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PubMed2004

Yeast two-hybrid systems confirm the membrane- association and oligomerization of BC1 but do not detect an interaction of the movement proteins BC1 and BV1 of Abutilon mosaic geminivirus.

Most of the plant begomoviruses use two proteins to transport their DNA from cell to cell, BV1 to shuttle it between nucleus and cytoplasm and BC1 to facilitate movement across plasmodesmata. In order to analyse their interaction for Abutilon mosaic geminivirus (AbMV) in yeast ( Saccharomyces cerevisiae), BC1 and BV1 genes were cloned into various plasmid vectors suitable for two-hybrid analysis. BC1 was fused to the binding domain (GBD) and BV1 to the activation domain (GAD) of the GAL4 transcription factor to check for interactions in the nucleus. Additionally, BC1 as well as BV1 were integrated into pMyr or pSos vectors to analyze protein binding at the plasma membrane using the CytoTraptrade mark system. Using freeze-fracture immuno-labelling (FreeFI), singly-expressed GBD:BC1 was localized at the plasma membrane although it was fused to a nuclear localization signal provided by the construct. GAD:BV1 was found in the nucleus of transformed cells as expected. Upon co-transformation of both constructs, cells grew poorly and exhibited symptoms of autolysis without any detectable level of GBD:BC1 or GAD:BV1, as revealed by FreeFI. In conclusion, both fusion proteins did not meet in the same compartment and appeared to be harmful to yeast if constitutively co-expressed. When expressed from pSos vector, BC1 induced the CytoTrap detection signal in the absence of pMyr indicating that BC1 protein alone is able to target the effector protein to the inner face of the plasma membrane. A mutated form of BC1 (DeltaBC1) lacking the previously identified membrane-binding domain was no longer able to auto-induce the CytoTrap signal cascade. Using DeltaBC1, an N-terminal, or a C-terminal third of BC1 revealed a homo-oligomerization of the C-terminal region of BC1 in two-hybrid analysis, but no interaction of BC1 with BV1.

Biological Transport↗

PubMed1999

NATURAL GENOMIC AND ANTIGENIC VARIATION IN WHITEFLY-TRANSMITTED GEMINIVIRUSES (BEGOMOVIRUSES).

Begomoviruses have circular single-stranded DNA genomes, cause many diseases of dicotyledons in areas with warm climates and are transmitted by whiteflies of the Bemisia tabaci complex. Their genomic and antigenic variation represents geography-related lineages that have little relation to host range. Genomic variation resulting from mutation is amplified by acquisition of extra DNA components, pseudo-recombination and recombination, both intraspecific and interspecific. Recombination, especially interspecific recombination, seems the key mechanism for generating novel virus forms, for enhancing biological fitness of pseudo-recombinants derived from closely related species and for maintaining the flow of genetic material among different geminiviruses occurring in the same geographical region. Recent begomovirus epidemics reflect favorable conjunctions of plant, vector, and viral (e.g. emergence of a novel recombinant virus) factors. Such epidemics typically result in co-infection of plants with different begomoviruses, leading to the appearance of further variants, especially recombinants. In their patterns of variation and evolution, begomoviruses differ greatly from plant viruses with RNA genomes.

Journal Article↗

PubMed1996

Infectivity and complete nucleotide sequence of the genome of a genetically distinct strain of maize streak virus from Reunion Island.

A complete infectious genome of an isolate of maize streak subgroup 1 geminivirus from Reunion Island (MSV-R) was cloned and sequenced. Using an Agrobacterium tumefaciens Ti plasmid delivery system, the cloned 2.7 kb circular DNA was shown to be infectious in maize. The agroinfected virus could be transmitted by Cicadulina mbila, the most common vector species of MSV in Reunion. Analysis of open reading frames (ORFs) revealed seven potential coding regions including the 4 ORFs conserved in all geminiviruses infecting monocotyledonous plants, the 2 on the viral "+" strand (MP, CP), and the 2 on the complementary "-" strand (RepA, RepB). The nucleotide sequence of MSV-R was compared to previously determined sequence of three African clones from Nigeria (MSV-N), Kenya (MSV-K), and South Africa (MSV-S). More similarity was found between the African clones (97.0-97.3%) than between these and MSV-R (94.4-95.3%). Nucleotide substitutions were frequent in the large intergenic region, particularly in and around the most likely TATA box for the complementary sense genes, and in the 5' end of ORF V1. The comparison of the predicted peptide sequences of the proteins encoded by ORFs MP, RepA and RepB confirmed the higher similarity between the African clones (97.8-99.3%) than between these and MSV-R (95.1-97.1%). However the amino acid sequences of the protein encoded by ORF CP (capsid protein) were very conserved among all the 4 clones, suggesting a high selection pressure on this ORF.

Base Sequence↗

PubMed1991

In vivo analysis of plant pre-mRNA splicing using an autonomously replicating vector.

In this paper, we demonstrate that an autonomously replicating plant expression vector can be used for analysis of pre-mRNA splicing determinants in intact dicot cells. This vector system relies on the Agrobacterium-mediated transfection of leaf discs with the A component of the geminivirus tomato golden mosaic virus (TGMV). Insertion of intron sequences between viral promoter and terminator sequences results in the production of high levels of pre-mRNA transcripts that are effectively and accurately spliced in vivo. Introns from the soybean B-conglycinin gene are spliced at greater than 95% efficiency indicating that the high expression levels of precursor RNA do not exceed the intron splicing capacity of these cells. Introns from the pea and wheat rbcS genes are spliced at 85% and 73% efficiency, respectively, indicating that tobacco leaf disc nuclei are capable of effectively and accurately processing particular dicot and monocot introns. Inclusion of a dicot intron in an engineered construct results in a five-fold enhancement of the level of mRNA stably expressed in dicot nuclei.

Base Sequence↗

PubMed2004

A geminivirus-induced gene silencing system for gene function validation in cassava.

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacum sulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow-white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2 endogenous gene, sharing 89% homology with CYP79D1 endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1 and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.

Capsid Proteins↗

PubMedDate not supplied

Use of digoxigenin-labelled probes for detection and host-range studies of tomato yellow leaf curl geminivirus.

We studied the host range of tomato yellow leaf curl virus (TYLCV) in some agronomically important tomato species. Transmission tests with the natural vector Bemisia tabaci from tomato to sweet pepper, eggplant, cucumber, melon, zucchini and spinach showed that these species did not develop symptoms and did not support viral replication. These species therefore do not constitutive a reservoir of the virus and can be cultivated as alternatives to tomato in the most affected areas. For host-range studies, we used a quick and sensitive dot-blot assay employing non-radioactive DNA probes. This technique, developed for detecting TYLCV in plant extracts, is easily used for diagnosis. The sensitivity of this non-radioactive test was comparable to that of radiolabelled probes.

Animals↗

PubMed1990

Wheat dwarf virus Ac/Ds vectors: expression and excision of transposable elements introduced into various cereals by a viral replicon.

The maize transposon Activator (Ac) and in vitro-generated nonautonomous derivatives thereof [Ac delta or Dissociation (Ds) elements] were inserted into the genome of a geminivirus of graminaceous plants, wheat dwarf virus, at a site that does not interfere with viral replication. These recombinant viral genomes were introduced into wheat, maize, and rice protoplasts, where rapid and efficient excision of Ac was observed. Excision was detected only in vectors in which, after transfection, the virus could replicate. This result is not restricted to the autonomous Ac; excision of Ds elements was also induced by transposase activity provided in trans by plasmids expressing the cDNA of Ac. The potential of this combination of a transposon with a viral replicon for plant molecular genetic engineering is discussed.

Base Sequence↗

PubMed1988

Delimitation of essential genes of cassava latent virus DNA 2.

Insertion and deletion mutagenesis of both extended open reading frames (ORFs) of cassava latent virus DNA 2 destroys infectivity. Infectivity is restored by coinoculating constructs that contain single mutations within different ORFs. Although frequent intermolecular recombination produces dominant parental-type virus, mutants can be retained within the virus population indicating that they are competent for replication and suggesting that rescue can occur by complementation of trans acting gene products. By cloning specific fragments into DNA 1 coat protein deletion vectors we have delimited the DNA 2 coding regions and provide substantive evidence that both are essential for virus infection. Although a DNA 2 component is unique to whitefly-transmitted geminiviruses, the results demonstrate that neither coding region is involved solely in insect transmission. The requirement for a bipartite genome for whitefly-transmitted geminiviruses is discussed.

Amino Acid Sequence↗

PubMed2000

Cotton leaf curl virus disease.

Cotton is one of the most important crops of Pakistan, accounting for over 60% of foreign exchange earnings. The present epidemic of cotton leaf curl disease (CLCuD) originated in the Punjab region near the city of Multan and was first reported in 1985, although it was noted in this region as early as 1967. By the early 1990s, CLCuD had become the major limitation to cotton production in Pakistan and it has now spread into India and, more recently, south and west into other provinces of Pakistan. The very characteristic symptoms include leaf curling, darkened veins, vein swelling and enations that frequently develop into cup-shaped, leaf-like structures on the undersides of leaves. Identification of the vector of CLCuD as the whitefly Bemisia tabaci (Genn.) quickly led to the suggestion that the causative agent of the disease is a geminivirus. Researchers soon confirmed the presence of such a virus that is currently ascribed to the genus Begomovirus of the family Geminiviridae, However, in 1999, the aetiology of the disease was shown to be more complex than was originally assumed. Despite the identification of both a begomovirus and a so-called nanovirus-like component, the precise causal agent of CLCuD remains uncertain.

Animals↗

PubMed2001

Exchange of three amino acids in the coat protein results in efficient whitefly transmission of a nontransmissible Abutilon mosaic virus isolate.

Geminiviruses are transmitted in a circulative manner by whiteflies, leafhoppers, or treehoppers. The whitefly species Bemisia tabaci (Genn.) is the vector for members of the genus Begomovirus. The closely related bipartite Central American begomoviruses Abutilon mosaic virus (AbMV), Sida golden mosaic virus originating from Costa Rica (SiGMV-CR), and Sida golden mosaic virus originating from Honduras (SiGMV-Hoyv) were used to study transmission by their insect vector. The AbMV isolate is defective in transmission, whereas the two Sida-infecting viruses are readily transmitted by B. tabaci. These three viruses are able to form pseudorecombinant viruses by exchange of genomic components. The pseudorecombinant virus SiGMV-Hoyv A/AbMV B was transmissible, whereas the reciprocal pseudorecombinant virus AbMV A/SiGMV-Hoyv B was not transmitted, indicating that DNA B is not involved in the transmission defect. However, the uptake of the pseudorecombinant virus AbMV A/SiGMV-Hoyv B was much better than AbMV itself, indicating that DNA B or DNA B gene products enhance uptake of viral DNA. Exchange of AbMV coat protein with that of SiGMV-CR resulted in a transmissible chimeric AbMV. Mutagenesis of the AbMV coat protein showed that the exchange of two amino acids, at positions 124 and 149, was sufficient to obtain a whitefly-transmissible AbMV mutant. However, when amino acid 174 was altered in addition to amino acids 124 and 149 AbMV was readily transmitted by B. tabaci. From this we conclude that it is not a concise motif, such as the amino acid triplet, aspartate-alanine-glycine (DAG), involved in aphid transmission of potyviruses, that determines transmissibility of begomoviruses by B. tabaci. Instead it is the composition of the coat protein domain from amino acid 123 to 149, as a minimal transmission domain, with the contribution of amino acids 149 to 174 for efficient transmission.

Amino Acid Sequence↗

PubMed1998

Gene silencing from plant DNA carried by a Geminivirus.

The geminivirus tomato golden mosaic virus (TGMV) replicates in nuclei and expresses genes from high copy number DNA episomes. The authors used TGMV as a vector to determine whether episomal DNA can cause silencing of homologous, chromosomal genes. Two markers were used to asses silencing: (1) the sulfur allele (su) of magnesium chelatase, an enzyme required for chlorophyll formation; and (2) the firefly luciferase gene (luc). Various portions of both marker genes were inserted into TGMV in place of the coat protein open-reading frame and the constructs were introduced into intact plants using particle bombardment. When TGMV vectors carrying fragments of su (TGMV::su) were introduced into leaves of wild type Nicotiana benthamiana, circular, yellow spots with an area of several hundred cells formed after 3-5 days. Systemic movement of TGMV::su subsequently produced varigated leaf and stem tissue. Fragments that caused silencing included a 786 bp 5' fragment of the 1392 bp su cDNA in sense and anti-sense orientation, and a 403 bp 3' fragment. TGMV::su-induced silencing was propogated through tissue culture, along with the viral episome, but was not retained through meiosis. Systemic downregulation of a constitutively expresse luciferase transgene in plants was achieved following infection with TGMV vectors carrying a 623 bp portion of luc in sense or anti-sense orientation. These results establish that homologous DNA sequences localized in nuclear episomes can modulate the expression of active chromosomal genes.

Journal Article↗

PubMed2001

The emergence and dissemination of whitefly-transmitted geminiviruses in Latin America.

The proliferation and rapid dissemination of whitefly-transmitted viruses of important food and industrial crops in Latin America, have been the consequence of drastic changes in traditional cropping systems. Some of the expanding non-traditional cash and export crops, such as soybean and several vegetables, have served as suitable reproductive hosts for the whitefly Bemisia tabaci. This insect pest has been shown to transmit at least 20 different geminiviruses that affect different commercial and basic food crops in Latin America. This review summarizes the existing knowledge on this important group of viruses and their vector in this region.

Animals↗

PubMed1991

Identification of the coat protein gene of bean golden mosaic geminivirus.

An open-reading frame designated 1R1, on DNA component 1 of bean golden mosaic geminivirus (BGMV), has been identified as the coat protein gene. A DraI restriction fragment of BGMV DNA1 that includes 1R1 was inserted into the SmaI pKK223-3 expression vector. The 32 kD protein expressed in Escherichia coli cells reacted with antibodies to the BGMV capsid polypeptide and behaved identically to purified capsid protein in western blots.

Animals↗

PubMed2006

Molecular ecology and emergence of tropical plant viruses.

An appreciation of the risks caused by emergent plant viruses is critical in tropical areas that rely heavily on agriculture for subsistence and rural livelihood. Molecular ecology, within 10 years, has unraveled the factors responsible for the emergence of several of the economically most important tropical plant viruses: Rice yellow mottle virus (RYMV), Cassava mosaic geminiviruses (CMGs), Maize streak virus (MSV), and Banana streak virus (BSV). A large range of mechanisms--most unsuspected until recently--were involved: recombination and synergism between virus species, new vector biotypes, genome integration of the virus, host adaptation, and long-distance dispersal. A complex chain of molecular and ecological events resulted in novel virus-vector-plant-environment interactions that led to virus emergence. It invariably involved a major agricultural change: crop introduction, cultural intensification, germplasm movement, and new genotypes. A current challenge is now to complement the analysis of the causes by an assessment of the risks of emergence. Recent attempts to assess the risks of emergence of virulent virus strains are described.

Ecosystem↗

PubMed2004

Trapping of Tomato yellow leaf curl virus (TYLCV) and other plant viruses with a GroEL homologue from the whitefly Bemisia tabaci.

To avoid destruction in the haemolymph of their vector, many plant circulative viruses interact with GroEL homologues produced by insect endosymbiotic bacteria. We have exploited this phenomenon to devise tools allowing trapping of plant viruses by either GroEL purified from the whitefly Bemisia tabaci or by whitefly GroEL over-expressed in E. coli. PCR tubes or 96-well plates coated with a GroEL preparation were incubated with cleared sap of virus infected plant leaves or insect vectors. GroEL-bound viruses were then identified by PCR or RT-PCR using virus-specific primers or by ELISA with virus specific antibodies. In this way Tomato yellow leaf curl virus (TYLCV) - a whitefly-transmitted geminivirus - was detected in plant sap, in extracts of leaf squashes and in homogenates of individual viruliferous whiteflies. Anti-GroEL antibody prevented TYLCV binding to GroEL. GroEL-bound virus was also detected by ELISA. GroEL was much more potent in binding TYLCV than commercial anti-TYLCV antibodies. In addition to several other geminiviruses, these procedures allowed detecting a variety of RNA viruses such as Cucumber mosaic virus (CMV), Prune dwarf virus (PDV) and Tomato spotted wilt (TSWV), but not Potato virus X and Potato virus Y (PVX and PVY), Grapevine leafroll-associated viruses (GLRV) and Tobacco mosaic virus (TMV). Predictions pertaining to viruses that do, or do not bind to GroEL, and applications in plant virus diagnosis, are presented.

Animals↗

PubMed1993

Nucleotide sequence and genome organization of tomato leaf curl geminivirus.

The genome of tomato leaf curl virus (TLCV) from Australia was cloned and its complete nucleotide sequence determined. It is a single circular ssDNA of 2766 nucleotides containing the consensus nonanucleotide sequence present in all geminiviruses. It has six open reading frames with an organization resembling that of certain other dicotyledonous plant-infecting monopartite geminiviruses, i.e. tomato yellow leaf curl and beet curly top viruses. The regulatory sequences present indicate a bidirectional mode of transcription. A dimeric TLCV DNA clone was constructed in a binary vector and used to agroinoculate three different host species. Typical virus infections were produced, confirming that the single DNA component is sufficient for infectivity.

Amino Acid Sequence↗

PubMed1988

Molecular variation in vector-borne plant viruses: epidemiological significance.

Patterns of variation are examined in four groups of plant viruses, with special reference to their particle proteins and to changes in vector transmissibility and specificity. In the nepoviruses and potyviruses, non-circulative transmission, by nematodes and aphids respectively, seems dependent on structural features on the surface of the virus particles. The N-terminal part of the particle protein may play the key role in potyviruses. Similarly in the luteoviruses, and possibly in the geminiviruses, specificity of circulative transmission by aphids, whiteflies and leafhoppers is linked to the antigenic specificity of the virus particles. Among naturally occurring isolates of the same virus, variation seems often to be discontinuous, and is predominantly of two sorts. Minor variations, characterized by loss of an epitope or substitutions of a few amino acids, can be associated with loss of transmissibility in luteoviruses and potyviruses, or have no effect. Major variations are associated with differences in vector specificity and seem likely to involve radical genetic changes that have evolved over long periods. The adaptation of virus particle proteins for transmission by vectors probably results in conservation of the genes that encode them, and in greater conservation of some parts of these genes than of others.

Amino Acid Sequence↗

PubMed1985

S1-sensitive sites in the supercoiled double-stranded form of tomato golden mosaic virus DNA component B: identification of regions of potential alternative secondary structure and regulatory function.

The sensitivity of the supercoiled double-stranded form of the DNA of tomato golden mosaic virus (TGMV), a geminivirus, to the single-strand specific enzyme S1 nuclease has been demonstrated. Specific S1 cleavage sites were identified in TGMV DNA component B by cloning into the single-strand bacteriophage vector M13 mp8 and sequencing of the inserted DNA. Analysis of the DNA sequence at the sites of S1 sensitivity in TGMV DNA component B revealed several possible regions of alternative secondary structure which were clustered in an intergenic region upstream of the starts of the two major open reading frames which are in opposite orientations. This region contains putative transcriptional promoter and modulatory sequences and a possible replication origin. The extreme S1 sensitivity of the supercoiled form of TGMV DNA component A precluded its cloning under the conditions employed for selective cleavage of DNA component B.

Base Sequence↗