Single-stranded DNA of Tomato leaf curl virus accumulates in the cytoplasm of phloem cells

Geminiviruses have been reported to replicate in, and localize to, the nuclei of host plant cells. We have investigated the tissue and intracellular distribution of the monopartite Tomato leaf curl virus (TLCV) by in situ hybridization. Contrary to the current understanding of geminiviral localization, single-stranded (ss) DNA of TLCV accumulated in the cytoplasm. TLCV ssDNA was also found in the nucleus, as was lower levels of replicative form double-stranded (ds) DNA. Under the same conditions, Tomato golden mosaic virus (TGMV) ssDNA and dsDNA were found in nuclei. ssDNA of TLCV, TGMV, and Tomato yellow leaf curl Sardinia virus (TYLCSV) was detected in some xylem vessels under specific hybridization conditions. Tissue specificity of TLCV was partially released by co-infection with TGMV. Our observations suggest that the mechanism of TLCV movement may differ from that of bipartite begomoviruses.     

Genetic analysis of bipartite geminivirus tissue tropism

The bipartite geminiviruses bean golden mosaic virus (BGMV), cabbage leaf curl virus (CabLCV), and tomato golden mosaic virus (TGMV) exhibit differential tissue tropism in Nicotiana benthamiana. In systemically infected leaves, BGMV remains largely confined to vascular-associated cells (phloem-limited), whereas CabLCV and TGMV can escape into the surrounding mesophyll. Previous work established that TGMV BRi, the noncoding region upstream from the BR1 open reading frame (ORF), is required for mesophyll invasion, but the virus must also contain the TGMV AL23 or BL1/BR1 ORFs. Here we show that, in a BGMV-based hybrid virus, CabLCV AL23 also directed efficient mesophyll invasion in conjunction with TGMV BRi, which suggests that host-adaptation of AL23 is important for the phenotype. Cis-acting elements required for mesophyll invasion were delineated by analyzing BGMV-based hybrid viruses in which various parts of BRi were exchanged with those of TGMV. Interestingly, mesophyll invasion efficiency of hybrid viruses was not correlated with the extent of viral DNA accumulation. In conjunction with TGMV AL23, a 52-bp region of TGMV BRi with sequence homology to DNA A was sufficient for mesophyll invasion. This 52-bp sequence also directed mesophyll invasion in combination with the TGMV BL1/BR1 ORFs. Overall, these results are consistent with a model for mesophyll invasion in which AL2 protein, in association with host factors, acts through the 52-bp region in TGMV BRi to affect expression of the BR1 gene.     

Dual interaction of plant PCNA with geminivirus replication accessory protein (Ren) and viral replication protein (Rep)

Geminiviruses replicate their small, single-stranded DNA genomes in plant nuclei using host replication machinery. Similar to most dicotyledonous plant-infecting geminiviruses, Tomato yellow leaf curl Sardinia virus (TYLCSV) encodes a protein, REn, that enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that REn protein from another geminivirus, Tomato golden mosaic virus (TGMV), forms oligomers and interacts with Rep protein, the only viral protein essential for replication. It has been shown that both proteins from TGMV also interact with a plant homolog of the mammalian tumor suppressor retinoblastoma protein (RBR). By using yeast two-hybrid technology and the TYLCSV REn protein as bait, we have isolated three clones of the proliferating cell nuclear antigen (PCNA) of Arabidopsis thaliana, a ring-shaped protein that encircles DNA and plays an essential role in eukaryotic chromosomal DNA replication. We also demonstrate by the two-hybrid system and a pull-down assay that REn interacts with tomato PCNA (LePCNA). Analysis of truncated proteins has located the REn-binding domain of LePCNA between amino acids 132 and 187, whereas all REn deletions used abolished or decreased dramatically its ability to interact with PCNA. Tomato PCNA also interacts with TYLCSV Rep. We propose that the interaction between PCNA and REn/Rep takes place during virus infection, inducing the assembly of the plant replication complex (replisome) close to the virus origin of replication.     

Host-specific adaptation of diverse betasatellites associated with distinct Indian tomato-infecting begomoviruses

Tomato leaf curl viruses cause major crop loss hindering tomato cultivation worldwide. The ‘Old World’ begomoviruses are often associated with circular ssDNA satellite molecules called betasatellites. In the present study, replication compatibility of five different betasatellites with three distinct Indian tomato-infecting begomoviruses representing each of a monopartite, a mono-bipartite and a bipartite begomoviruses was studied. All the betasatellites could be trans-replicated by the begomoviruses in Nicotiana benthamiana plants, however, not uniformly in tomato. Tomato leaf curl Joydebpur betasatellite—Magrahat could not induce symptom with any of these begomoviruses in tomato, whereas only Tomato leaf curl Gujarat virus could trans-replicate Radish leaf curl betasatellite in this plant species. However, none of the betasatellites were found to complement the movement function of a bipartite begomovirus in tomato. Unlike tomato, the trans-replication/maintenance of betasatellites by these begomoviruses in N. benthamiana could be due to its compromised host defence machinery. Co-infection of betasatellites with these viruses did not enhance the helper virus accumulation, but the incubation period was reduced. The possible factors involved in this host-driven adaptability of betasatellites were also discussed.     

Genetic analysis of tomato golden mosaic virus: ORF AL2 is required for coat protein accumulation while ORF AL3 is necessary for efficient DNA replication

Tomato golden mosaic virus (TGMV) is a geminivirus whose genome is divided between two DNA components, designated A and B. The TGMV genome contains six open reading frames (ORFs) which can encode proteins of greater than 10 kDa. We have used a protoplast transfection system to determine the effects of viral proteins, as defined by these ORFs, on the accumulation of viral DNA in infected cells. The accumulation of cost protein was also examined in leaf discs. Our results indicate that mutations in ORFs AR1 and AL2 do not affect viral double-stranded DNA (dsDNA) levels, although AR1 and AL2 mutants accumulate only small amounts of single-stranded viral DNA (ssDNA). In contrast, a large reduction in both ss- and dsDNA levels is observed when a mutation is introduced into ORF AL3. Mutations within either of the two DNA B ORFs do not affect DNA replication. The AL3, BR1, and BL1 mutants are capable of synthesizing coat protein; however, coat protein is not detected in leaf discs inoculated with AR1 or AL2 mutants. Testable models are proposed to explain the influence of AL2 protein on coat protein accumulation and to account for the stimulation of viral DNA synthesis mediated by the AL3 gene product.     

Characterization and distribution of tomato yellow margin leaf curl virus, a begomovirus from Venezuela

A begomovirus causing mottling and leaf deformation in tomato from the State of Mérida was cloned and sequenced. The virus has a bipartite genome comprised of a DNA-A (2,572 nucleotides) and a DNA-B (2,543 nucleotides) with a genome organization typical of New World begomoviruses. Both components share a common region of 115 nucleotides with 98 % sequence identity. Phylogenetic analysis indicated that while no virus sequences were closely related, the A component was distantly related to those of two other tomato-infecting viruses, tomato leaf deformation virus and Merremia mosaic virus; and the DNA-B, to those of pepper huasteco yellow vein virus and Rhynchosia golden mosaic Yucatan virus. The DNA-A and DNA-B sequences were submitted to GenBank (accession no. AY508993 and AY508994, respectively) and later accepted by the International Committee on Taxonomy of Viruses as the genome of a member of a unique virus species with the name Tomato yellow margin leaf curl virus (TYMLCV). Tomato (Solanum lycopersicum L. ‘Fl. Lanai’) plants inoculated with cloned TYMLCV DNA-A and DNA-B became systemically infected and showed chlorotic margins and leaf curling. The distribution of TYMLCV in tomato-producing states in Venezuela was determined by nucleic acid spot hybridization analysis of 334 tomato leaf samples collected from ten states using a TYMLCV-specific probe and confirmed by PCR and sequencing of the PCR fragment. TYMLCV was detected in samples from the states of Aragua, Guárico, and Mérida, suggesting that TYMLCV is widely distributed in Venezuela.     

Tomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: Implications for the delimitation of <Emphasis Type="Italic">cis</Emphasis>- and <Emphasis Type="Italic">trans</Emphasis>-acting replication specificity determinants

Summary. Over the last decade, the tomato production in Cuba has been affected by new whitefly-associated diseases. In addition to the well-documented presence of Tomato yellow leaf curl virus (TYLCV) along the island, the occurrence of bipartite begomoviruses has also been reported. One of them, tentatively named Tomato mottle Taino virus (ToMoTV), has now been cloned and characterized at the molecular level. Its genomic organization is similar to other bipartite geminiviruses. Phylogenetic analyses placed ToMoTV in a subcluster with other geminiviruses isolated in the Caribbean Basin: Tomato mottle virus (ToMoV), Bean dwarf mosaic virus, Abutilon mosaic virus, Sida golden mosaic virus and Potato yellow mosaic virus (PYMV). Biolistic inoculation of tobacco and tomato plants with cloned viral DNA showed that ToMoTV pseudorecombines with PYMV-GP as predicted by the identity of their iterative elements, whereas it does not show the same ability with ToMoV, even when their replication-associated proteins (Rep and REn) show the highest percentage of similarity. A comparative analysis of Rep proteins from begomoviruses that are able to produce viable reassortants suggests that some key elements for virus replication specificity are located in the first ten amino acids of this protein.Received October 18, 2002; accepted April 22, 2003 Published online July 2, 2003     

Infectivity and complete nucleotide sequence of the cloned genomic components of a bipartite squash leaf curl geminivirus with a broad host range phenotype

Through cloning and molecular analysis we have identified two highly homologous bipartite geminiviruses as causing squash leaf curl disease. Mechanical and Agrobacterium-mediated inoculation of plants with cloned viral DNA components identified the two genomic components of SqLCV-E, a squash leaf curl virus with an unexpectedly broad host range for a whitefly-transmitted geminivirus. Nucleotide sequence analysis of the genome of this virus showed it to have the same bipartite component organization characteristic of other whitefly-transmitted geminiviruses. Sequence comparison with the genomic components of tomato golden mosaic virus and bean golden mosaic virus revealed a close evolutionary relationship with these two bipartite geminiviruses, with which SqLCV-E shares common hosts. These studies provide clear molecular evidence for the assignment of SqLCV to the subfamily of bipartite geminiviruses.     

Characterization of Rhynchosia yellow mosaic Yucatan virus, a new recombinant begomovirus associated with two fabaceous weeds in Yucatan, Mexico

Rhynchosia minima (L.) DC. (Fabaceae) plants exhibiting bright golden mosaic symptoms were previously associated with begomovirus infection in Yucatan, México [1]. To characterize the begomovirus infecting these plants, the complete bipartite genome was cloned and sequenced. Sequence comparisons indicated that the virus was distinct from all other begomoviruses known to date, including those previously identified from symptomatic R. minima, and the name Rhynchosia yellow mosaic Yucatan virus (RhYMYuV) is proposed. Pairwise comparisons indicated that RhYMYuV DNA-A [2,597 nt, (EU021216)] and DNA-B [2,542 nt, (FJ792608)] components shared the highest nt sequence identity with Cabbage leaf curl virus (CaLCuV), 87% for component A and 71% for component B. Phylogenetic analysis indicated that both components of RhYMYuV are most closely related to other New World begomoviruses, having as closest relatives immediate outliers to the major Squash leaf curl virus (SLCV) clade. Recombination analysis of the RhYMYuV genome indicated that the DNA-A component has arisen through intermolecular recombination. R. minima plants inoculated with the monomeric clones developed a bright yellow mosaic similar to symptoms observed in naturally infected plants, confirming that the clones were infectious. Nicotiana benthamiana plants biolistically inoculated with monomeric clones developed curling and chlorosis in the newly emerging leaves. RhYMYuV was also detected in symptomatic Desmodium sect. Scorpiurus Benth. (Fabaceae) that were collected near the RhYMYuV-infected plants.     

Further characterization of tomato-infecting begomoviruses in Brazil

Tomato cultivation in Brazil is threatened by a number of tomato-infecting viruses belonging to the genus Begomovirus of the family Geminiviridae. Here, we report the full DNA-A sequences of three Brazilian begomoviruses: a potentially new tomato-infecting viruses, tomato interveinal chlorosis virus (ToICV), and two previously proposed begomoviruses for which only partial DNA-A sequences are available in the databases: tomato mottle leaf curl virus (TMoLCV) and tomato golden vein virus (TGVV). The complete sequences of the DNA-B components of TMoLCV and TGVV and the DNA-A components of a number of tomato severe rugose virus variants are also presented. Collectively, all of the analyzed sequences were phylogenetically clustered within the two major groups of Brazilian tomato-infecting begomoviruses.     

Genetic diversity and distribution of tomato-infecting begomoviruses in Iran

The incidence and severity of tomato leaf curl disease (TLCD) is increasing worldwide. Here we assess the diversity and distribution within tomato producing areas of Iran of begomoviruses that cause this disease. Tomato with typical TLCD symptoms and asymptomatic weeds were collected in 2005 and 2006 and tested for the presence of begomovirus DNA using polymerase chain reaction (PCR). Analysis of cloned and sequenced PCR products revealed that both mono- and bipartite begomoviruses are associated with TLCD in Iran. Furthermore, our results confirmed the symptomless infection with mono- and bipartite begomoviruses of two weed species, Chrozophora hierosolymitana Spreng (Euphobiaceae) and Herniaria sp. (Caryophyllaceae). Eighteen Iranian begomovirus isolates were classified into two major groups and two or three subgroups according to the 5′-proximal 200 nucleotides of the coat protein (CP) gene or the N-terminal 600 nucleotides of the Rep gene. Whereas most of the monopartite isolates showed closest similarity to tomato yellow leaf curl virus-Gezira (TYLCV-Ge), the three bipartite isolates were most similar to Tomato leaf curl New Delhi virus (ToLCNDV). Mixed mono- and a bipartite begomovirus infections were detected in both tomato and C. hierosolymitana. Our results indicate that the tomato producing areas in central, southern, and southeastern Iran are threatened by begomoviruses originating from both the Mediterranean basin and the Indian subcontinent.     

Complete nucleotide sequences of a distinct bipartite begomovirus, bitter gourd yellow vein virus, infecting Momordica charantia

Momordica charantia (Cucurbitaceae), a vegetable crop commonly cultivated throughout Pakistan, and begomoviruses, a serious threat to crop plants, are natives of tropical and subtropical regions of the world. Leaf samples of M. charantia with yellow vein symptoms typical of begomovirus infections and samples from apparently healthy plants were collected from areas around Lahore in 2004. Full-length clones of a bipartite begomovirus were isolated from symptomatic samples. The complete nucleotide sequences of the components of one isolate were determined, and these showed the arrangement of genes typical of Old World begomoviruses. The complete nucleotides sequence of DNA A showed the highest nucleotide sequence identity (86.9%) to an isolate of Tomato leaf curl New Delhi virus (ToLCNDV), confirming it to belong to a distinct species of begomovirus, for which the name Bitter gourd yellow vein virus (BGYVV) is proposed. Sequence comparisons showed that BGYVV likely emerged as a result of inter-specific recombination between ToLCNDV and tomato leaf curl Bangladesh virus (ToLCBDV). The complete nucleotide sequence of DNA B showed 97.2% nucleotide sequence identity to that of an Indian strain of Squash leaf curl China virus.     

Genetic variability of begomoviruses associated with cotton leaf curl disease originating from India

Summary. Cotton leaf curl disease (CLCuD) causing viruses belong to the Begomovirus genus of the family Geminiviridae. Most begomoviruses are bipartite with two molecules of circular single stranded DNA (A and B) encapsidated in icosahedral geminate particles. However, the begomoviruses associated with CLCuD have DNA- instead of DNA-B. In this communication we report the complete genomic sequence of DNA-A component of two CLCuD-causing begomoviruses, cotton leaf curl Kokhran virus-Dabawali (CLCuKV-Dab), tomato leaf curl Bangalore virus-Cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequences of two other isolates cotton leaf curl Rajasthan virus-Bangalore (CLCuRV-Ban) and cotton leaf curl Kokhran virus-Ganganagar (CLCuKV-Gang). A phylogenetic analysis of these isolates along with other related begomoviruses showed that ToLCBV-Cotton [Fat] isolate was closest to the tomato leaf curl Bangalore virus-5 (ToLCBV-Ban5) where as CLCuKV-Dab isolate was close to the cotton leaf curl Kokhran virus-Faisalabad1 (CLCuKV-Fai1), cotton leaf curl Kokhran virus-72b (CLCuKV-72b) and cotton leaf curl Kokhran virus-806b (CLCuKV-806b) isolates from Pakistan. The phylogenetic analysis further showed that the ToLCBV-Cotton [Fat] and CLCuKV-Dab isolates along with CLCuKV-Fai1, CLCuKV-72b and CLCuKV-806b are closer to the ToLCBV, tomato leaf curl Gujarat virus (ToLCGV), tomato leaf curl Gujarat virus-Varanasi (ToLCGV-Var) and tomato leaf curl Sri Lanka virus (ToLCSLV) isolates, where as cotton leaf curl Alabad virus-804a (CLCuAV-804a), cotton leaf curl Multhan virus (CLCuMV) cluster with the isolates from cotton leaf curl Rajasthan virus (CLCuRV) and okra yellow vein mosaic virus (OYVMV). These results demonstrate the extensive variability observed in this group of viruses. The AC4 ORF is the least conserved among these viruses. In order to further asses the variability in the CLCuD-causing begomoviruses, the region showing minimum similarity in the DNA-A sequence was first determined by a comparison of segments of different lengths of the aligned sequences. The results indicated that region 2411–424 (771nt) was the least conserved. A phylogenetic tree constructed using the sequences of all the CLCuD causing begomoviruses, corresponding to the least conserved region showed that they form two distinct clusters.     

Molecular characterization and experimental host range of an isolate of <Emphasis Type="Italic">Wissadula golden mosaic St. Thomas virus</Emphasis>

Partial genome segments of a begomovirus were previously amplified from Wissadula amplissima exhibiting yellow-mosaic and leaf-curl symptoms in the parish of St. Thomas, Jamaica and this isolate assigned to a tentative begomovirus species, Wissadula golden mosaic St. Thomas virus. To clone the complete genome of this isolate of Wissadula golden mosaic St. Thomas virus, abutting primers were designed to PCR amplify its full-length DNA-A and DNA-B components. Sequence analysis of the complete begomovirus genome obtained, confirmed that it belongs to a distinct begomovirus species and this isolate was named Wissadula golden mosaic St. Thomas virus-[Jamaica:Albion:2005] (WGMSTV-[JM:Alb:05]). The genome of WGMSTV-[JM:Alb:05] is organized similar to that of other bipartite Western Hemisphere begomoviruses. Phylogenetic analyses placed the genome components of WGMSTV-[JM:Alb:05] in the Abutilon mosaic virus clade and showed that the DNA-A component is most closely related to four begomovirus species from Cuba, Tobacco leaf curl Cuba virus, Tobacco leaf rugose virus, Tobacco mottle leaf curl virus, and Tomato yellow distortion leaf virus. The putative Rep-binding-site motif in the common region of WGMSTV-[JM:Alb:05] was observed to be identical to that of Chino del tomate virus-Tomato [Mexico:Sinaloa:1983], Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005], and Tomato leaf curl Sinaloa virus-[Nicaragua:Santa Lucia], suggesting that WGMSTV-[JM:Alb:05] is capable of forming viable pseudo-recombinants with these begomoviruses, but not with other members of the Abutilon mosaic virus clade. Biolistic inoculation of test plant species with partial dimers of the WGMSTV-[JM:Alb:05] DNA-A and DNA-B components showed that the virus was infectious to Nicotiana benthamiana and W. amplissima and the cultivated species Phaseolus vulgaris (kidney bean) and Lycopersicon esculentum (tomato). Infected W. amplissima plants developed symptoms similar to symptoms observed under field conditions, confirming that this virus is a causal agent of Wissadula yellow mosaic disease in W. amplissima.     

Molecular analysis of new isolates of Tomato leaf curl Philippines virus and an associated betasatellite occurring in the Philippines

Three new begomovirus isolates and one betasatellite were obtained from a tomato plant exhibiting leaf curl symptom in Laguna, the Philippines. Typical begomovirus DNA components representing the three isolates (PH01, PH02 and PH03) were cloned, and their full-length sequences were determined to be 2754 to 2746 nucleotides. The genome organizations of these isolates were similar to those of other Old World monopartite begomoviruses. The sequence data indicated that PH01 and PH02 were variants of strain B of the species Tomato leaf curl Philippines virus, while PH03 was a variant of strain A of the species Tomato leaf curl Philippines virus. These isolates were designated ToLCPV-B[PH:Lag1:06], ToLCPV-B[PH:Lag2:06], and ToLCPV-A[PH:Lag3:06], respectively. Phylogenetic analysis revealed that the present isolates form a separate monophyletic cluster with indigenous begomoviruses reported earlier in the Philippines. A betasatellite isolated from same sample belongs to the betasatellite species Tomato leaf curl Philippines betasatellite and designated Tomato leaf curl Philippines betasatellite-[Philippines:Laguna1:2006], ToLCPHB-[PH:Lag1:06]. When co-inoculated with this betasatellite, tomato leaf curl Philippines virus induced severe symptoms in N. benthamiana and Solanum lycopersicum plants. Using a PVX-mediated transient assay, we found that the C4 and C2 proteins of tomato leaf curl Philippines virus and the βC1 protein of ToLCPHB-[PH:Lag1:06] function as a suppressor of RNA silencing.     

Molecular characterization of a distinct bipartite begomovirus species infecting tomato in India

A distinct bipartite begomovirus was found associated with tomato plants showing yellowing, curling, and crumpling of the leaves, in a sub-temperate region in India. The complete DNA-A and DNA-B components were amplified through rolling circle amplification (RCA) using Φ-29 DNA polymerase and characterized. The DNA-A of the isolate was comprised of 2,756 nucleotides, encoding six open reading frames (ORFs) and DNA-B that of 2,725 nucleotides, encoding two ORFs. Genome organization of the isolate was typical of an old world bipartite begomovirus. Comparisons showed that DNA-A and its intergenic region (IR) have the highest sequence identity (86% and 84%, respectively) with the Tomato leaf curl New Delhi virus (ToLCNDV; DQ116885) and some other begomoviruses (>84%) reported from cucurbits and tomato. This data suggested that the isolate is a distinct begomovirus species for which a name Tomato leaf curl Palampur virus (ToLCPMV) is proposed. DNA-B showed the maximum sequence identity (73%) with Tomato leaf curl New Delhi virus-India-[Pakistan:Dargai:T5/6:2001] (AY150305). The common region (CR) of DNA-A and DNA-B showed 94% sequence similarity with each other. In the present study, phylogenetic relationship of this new species was also established with different begomoviruses reported from tomato and other begomoviruses showing highest homologies with complete DNA-A and DNA-B sequences. ToLCPMV is being reported from a sub-temperate region in India which was previously unaffected by begomoviruses and its whitefly vector. An erratum to this article can be found at