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Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus.

The functional properties of proteins [capsid protein (CP), V1, and C4] potentially involved with movement of the monopartite begomovirus, Tomato yellow leaf curl virus (TYLCV), were investigated using microinjection of Escherichia coli expressed proteins and transient expression of GFP fusion proteins. The TYLCV CP localized to the nucleus and nucleolus and acted as a nuclear shuttle, facilitating import and export of DNA. Thus, the CP serves as the functional homolog of the bipartite begomovirus BV1. The TYLCV V1 localized around the nucleus and at the cell periphery and colocalized with the endoplasmic reticulum, whereas C4 was localized to the cell periphery. Together, these patterns of localization were similar to that of the bipartite begomovirus BC1, known to mediate cell-to-cell movement. However, in contrast to BC1, V1 and C4, alone or in combination, had a limited capacity to move and mediate macromolecular trafficking through mesophyll or epidermal plasmodesmata. Immunolocalization and in situ PCR experiments, conducted with tomato plants at three stages of development, established that TYLCV infection was limited to phloem cells of shoot apical, leaf, stem, and floral tissues. Thus, the V1 and/or C4 may be analogs of the bipartite begomovirus BC1 that have evolved to mediate TYLCV movement within phloem tissue.

Active Transport, Cell Nucleus↗

Molecular characterization of squash leaf curl Yunnan virus, a new begomovirus and evidence for recombination.

Virus isolate Y23V, obtained from squash showing leaf curl symptoms in Yunnan, China, was readily differentiated from four studied Chinese begomovirus isolates in reactions with a set of monoclonal antibodies raised against begomoviruses. The complete nucleotide sequence (2714 nts) of the DNA-A-like molecule of Y23V was determined. The DNA-A of Y23V is most closely related to that of tomato yellow leaf curl Thailand virus-[1] (TYLCTHV-[1]) (84% sequence identity). However, the AC1 and AC4 gene of Y23V DNA-A resembled to Pepper leaf curl virus from Bangladesh (PepLCBDV). The DNA-A of Y23V has three distinct regions: the region from 74-2071 nts is 95% identical to TYLCTHV-[1] excluding a 27 nt deletion; the following 386 nts are 91% identical to PepLCBDV and the rest of the DNA-A is not closely related to any reported begomovirus. Y23V, therefore, is considered to have arisen by recombination. The 84% sequence identity of Y23V with TYLCTHV-[1] allows Y23V to be considered as a distinct begomovirus species, for which the name squash leaf curl Yunnan virus (SLCYNV) is proposed.

China↗

Molecular characterization of tomato-infecting begomoviruses in Yunnan, China.

The importance of diseases of tomato caused by begomoviruses is increasing worldwide. Here, we report that several begomoviruses are associated with tomato leaf curl disease in Yunnan province, China. 14 tomato samples showing leaf curl symptoms were collected in three districts in Yunnan, and they fell into four groups according to their reaction with a panel of 16 monoclonal antibodies in TAS-ELISA. Comparison of partial DNA-A sequences amplified with degenerate primers confirmed the existence of several types of begomoviruses. The complete DNA-A sequences of 4 isolates (Y25, Y41, Y72, Y161), corresponding to the four groups, were determined. Sequence comparisons and phylogenetic analysis revealed that they corresponded to four previously identified begomoviruses. Groups I, II and IV are most closely related to Tomato yellow leaf curl China virus (TYLCCNV), Tobacco curly shoot virus (TbCSV) and Tobacco leaf curl Yunnan virus (TbLCYNV), respectively, while Group III shows close relationships with Tomato yellow leaf curl Thailand virus (TYLCTHV). In addition, all isolates in Groups I and III were found to be associated with DNAbeta molecules, while satellite DNA was not found in virus isolates in Groups II and IV. The complete DNAbeta sequences of three isolates from Group III (Y72, Y77, Y79) were determined. Sequence analysis showed that Y72beta, Y77beta and Y79beta seem to be different from other characterised DNAbeta, sharing the highest nucleotide sequence identity with DNAbeta of TbCSV.

Antigens, Viral↗

Molecular characterization of a new begomovirus infecting Sida cordifolia and its associated satellite DNA molecules.

Two virus isolates Hn57 and Hn60 were obtained from Sida cordifolia showing mild upward leaf-curling symptoms in Hainan province of China. Comparison of partial sequences of DNA-A like molecule confirmed the existence of a single type of begomovirus. The complete nucleotide sequence of DNA-A of Hn57 was determined to be 2757 nucleotides, with a genomic organization typical of begomoviruses. Complete sequence comparison with other reported begomoviruses revealed that Hn57 DNA-A has the highest sequence identity (71.0%) with that of Tobacco leaf curl Yunnan virus. Consequently, Hn57 was considered to be a new begomovirus species, for which the name Sida leaf curl virus (SiLCV) is proposed. In addition to DNA-A molecule, two additional circular single-stranded satellite DNA molecules corresponding to DNAbeta and DNA1 were found to be associated with SiLCV isolates. Both DNAbeta and DNA1 were approximately half the size of their cognate genomic DNA. Sequence analysis shows that DNAbeta of Hn57 and Hn60 share 93.8% nucleotide sequence identity, and they have the highest sequence identity (58.5%) with DNAbeta associated with Ageratum leaf curl disease (AJ316027). The nucleotide sequence identity between DNA1 of Hn57 and that of Hn60 was 83.8%, they share 58.2-79.3% nucleotide sequence identities in comparison with other previously reported DNAl.

DNA, Satellite↗

Differential transcriptional activity of plant-pathogenic begomoviruses in their whitefly vector (Bemisia tabaci, Gennadius: Hemiptera Aleyrodidae).

Plant-pathogenic begomoviruses have a complex association with their whitefly vector and aspects concerning virus genetic activity (genome replication and gene transcription) within the insect remain highly controversial. Virus transcript abundance was assessed by quantifying selected gene transcripts of Tomato mottle virus (ToMoV, a New World bipartite begomovirus) and Tomato yellow leaf curl virus (TYLCV, an Old World monopartite begomovirus) in whiteflies (Bemisia tabaci biotype B) after feeding on virus-infected tomato plants and after subsequent transfer to cotton, a plant that is immune to the selected begomoviruses. Real-time RT-PCR was performed using specific primers for three ToMoV genes (AV1, BC1 and BV1) and three TYLCV genes (V1, V2 and C3). The ToMoV gene transcripts rapidly became undetectable in whiteflies following transfer from tomato to cotton, probably because degradation was not accompanied by new synthesis. On the other hand, TYLCV transcripts increased after transfer of whiteflies to cotton, indicating active TYLCV transcription. Interestingly, the difference observed in ToMoV and TYLCV transcripts in the vector parallel observations on the different biological effects of these viruses on whiteflies, i.e. TYLCV, but not ToMoV, reduces whitefly fitness.

Animals↗

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↗

Molecular characterization of a distinct begomovirus species and its associated satellite DNA isolated from Malvastrum coromandelianum in China.

A virus isolate Y160 was obtained from Malvastrum coromandelianum showing yellow vein symptoms in Baoshan, Yunnan province of China. The complete nucleotide sequence of DNA-A was determined, it contains 2747 nucleotides and has typical genomic organization of a begomovirus. Comparisons show that the total DNA-A of Y160 has the highest sequence identity (82.2%) with that of Malvastrum yellow vein virus-[Y47] (AJ457824), while less than 77.6% identities are found when compared with the other begomoviruses. The molecular data show that Y160 is a distinct begomovirus species, for which the name Malvastrum yellow vein Yunnan virus (MYVYNV) is proposed. Satellite DNA molecule (Y160beta) was found to be associated with Y160 using the abutting primers beta 0l and beta 02 specific for DNA beta. Y160beta consists of 1350 nucleotides, with a functional ORF (beta C1) in complementary-sense DNA. Y160beta has the highest sequence identity (73.5%) with DNA beta molecule associated with Malvastrum yellow vein virus-[Y47] (AJ421482).

Begomovirus↗

Characterisation of Sri Lankan cassava mosaic virus and Indian cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus.

Two bipartite begomoviruses, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), have been isolated from mosaic-diseased cassava originating from central India and Sri Lanka, respectively. ICMV was transmitted with low efficiency from cassava to Nicotiana benthamiana by sap inoculation to give leaf curl symptoms. SLCMV was much more virulent in this host, producing severe stunting, leaf curl, and chlorosis. These symptoms were reproduced when their cloned genomic components (DNAs A and B) were introduced into N. benthamiana by either mechanical or Agrobacterium-mediated inoculation (agroinoculation). SLCMV is more closely related to ICMV (DNA A, 84%; DNA B, 94% nucleotide identity) than African cassava mosaic virus (ACMV) (DNA A, 74%; DNA B, 47% nucleotide identity). Sequence comparisons suggest that SLCMV DNA B originated from ICMV DNA B by a recombination event involving the SLCMV DNA A intergenic region. Pseudorecombinants produced by reassortment of the cloned components of ICMV and ACMV were not infectious in N. benthamiana, emphasising their status as distinct virus species. In contrast, a pseudorecombinant between ACMV DNA A and SLCMV DNA B was infectious. Consistent with these observations, iteron motifs located within the intergenic region that may be involved in the initiation of viral DNA replication are conserved between SLCMV and ACMV but not ICMV. When introduced into N. benthamiana by agroinoculation, SLCMV DNA A alone produced a severe upward leaf roll symptom, reminiscent of the phenotype associated with some monopartite begomoviruses. Furthermore, coinoculation of SLCMV DNA A and the satellite DNA beta associated with ageratum yellow vein virus (AYVV) produced severe downward leaf curl in N. glutinosa and yellow vein symptoms in Ageratum conyzoides, resembling the phenotypes associated with AYVV DNA A and DNA beta infection in these hosts. Thus, SLCMV DNA A has biological characteristics of a monopartite begomovirus, and the virus probably evolved by acquisition of a DNA B component from ICMV.

Base Sequence↗

Bhendi yellow vein mosaic disease in India is caused by association of a DNA Beta satellite with a begomovirus.

Yellow vein mosaic disease is the major limitation in the production of bhendi or okra (Abelmoschus esculentus), an important vegetable crop of India. This disease is caused by a complex consisting of the monopartite begomovirus Bhendi yellow vein mosaic virus (BYVMV, family: Geminiviridae) and a small satellite DNA beta component. BYVMV can systemically infect bhendi upon agroinoculation but produces only mild leaf curling in this host. DNA beta induces typical symptoms of bhendi yellow vein mosaic disease (BYVMD) when co-agroinoculated with the begomovirus to bhendi. The DNA beta component associated with BYVMD has a number of features in common with those reported for ageratum yellow vein disease and cotton leaf curl disease. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction.

Abelmoschus↗

Sida micrantha mosaic is associated with a complex infection of begomoviruses different from Abutilon mosaic virus.

We report on the nucleotide sequences of geminiviruses of the genus Bemogovirus infecting Sida micrantha Schr., a common weed in Brazil. For decades, the mosaic frequently associated with Sida plants was considered to be caused by a Brazilian strain of Abutilon mosaic virus (AbMV). By infection studies and sequence comparisons, we demonstrate that it is associated with a complex of at least two begomoviruses as different from AbMV as most South American geminiviruses. Two molecules of DNA A (A1, A2) and three of DNA B (B1, B2, B3) were cloned and sequenced. According to the high homology in their common regions, DNA A1 and DNA B3, as well as DNA A2 and DNA B2, are cognate components of two begomoviruses, which were infectious in Nicotiana benthamiana plants. No trans-replication was found for any other A/B combination. The intergenic region of DNA B2 appears to be the product of the recombination between DNA B1 and DNA A2. These results show that a coinfection of begomoviruses can persist over decades, producing a reservoir of partially recombined but distinct geminiviruses.

Base Sequence↗

East African cassava mosaic Zanzibar virus - a recombinant begomovirus species with a mild phenotype.

Cassava plants exhibiting mild symptoms of cassava mosaic disease (CMD) were collected from Unguja island, Zanzibar. Cuttings grown from these plants in the glasshouse produced similar symptoms, which were milder than those caused by other known cassava mosaic geminiviruses (CMGs). The whitefly vector, Bemisia tabaci (Gennadius), transmitted the putative virus to 27.7% (n = 18) of target plants. Total DNA extracted from diseased leaves did not yield diagnostic PCR-bands using virus-specific primers to known CMGs. Degenerate primers, however, produced a diagnostic band indicating the presence of a begomovirus. Full-length DNA-A (2785 nucleotides) and DNA-B (2763 nucleotides) components were subsequently PCR-amplified, cloned and sequenced. Phylogenetic analyses of DNA-A and -B sequences showed that they were most similar to strains of East African cassava mosaic virus from Tanzania and Uganda at 83% and 86% nucleotide identities, respectively. The number and arrangement of open reading frames were similar to those of bipartite begomoviruses from the Old World. DNA-A was predicted to have recombined in the intergenic region (IR), AC1 and AC4 genes, and DNA-B in the IR. A maximum nucleotide identity of 83% in the DNA-A component with other sequenced begomoviruses, together with different biological properties allows this virus to be recognised as belonging to a new species named East African cassava mosaic Zanzibar virus (EACMZV).

Animals↗

Stachytarpheta leaf curl virus is a novel monopartite begomovirus species.

Begomovirus isolates were obtained from Stachytarpheta jamaicensis plants showing leaf curl and chlorosis symptoms collected in the Hainan province of China. The complete sequences of isolates Hn5-4, Hn6-1, Hn30 and Hn34 were determined to be 2748, 2751, 2748 and 2748 nucleotides long, respectively. The complete sequences of the four isolates share more than 94.9% nucleotide sequence identity, but all of them have less than 86% nucleotide sequence identity with other reported begomoviruses. The molecular data show that Hn5-4, Hn6-1, Hn30 and Hn34 are isolates of a distinct begomovirus species, for which the name Stachytarpheta leaf curl virus (StaLCV) is proposed. PCR and Southern blot analyses demonstrate that all the collected field samples are not associated with DNAbeta or DNA-B components. An infectious clone of StaLCV isolate Hn5-4 was constructed, and could efficiently infect Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Lycopersicon esculentum and Petunia hybrida plants, inducing upward leaf roll and vein swelling symptoms. In addition, we illustrate that StaLCV can functionally interact with distinct DNAbeta molecules in plants.

China↗

Identification of a second begomovirus, Sri Lankan cassava mosaic virus, causing cassava mosaic disease in India.

The DNA A and DNA B components of a begomovirus associated with cassava mosaic disease (CMD) originating from Kerala, India, were cloned. Biolistically inoculated clones induced symptoms typical of CMD in cassava. Sequence comparisons showed the virus to be an isolate of Sri Lankan cassava mosaic virus (SLCMV). This is the first time this begomovirus species has been identified in India and only the second species shown to cause CMD in the country. The implication of these findings on our understanding of the diversity and geographic distribution of CMD-associated begomoviruses in the region and on efforts to obtain resistance to CMD are discussed.

Base Sequence↗

Diversity of DNA 1: a satellite-like molecule associated with monopartite begomovirus-DNA beta complexes.

DNA 1 components are satellite-like, single-stranded DNA molecules associated with begomoviruses (family Geminiviridae) that require the satellite molecule DNA beta to induce authentic disease symptoms in some hosts. They have been shown to be present in the begomovirus-DNA beta complexes causing cotton leaf curl disease (CLCuD) and okra leaf curl disease (OLCD) in Pakistan as well as Ageratum yellow vein disease (AYVD) in Singapore. We have cloned and sequenced a further 17 DNA 1 molecules from a diverse range of plant species and geographical origins. The analysis shows that DNA 1 components are associated with the majority of begomovirus-DNA beta complexes, being absent from only two of the complexes examined, both of which have their origins in Far East Asia. The sequences showed a high level of conservation as well as a common organization consisting of a single open reading frame (ORF) in the virion sense, a region of sequence rich in adenine and a predicted hairpin structure. In phylogenetic analyses, there was some evidence of grouping of DNA 1 molecules according to geographic origin, but less evidence for grouping according to host plant origin. The possible origin and function of DNA 1 components are discussed in light of these findings.

Amino Acid Sequence↗

Begomovirus genetic diversity in the native plant reservoir Solanum nigrum: Evidence for the presence of a new virus species of recombinant nature.

We examined the native plant host Solanum nigrum as reservoir of genetic diversity of begomoviruses that cause the tomato yellow leaf curl disease (TYLCD) emerging in southern Spain. Presence of isolates of all the species and strains found associated with TYLCD in this area was demonstrated. Mixed infections were common, which is a prerequisite for recombination to occur. In fact, presence of a novel recombinant begomovirus was demonstrated. Analysis of an infectious clone showed that it resulted from a genetic exchange between isolates of the ES strain of Tomato yellow leaf curl Sardinia virus and of the type strain of Tomato yellow leaf curl virus. The novel biological properties suggested that it is a step forward in the ecological adaptation to the invaded area. This recombinant represents an isolate of a new begomovirus species for which the name Tomato yellow leaf curl Axarquia virus is proposed. Spread into commercial tomatoes is shown.

Base Sequence↗

Diversity of DNA beta, a satellite molecule associated with some monopartite begomoviruses.

DNA beta molecules are symptom-modulating, single-stranded DNA satellites associated with monopartite begomoviruses (family Geminiviridae). Such molecules have thus far been shown to be associated with Ageratum yellow vein virus from Singapore and Cotton leaf curl Multan virus from Pakistan. Here, 26 additional DNA beta molecules, associated with diverse plant species obtained from different geographical locations, were cloned and sequenced. These molecules were shown to be widespread in the Old World, where monopartite begomoviruses are known to occur. Analysis of the sequences revealed a highly conserved organization for DNA beta molecules consisting of a single conserved open reading frame, an adenine-rich region, and a region of high sequence conservation [the satellite conserved region (SCR)]. The SCR contains a potential hairpin structure with the loop sequence TAA/GTATTAC; similar to the origins of replication of geminiviruses and nanoviruses. Two major groups of DNA beta satellites were resolved by phylogenetic analyses. One group originated from hosts within the Malvaceae and the second from a more diverse group of plants within the Solanaceae and Compositae. Within the two clusters, DNA beta molecules showed relatedness based both on host and geographic origin. These findings strongly support coadaptation of DNA beta molecules with their respective helper begomoviruses.

Amino Acid Sequence↗

Genome organization of Tobacco leaf curl Zimbabwe virus, a new, distinct monopartite begomovirus associated with subgenomic defective DNA molecules.

The complete DNA A of the begomovirus Tobacco leaf curl Zimbabwe virus (TbLCZWV) was sequenced: it comprises 2767 nucleotides with six major open reading frames encoding proteins with molecular masses greater than 9 kDa. Full-length TbLCZWV DNA A tandem dimers, cloned in binary vectors (pBin19 and pBI121) and transformed into Agrobacterium tumefaciens, were systemically infectious upon agroinoculation of tobacco and tomato. Efforts to identify a DNA B component were unsuccessful. These findings suggest that TbLCZWV is a new member of the monopartite group of begomoviruses. Phylogenetic analysis identified TbLCZWV as a distinct begomovirus with its closest relative being Chayote mosaic virus. Abutting primer PCR amplified ca. 1300 bp molecules, and cloning and sequencing of two of these molecules revealed them to be subgenomic defective DNA molecules originating from TbLCZWV DNA A. Variable symptom severity associated with tobacco leaf curl disease and TbLCZWV is discussed.

Agrobacterium tumefaciens↗

Differential roles of C4 and betaC1 in mediating suppression of post-transcriptional gene silencing: evidence for transactivation by the C2 of Bhendi yellow vein mosaic virus, a monopartite begomovirus.

Bhendi yellow vein mosaic disease (BYVMD) is caused by the association of a DNA beta satellite with a begomovirus component. The begomovirus component has two promoters, one in the virion sense (V-sense) and the other in the complementary sense (C-sense) in the intergenic region (IR). To study the promoter activities of V-sense and C-sense promoters, mGFP gene fusion was made downstream to the promoters. Transient and stable expressions in N. benthamiana leaves showed significant GFP expression under C-sense promoter whereas the expression under the V-sense promoter was very weak in the absence of the transactivator C2. Untransformed N. benthamiana plants were agroinfiltrated with binary vector constructs containing V-sense-GFP alone or along with C1, C2, C4, V1, V2 or betaC1 (in both sense and antisense orientations) to understand the roles of these gene products in transactivation and/or suppression of post-transcriptional gene silencing (PTGS). The results showed strong suppression of gene silencing activities for C4 and betaC1 but a weak activity for C2. The suppression activities were also confirmed using gfp-silenced GFP16c/GFPi plants by agroinfiltration and agroinoculation. The expression of C4 and betaC1 as transgenes produced abnormal phenotypic growth compared to the other viral genes mentioned above, further supporting their suppressor function.

Begomovirus↗