Ambivalent effects of defective DNA in beet curly top virus-infected transgenic sugarbeet plants.

Beet curly top virus (BCTV) limits sugarbeet production considerably. Previous studies have shown that infections are associated with the generation of defective DNAs (D-DNA) which may attenuate symptoms. Transgenic sugarbeet lines were established carrying a partial direct repeat construct of D-DNA in order to examine whether they are useful as a means of generating tolerance against BCTV. Thirty four independent transgenic lines were challenged. Viral full-length and D-DNAs were monitored by polymerase chain reaction (PCR) or rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP). The differential accumulation of both DNA species was compared with symptom severity during the course of infection. RCA/RFLP allowed the discrimination of two D-DNA classes which were either derived from the transgenic construct (D(0)) or had been generated de novo (D(n)). The statistical analysis of the results showed that the presence of D(0)-DNA correlated with increased symptom severity, whereas D(n)-DNAs correlated with attenuated symptoms.

Biolistic infection of cassava using cloned components of Indian cassava mosaic virus.

Cassava mosaic disease (CMD) is a major constraint to cassava production in Africa and Asia. Of the two begomoviruses associated with CMD on the Indian subcontinent, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus, only the latter has been successfully reintroduced into cassava to resolve the aetiology of the disease. Here, we report the complete nucleotide sequence of an ICMV isolate from Maharashtra (ICMV-[Mah2]), central India. Biolistic inoculation of the cloned components produced a systemic infection and typical mosaic symptoms in cassava, thereby fulfilling Koch's postulates. The availability of infectious clones will provide a valuable tool to screen new cassava cultivars for disease resistance under defined conditions.

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.

Subcellular targeting of the coat protein of African cassava mosaic geminivirus.

The coat protein (CP) of geminiviruses is involved in a number of processes during the life cycle of the virus. The predominant function is encapsidation of single-stranded DNA and formation of the virus particle to protect viral DNA during transmission. The CP of monopartite geminiviruses is absolutely essential for virus movement, whereas CP mutants of bipartite geminiviruses are able to infect some host plants systemically, indicating an involvement of the CP in host specificity. During the life cycle of geminiviruses, the viral DNA enters the nucleus of the infected cell where virus replication, transcription, and encapsidation occur. For systemic infection, the virus moves cell-to-cell from the site of inoculation to vascular tissue and via phloem to other plant tissues. To move, viral DNA has to shuttle in and out of the nucleus and through plasmodesmata. Parts of the bipartite African cassava mosaic virus (ACMV) CP were fused with green fluorescent protein (GFP) or beta-glucuronidase (GUS). CP domains were identified that mediate both nuclear import and export, as well as targeting of CP-fusion proteins to the cell periphery. These results indicate that domains of the CP facilitate several aspects of geminivirus movement, including nuclear import and export and transport of the viral genome to the cell periphery.

Exchange of three amino acids in the coat protein results in efficient whitefly transmission of a nontransmissible Abutilon mosaic virus isolate.

Geminiviruses are transmitted in a circulative manner by whiteflies, leafhoppers, or treehoppers. The whitefly species Bemisia tabaci (Genn.) is the vector for members of the genus Begomovirus. The closely related bipartite Central American begomoviruses Abutilon mosaic virus (AbMV), Sida golden mosaic virus originating from Costa Rica (SiGMV-CR), and Sida golden mosaic virus originating from Honduras (SiGMV-Hoyv) were used to study transmission by their insect vector. The AbMV isolate is defective in transmission, whereas the two Sida-infecting viruses are readily transmitted by B. tabaci. These three viruses are able to form pseudorecombinant viruses by exchange of genomic components. The pseudorecombinant virus SiGMV-Hoyv A/AbMV B was transmissible, whereas the reciprocal pseudorecombinant virus AbMV A/SiGMV-Hoyv B was not transmitted, indicating that DNA B is not involved in the transmission defect. However, the uptake of the pseudorecombinant virus AbMV A/SiGMV-Hoyv B was much better than AbMV itself, indicating that DNA B or DNA B gene products enhance uptake of viral DNA. Exchange of AbMV coat protein with that of SiGMV-CR resulted in a transmissible chimeric AbMV. Mutagenesis of the AbMV coat protein showed that the exchange of two amino acids, at positions 124 and 149, was sufficient to obtain a whitefly-transmissible AbMV mutant. However, when amino acid 174 was altered in addition to amino acids 124 and 149 AbMV was readily transmitted by B. tabaci. From this we conclude that it is not a concise motif, such as the amino acid triplet, aspartate-alanine-glycine (DAG), involved in aphid transmission of potyviruses, that determines transmissibility of begomoviruses by B. tabaci. Instead it is the composition of the coat protein domain from amino acid 123 to 149, as a minimal transmission domain, with the contribution of amino acids 149 to 174 for efficient transmission.

Fulfilling Koch's postulates for Abutilon mosaic virus.

Cloned Abutilon mosaic geminivirus (AbMV) has been re-transmitted to Abutilon sellovianum (syn. striatum) plants by a two-step combined agroinfection/grafting method. The symptoms induced were indistinguishable from the characteristic mosaic of ornamental Abutilon plants. Therefore, we can exclude that a mixture of different AbMV variants is responsible for the striking variety of Abutilon leaf pattern elements. Analysis of the symptoms on consecutively infected leaves suggests that mosaic formation depends on routing and timing of super-imposed virus waves.

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/Ho(yv)), the pseudorecombinant viruses were infectious in various plant species. Three DNA B components (B(1), B(2), B(3)), different in a few nucleotides, were isolated from Sida rhombifolia naturally infected with SiGMV/Ho(yv). Only SiGMV/Ho(yv) DNA B(2) 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/Ho(yv) DNA B(1) component, indicating that impaired movement is involved in the deficiency of SiGMV/Ho(yv) DNA B(1) to form a pseudorecombinant virus with SiGMV/Co DNA A. Even after extensive mutation analysis of SiGMV/Ho(yv) DNA B(1) and B(2), we were unable to pinpoint differences in SiGMV/Ho(yv) DNA B(2) 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/Ho(yv) DNA B(1) and B(3) 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.

Molecular characterization of a new whitefly-transmissible bipartite geminivirus infecting tomato in Panama.

The nucleotide sequence of infectious clones of a tomato-infecting geminivirus from Panama [named tomato leaf curl virus (ToLCV-Pan) because of symptoms produced in infected tomato (plant stunting and mild leaf curling)] was determined. ToLCV-Pan has a bipartite genome (DNAs A and B) and computer analysis showed that the genome resembles that of other bipartite, whitefly-transmitted geminiviruses. DNA A (2584 nt) and B (2542 nt) have little sequence homology other than within the common region. ToLCV-Pan clones were introduced into Lycopersicon esculentum and infected plants developed the same symptoms as naturally infected tomatoes. Homology analysis of DNA A and B showed that ToLCV-Pan is most closely related to potato yellow mosaic virus (PYMV) from Venezuela. Pseudorecombination between ToLCV-Pan and PYMV did not give viable pseudorecombinant viruses. However, in some plants infected with the pseudorecombinant virus produced by ToLCV-Pan DNA A and PYMV DNA B, systemic movement of ToLCV-Pan DNA A was observed.

Recombination between viral DNA and the transgenic coat protein gene of African cassava mosaic geminivirus.

Nicotiana benthamiana was transformed with three different constructs (pCRA1, pCRA2 and pJC1) containing the coat protein coding sequence of African cassava mosaic virus (ACMV). Transformed plants were inoculated with a coat protein deletion mutant of ACMV that induces mild systemic symptoms in control plants. Several inoculated plants of transgenic lines CRA1/3, CRA1/4, CRA2/1 and CRA2/2 developed severe systemic symptoms typical of ACMV. DNA analysis revealed that, in these plants, recombination had occurred between the mutant viral DNA and the integrated construct DNA, resulting in the production of recombinant virus progeny with 'wild-type' characteristics. No reversion of mutant to 'wild-type' virus was observed in pJC1-transformed plants. Recombinant virus from several transgenic plants was analysed by PCR and parts of DNA A of virus progeny were cloned. Sequence analysis revealed that only a few nucleotides were changed from the published sequence.

Coat protein gene replacement results in whitefly transmission of an insect nontransmissible geminivirus isolate.

Geminiviruses are transmitted by whiteflies, leafhoppers, or treehoppers. The whitefly species Bemisia tabaci (Gennadius) is the most efficient vector of Subgroup III geminiviruses. An isolate of Abutilon mosaic virus (AbMV), a bipartite geminivirus, was not detectable in the DNA extract from insects by Southern blot analysis, nor was the isolate transmissible by the B-biotype of B. tabaci, although the virus DNA was amplified (by PCR) from some insects. In contrast, Sida golden mosaic virus (SiGMV-Co), a closely related geminivirus, was acquired and transmitted by B. tabaci to various host plants. The coat protein of AbMV was replaced with that of SiGMV-Co. The resulting chimeric AbMV was acquired and transmitted to various host plants by B. tabaci, indicating that the coat protein plays an essential role in the transmission process by B. tabaci.

Nucleotide sequence of a new bipartite geminivirus isolated from the common weed Sida rhombifolia in Costa Rica.

The nucleotide sequence of infectious clones of a geminivirus from Costa Rica that infects Sida rhombifolia was determined. Sida golden mosaic virus (SiGMV-Co) has a bipartite genome (DNAs A and B). Computer analysis showed that the bipartite genome of SiGMV-Co resembles that of other whitefly-transmitted geminiviruses. The DNA A (2605 nt) and DNA B (2587 nt) components have little sequence homology other than within the common region (CR). Analysis of DNAs A and B showed that SiGMV-Co is closely related to bean dwarf mosaic virus (BDMV). SiGMV-Co was introduced via agroinoculation into seven plant species, including tomato and bean.

Nucleotide sequence evidence for the occurrence of three distinct whitefly-transmitted, Sida-infecting bipartite geminiviruses in Central America.

The nucleotide sequences of two Sida-infecting geminiviruses from Honduras were determined. The symptoms of both viruses are identical in Sida rhombifolia but different in Nicotiana benthamiana. An additional symptom of one virus was yellow vein clearing on infected N. benthamiana leaves. Both Sida golden mosaic viruses (SiGMV-Ho and SiGMV-Ho(yv)) have bipartite genomes (DNAs A and B). From the SiGMV-Ho(yv)-infected S. rhombifolia plant two different DNA B molecules were isolated and cloned. They differ in length by 24 nucleotides [SiGMV-Ho(yv) B1 (2593 nt) and B2 (2569 nt)] and at eight nucleotide positions. Both proteins encoded by DNA B (BV1 and BC1) are affected by these substitutions. Computer analysis shows that the bipartite genomes resemble those of other whitefly-transmitted geminiviruses. From homology analyses we conclude that both viruses are closely related but distinct. Comparison with a Sida-infecting virus from Costa Rica (SiGMV-Co) showed that the two viruses from Honduras are more similar to each other than either of them are to SiGMV-Co. Exchange of SiGMV-Ho and SiGMV-Ho(yv) genomic components resulted in viable pseudorecombinant viruses. SiGMV-Ho DNA A was able to produce a viable pseudorecombinant with SiGMV-Co DNA B while the reciprocal exchange was not infectious in N. benthamiana. SiGMV-Ho(yv) DNA A and SiGMV-Co DNA B produced a viable pseudorecombinant virus whereas only pseudorecombination of SiGMV-Co DNA A with SiGMV-Ho(yv) DNA B2, and not with DNA B1, was infectious in N. benthamiana.

Beet curly top virus symptom amelioration in Nicotiana benthamiana transformed with a naturally occurring viral subgenomic DNA.

Beet curly top virus (BCTV) infection is associated with the de novo synthesis of a heterogeneous population of subgenomic viral DNAs. Nicotiana benthamiana plants transformed with a partial repeat of one such subgenomic DNA remain susceptible to infection but produce ameliorated symptoms when agroinoculated with BCTV. Transgenic plants contained from 10 to 30% of the amount of viral DNA detected in nontransformed control plants showing severe symptoms. Symptom amelioration is associated with the mobilization of subgenomic DNA from the integrated template and its amplification to approximately one third of the total amount of viral DNA. The amplification in transgenic plants of a specific subgenomic DNA rather than a heterogeneous population implies that mobilization from the integrated template frequently occurs during systemic infection, precluding the accumulation of other subgenomic DNA forms.

Transcriptional analysis of the virion-sense genes of the geminivirus beet curly top virus.

The genome of the geminivirus beet curly top virus (BCTV) consists of a single circular DNA containing overlapping open reading frames (ORFs) located on both the virion-sense and complementary-sense DNA strands. To investigate the expression of these ORFs, RNA extracted from infected Nicotiana benthamiana and Beta vulgaris has been examined for the presence of viral transcripts. An abundant 1.1-kb virion-sense polyadenylated RNA and four complementary-sense polyadenylated RNAs of 1.7, 1.5, 1.3, and 0.7 kb have been identified by northern blot hybridization, confirming the bidirectional transcription strategy implied by the arrangement of ORFs. We previously demonstrated that two overlapping virion-sense ORFs are involved in coat protein synthesis (ORF V1) and viral single-stranded DNA accumulation (ORF V2). Mutants of a third virion-sense ORF (ORF V3), located upstream and overlapping ORFs V1 and V2, retain the ability to replicate efficiently in N. benthamiana leaf discs but produce an asymptomatic infection in N. benthamiana and B. vulgaris at low frequency, associated with reduced levels of viral DNA compared to wild-type infection. Our data support the recent suggestion that ORF V3 participates in virus movement. The 1.1 kb virion-sense RNA comprises a population of overlapping transcripts with 5' termini suitably positioned for the expression of ORFs V1, V2, and V3. The overlapping arrangement of the transcripts and juxtaposition of putative regulatory elements could provide a means for the temporal control of virion-sense gene expression.

Specificity of bipartite geminivirus movement proteins.

Pseudorecombinants produced by exchanging genome components (DNAs A and B) of the geminiviruses African cassava mosaic virus (ACMV) and Indian cassava mosaic virus (ICMV), ACMV, and tomato golden mosaic virus (TGMV), and TGMV and abutilon mosaic virus (AbMV) are not infectious in their common host Nicotiana benthamiana. In each case, DNA A was unable to trans-replicate the heterogenomic DNA B component in a N. benthamiana leaf disc assay. The non-viability of the pseudorecombinants has been exploited to investigate the specificity of geminivirus movement proteins, encoded by DNA B, by co-inoculating N. benthamiana with both genome components of one virus and DNA A of a second virus. We demonstrate that ACMV can mediate the systemic movement of ICMV, TGMV and AbMV DNA A components. In reciprocal experiments, neither TGMV nor AbMV can mediate the systemic movement of ACMV DNA A although they can support the movement of each other's DNA A. The variation in movement protein specificity suggests evolutionary divergence of New and Old World geminiviruses. Co-inoculation of combinations of ACMV and ICMV genome components into discriminating hosts and comparison of their behavior in N. benthamiana and N. tabacum leaf disc assays suggests that the host range of ICMV, a subset of that of ACMV, is restricted by impaired viral DNA replication rather than the inability of the virus to spread in non-host backgrounds.

Detection and possible functions of African cassava mosaic virus DNA B gene products.

Polyclonal antisera raised against synthetic oligopeptides have been used to detect the DNA B gene products BV1 and BC1 of the geminivirus African cassava mosaic virus following SDS-PAGE fractionation of Nicotiana benthamiana extracts. BV1 antiserum detected a soluble protein of 29 kDa, consistent with the size predicted from sequence data. BC1 antiserum detected proteins of 37, 39, and 42 kDa in addition to variable, less abundant species, all of which are larger than the predicted size of 34 kDa. BC1 antiserum detected a single protein of 35-36 kDa following in vitro translation in reticulocyte lysate, suggesting that BC1 is post-translationally modified in plants. The nature of the modification was not resolved, although neither glycosylation nor association with nucleic acids is involved. In common with putative spread proteins of several other plants viruses, BC1 co-fractionated with the cell wall. The replication of both genomic components in N. tabacum protoplasts was unaffected by the introduction of frameshift mutations into BV1 and BC1 coding regions. In inoculated N. benthamiana leaves, however, the accumulation of a BV1 mutant was significantly reduced compared to the levels attained by co-inoculated, complementing BV1 and BC1 mutants. In contrast, the accumulation of a BC1 mutant was unaffected, although symptom induction in inoculated leaves and systemic infection occurred only in the presence of both BV1 and BC1. The results are consistent with a role for BV1 in localized cell-to-cell spread and for BC1, possibly together with BV1, in long-distance vascular spread of the virus.

Characterization of beet curly top virus subgenomic DNA localizes sequences required for replication.

Subgenomic viral DNA is accumulated in Nicotiana benthamiana and Beta vulgaris plants agroinoculated with the geminivirus beet curly top virus. The subgenomic DNA is more abundant in N. benthamiana and is distributed between two broad size groups in this host. Six unique examples, ranging in size from 887 to 1311 nucleotides, have been cloned from viral double-stranded DNA purified from N. benthamiana and analyzed by sequence determination. Deletions are distributed throughout most of the genome and only nucleotides 2946-410 are represented in all subgenomic DNAs. Comparison with a previously characterized subgenomic DNA suggests that cis-acting signals necessary for viral DNA replication are located in a predominantly intergenic region between nucleotides 2946-308.

Transcript mapping of Abutilon mosaic virus, a geminivirus.

The transcripts of the DNA of Abutilon mosaic virus, a geminivirus with a bipartite genome (DNA A and DNA B), were characterized by Northern blot hybridization, S1 nuclease assay, primer extension analysis, and sequencing of the 3' termini of cDNA clones. It was shown that transcription is bidirectional and that the transcripts are polyadenylated. Two overlapping transcripts of 1.6 and 0.7 kb were mapped to the complementary strand of DNA A and two of 1.3 and 1.2 kb to the complementary strand of DNA B. One transcript of 0.9 kb was mapped to the viral sense in DNA A and one of 1.0 kb to the viral sense in DNA B. The ends of complementary and viral mRNA overlapped in both genome parts in regions rich in polyadenylation signals.

African cassava mosaic virus DI DNA interferes with the replication of both genomic components.

Natural infections of the geminivirus African cassava mosaic virus (ACMV) are known to be associated with low levels of defective interfering (DI) DNAs. Recently it has been demonstrated that extrachromosomal copies of the DI DNA, mobilized and amplified from an integrated DI DNA dimer, can ameliorate ACMV symptoms in transformed Nicotiana benthamiana, providing a possible means for the control of cassava mosaic disease. To further understand the molecular basis of the interference phenomenon, we have compared the ability of ACMV and tomato golden mosaic virus (TGMV) genomic components to replicate in leaf discs derived from DI DNA-transformed and control plants. Results indicate that the ACMV DI DNA interferes with the replication of both genomic components of ACMV to a similar extent. TGMV DNA A replicates to normal levels in transformed leaf discs and plants because it is unable to mobilize and amplify ACMV DI DNA. Differences in the relative levels of ACMV genomic components in transformed leaf discs and plants are discussed in terms of DNA replication and the availability of the genomic components for spread throughout the plant.