Bipartite Geminivirus Research Articles

Functional equivalence of late gene promoters in bean golden mosaic virus with those in tomato golden mosaic virus.

In the bipartite geminivirus tomato golden mosaic virus (TGMV), the activity of late gene promoters is up-regulated by the multifunctional viral protein AL2. Cis-acting sequences required for AL2-mediated promoter responses have not been well characterized. However, nucleotide sequence analysis has implicated a motif termed the conserved late element (CLE). The CLE is present in TGMV and many other begomoviruses, although it is not ubiquitous. Here we analysed the regulation of late gene expression in bean golden mosaic virus (BGMV), one of the begomoviruses which lacks the CLE. Transient reporter gene assays showed that BGMV late gene promoters were trans-activated in Nicotiana benthamiana protoplasts, both by the homologous BGMV AL2 protein and by the heterologous TGMV AL2 protein. The BGMV AL2 protein also trans-activated TGMV late gene promoters. Consistent with these results, we found that hybrid viruses with the late gene promoters exchanged between BGMV and TGMV were viable in planta.

Sources of resistance in Lycopersicon spp. to a bipartite whitefly-transmitted geminivirus from Brazil

Accessions of Lycopersicon chilense, L. peruvianum, L.hirsutum and sixteen L. esculentum genotypes were evaluated undergreenhouse conditions for resistance to a whitefly-transmitted geminivirusisolate from Brasilia-DF (DF1). Artificial cage inoculation oftomato plants at the two true-leaf stage, using 20 viruliferous whiteflies perplant in individual insect-proof cages, ensured 100% infection ofsusceptible tomato plants. Virus infection was confirmed by symptomdevelopment and dot-blot or squash-blot hybridization. In advanced testing,accessions of L. chilense (LA 1967), L. peruvianum (CNPH784) and L. hirsutum (PI-127827) and three selected inbred lines(TY 197, TY 198 and Tx 468-1) showed no symptoms and viral DNAwas barely detectable four weeks after inoculation, indicating good sourcesof resistance to the virus.

A New Tomato-Infecting Begomovirus in Barbados.

In September 1998, tomato plants in Barbados exhibited symptoms of severe leaf curling without marginal chlorosis. These symptoms were often associated with an increase in whitefly (Bemisia tabaci) populations. DNA was extracted from leaf tissue from symptomatic tomato plants. Polymerase chain reaction (PCR) was performed with DNA-A degenerate primer pair PAC1v1978/PAV1c715, which amplifies part of the rep gene, the cp gene, and the common region (CR), and with DNA-B primer pair PBC1v2039/PBV1c800, which amplifies part of the bc1 and bv1 genes and the CR (2). The amplified PCR fragments of DNA-A and DNA-B were 1.3 and 1.4 kb, respectively, which are the expected sizes from bipartite, whitefly-transmitted geminiviruses of the Western Hemisphere (2). DNA sequence of the cloned fragments of DNA-A and DNA-B are available as GenBank No. AF213013 and AF213014, respectively. The 181 nucleotides of the CR of DNA-A had a nucleotide identity of 96% with the CR of DNA-B, which indicates that this is a bipartite begomovirus. Pairwise comparisons using DNASTAR (DNASTAR, Madison, WI) of the sequenced part of DNA-A was most similar to Cabbage leaf curl virus (CaLCuV, 69%, U65529) and Squash leaf curl virus extended host range isolate (SqLCV-E, 64%, M38183), and <59% to 13 other bipartite Western Hemisphere geminiviruses and Tomato yellow leaf curl virus from Israel (X15656). Pairwise comparisons of the DNA-B fragment sequence was 59 and 55% similar to CaLCuV (U65530) and SqLCV-E (M38182), respectively. Phylogenetic analysis of DNA-A of the major groups of Western Hemisphere begomoviruses placed the Barbados tomato-infecting geminivirus in the cluster with CaLCuV and SqLCV-E (1), while DNA-B analysis placed it with CaLCuV. The DNA-A amplified fragment was used as a probe at high stringency with the dot blot hybridization assay using the Genius II labeling and detection kit (Boeringer Mannheim) to detect this geminivirus in tomato and several other plant species, which had typical geminiviral symptoms. Strong hybridization signals were obtained for all 23 tomato plants with symptoms, weak signals were observed for two of three muskmelon and two of seven watermelon plants, all with leaf curling symptoms. No hybridization signals were observed for peppers with leaf curling symptoms and two weed species, Macroptilium lathyroides and Rhynchosia minima, with golden mosaic symptoms or with the symptomless plant species used as negative controls. The weak signals observed from watermelon and muskmelon samples indicated the presence of low virus titer or geminiviruses distinct from this tomato virus. The presence of viral DNA in these two plant species was confirmed by PCR with degenerate primers described above. Resulting database searches of sequences in the GenBank revealed that the Barbados tomato virus appears to be a previously unreported virus. This new virus is given the provisional name Tomato leaf curl Barbados virus (ToLCBBV).

Bipartite Geminivirus Host Adaptation Determined Cooperatively by Coding and Noncoding Sequences of the Genome

Bipartite geminiviruses are small, plant-infecting viruses with genomes composed of circular, single-stranded DNA molecules, designated A and B. Although they are closely related genetically, individual bipartite geminiviruses frequently exhibit host-specific adaptation. Two such viruses are bean golden mosaic virus (BGMV) and tomato golden mosaic virus (TGMV), which are well adapted to common bean (Phaseolus vulgaris) and Nicotiana benthamiana, respectively. In previous studies, partial host adaptation was conferred on BGMV-based or TGMV-based hybrid viruses by separately exchanging open reading frames (ORFs) on DNA A or DNA B. Here we analyzed hybrid viruses in which all of the ORFs on both DNAs were exchanged except for AL1, which encodes a protein with strictly virus-specific activity. These hybrid viruses exhibited partial transfer of host-adapted phenotypes. In contrast, exchange of noncoding regions (NCRs) upstream from the AR1 and BR1 ORFs did not confer any host-specific gain of function on hybrid viruses. However, when the exchangeable ORFs and NCRs from TGMV were combined in a single BGMV-based hybrid virus, complete transfer of TGMV-like adaptation to N. benthamiana was achieved. Interestingly, the reciprocal TGMV-based hybrid virus displayed only partial gain of function in bean. This may be, in part, the result of defective virus-specific interactions between TGMV and BGMV sequences present in the hybrid, although a potential role in adaptation to bean for additional regions of the BGMV genome cannot be ruled out.

Bean golden yellow mosaic virus from Chiapas, Mexico: Characterization, Pseudorecombination with Other Bean-Infecting Geminiviruses and Germ Plasm Screening

ABSTRACT The complete nucleotide (nt) sequences of the cloned DNA-A (2644 nts) and DNA-B (2609 nts) components of Bean golden yellow mosaic virus (BGYMV-MX) from Chiapas, Mexico were determined. The genome organization of BGYMV-MX is similar to that of other Western Hemisphere bipartite geminiviruses (genus Begomovirus). Infectivity of the cloned BGYMV-MX DNA components in common bean (Phaseolus vulgaris) plants was demonstrated by particle bombardment and agroinoculation. BGYMV-MX was identified as a BGYMV (previously type II BGMV) isolate based on sequence analyses, sap-transmissibility, and pseudorecombination experiments with other bean-infecting begomoviruses. On the basis of differences in the DNA-B hypervariable region, symptom phenotype, and properties of infectious pseudorecombinants, BGYMV-MX may represent a distinct strain of BGYMV. Pseudorecombination experiments further established that BGYMV symptom determinants mapped to DNA-B, and that BGYMV-MX was most closely related to BGYMV from Guatemala. A Tomato leaf crumple virus (TLCrV) DNA-A/BGYMV-MX DNA-B pseudorecombinant was infectious in bean, establishing that a viable reassortant can be formed between begomovirus species from different phylogenetic clusters. Bean germ plasm representing the two major gene pools (Andean and Mesoamerican) was screened for response to BGYMV-MX with three methods of inoculation: sap-inoculation, particle bombardment, and agroinoculation. Andean germ plasm was very susceptible and similar results were obtained with all three methods, whereas Mesoamerican germ plasm showed resistance to BGYMV-MX, particularly with agroinoculation.

Virus-Specific Adaptations for the Production of a Pseudorecombinant Virus Formed by Two Distinct Bipartite Geminiviruses from Central America

Most whitefly-transmitted geminiviruses possess bipartite genomes comprising DNAs A and B. The production of viable pseudorecombinants by reassortment of infectious cloned components is generally limited to isolates/strains of a particular virus. Following exchange of cloned genomic components of Sida golden mosaic virus from Costa Rica (SiGMV/Co) and Sida golden mosaic virus from Honduras (SiGMV/Hoyv), the pseudorecombinant viruses were infectious in various plant species. Three DNA B components (B1, B2, B3), different in a few nucleotides, were isolated from Sida rhombifolia naturally infected with SiGMV/Hoyv. Only SiGMV/Hoyv DNA B2 was able to form a viable pseudorecombinant with SiGMV/Co DNA A. In protoplasts, as well as in inoculated leaves, SiGMV/Co DNA A trans-replicated the heterogenomic SiGMV/Hoyv DNA B1 component, indicating that impaired movement is involved in the deficiency of SiGMV/Hoyv DNA B1 to form a pseudorecombinant virus with SiGMV/Co DNA A. Even after extensive mutation analysis of SiGMV/Hoyv DNA B1 and B2, we were unable to pinpoint differences in SiGMV/Hoyv DNA B2 that allowed the formation of a pseudorecombinant virus with SiGMV/Co DNA A. We observed a gradual increase of infectivity from noninfectious SiGMV/Co DNA A/SiGMV/Hoyv DNA B1 and B3 pseudorecombinant virus to pseudorecombinant viruses showing normal systemic spread of both genomic components associated with symptomatic plants.

Host range and symptom variation of pseudorecombinant virus produced by two distinct bipartite geminiviruses.

Within the whitefly group only the species Bemisia tabaci (Gennadius) is the vector. Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, DNAs A and B. Although they are closely related to each other, the production of viable pseudorecombinants between bipartite geminiviruses by reassortment of infectious cloned components is generally limited to strains of a particular virus. Following exchange of cloned genomic components of Sida golden mosaic virus (SiGMV/Hoyv) and Abutilon mosaic virus (AbMV), the pseudorecombinant viruses were infectious in various host plants. The symptom type of pseudorecombinant virus was in most cases determined by DNA B. However, in some host plants also DNA A of the pseudorecombinant virus was involved in the symptom phenotype.

Identification of Tomato yellow leaf curl virus-Is in The Bahamas.

In December 1996, symptoms of stunting, curling, and marginal chlorosis of leaves, reduced leaf size, and marked reduction in number of fruits were first seen in tomato (Lycopersicon esculentum) plants on the island of North Andros, The Bahamas. Similar symptoms were observed for the first time during fall 1997 in tomatoes on the island of Eleuthera. Incidences of symptomatic plants were as high as 100% in some fields. Leaves from one symptomatic plant from each island were collected during April 1998. DNA was extracted from the samples and tested by polymerase chain reaction (PCR) amplification for the presence of one or more geminiviruses (2). Three sets of primers were used to amplify the extracts: PAL1c496 and PAL1v1978, which amplify an ≈1,100- or ≈1,300-bp DNA product from the A component of a wide range of bipartite and monopartite begomoviruses, respectively; primers PCRc154 and PBL1v2042, which amplify an ≈600-bp DNA fragment from the B component of a wide range of bipartite geminiviruses; and primers PCRc154 and PTYC1v2180 (5'ACTACCATGGCCGC-GCAGCGGAATAC3'), which preferentially amplify Tomato yellow leaf curl virus (TYLCV-Is) (1,2). DNA products of ≈1,300 and ≈780 bp were amplified with PAR1c496 and PAL1v1978 and PCRc154 and PTYC1v2180, respectively, from one sample from each island. No product was obtained from primers PCRc154 and PBL1v2042. The symptoms and PCR results are consistent for the presence of TYLCV. PCR products generated by primers PAL1c496 and PAL1v1978 from each sample were cloned into a pGEM-T Easy Vector (Promega, Madison, WI), and one clone from each was sequenced with vector primers. The sequences of the two 1,300-nt Bahamian clones were identical. The Bahamian clones shared 98.9% sequence homology with equivalent sequences of a TYLCV-Is clone from Florida, 99.2 and 99.4% homology with two TYLCV-Is clones from the Dominican Republic (GenBank Accession no. AF024715, and a full-length infectious clone submitted to GenBank), 98.7% homology with a clone from Cuba (GenBank Accession no. AJ223505), and 98.0% homology with the type sequence from Israel (GenBank Accession no. X15656). A deletion (28 or 29 nt) in the intergenic region was shared by clones from The Bahamas and Florida but was not present in clones from Cuba (AJ223505), the Dominican Republic (AF024715), Egypt (GenBank Accession no. L12219), Israel (X15656), Jamaica (GenBank Accession no. U84146), Lebanon (GenBank Accession no. AF160875), Mexico (GenBank Accession no. AF168709), and Spain (GenBank Accession no. AJ223505). TYLCV-Is appeared in The Bahamas and Florida at almost the same time (1). Because clones from both locations share an unusual deletion, there may be a common source for both introductions. This is the first report of TYLCV-Is in The Bahamas. Reference: (1) J. E. Polston et al. Plant Dis. 83:984, 1999. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

A New Bipartite Geminivirus (Begomovirus) Causing Leaf Curl and Crumpling in Cucurbits in the Imperial Valley of California.

During fall 1998, volunteer watermelons (Citrullus lunatus L. (Thunb.) Matsum. & Nakai) showing leaf curl, crumpling, and yellowing symptoms were found in a commercial honeydew melon (Cucumis melo L. subsp. melo Inodorus group) field in the Imperial Valley of California. The plants were infected with a begomovirus (family Geminiviridae, genus Begomovirus) based on (i) a positive response in squash blots probed with a general begomovirus DNA probe (1) and (ii) amplification of DNA-A (≈1.2 kb) and DNA-B (≈1.4 kb) fragments by polymerase chain reaction (PCR) with degenerate DNA-A (PAL1v1978/PAR1c496) and DNA-B (PBL1v2040/PBR1c970) primers, respectively (3). The DNA-A and -B fragments were cloned and sequenced (GenBank accession nos. AF224760 [DNA-A] and AF224761 [DNA-B]). The DNA-A and -B fragments had a nearly identical (99.5%) common region (CR) of 186 (DNA-A) and 187 (DNA-B) nucleotides, indicating they were from the same begomovirus. Database searches conducted with these sequences revealed no high degree of sequence identity (i.e., >90%) with other begomoviruses, including Squash leaf curl virus (SqLCV [2]) from southern California. The partial AC1 sequence (669 nt) was most identical to Tomato severe leaf curl virus (ToSLCV) from Guatemala (83%) and SqLCV (81%), the partial AV1 sequence (135 nt) was most identical to Tomato golden mosaic virus from Brazil (84%) and SqLCV (81%), and the CR was most identical to Squash yellow mottle virus from Costa Rica (81%), ToSLCV (81%), and SqLCV (77%). The partial BV1 sequence (465 nt) was most identical to Bean calico mosaic virus and SqLCV (72%), and the partial BC1 sequence (158 nt) was most identical to SqLCV (75%). Watermelon seedlings bombarded with a DNA extract from infected watermelon volunteers developed crumpling and distortion symptoms, whereas seedlings bombarded with gold particles alone developed no symptoms. Geminivirus infection in symptomatic seedlings was confirmed by PCR. These results suggest a new begomovirus caused the disease symptoms in the watermelon volunteers. Leaf crumpling and curling symptoms were not observed in spring melons in the Imperial Valley in 1999, but on 2 July and 17 August 1999, cantaloupe (C. melo L. subsp. melo Cantalupensis group), muskmelon (C. melo L. subsp. melo Cantalupensis group), and watermelon plants with leaf crumpling and yellowing were found. These plants were infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments (97 to 98% identity for CR and partial AC1 sequence). A survey of fall melons, conducted 23 to 24 September 1999, revealed widespread symptoms of leaf curl and crumpling on new growth of muskmelon plants in all seven commercial fields examined (estimated incidence 25 to 50%) and on watermelon volunteers. No such symptoms were observed on leaves of honeydew melons. Symptomatic muskmelon and watermelon leaves, collected from eight locations throughout the Imperial Valley, were infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments. Thus, a new begomovirus has emerged in the Imperial Valley; the name Cucurbit leaf crumple virus (CuLCrV) is proposed.

Yellow Leaf Disease of Cantaloupe and Wax Gourd from Thailand Caused by Tomato leaf curl virus.

Cantaloupe (Cucumis melo var. reliculatus) and wax gourd (Benincasa hispida Cogn.) plants showing a yellow leaf disease have been observed in central Thailand since 1993. The disease has been transmitted to cantaloupe and wax gourd plants by grafting and whiteflies (Bemisia tabaci Genn.) (K. Kittipakorn, unpublished data). Because of the leaf yellowing symptoms and transmission by whiteflies, a geminivirus was suspected. Three naturally infected cantaloupe and four naturally infected wax gourd plants showing leaf yellowing were collected from fields at Kamphaengsaen, Nakorn Pathom, Thailand. Virus cultures were maintained in cantaloupe and wax gourd plants. Inoculations were done with a whitefly vector. Symptoms in cantaloupe and wax gourd plants were the same as those observed in the field. Virus DNA associated with yellow leaf disease of cantaloupe and wax gourd plants was amplified by polymerase chain reaction with geminivirus-specific degenerate primers, described by Rojas et al. (2), that anneal within the AC1 open reading frame (ORF; replication associated protein gene) and the AV1 ORF (coat protein gene) of bipartite geminiviruses and the C1 and V2 ORFs of monopartite geminiviruses (2). Fragments (1.2 kbp) were amplified and cloned from affected cantaloupe and wax gourd plants. Three independent clones were sequenced and analyzed, and identical 32-base stem loop regions were found in the amplified fragments from both isolates, including the conserved nonanucleotide sequence TAATATTAC, which is present in all geminiviruses. The iterative sequence GGCGTC also was found in the intergenic region (IR) (434 bp) of the amplified fragments from both isolates. The B component could not be detected. The nucleotide sequences of the IR and V1 were compared with 28 well-studied whitefly-transmitted geminiviruses and revealed ≈96% sequence identity with DNA A of Tomato leaf curl virus of the genus Geminivirus from India (ToLCV-In 2) (1) for both isolates. The iterative sequence GGCGTC in the IR also was identical to the sequence in ToLCV-In 2. These results establish the provisional identity of the pathogen causing yellow leaf disease on cantaloupe and wax gourds in Thailand as ToLCV or closely related strains.

A Nanovirus-like DNA Component Associated with Yellow Vein Disease of Ageratum conyzoides: Evidence for Interfamilial Recombination between Plant DNA Viruses

Yellow vein disease of Ageratum conyzoides, a weed species that is widely distributed throughout Asia, has been attributed to infection by the geminivirus Ageratum yellow vein virus (AYVV). In addition to a single AYVV genomic component (DNA A), we have previously demonstrated that infected plants contain chimeric defective viral components, comprising DNA A and nongeminiviral sequences, that act as defective interfering DNAs. A database search has revealed that the nongeminiviral sequences of one such defective component (def19) show significant homology with sequences of nanovirus components that encode replication-associated proteins (Reps). Primers designed to hybridise to the nongeminiviral DNA were used to PCR-amplify a full-length nanovirus-like component, referred to as DNA 1, from an extract of infected A. conyzoides. DNA 1 is unrelated to AYVV DNA A but resembles nanovirus components that encode Reps and is most closely related (73% identity) to a nanovirus-like DNA recently isolated from geminivirus-infected cotton. DNA 1 is dependent on AYVV DNA A for systemic infection of A. conyzoides and Nicotiana benthamiana and can systemically infect N. benthamiana in the presence of the bipartite geminivirus African cassava mosaic virus. A. conyzoides plants coinfected with AYVV DNA A and DNA 1 remain asymptomatic, indicating that additional factors are required to elicit yellow vein disease. Our results provide direct evidence for recombination between distinct families of plant single-stranded DNA viruses and suggest that coinfection by geminivirus and nanovirus-like pathogens may be a widespread phenomenon. The ability of plant DNA viruses to recombine in this way may greatly increase their scope for diversification.

Tomato dwarf leaf curl virus, a new bipartite geminivirus associated with tomatoes and peppers in Jamaica and mixed infection with tomato yellow leaf curl virus

Genomic characterization using nonradioactive probes, polymerase chain reaction with degenerate primers for whitefly transmitted geminiviruses and nucleotide sequencing were used to describe a new bipartite geminivirus, associated with dwarfing and leaf curling of tomatoes and peppers in Jamaica. Partial DNA‐A and DNA‐B clones were obtained. DNA sequence analysis showed that tomato and pepper samples have a similar geminivirus associated with them. Nucleotide sequence identity &gt; 92% between the common regions of DNA‐A and DNA‐B confirmed the bipartite nature of the Jamaican geminivirus isolates. Nucleotide sequence comparisons of DNA‐A and DNA‐B with those of geminiviruses representing the major phylogenetic groups of Western Hemisphere geminiviruses showed the greatest similarity to potato yellow mosaic virus and members of the Abutilon mosaic virus cluster of geminiviruses. This new virus is given the name tomato dwarf leaf curl virus (TDLCV) because of the dwarfing and leaf curling symptoms associated with infected tomato plants. Polymerase chain reaction and Southern hybridization showed mixed infections of TDLCV with tomato yellow leaf curl virus from Israel in 16% of the field samples of tomatoes and peppers.

The bipartite geminivirus coat protein aids BR1 function in viral movement by affecting the accumulation of viral single-stranded DNA.

The movement of bipartite geminiviruses such as squash leaf curl virus (SqLCV) requires the cooperative interaction of two essential virus-encoded movement proteins, BR1 and BL1. While the viral coat protein AR1 is not essential for systemic infection, genetic studies demonstrate that its presence masks the defective phenotype of certain BR1 missense mutants, thus suggesting that coat protein does interact with the viral movement pathway. To further examine the mechanism of this interaction, we have constructed alanine-scanning mutants of AR1 and studied them for the ability to mask the infectivity defects of appropriate BR1 mutants, for the ability to target to the nucleus and to bind viral single-stranded DNA (ssDNA) and multimerize, and for effects on the accumulation of replicated viral ssDNA. We identified a specific region of AR1 required for masking of appropriate BR1 mutants and showed that this same region of AR1 was also important for ssDNA binding and the accumulation of viral replicated ssDNA. This region of AR1 also overlapped that involved in multimerization of the coat protein. We also found that the accumulation in protoplasts of single-stranded forms of a recombinant plasmid that included the SqLCV replication origin but was too large to be encapsidated was dependent on the presence of AR1 but did not appear to require encapsidation. These findings extend our model for SqLCV movement, demonstrating that coat protein affects viral movement through its ability to induce the accumulation of replicated viral ssDNA genomes. They further suggested that encapsidation was not required for the AR1-dependent accumulation of viral ssDNA.

Asymmetric Infectivity of Pseudorecombinants of Cabbage Leaf Curl Virus and Squash Leaf Curl Virus: Implications for Bipartite Geminivirus Evolution and Movement

The bipartite geminiviruses squash leaf curl virus (SqLCV) and cabbage leaf curl virus (CLCV) have distinct host ranges. SqLCV infects a broad range of plants within the Cucurbitaceae, including pumpkin and squash, and CLCV has a broad host range within Brassicaceae that includes cabbage andArabidopsis thaliana.Despite this, the genomic A components of these viruses share a high degree of sequence identity, particularly in the gene encoding the replication protein AL1, and their common regions are 77% identical. However, there is unexpected sequence diversity in the common regions of the two CLCV genomic A and B components, these being only 80% identical. Based on these sequence similarities, we investigated the host range properties of pseudorecombinants of SqLCV and CLCV. We found that in a pseudorecombinant virus consisting of the A component of CLCV and the B component of SqLCV, both components replicated in tobacco protoplasts, and this pseudorecombinant was infectious and caused systemic disease inNicotiana benthamiana, a common host to all bipartite geminiviruses. However, this pseudorecombinant did not move systemically in pumpkin orArabidopsis, despite the demonstrated replication compatibility of the genome components. As a result of the greater sequence differences between the common regions, the pseudorecombinant of SqLCV A and CLCV B components neither replicated the CLCV B component nor systemically infected any of the hosts tested. These findings demonstrate that for different geminiviruses with distinct host ranges, the replication origins and AL1 proteins can be sufficiently similar to permit infectious pseudorecombinants, but replication alone is not sufficient to cause systemic disease, and host range may ultimately be limited at the level of movement. The results of this study further suggest that CLCV is an evolving virus that can provide insights into how new bipartite geminiviruses arise from mixed infections.

Bean Dwarf Mosaic Geminivirus Movement Proteins Recognize DNA in a Form- and Size-Specific Manner

Plant viral movement proteins mediate the cell-to-cell movement of nucleic acids. This involves either a direct interaction between the viral movement protein and the nucleic acid or an indirect interaction involving host factors. The bipartite geminiviruses possess two movement proteins, BV1 and BC1, that coordinate movement of viral DNA across nuclear and plasmodesmal boundaries, respectively. Here, we demonstrate that both BV1 and BC1 interact directly with DNA and, in addition, that they have the unique property to recognize DNA on the basis of form and size rather than sequence. This is a novel feature for plant virus movement proteins and raises the possibility that BV1 and BC1 may be determinants of genome size in the bipartite geminiviruses.

Havana tomato virus, a new bipartite geminivirus infecting tomatoes in Cuba.

The cloning and nucleotide sequence of a new bipartite geminivirus found in Cuba is described. DNA A (2620 nt) and DNA B (2586 nt) presented a genomic structure resembling that of other geminiviruses transmitted by Bemisia tabaci. Both components had a common region of 168 nt with a 95% identity. Typical elements involved in replication and transcription were found in this region, though group-characteristic arrangement of iterons was not conserved in this virus. Sequence was compared with geminivirus sequences deposited in the GenBank. Interestingly, when total DNAs or individual ORFs and deduced amino acid sequences were compared, the highest scores were for different viruses. It showed to be most closely related to tomato mottle virus (81.9% and 65.5% similarity with DNAs A and B, respectively) and a member of the abutilon mosaic cluster of New World Begomoviruses. When clones A and B were co-agroinoculated they resulted highly infectious and induced symptoms in Nicotiana benthamiana plants. The A component alone was infectious but induced only mild symptoms, while the B component was not infectious. The presence of viral DNA in N. benthamiana plants was confirmed by dot-blot hybridization using specific probes. These data show that the cuban isolate is a new geminivirus for which the name of Havana tomato virus is proposed.

First Report of Tomato Mottle Geminivirus Infecting Tomatoes in Yucatan, Mexico

Whitefly-transmitted geminiviruses are a major constraint on tomato production in Mexico (3). In the Yucatan State, these viruses can cause serious losses in late season plantings. As part of an effort to characterize these viruses, leaf samples from four tomato plants showing symptoms of geminivirus infection, such as stunted growth and leaf mottling and deformation, were collected from a single field in the Yucatan State in February, 1996. Geminivirus nucleic acids were detected in leaf samples from all four plants by squash blot hybridization analysis with a general DNA probe for Western Hemisphere whitefly-transmitted geminiviruses (2). Nicotiana benthamiana plants inoculated with sap prepared with leaf tissue from one plant developed stunted growth and leaf mottling and deformation. When graft-transmitted from N. benthamiana to tomato, the geminivirus(es) induced leaf mottling and deformation, which were similar to symptoms in the field-collected tomato plants. The presence of geminivirus DNA in the sap- and graft-inoculated plants was confirmed with the polymerase chain reaction (PCR) and degenerate primers for the DNA-A (PAL1v1978 and PAR1c496) or DNA-B (PBL1v2040 and PCRc1) components of whitefly-transmitted geminiviruses (4). Using PCR and these degenerate primers, approximately 1.1-kb DNA-A and approximately 0.6-kb DNA-B fragments were amplified from DNA extracts prepared from leaves of each of the four Yucatan tomato plants. No DNA fragments were amplified from these extracts with primers for pepper huasteco geminivirus (pAL1c2329 and pAL1v1471, or pBR1c840 and pBL1v1830). To determine the identity of the geminivirus(es) infecting these tomato plants, the PCR-amplified DNA-A and DNA-B fragments from one of the samples were cloned and sequenced. Comparisons made with these sequences revealed two distinct types of DNA-A and DNA-B clones, indicating a mixed infection of at least two bipartite geminiviruses. DNA-A and DNA-B sequences of one set of clones were >97% identical to sequences of tomato mottle geminivirus (ToMoV) from Florida (1). The presence of ToMoV in all four tomato leaf samples was demonstrated by the PCR-mediated amplification of a 0.9-kb DNA-A fragment with ToMoV-specific primers (pAL1v2295 and pAR1c580). The identity of this 0.9-kb DNA fragment was further confirmed based upon its hybridization with a full-length clone of ToMoV DNA-A under high stringency conditions (2). A data base search made with the sequence of the other type of DNA-A clone revealed sequence identities of <70% with various bipartite geminiviruses (e.g., identities of 70% with tomato mottle, 69% with Sida golden mosaic, 67% with bean dwarf mosaic, and 66% with taino tomato mottle and with potato yellow mosaic), which confirmed that a second geminivirus was present in a mixed infection with ToMoV in this tomato leaf sample. To confirm the bipartite nature of this geminivirus, a DNA-B fragment that contained the common region (CR) sequence was amplified from the same sample with PCR and primers PBL1v2040 and PBR1c970 (a degenerate primer that anneals within the BV1 open reading frame; F. M. Zerbini and R. L. Gil-bertson, unpublished data), cloned, and sequenced. The CR sequence of this DNA-B fragment was 96% identical to that of the DNA-A fragment, which establishes the presence of another bipartite geminivirus in this sample. This is the first report of ToMoV in Mexico. These results also suggest that at least two bipartite geminiviruses may infect tomatoes in the Yucatan Peninsula.

Dynamics of Bean Dwarf Mosaic Geminivirus Cell-to-Cell and Long-Distance Movement in Phaseolus vulgaris Revealed, Using the Green Fluorescent Protein

The cell-to-cell and long-distance movement of the bipartite geminivirus, bean dwarf mosaic (BDMV), in Phaseolus vulgaris plants was examined with the noninvasive reporter, the green fluorescent protein (GFP). A modified GFP gene (mGFP4) was inserted into the BDMV DNA-A component in place of the coat protein gene (BDMVA-mGFP4), and particle bombardment was used to introduce viral DNA into bean seedlings (radicle and hypocotyl tissues). Fluorescence analysis of GFP expressed from BDMVA-mGFP4 established that particle bombardment introduced viral DNA only into epidermal cells, and the requirement for the DNA-B-encoded proteins (BV1 and BC1) in the cell-to-cell movement of BDMVA-mGFP4. This GFP reporter system was used to follow the viral infection process from the seedling stage throughout the entire plant life cycle. In inoculated hypocotyls, BDMV moved from cell to cell through the cortex and showed a striking phloem tropism. Upon entry into phloem tissues, BDMV moved rapidly toward the root via the long-distance transport system, and toward the shoot apex by a combination of cell-to-cell and long-distance movement. Analysis of GFP distribution in systemically infected tissues revealed that BDMV was restricted to phloem cells in both roots and stems. In systemically infected primary and trifoliolate leaves, BDMV infected phloem cells associated with all vein orders (first through fifth), and the capacity of BDMV to exit from phloem tissue into nonphloem cells was correlated with the stage of plant development. Finally, fluorescence analysis of GFP in reproductive tissues established that BDMV infected flower, pod, and seed-coat tissues, but was excluded from the embryo.

Host Adaptation and Replication Properties of Two Bipartite Geminiviruses and Their Pseudorecombinants

To investigate factors involved in host adaptation and specificity of bipartite geminiviruses, the infectivity of bean dwarf mosaic (BDMV) and tomato mottle (ToMoV) geminiviruses and the BDMV/ToMoV pseudorecombinants [BDMV DNA-A + ToMoV DNA-B (BA+TB) and ToMoV DNA-A + BDMV DNA-B (TA+BB)] in Phaseolus vulgaris, Lycopersicon esculentum, Nicotiana benthamiana, and N. tabacum plants was determined. Additionally, replication of these viruses was examined in protoplasts prepared from N. tabacum BY2 and Xanthi-nc cells. In adapted hosts and the permissive experimental host, N. benthamiana, BDMV and ToMoV infected nearly 100% of inoculated plants, induced severe symptoms, and had high levels of both DNA components. In nonadapted hosts, BDMV and ToMoV infected approximately 40% of inoculated plants, induced no symptoms, and had reduced levels of both DNA components. For the pseudorecombinants, symptoms were observed only in TA+BB-infected N. benthamiana and P. vulgaris plants. In the other pseudorecombinant/host combinations, symptomless infections were detected and some plants were infected with the DNA-A component only. Symptom development and/or higher infection rates for the pseudorecombinants were correlated with the host-adapted DNA-B component, and pseudorecombinant-infected plants had reduced levels of DNA-B. Protoplast replication assays revealed inefficient DNA-B replication for the pseudorecombinants, and differences in viral replication properties in the two N. tabacum cell lines.

Leaf Curl Disease of Carica papaya from India May Be Caused by a Bipartite Geminivirus.

Papaya has considerable economic importance to agriculture in India. Papaya leaf curl disease was first reported in 1939 by Thomas and Krishnaswamy (3). This disease is of moderate incidence and widely distributed in India. Recent observations of papaya fields in India indicated that there has been a continued increase in the incidence of papaya leaf curl disease (as shown by symptoms), resulting in severe economic losses. The disease is characterized by downward curling and cupping of leaves followed by vein clearing and thickening. Enations develop in the form of frills on green veins. The affected leaves become leathery and brittle and the petioles become twisted in a zig-zag manner. Diseased plants may bear a few small fruits, which are distorted in shape and tend to fall prematurely. The disease could be transmitted by the whitefly Bemisia tabaci Genn. Therefore, possible involvement of a geminivirus was suspected. Three different cloned geminiviral DNAs, Indian tomato leaf curl virus (ITLCV) (2), tomato yellow leaf curl virus from Sardinia (TYLCV Sar), and tomato golden mosaic virus (TGMV), were used as probes (with radioactive labeling) to detect the presence of geminiviral DNA from infected papaya tissue in both slot-blot and Southern blot hybridization studies with high stringency washes. These DNA probes gave strong signals with DNA isolated from infected papaya tissue whereas they did not give any signals with DNA from healthy tissue. Further, successful polymerase chain reaction (PCR)-based amplification of fragments from both DNA-A and DNA-B components with geminivirus degenerate primers (1) was accomplished only from the DNA of infected papaya plants. The PCR-amplified DNA fragments gave positive signals in Southern blot hybridization with the three geminiviral DNA probes. These results suggest that the causal agent of papaya leaf curl disease is a bipartite geminivirus that may be provisionally called papaya leaf curl virus (PLCV).