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Evidence for transovarial transmission of tomato yellow leaf curl virus by its vector, the whitefly Bemisia tabaci.

The whitefly Bemisia tabaci is the only vector of the tomato yellow leaf curl geminivirus (TYLCV). The insect transmits the virus in a persistent-circulative manner. TYLCV DNA was detected by polymerase chain reaction and by Southern blot hybridization in progeny (eggs, first and second instars, adults) of single viruliferous whiteflies that developed on eggplant or on cotton (two TYLCV nonhost plants). Furthermore, TYLCV DNA was present in the progeny of insects that had acquired the virus through the egg. The adult progeny of the viruliferous insects and their own progeny were able to infect tomato test plants, producing typical disease symptoms. Ovaries and maturing eggs of viruliferous insects contained viral DNA, as did eggs laid by viruliferous insects maintained on an artificial diet Eggs laid by nonviruliferous whiteflies on cotton plants previously caged with viruliferous insects did not acquire viral DNA from the plant. Hence, TYLCV can be transmitted through the egg for at least two generations. In the absence of an available plant host, the whitefly may serve as a reservoir of the virus between growing seasons.

Animals↗

Trans replication and high copy numbers of wheat dwarf virus vectors in maize cells.

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.

Blotting, Southern↗

Improvement of the print-capture polymerase chain reaction procedure for efficient amplification of DNA virus genomes from plants and insect vectors.

A rapid and simple procedure is described to amplify efficiently geminivirus DNA genomes by improving the print-capture polymerase chain reaction (PCR) procedure reported recently for RNA viruses. This method, termed print-PCR (P-PCR), allows direct amplification of DNA from infected plant or whitefly tissues printed directly on Whatman 3MM paper, without the need of any grinding, incubation, or washing steps previous to the amplification reaction. P-PCR reduces sample manipulation and avoids previous extraction of nucleic acids, thereby diminishing the possibilities of cross-contamination between samples. P-PCR has been successfully applied to whiteflies and various plant species infected by two different tomato yellow leaf curl viruses, TYLCV-Sr and TYLCV-Is, and for the amplification of the full-length genome of TYLCV-Is from infected plants.

Animals↗

Expression of a bacterial gene in plants mediated by infectious geminivirus DNA.

A viable coat protein deletion mutant of cassava latent virus (CLV) DNA 1 has been isolated, suggesting that this geminivirus might be exploited as a gene replacement vector. An extensive deletion of 727 nucleotides within the coat protein gene renders DNA 1 non-infectious. Chimeric clones have been constructed in which the deleted coat protein open reading frame has been replaced by the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene. Infectivity is restored to DNA 1 when the CAT gene is inserted in either orientation, producing symptoms typical of CLV infection. The results demonstrate that the coat protein plays no essential role in virus spread throughout the host. Levels of CAT expression of 80 U/mg soluble protein occur in systemically infected Nicotiana benthamiana leaves when the CAT gene is fused in-frame to the amino terminus of the coat protein, providing a sensitive assay for viral DNA replication.

Journal Article↗

Expression of a vaccine protein in a plant cell line using a geminivirus-based replicon system.

Edible vaccines have been generated from both transgenic plants as well as from plant viral vectors. Here, we have taken the best attributions of both systems and designed a minimalized version of the bean yellow dwarf geminivirus (BeYDV)-based replicon consisting of the cis-acting elements required for BeYDV replication as a means to express foreign genes in a plant cell line. Replication can be switched on at high levels upon expression of the BeYDV Rep protein, and gene expression enhanced enormously. Construction of an expression cassette encoding a synthetic vaccine gene and analysis of expression levels of a vaccine protein in a plant cell line system are described.

Antigens, Bacterial↗

The use of African cassava mosaic virus as a vector system for plants.

This paper describes the development of a gene-displacement vector based on DNA1, one of two single stranded circular genomic components of a bipartite geminivirus, African cassava mosaic virus (ACMV). The DNA1 molecules of ACMV were cloned as dimers into a plant transformation vector and the constructs have been integrated into tobacco protoplasts by PEG-mediated DNA transfer. In transgenic plants extrachromosomal copies of DNA1 monomers could be detected. Deletion of the coat protein-encoding gene in chimeric constructs resulted in free DNA1 copies of reduced size, and extrachromosomal recombinant molecules were detected after displacement of the coat protein-encoding region by foreign DNA fragments of comparable size. Due to the absence of the second component of ACMV, DNA2, the transgenic plants are free from viral infection symptoms which allows the establishment of healthy transformants that carry a recombinant construct in an extrachromosomal form.

Blotting, Southern↗

Novel system for the simultaneous analysis of geminivirus DNA replication and plant interactions in Nicotiana benthamiana.

The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replication-associated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R(2), R(5), R(7) and K(11) or H(56), L(57) and H(58) prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.

DNA Replication↗

Tomato leaf curl virus from Bangalore (ToLCV-Ban4): sequence comparison with Indian ToLCV isolates, detection in plants and insects, and vector relationships.

Tomato leaf curl virus (ToLCV) is a whitefly (Bemisia tabaci) transmitted geminivirus (family Geminiviridae, genus Begomovirus) causing a destructive disease of tomato in many regions of India, East Asia and Australia. While ToLCV isolates from Australia and Taiwan have a single genomic component (designated DNA-A), those from Northern India have two components (DNA-A and DNA-B). The ToLCV isolates from Southern India (Bangalore) previously cloned seem to have a DNA-A-like monopartite genome. We have used degenerate DNA-A-specific PCR primers to clone the genome of a ToLCV isolate (named ToLCV-Ban4) from field-infected tomato plants growing in Bangalore, India, in 1997. Degenerate DNA-B-specific PCR primers have not allowed to amplify a putative DNA-B from infected tomato, at the time when DNA-B fragments were amplified from plants infected by known bipartite begomoviruses. The full-length 2759 nucleotide-long DNA-A-like viral genome was sequenced. Similarly to other monopartite ToLCV and TYLCV isolates, ToLCV-Ban4 contains six open reading frames, two on the virion strand and four on the complementary strand. Sequence comparisons indicated that ToLCV-Ban4 is similar to the other three isolates from Bangalore previously sequenced, and is closely related to ToLCV-Ban2 (approximately 91% nucleotide sequence identity). Phylogenetic analysis showed that the ToLCV isolates from Bangalore constitute a group of viruses separated from those of Northern India. ToLCV-Ban4 was detected in tomato and in its whitefly vector Bemisia tabaci by one or by a combination of ELISA, Southern blot hybridization and PCR. Parameters of virus acquisition, retention and transmission by the whitefly vector were investigated in the laboratory. Single whiteflies were able to acquire ToLCV-Ban4 from infected tomato and to transmit the virus to tomato test plants, but five insects were necessary to achieve 100% transmission. Minimum acquisition access and inoculation access periods were 10 min and 20 min, respectively. A latent period of 6 h was required for B. tabaci to efficiently infect tomato test plants. Following a 24 h acquisition access period the insect retained its ability to infect tomato test plants for 12 days, but not for its entire life. In one insect/one plant inoculation tests, female whiteflies were more efficient (approximately 95%) than males (approximately 25%) in transmitting the virus.

Animals↗

The nucleotide sequence of an infectious clone of the geminivirus beet curly top virus.

A number of infectious clones of a Californian isolate of the leafhopper-transmitted geminivirus beet curly top virus (BCTV) have been constructed from virus-specific double-stranded DNA isolated from infected Beta vulgaris and used to demonstrate a single component genome. The nucleotide sequence of one infectious clone has been determined (2993 nucleotides). Comparison with other geminiviruses has shown that the organisation of the genome closely resembles DNA 1 of the whitefly-transmitted members. The four conserved coding regions of DNA 1 have highly homologous counterparts in BCTV with the exception of the putative coat protein which is more closely related to those of the leafhopper-transmitted geminiviruses suggesting a strong interrelationship between coat protein and insect vector. A BCTV component equivalent to DNA 2 is not required for virus infection or transmission and has not been isolated from infected plants.

Journal Article↗

Analysis of the potential promoter sequences of African cassava mosaic virus by transient expression of the beta-glucuronidase gene.

DNA fragments from promoter regions of the geminivirus, African cassava mosaic virus, were cloned into pG1, a vector based on pUC18, producing transcriptional fusions with the beta-glucuronidase (GUS) gene and nopaline synthase termination sequence. The activity of each promoter construct was assessed by analysing the transient expression of GUS in Nicotiana clevelandii protoplasts. The results demonstrated that constructs containing the common region of DNA A showed much stronger promoter activity in the complementary sense than in the viral sense. These results were supported by the analysis of promoter activity in transgenic N. benthamiana plants. In comparison, in protoplasts a region upstream of the AC2 open reading frame was shown to have moderate promoter activity. Unlike DNA A, the complementary sense DNA B promoter constructs had weak activity; the viral sense DNA B promoter constructs appeared to be regulated by host factors. The implications of these results for the regulation of early and late genes are discussed.

Base Sequence↗

Cassava mosaic geminiviruses in Africa.

Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) (Geminiviridae:Begomovirus) is undoubtedly the most important constraint to the production of cassava in Africa at the outset of the 21st century. Although the disease was recorded for the first time in the latter part of the 19th century, for much of the intervening period it has been relatively benign in most of the areas where it occurs and has generally been considered to be of minor economic significance. Towards the end of the 20th century, however, the inherent dynamism of the causal viruses was demonstrated, as a recombinant hybrid of the two principal species was identified, initially from Uganda, and shown to be associated with an unusually severe and rapidly spreading epidemic of CMD. Subsequent spread throughout East and Central Africa, the consequent devastation of production of the cassava crop, a key staple in much of this region, and the observation of similar recombination events elsewhere, has once again demonstrated the inherent danger posed to man by the capacity of these viruses to adapt to their environment and optimally exploit their relationships with the whitefly vector, plant host and human cultivator. In this review of cassava mosaic geminiviruses in Africa, we examine each of these relationships, and highlight the ways in which the CMGs have exploited them to their own advantage.

Africa↗

Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus.

The genome of an isolate of tomato yellow leaf curl virus from Sardinia, Italy (TYLCV-S), a geminivirus transmitted by the whitefly Bemisia tabaci, has been cloned and sequenced. The single circular DNA molecule comprises 2770 nucleotides. Genome organisation closely resembles that of the DNA A component of the whitefly-transmitted geminiviruses with a bipartite genome. A 1.8 mer of the TYLCV-S genome in a binary vector of Agrobacterium tumefaciens is infectious upon agroinoculation of tomato plants. Typical tomato yellow leaf curl disease symptoms developed about three weeks after inoculation. The disease was transmitted by the natural vector B.tabaci from agroinfected plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-S consists of only one circular single-stranded DNA molecule. Contrary to the other whitefly-transmitted geminiviruses described so far, there is no evidence for the existence nor the necessity of a second component (B DNA) in the TYLCV-S genome.

Agrobacterium tumefaciens↗

The activity of the coat protein promoter of chloris striate mosaic virus is enhanced by its own and C1-C2 gene products.

DNA fragments from the bidirectional promoter region of the geminivirus chloris striate mosaic virus (CSMV) were cloned into the pUC18-based vector, pG1 producing transcriptional fusions with the beta-glucuronidase (GUS) gene and nopaline synthase terminator sequence. The relative activity of each promoter construct was analyzed by a GUS expression assay of extracts from Zea mays (maize) Black Mexican Sweet protoplasts coelectroporated with the GUS reporter constructs and constructs in which individual CSMV open reading frames (ORFs) were placed under control of a cauliflower mosaic virus 35 S promoter. Weak promoter activity was observed for the promoter of the C1 and C2 ORFs (C1-C2 gene) and for the promoter of the V1 ORF. The activity of these promoters was unaffected by coelectroporation with the CSMV ORF constructs. Moderate activity was observed for the promoter of the V2 ORF (coat protein gene) which was enhanced by coelectroporation of the C1-C2 ORF construct. Sequences within the C1-C2 gene responsible for transactivation of the V2 ORF promoter were mapped close to the A site of a conserved NTP-binding sequence pattern within the C2 ORF. To a lesser extent activity for the promoter of the V2 ORF was enhanced by the V2 ORF construct providing evidence for positive autoregulation of the CSMV coat protein gene.

Base Sequence↗

Regulation of the activities of African cassava mosaic virus promoters by the AC1, AC2, and AC3 gene products.

DNA fragments comprising each of the promoter regions from the geminivirus African cassava mosaic virus (ACMV) were cloned into the pUC18-based vector, pG1, producing transcriptional fusions with the beta-glucuronidase gene (GUS) and nopaline synthase terminator sequence. The relative activity of each promoter construct was analyzed by a GUS expression assay of extracts from Nicotiana clevelandii protoplasts coelectroporated with the GUS reporter constructs and constructs in which individual ACMV open reading frames (ORFs) were placed under control of a cauliflower mosaic virus 35 S promoter. Results suggest repression of the AC1 gene by its gene product, which is required for ACMV DNA synthesis. The promoter activity observed for the single promoter for the DNA A genes encoding functions of spread and the regulation of replication (AC2 and AC3 ORFs) was unaffected by coelectroporation with any of the ACMV ORF constructs. Promoters for the AV1 (coat protein) gene and the two DNA B genes (BV1 and BC1) were activated by electroporation of the AC2 ORF construct. To a lesser extent promoters for the AV1 and BV1 genes were activated with the AC3 ORF construct. The same pattern of promoter repression and activation was observed when transgenic N. benthamiana plants expressing the GUS reporter constructions were inoculated with ACMV DNA A.

DNA-Directed DNA Polymerase↗

The nucleotide sequence of an infectious insect-transmissible clone of the geminivirus Panicum streak virus.

The infectious genome of a Kenyan isolate of Panicum streak virus (PSV) has been cloned and sequenced. Infection of host plants was done using an Agrobacterium binary vector containing a partial repeat of the genome. Progeny virus from resultant infections proved to be transmissible by the leafhopper Cicadulina mbila (Naude). Comparisons of the amino acid sequences of PSV DNA-encoded proteins with those of previously characterized geminiviruses infecting monocotyledonous plants, including maize streak virus, revealed high levels of identity. The evolutionary relationship between PSV and other geminiviruses infecting monocotyledons is discussed.

Amino Acid Sequence↗

Agrobacterium-mediated infectivity of cloned digitaria streak virus DNA.

A monomeric clone of double-stranded DNA synthesized in vitro DNA of the geminivirus Digitaria streak (DSV) was subcloned as a tandem dimeric unit into a binary vector of Agrobacterium tumefaciens, creating a plasmid pDS2. Inoculation of digitaria sanguinalis with A. tumefaciens carrying pDS2 resulted in viral infection. The symptoms, virus particles, and DNA forms obtained were indistinguishable from those of a natural DSV infection of D. sanguinalis. Inoculations have also induced infections in Zea mays and Avena sativa. The sequence of the Agrobacterium-mediated infectious clone of DSV has been determined.

Cloning, Molecular↗

The nucleotide sequence and genome structure of the geminivirus miscanthus streak virus.

A tandem dimer of miscanthus streak virus (MiSV) DNA was inserted into the T-DNA of the binary plasmid vector pBIN19 and agroinoculated into several monocotyledonous plants (monocots) using Agrobacterium tumefaciens or A. rhizogenes. Disease symptoms and geminate particles were produced in maize and Panicum milaceum plants, and MiSV-specific double-stranded and single-stranded DNAs were found in these plants. The nucleotide sequence of the infectious MiSV clone, consisting of 2672 nucleotides, was determined. Four open reading frames (ORFs) for proteins of Mr greater than 10K were identified, two (V0 and V2) in the virus (+) sense and two (C1 and C2) in the complementary (-) sense, although C2 did not have an ATG start codon. Unlike other geminiviruses infecting monocots, complementary-sense ORFs did not overlap. Potential splicing donor and acceptor sites were identified in the sequence of the border region between the C terminus of ORF C1 and the N terminus of ORF C2. Amino acid sequences predicted from three (V2, C1 and C2) of these ORFs showed significant homology with the corresponding ORFs of other geminiviruses infecting monocots. A fifth ORF (V1), which showed some homology with ORF V1 of other monocot-infecting geminiviruses despite having a coding capacity for a product of Mr 8.8K, was found just upstream of ORF V2 as observed in those geminiviruses. ORF V0 showed no significant homology with ORFs present in any other geminiviruses. A mutation of V0 indicated that the C-terminal 30% of this ORF was not necessary for infection in maize, but that sequences around the mutated LspI site might have some regulatory role.

Amino Acid Sequence↗