asymmetric infectivity of pseudorecombinants of cabbage leaf curl virus and squash leaf curl virus: implications for bipartite geminivirus evolution and movement.the bipartite geminiviruses squash leaf curl virus (sqlcv) and cabbage leaf curl virus (clcv) have distinct host ranges. sqlcv infects a broad range of plants within the cucurbitaceae, including pumpkin and squash, and clcv has a broad host range within brassicaceae that includes cabbage and arabidopsis thaliana. despite this, the genomic a components of these viruses share a high degree of sequence identity, particularly in the gene encoding the replication protein al1, and their common regions ...19989792839
characterization of tobacco geminiviruses in the old and new world.biological differences and molecular variability between six phenotypically distinct tobacco-infecting geminivirus isolates from southern africa (zimbabwe) and mexico were investigated. host range studies conducted with tobacco virus isolates zim h from zimbabwe and mex 15 and mex 32 from mexico indicated all had narrow host ranges restricted to the solanaceae. alignment of coat protein gene (cp) and common region (cr) sequences obtained by pcr, and phylogenetic analysis of the cp sequences indi ...199910365162
genetic analysis of bipartite geminivirus tissue tropism.the bipartite geminiviruses bean golden mosaic virus (bgmv), cabbage leaf curl virus (cablcv), and tomato golden mosaic virus (tgmv) exhibit differential tissue tropism in nicotiana benthamiana. in systemically infected leaves, bgmv remains largely confined to vascular-associated cells (phloem-limited), whereas cablcv and tgmv can escape into the surrounding mesophyll. previous work established that tgmv bri, the noncoding region upstream from the br1 open reading frame (orf), is required for me ...200111878900
a novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein.the geminivirus replication factor al1 interacts with the plant retinoblastoma-related protein (prbr) to modulate host gene expression. the al1 protein of tomato golden mosaic virus (tgmv) binds to prbr through an 80-amino-acid region that contains two highly predicted alpha-helices designated 3 and 4. earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in prbr binding. we generated a series of al ...200415078963
geminivirus vectors for transient gene silencing in plants.both rna and dna viruses have been engineered to serve as vectors for transient silencing in intact plants. host gene sequences carried by the virus are seen by the plant as "foreign," and homologous gene-silencing machinery acts on both the viral vector rna and the endogenous host gene mrna. dna viruses, such as geminiviruses, are advantageous for silencing because only their mrnas are silenced and their dna genomes continue to replicate and move. the conserved genome organization of geminiviru ...200415103071
interaction of the movement protein nsp and the arabidopsis acetyltransferase atnsi is necessary for cabbage leaf curl geminivirus infection and pathogenicity.dna viruses can modulate the activity of cellular acetyltransferases to regulate virus gene expression and to affect cell cycle progression in order to support virus replication. a role for protein acetylation in regulating the nuclear export of the bipartite geminivirus dna genome was recently suggested by the findings that the viral movement protein nsp, which shuttles the viral genome between the nucleus and the cytoplasm, interacts with a novel arabidopsis acetyltransferase, atnsi, and the i ...200415452236
the geminivirus nuclear shuttle protein nsp inhibits the activity of atnsi, a vascular-expressed arabidopsis acetyltransferase regulated with the sink-to-source transition.dna viruses can suppress or enhance the activity of cellular acetyltransferases to regulate virus gene expression and to affect cell cycle progression in support of virus replication. a role for protein acetylation in regulating the nuclear export of the bipartite geminivirus (begomovirus) dna genome was recently suggested by the findings that the viral movement protein nsp, a nuclear shuttle protein, interacts with the arabidopsis (arabidopsis thaliana) nuclear acetyltransferase atnsi (nuclear ...200616461385
peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells.the al1 protein of tomato golden mosaic virus (tgmv), a member of the geminivirus family, is essential for viral replication in plants. its n terminus contains three conserved motifs that mediate origin recognition and dna cleavage during the initiation of rolling-circle replication. we used the n-terminal domain of tgmv al1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin a (trxa) gene. the screen selected 88 trxa peptide ...200616731923
a perk-like receptor kinase interacts with the geminivirus nuclear shuttle protein and potentiates viral infection.the nuclear shuttle protein (nsp) from bipartite geminiviruses facilitates the intracellular transport of viral dna from the nucleus to the cytoplasm and acts in concert with the movement protein (mp) to promote the cell-to-cell spread of the viral dna. a proline-rich extensin-like receptor protein kinase (perk) was found to interact specifically with nsp of cabbage leaf curl virus (calcuv) and of tomato-infecting geminiviruses through a yeast two-hybrid screening. the perk-like protein, which w ...200616775352
dna abrasion onto plants is an effective method for geminivirus infection and virus-induced gene silencing.geminiviruses belong to a rapidly growing group of plant pathogens that contribute to crop losses in tropical and subtropical areas of the world. geminivirus infection is a model for plant dna replication and virus/host interactions. geminiviruses are also used as vectors to induce silencing of endogenous genes in several plant species. a method was analyzed for inoculating geminiviruses using plasmid dna rubbed onto leaves in the presence of an abrasive (dna abrasion). although the use of dna a ...200717337069
high-frequency reversion of geminivirus replication protein mutants during infection.the geminivirus replication protein al1 interacts with retinoblastoma-related protein (rbr), a key regulator of the plant division cell cycle, to induce conditions permissive for viral dna replication. previous studies of tomato golden mosaic virus (tgmv) al1 showed that amino acid l148 in the conserved helix 4 motif is critical for rbr binding. in this work, we examined the effect of an l148v mutation on tgmv replication in tobacco cells and during infection of nicotiana benthamiana plants. the ...200717670823
functional analysis of bipartite begomovirus coat protein promoter sequences.we demonstrate that the al2 gene of cabbage leaf curl virus (calcuv) activates the cp promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for tomato golden mosaic virus (tgmv). binding studies indicate that sequences mediating repression and activation of the tgmv and calcuv cp promoter specifically bind different nuclear factors common to nicotiana benthamiana, spinach and tomato. however, chromatin immunoprecipitation demonstrates that tgmv al2 ...200818430450
genetic and phenotypic variation of the pepper golden mosaic virus complex.abstract three isolates of the bipartite begomovirus pepper golden mosaic virus (pepgmv) were characterized for genomic and biological properties. the complete nucleotide sequences of the dna-a and dna-b components were determined from infectious clones of pepgmv-serrano (pepgmv-ser), pepgmv-mosaic (pepgmv-mo), and pepgmv-distortion (pepgmv-d). nucleotide sequence identity among pepgmv components ranged from 91 to 96% for dna-a and from 84 to 99% for dna-b, with each pepgmv component most closel ...200518943475
biotic, molecular, and phylogenetic characterization of bean calico mosaic virus, a distinct begomovirus species with affiliation in the squash leaf curl virus cluster.abstract bean calico mosaic virus (bcmov), a whitefly-transmitted geminivirus from sonora, mexico, was purified, and the genome components were cloned and sequenced. purified viral fractions and cloned genome components were infectious by biolistic inoculation to bean, completing koch's postulates for both. the b biotype of the whitefly bemisia tabaci efficiently transmitted both native virus and progeny virus derived from cloned dna inoculum. host ranges of native virus and of progeny virus der ...199918944770
maintenance of an old world betasatellite by a new world helper begomovirus and possible rapid adaptation of the betasatellite.begomoviruses (family geminiviridae) cause major losses to crops throughout the tropical regions of the world. begomoviruses originating from the new world (nw) and the old world (ow) are genetically distinct. whereas the majority of ow begomoviruses have monopartite genomes and whereas most of these associate with a class of symptom-modulating satellites (known as betasatellites), the genomes of nw begomoviruses are exclusively bipartite and do not associate with satellites. here, we show for t ...200919570867
arabidopsis synaptotagmin syta regulates endocytosis and virus movement protein cell-to-cell transport.synaptotagmins are calcium sensors that regulate synaptic vesicle exo/endocytosis. thought to be exclusive to animals, they have recently been characterized in plants. we show that arabidopsis synaptotagmin syta regulates endosome recycling and movement protein (mp)-mediated trafficking of plant virus genomes through plasmodesmata. syta localizes to endosomes in plant cells and directly binds the distinct cabbage leaf curl virus (calcuv) and tobacco mosaic virus (tmv) cell-to-cell movement prote ...201020133785
virus-based microrna expression for gene functional analysis in plants.traditional virus-induced gene silencing (vigs) is a powerful virus-based short interfering rna-mediated rna silencing technique for plant functional genomics. besides short interfering rnas, micrornas (mirnas) have also been shown to regulate gene expression by rna silencing in various organisms. however, plant virus-based mirna silencing has not been reported. in addition, a number of plant mirnas have been identified or predicted, while their functions are largely unknown. thus, there is an u ...201020388670
inoculation of plants with begomoviruses by particle bombardment without cloning: using rolling circle amplification of total dna from infected plants and whiteflies.a new system for inoculation of plants with begomoviral dna without cloning or the use insect vectors is described. total dna extracted from begomovirus-infected plants was amplified by rolling circle amplification (rca) using the bacteriophage phi29 dna polymerase, and inoculated to plants by particle bombardment. infection rates of up to 100% were obtained using this technique. this technique successfully inoculated all the begomoviruses evaluated: five bipartite (bean golden yellow mosaic vir ...201020447420
characterization of rhynchosia yellow mosaic yucatan virus, a new recombinant begomovirus associated with two fabaceous weeds in yucatan, mexico.rhynchosia minima (l.) dc. (fabaceae) plants exhibiting bright golden mosaic symptoms were previously associated with begomovirus infection in yucatan, méxico [1]. to characterize the begomovirus infecting these plants, the complete bipartite genome was cloned and sequenced. sequence comparisons indicated that the virus was distinct from all other begomoviruses known to date, including those previously identified from symptomatic r. minima, and the name rhynchosia yellow mosaic yucatan virus (rh ...201020574644
a common set of developmental mirnas are upregulated in nicotiana benthamiana by diverse begomoviruses.begomoviruses are single-stranded dna viruses that cause economically important diseases of many crops throughout the world and induce symptoms in plants, including enations, leaf curling and stunting, that resemble developmental abnormalities. micrornas (mirnas) are small endogenous rnas that are involved in a variety of activities, including plant development, signal transduction and protein degradation, as well as response to environmental stress, and pathogen invasion.201121447165
comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in nicotiana benthamiana and their correlation with the levels of developmental mirnas.abstract: background: whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. micrornas (mirnas) are small endogenous rnas that regulate plant growth and development. the study described ...201121592402
a novel arabidopsis acetyltransferase interacts with the geminivirus movement protein nsp.protein acetylation is important in regulating dna-templated processes specifically and protein-protein interactions more generally in eukaryotes. the geminivirus movement protein nsp is essential for virus movement, shuttling the viral dna genome between the nucleus and the cytoplasm. we have identified a novel arabidopsis protein, atnsi, that interacts with nsp. atnsi is highly conserved among widely divergent plants. biochemical studies show that its interaction with nsp is direct and that at ...200312837950
the infective cycle of cabbage leaf curl virus (calcuv) is affected by crumpled leaf (crl) gene in arabidopsis thaliana.geminiviruses are single-stranded dna viruses that cause serious crop losses worldwide. successful infection by these pathogens depends extensively on virus-host intermolecular interactions that allow them to express their gene products, to replicate their genomes and to move to adjacent cells and throughout the plant.200919840398
analysis of geminivirus al2 and l2 proteins reveals a novel al2 silencing suppressor activity.both posttranscriptional and transcriptional gene silencing (ptgs and tgs, respectively) participate in defense against the dna-containing geminiviruses. as a countermeasure, members of the genus begomovirus (e.g., cabbage leaf curl virus) encode an al2 protein that is both a transcriptional activator and a silencing suppressor. the related l2 protein of beet curly top virus (genus curtovirus) lacks transcription activation activity. previous studies showed that both al2 and l2 suppress silencin ...201525552721
a geminivirus-based guide rna delivery system for crispr/cas9 mediated plant genome editing.crispr/cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. however, delivery of genome editing reagents remains a challenge in plants. here, we report a virus-based guide rna (grna) delivery system for crispr/cas9 mediated plant genome editing (vige) that can be used to precisely target genome locations and cause mutations. vige is performed by using a modified cabbage leaf curl virus (calcuv) vector to express ...201526450012
persistent virus-induced gene silencing in asymptomatic accessions of arabidopsis.coupled with the advantages afforded by the model plant arabidopsis, virus-induced gene silencing (vigs) offers a rapid means to assess gene function. the geminivirus vector based on cabbage leaf curl virus described here has the benefits of small insert size and persistent silencing of the target gene through the life cycle of the plant. here, we show that genetic variation in the vast collection of arabidopsis accessions can be leveraged to ameliorate viral symptomology that accompanies the vi ...201525757779
the arabidopsis synaptotagmin syta regulates the cell-to-cell movement of diverse plant viruses.synaptotagmins are a large gene family in animals that have been extensively characterized due to their role as calcium sensors to regulate synaptic vesicle exocytosis and endocytosis in neurons, and dense core vesicle exocytosis for hormone secretion from neuroendocrine cells. thought to be exclusive to animals, synaptotagmins have recently been characterized in arabidopsis thaliana, in which they comprise a five gene family. using infectivity and leaf-based functional assays, we have shown tha ...201425414709
altered expression of arabidopsis genes in response to a multifunctional geminivirus pathogenicity protein.geminivirus ac2 is a multifunctional protein that acts as a pathogenicity factor. transcriptional regulation by ac2 appears to be mediated through interaction with a plant specific dna binding protein, peapod2 (ppd2), that specifically binds to sequences known to mediate activation of the cp promoter of cabbage leaf curl virus (calcuv) and tomato golden mosaic virus (tgmv). suppression of both basal and innate immune responses by ac2 in plants is mediated through inactivation of snrk1.2, an arab ...201425403083
interaction between the transcription factor attify4b and begomovirus al2 protein impacts pathogenicity.the begomovirus al2 protein is a transcriptional activator, a silencing suppressor, and inhibitor of basal defense. al2 forms a complex at the cp promoter, through interaction with a plant-specific dna-binding protein, arabidopsis peapod2 (also known as tify4b). attify4b has three domains (ppd, tify and cct_2) conserved between homologs from different plant species. we confirmed that the al2 protein from tomato golden mosaic virus and cabbage leaf curl virus interacts with tify4b from arabidopsi ...201425011501
snrk1 phosphorylation of al2 delays cabbage leaf curl virus infection in arabidopsis.geminivirus al2/c2 proteins play key roles in establishing infection and causing disease in their plant hosts. they are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with the host signaling involved in pathogen resistance. we report here that begomovirus and curtovirus al2/c2 proteins interact strongly with host geminivirus rep-interacting kinases (griks), which are upstream activating kinases of the protein kinase snrk1, a ...201424990996
assessing global transcriptome changes in response to south african cassava mosaic virus [za-99] infection in susceptible arabidopsis susceptible plant hosts, co-evolution has favoured viral strategies to evade host defenses and utilize resources to their own benefit. the degree of manipulation of host gene expression is dependent on host-virus specificity and certain abiotic factors. in order to gain insight into global transcriptome changes for a geminivirus pathosystem, south african cassava mosaic virus [za:99] and arabidopsis thaliana, 4×44k agilent microarrays were adopted. after normalization, a log2 fold change filt ...201323826319
identification of sequences required for al2-mediated activation of the tomato golden mosaic virus-yellow vein br1 promoter.a 108 bp sequence has been identified in the tomato golden mosaic virus-yellow vein (yvtgmv) b component that is necessary and sufficient for al2-mediated activation of the br1 promoter. the sequence appears to have a bipartite arrangement, with elements located between -144 to -77 and -59 to -36 from the transcription start site, with both being required for activation by al2. these sequences are located upstream of a tata box and bind nuclear proteins from spinach, tomato and arabidopsis. thes ...201323486662
virus-induced gene silencing using artificial mirnas in nicotiana benthamiana.virus-induced gene silencing using artificial micrornas (mir vigs) is a newly developed technique for plant reverse genetic studies. traditional virus-induced gene silencing (vigs) assays introduce a large gene fragment, which is expressed and then converted into small rnas by the endogenous sirna-based gene silencing machinery of the plant host. by contrast, mir vigs uses well-designed mirnas to induce rna-mediated silencing of the target gene. using a single artificial mirna can provide greate ...201323386298
primary and secondary sirnas in geminivirus-induced gene plants, rna silencing-based antiviral defense is mediated by dicer-like (dcl) proteins producing short interfering (si)rnas. in arabidopsis infected with the bipartite circular dna geminivirus cabbage leaf curl virus (calcuv), four distinct dcls produce 21, 22 and 24 nt viral sirnas. using deep sequencing and blot hybridization, we found that viral sirnas of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant sirnas correspond primarily to t ...201223028332
the arabidopsis peapod2 transcription factor interacts with geminivirus al2 protein and the coat protein promoter.the trap protein of bipartite begomoviruses activates the coat protein (cp) promoter in mesophyll and derepresses the promoter in vascular tissue through two sequences, located 60-125 bp and 1.2-1.5 kbp respectively, upstream of the cp gene. trap does not, however, exhibit specific binding to either sequence directly. we have identified a plant-specific dna-binding protein, arabidopsis peapod2 (ppd2), that specifically binds sequences mediating activation of the cp promoter of tomato golden mosa ...200919647846
global analysis of arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection.geminiviruses are small dna viruses that use plant replication machinery to amplify their genomes. microarray analysis of the arabidopsis (arabidopsis thaliana) transcriptome in response to cabbage leaf curl virus (calcuv) infection uncovered 5,365 genes (false discovery rate <0.005) differentially expressed in infected rosette leaves at 12 d postinoculation. data mining revealed that calcuv triggers a pathogen response via the salicylic acid pathway and induces expression of genes involved in p ...200818650403
geminivirus vigs of endogenous genes requires sgs2/sde1 and sgs3 and defines a new branch in the genetic pathway for silencing in plants.virus-induced gene silencing (vigs) is a sequence-specific rna degradation process that can be used to downregulate plant gene expression. both rna and dna viruses have been used for vigs, but they differ in their mode of replication, gene expression, and cellular location. this study examined silencing mediated by a dna virus, cabbage leaf curl virus (calcuv), in several silencing-deficient arabidopsis mutants. a dna vigs vector derived from calcuv, which silenced chlorata42 (chli) needed for c ...200415165191
geminivirus-based vectors for gene silencing in arabidopsis.gene silencing, or rna interference, is a powerful tool for elucidating gene function in caenorhabditis elegans and drosophila melanogaster. the vast genetic, developmental and sequence information available for arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. we have developed a system based on the bipartite geminivirus cabbage leaf curl virus (cblcv) that allows silencing of endogenous genes singly or in combinations ...200211967097
viral vectors for plant genome engineering.recent advances in genome engineering (ge) has made it possible to precisely alter dna sequences in plant cells, providing specifically engineered plants with traits of interest. gene targeting efficiency depends on the delivery-method of both sequence-specific nucleases and repair templates, to plant cells. typically, this is achieved using agrobacterium mediated transformation or particle bombardment, both of which transform only a subset of cells in treated tissues. the alternate in planta ap ...201728443125
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