, Ferhan Ayaydin, Taras Pasternak, Dénes Dudits, Gábor V. HorváthAbstract:It is believed that geminiviral DNA replication is coupled to the cell-cycle regulatory complex of the plant cell and that the Virus-early (complementary or C sense) gene products REP and REPA may be able to manipulate the regulation of the cycle. In this study, we examined expression from the promoters of Maize Streak Virus (MSV) in transgenic Maize plants and cells to determine whether they showed cell-cycle specificity. Histochemical staining of plant roots containing “long and short” C-sense promoter sequences upstream of the GUS (β-glucuronidase) reporter gene showed that promoter activity was restricted to the meristematic region of the roots and was enhanced by 2,4-dichlorophenoxy acetic acid (2,4-D) treatment. Analysis of reporter gene and cell-cycle-specific gene transcript levels coupled with flow cytometric data in synchronized transgenic Maize cells revealed that all of the MSV promoters showed cell-cycle specificity. The coat protein gene promoter showed highest activity in early G2, whereas ...
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Maize Streak Virus coat protein is karyophyllic and facilitates nuclear transport of viral dna
Molecular Plant-microbe Interactions, 1999Co-Authors: Huanting Liu, Margaret I. Boulton, C L Thomas, D A M Prior, Karl J Oparka, Jeffrey W. DaviesAbstract:Transport of Maize Streak Virus (MSV) DNA into the nucleus of host cells is essential for Virus replication and the presence of Virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculoVirus vector system, and also in tobacco protoplasts with a cauliflower mosaic Virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1labeled viral ss or ds DNA, was microinjected into Maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.
