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The ecology and epidemiology of whitefly-transmitted viruses in Latin America.

Whitefly-transmitted geminiviruses are the most important constraint to the production of common bean and vegetable crops in the tropical lowlands and mid-altitude valleys of Latin America. Currently, over 30 distinct species of geminiviruses that are transmitted by the whitefly Bemisia tabaci infect common bean, tomato, pepper, cucurbits and other horticultural crops. A climate probability model (using FloraMap) was obtained using data from 304 geo-referenced locations where B. tabaci and geminiviruses cause significant damage. Clustering of the 304 points produced a simple model with two climatic variables: a dry season of at least 4 months each with less than 80 mm of rain and a mean temperature of the hottest month exceeding 21 degrees C. A modified Koeppen climate classification showed that 55% of the geminivirus-affected localities are in the tropical wet/dry region, 22% in the tropical and subtropical dry/humid climates and the remaining locations belonged to the wet equatorial and trade wind litoral climates. These findings contribute to understanding whitefly/geminivirus epidemics and implementation of sustainable integrated pest and disease management practices.

Animals↗

Geminivirus coat protein gene replacement alters insect specificity.

Chimeric clones have been constructed in which the coat protein encoded by DNA A of the bipartite genome of the geminivirus African cassava mosaic virus (ACMV) has been replaced by that of beet curly top virus (BCTV). Constructs containing the coding region inserted in either orientation were infectious when co-inoculated with ACMV DNA B onto Nicotiana benthamiana, producing symptoms typical of ACMV infection. The onset of symptom production was delayed relative to plants inoculated with parental ACMV clones and remission of symptoms was observed. When inserted in the correct orientation for expression from the ACMV coat protein promoter, the BCTV gene was expressed in plants and the coat protein synthesized encapsidated ssDNA of both ACMV genomic components. The BCTV leafhopper vector, Circulifer tenellus (Baker), transmitted both BCTV and the chimeric virus but not ACMV when injected with virus preparations and transferred to N. benthamiana seedlings. The results show that the specificity of leafhopper transmission from insect to plant resides with the coat protein.

Animals↗

Serological relationships and epitope profiles of isolates of okra leaf curl geminivirus from Africa and the Middle East.

A panel of seven murine monoclonal antibodies (mAbs) was raised against particles of okra leaf curl virus (OLCV), a whitefly-transmitted geminivirus which is prevalent in West Africa. The mAbs detected at least six distinguishable epitopes, two continuous and four discontinuous. In tests with these mAbs, relatively little antigenic variation was found among 24 OLCV isolates from Burkina Faso, Chad, Ghana, Ivory Coast, Nigeria, Oman and Saudi Arabia, each virus isolate reacting with at least five of them. However, on the basis of their reactions with 17 mAbs raised against particles of a group A isolate of African cassava mosaic virus (ACMV), the OLCV isolates could be assigned to two groups, which have geographical distributions that overlap in West Africa. A network of antigenic relationships was revealed by reactions between the OLCV mAbs and other whitefly-transmitted geminiviruses from six plant species in 12 countries. OLCV shared the most epitopes with tobacco leaf curl (African isolates) virus, ACMV, Indian cassava mosaic virus and bean golden mosaic virus. Selected OLCV mAbs are suitable for routine detection of OLCV, and a panel of three mAbs can be used to identify it.

Africa, Western↗

Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development.

The geminiviruses are a unique group of higher plant viruses that are composed of twin isometric particles which contain circular, single-stranded DNA. Tomato golden mosaic virus (TGMV), a whitefly-transmitted agent, belongs to the subgroup of geminiviruses whose members possess a bipartite genome. The TGMV A genome component has the capacity to encode at least four proteins. One of these is the viral coat protein, as inferred by homology with coat-protein, genes of other geminiviruses and by the observation of typical geminate particles in transgenic plants that contain inserts of TGMV A DNA. We have investigated the role of the coat protein in TGMV replication and report here that its coding sequence may be interrupted or substantially deleted without loss of infectivity. However, certain coat-protein mutants showed reproducible delays in time of symptom appearance as well as reduced symptom development, when inoculated onto transgenic Nicotiana benthamiana plants containing the TGMV B component. The most attenuated symptoms were seen with a mutant in which the coat-protein coding sequence was almost entirely deleted. The significance of these findings for the development of plant vectors from TGMV DNA is discussed.

Journal Article↗

Single-stranded DNA from abutilon mosaic virus is present in the plastids of infected Abutilon sellovianum.

The leaf mosaic of Abutilon sellovianum is caused by abutilon mosaic virus (AbMV), a geminivirus that has a circular single-stranded DNA genome. DNA was isolated from intact plastids of AbMV-infected and noninfected plants. Plastids from infected plants were shown to contain the single-stranded AbMV DNA by Southern-blot hybridization experiments that used a probe made from highly purified AbMV DNA. The possibility of adsorption of AbMV virions or viral DNA onto the plastid envelope was ruled out by several in vitro experiments with DNase I and protease. Furthermore, the lamellar system of plastids from AbMV-infected plants was degenerated and substituted by amorphous electron-dense material. The transport of AbMV DNA across the plastid envelope has implications for the development of a chloroplast transformation vector.

Journal Article↗

Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins.

Tobacco plants were transformed with the movement protein (pathogenicity) gene (BC1) from tomato mottle geminivirus (TMoV), using Agrobacterium-mediated transformation. Different transgenic tobacco lines that expressed high levels of the BC1 protein had phenotypes ranging from plants with severe stunting and leaf mottling (resembling geminivirus symptoms) to plants with no visible symptoms. The sequence data for the BC1 transgene from the transgenic plants with the different phenotypes indicated an association of spontaneously mutated forms of the BC1 gene in the transformed tobacco with phenotype variations. One mutated transgene associated with an asymptomatic phenotype had a major deletion at the C terminus of 119 amino acid residues with a recombination resulting in the addition of 26 amino acid residues of unidentified origin. This asymptomatic, mutated BC1 attenuated the phenotypic expression of the symptomatic BC1 in a tobacco line containing both copies of the BC1 gene. Another mutated form of the BC1 gene amplified from an asymptomatic, multicopy transgenic tobacco plant did not induce symptoms when transiently expressed in tobacco via a virus vector. The symptom attenuation in the transgenic tobacco by the asymptomatic BC1 may involve trans-dominant negative interference.

Amino Acid Sequence↗

The geminivirus BR1 movement protein binds single-stranded DNA and localizes to the cell nucleus.

Plant viruses encode movement proteins that are essential for infection of the host but are not required for viral replication or encapsidation. Squash leaf curl virus (SqLCV), a bipartite geminivirus with a single-stranded DNA genome, encodes two movement proteins, BR1 and BL1, that have been implicated in separate functions in viral movement. To further elucidate these functions, we have investigated the nucleic acid binding properties and cellular localization of BR1 and BL1. In this study, we showed that BR1 binds strongly to single-stranded nucleic acids, with a higher affinity for single-stranded DNA than RNA, and is localized to the nucleus of SqLCV-infected plant cells. In contrast, BL1 binds only weakly to single-stranded nucleic acids and not at all to double-stranded DNA. The nuclear localization of BR1 and the previously demonstrated plasma membrane localization of BL1 were also observed when these proteins were expressed from baculovirus vectors in Spodoptera frugiperda insect cells. The biochemical properties and cellular locations of BR1 and BL1 suggest a model for SqLCV movement whereby BR1 is involved in the shuttling of the genome in and/or out of the nucleus and BL1 acts at the plasma membrane/cell wall to facilitate viral movement across cell boundaries.

Animals↗

Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide.

Tomato yellow leaf curl (TYLC) is one of the most devastating viral diseases of cultivated tomato (Lycopersicon esculentum) in tropical and subtropical regions worldwide, and losses of up to 100% are frequent. In many regions, TYLC is the main limiting factor in tomato production. The causal agents are a group of geminivirus species belonging to the genus Begomovirus of the family Geminiviridae, all of them named Tomato yellow leaf curl virus (TYLCV) (sensu lato). There has been almost 40 years of research on TYLCV epidemics and intensive research programmes have been conducted to find solutions to the severe problem caused by these viruses. This paper provides an overview of the most outstanding achievements in the research on the TYLCV complex that could lead to more effective control strategies.

Animals↗

Organization of the cis-acting element required for wheat dwarf geminivirus DNA replication and visualization of a rep protein-DNA complex.

Initiation of geminivirus DNA replication depends on the activity of the initiator protein (Rep) upon interaction with DNA sequences present in the intergenic region of the viral DNA. In this study, we have analyzed the DNA sequences present in the large intergenic region (LIR) of wheat dwarf virus (WDV), a subgroup I member of the geminivirus family, which are required for viral DNA replication. We have (i) defined the boundaries of the viral cis-acting DNA replication element, (ii) determined the contribution of different domains of the LIR to DNA replication efficiency, and (iii) visualized WDV Rep-DNA complexes. Analysis of unidirectional deletions from both sides of the LIR leads us to establish that a approximately 200-bp cis-acting element (core) is essential for viral DNA replication. It spans approximately 170 and 28 bp upstream and downstream, respectively, from the initiation site (+1), located in the invariant loop. This core element is flanked, at each side, by auxiliary regions (5'-aux and 3'-aux, approximately 70 and approximately 25 bp long, respectively), which contain DNA sequences that stimulate DNA replication. Competition experiments using viral replicating vectors bearing wild-type or mutant WDV LIRs suggest that the auxiliary regions may contribute to the stabilization and/or activity of the initiation complex formed by WDV Rep at the origin. We have visualized DNA-protein complexes by electron microscopy and a high-affinity binding site of WDV Rep protein within the core element has been mapped to approximately 144 +/- 18 bp upstream from the initiation site, between the start site for complementary-sense transcription and the TATA box. Our studies (i) establish the modular structure of the WDV DNA replication cis-acting element and (ii) provide direct evidence for the formation in vitro of a large nucleoprotein complex within the essential cis-acting element.

DNA Helicases↗

Rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in Escherichia coli.

A bacterial plasmid containing 1.75 copies of double-stranded porcine circovirus (PCV) DNA in tandem (0.8 copy of PCV type 1 [PCV1], 0.95 copy of PCV2) with two origins of DNA replication (Ori) yielded three different DNA species when transformed into Escherichia coli: the input construct, a unit-length chimeric PCV1(Rep)/PCV2(Cap) genome with a composite Ori but lacking the plasmid vector, and a molecule consisting of the remaining 0.75 copy PCV1(Cap)/PCV2(Rep) genome with a different composite Ori together with the bacterial plasmid. Replication of the input construct was presumably via the theta replication mechanism utilizing the ColE(1) Ori, while characteristics of the other two DNA species, including a requirement of two PCV Oris and the virus-encoded replication initiator Rep protein, suggest they were generated via the rolling-circle copy-release mechanism. Interestingly, the PCV-encoded Rep' protein essential for PCV DNA replication in mammalian cells was not required in bacteria. The fact that the Rep' protein function(s) can be compensated by the bacterial replication machinery to support the PCV DNA replication process echoes previous suggestions that circular single-stranded DNA animal circoviruses, plant geminiviruses, and nanoviruses may have evolved from prokaryotic episomal replicons.

Animals↗

Quantitative determination of tomato yellow leaf curl geminivirus DNA by chemiluminescent assay using digoxigenin-labeled probes.

A quantitative dot-blot hybridisation assay was developed for tomato yellow leaf curl geminivirus (TYLCV). The assay is based on chemiluminescent detection of viral and plasmid DNA using digoxigenin-labeled probes on nylon membranes. The response-error relationship was studied and a square root transformation was found to stabilise the variance of the response. An asymmetric sigmoid (gompertz) curve was used to describe the dose-response relationship. The detection limits and the precision profiles of the curves were studied. A method is suggested for setting an upper limit on the maximum DNA amount that can be discriminated from the upper asymptote. With respect to different times of exposure of an X-ray film to a membrane, the shortest times gave better upper limits and the longest times provided better detection limits. Purified virus and plasmid preparations were studied in various dilution media, such as TYLCV-free Bemisia tabaci (the whitefly vector) and tomato extracts, with particular attention to parallelism with standard calibration curves. Plasmid diluted in buffer was found useful for calibration of purified virus and virus in the vector, while extraction of known amounts of virus, in parallel with samples to be examined, was needed to quantify viral DNA in plant hosts.

DNA Probes↗

The DNA beta satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein (betaC1).

Ageratum yellow vein disease (AYVD) is caused by the geminivirus ageratum yellow vein virus (AYVV) and an associated DNA beta satellite. We have mapped a DNA beta transcript to a highly conserved open reading frame (betaC1 ORF). The most abundant transcript 5'-terminus is located 8 bases upstream of the betaC1 ORF putative initiation codon while the transcript terminates at multiple sites downstream from the putative termination codon. Disruption of betaC1 protein expression by the introduction of an internal nonsense codon prevented infection of the AYVV-satellite complex in ageratum and altered the phenotype in Nicotiana benthamiana to that produced by AYVV alone although the mutant was maintained in systemically infected tissues. Modification of the putative initiation codon to a nonsense codon produced an intermediate phenotype in N. benthamiana and a mild yellow vein phenotype in ageratum, suggesting that betaC1 protein expression could be initiated from an alternative site. N. benthamiana plants containing a dimeric DNA beta transgene produced severe developmental abnormalities, vein-greening, and cell proliferation in the vascular bundles. Expression of betaC1 protein from a potato virus X (PVX) vector also induced abnormal plant growth. Our results demonstrate that the satellite encodes at least one protein that plays a major role in symptom development and is essential for disease progression in ageratum, the natural host of the AYVD complex.

Ageratum↗

Mutational analysis of the small intergenic region of maize streak virus.

Maize streak virus (MSV) is a leafhopper-transmitted geminivirus containing one molecule of circular single-stranded DNA of about 2.7 kb. We have tested the infectivity of MSV mutants. A deletion of 29 bases in the small intergenic region (SIR) did not affect the infectivity of MSV. Mutants containing insertions of oligonucleotides of up to 32 bases at the AsnI site in SIR were also infectious. However, the infection efficiency of the insertion mutants decreased as the size of the oligonucleotides increased. This reduced virulence may be due to a decreased efficiency of 3'-end formation of the complementary sense mRNA(s). The stability of MSV mutants in infected maize plants was analyzed by polymerase chain reaction (PCR). Some oligonucleotide-insertion mutants were completely stable while others obtained deletions. Infectivity of the mutants, however, did not require deletion formation. Deletion rearrangement was shown to be dependent on the sequence of the inserted oligonucleotide. The AsnI site is the only site known to permit insertion of nonviral DNA without abolishing MSV infectivity. The system described may allow the use of MSV as a delivery vector to introduce at least small segments of DNA into maize.

Base Sequence↗

Interaction between a geminivirus replication protein and origin DNA is essential for viral replication.

The geminivirus, tomato golden mosaic virus (TGMV), encodes one protein, AL1, that is absolutely required for viral DNA replication. AL1 interacts with the TGMV DNA genome by binding specifically to the viral origin of replication. We have investigated the nature and significance of AL1/origin interactions in vitro and in vivo by using competitive DNA binding and transient replication assays. Competition assays established that a 13-base pair (bp) element (5'-GGTAGTAAGGTAG) containing two 5-bp direct repeat motifs separated by a 3-bp central core constitutes a high affinity AL1 binding site. DNAs containing intact 3' repeat sequences plus core (TAAGGTAG and ccTAGTAAGGTAG) were stronger competitors for AL1 binding than DNAs containing intact 5' repeat sequences plus core (GGTAGTAA and GGTAGTA-AccTAG), thereby demonstrating that AL1 interacts differently with the repeat motifs. Replication in tobacco protoplasts established that the AL1 binding site is an essential cis-acting element for viral replication. No replication was detected for DNAs containing mutations in either of the repeat motifs of the AL1 recognition sequence when AL1 was provided in trans from a plant gene expression vector. In contrast, a DNA with a mutation in the 5' repeat motif (ccTAGTAAGGTAG) replicated when both AL1 and AL3, a TGMV protein involved in viral DNA accumulation, were provided in trans. No replication was detected for a DNA containing a mutation in the 3' repeat motif (GGTAGTAAccTAG) in the presence of AL1 and AL3. The in vitro and in vivo results suggest that binding of AL1 to the 3' repeat element is an essential step in DNA replication, while binding to the 5' repeat element may serve to enhance viral replication.

Base Sequence↗

Identification of genes directly and indirectly involved in the insect transmission of African cassava mosaic geminivirus by Bemisia tabaci.

The inability to transmit progeny virus resulting from the cloned components of an isolate of African cassava mosaic virus originating from Kenya (ACMV-K) has been shown to be due to defects in both genomic components. This was achieved by the production of infectious pseudorecombinants between ACMV-K and the cloned components of a whitefly-transmissible ACMV isolate originating from Nigeria (ACMV-NOg). The exchange of gene fragments between ACMV-K and ACMV-NOg has been used to demonstrate that the defects responsible for lack of transmissibility reside on the coat protein and DNA B C1 gene of ACMV-K. The significance of these finding with respect to the present understanding of the function of these gene products are discussed.

Animals↗

Geminate structures of African cassava mosaic virus.

Two types of geminate structures were purified from African cassava mosaic geminivirus (ACMV)-infected Nicotiana benthamiana plants and analyzed by electron cryomicroscopy and image reconstruction. After cesium sulfate density gradient centrifugation, they were separated into lighter top (T) and heavier bottom (B) components. T particles comigrated with host proteins, whereas B particles were concentrated in a cesium density typical for complete virions. Both particles were composed of two incomplete icosahedra of 11 capsomers each, but T particles were slightly larger (diameter, 22.5 nm) and less dense in the interior than B particles (diameter, 21.5 nm). T particles were frequently associated with small globules of approximately 14 nm diameter of unknown origin. The overall structure of ACMV, a begomovirus transmitted by whiteflies, was similar to that of Maize streak virus (MSV), a mastrevirus transmitted by leafhoppers, although the vertices of the icosahedra were less pronounced. Models of ACMV coat proteins based on Satellite tobacco necrosis virus support the exposure of parts of the molecule essential for transmission specificity by whiteflies and provide possible structural explanations for the smaller protrusion of the ACMV capsid relative to MSV. The differences of ACMV and MSV virion shapes are discussed with reference to their different animal vectors.

Amino Acid Sequence↗

Cassava mosaic virus disease in East Africa: a dynamic disease in a changing environment.

Cassava mosaic disease (CMD), now known to be caused by cassava mosaic geminiviruses (Family Geminiviridae; Genus Begomovirus), was first reported in East Africa in 1894. Epidemics occurred in Madagascar and Uganda in the 1930s and 1940s, and more localised rapid spread of CMD was observed in parts of coastal Tanzania in the 1930s and coastal Kenya in the 1970s. During the 1990s, a major regional pandemic of an unusually severe form of CMD has expanded to affect parts of at least five countries, causing massive economic losses and destabilising food security. Mechanisms responsible for the development and progress of the pandemic have been described, and comparisons of epidemiological data for varieties grown throughout the period under review suggest that the recent pandemic has been characterised by rapid rates of CMD spread hitherto unknown in East Africa. A key factor in the genesis and spread of the pandemic has been the recombination between two distinct cassava mosaic geminiviruses to produce a novel and more virulent hybrid. Although such events may be common, the known history of CMD in East Africa suggests that the frequency with which they become epidemiologically significant is low. A corollary of this is that resistance, developed originally in Tanzania between 1934 and 1960, and utilized and supplemented at the International Institute of Tropical Agriculture, Nigeria, since 1971, is providing effective CMD control in current pandemic-affected areas of East Africa. Consequently, it is concluded that prospects for managing CMD in the 21st century are good, and that the approach adopted should build on the model of collaborative research and implementation that has been established in tackling the current CMD pandemic.

Africa, Eastern↗

Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein.

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1-120) and oligomerization (amino acids 134-181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitro experiments demonstrated that a pair of predicted alpha-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions.

Amino Acid Sequence↗