Tomato Golden Mosaic Virus Replication Research Articles

High-Frequency Reversion of Geminivirus Replication Protein Mutants during Infection

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.

A DNA structure is required for geminivirus replication origin function

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.

A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.

Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.

A DNA sequence required for geminivirus replication also mediates transcriptional regulation.

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, requires a single virus-encoded protein for DNA replication. We show that the TGMV replication protein, AL1, also acts during transcription to specifically repress the activity of its promoter. An earlier study established that AL1 binds to a 13-bp sequence (5'-GGTAGTAAGGTAG) that is essential for activity of the TGMV replication origin. Analysis of AL1 binding site mutants in transient expression assays demonstrated that the same site, which is located between the transcription start site and TATA box in the AL1 promoter, also mediates transcriptional repression. These experiments revealed that the repeated motifs in the AL1 binding site contribute differentially to repression, as has been observed previously for AL1-DNA binding and viral replication. Introduction of the AL1 binding site into the 35S promoter of the cauliflower mosaic virus was sufficient to confer AL1-mediated repression to the heterologous promoter. Analysis of a truncated AL1 promoter and of mutant AL1 proteins showed that repression does not require a replication-competent template or a replication-competent AL1 protein. Transient expression studies using two different Nicotiana cell lines revealed that, although the two lines replicate plasmids containing the TGMV origin similarly, they support very different levels of AL1-mediated repression. These results suggest that geminivirus transcriptional repression and replication may be independent processes.

TGMV replication protein AL1 preferentially binds to single-stranded DNA from the common region

The AL1 protein of tomato golden mosaic virus (TGMV) is encoded by the viral DNA and has been shown to be essential for viral DNA replication. We have over-expressed the AL1 open reading frame in E. coli and purified the protein from bacterial extracts to near homogeneity. Using various different techniques we have studied the interaction of the AL1 protein with DNA. The AL1 protein is able to bind to DNA containing the common region of the viral genome, which can be demonstrated by photochemical cross-linking. Binding is 4-fold stronger to single-stranded than to double-stranded DNA. Antibodies against the AL1 protein can be used to precipitate the protein-DNA complex. The binding to single- and double-stranded DNA is specifically to the common region since a DNA fragment unrelated to TGMV is not shifted in a gel retardation assay.

The behaviour of tomato golden mosaic virus DNA in cultured cells isolated from systemically infected tobacco leaves

When callus tissue was cultured from leaf pieces taken from a Nicotiana tabacum cv. Xanthi nc. plant systemically infected with tomato golden mosaic virus (TGMV), TGMV-specific DNA persisted for up to 6 months in culture. Analysis of TGMV-specific intracellular DNA forms indicated a decrease in double-stranded relative to single-stranded forms and an increase in sub-genomic relative to genomic single-stranded DNA species in the callus tissue compared to those in the original leaf explant. The implications of the results with regard to TGMV replication are discussed.

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

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

Independent encapsidation of tomato golden mosaic virus A component DNA in transgenic plants

Tomato golden mosaic virus (TGMV), a member of the geminivirus group, has a genome consisting of two DNA molecules designated the A and B components. Both are required for infectivity in healthy plants, although the former has been shown to replicate independently in transgenic plants containing tandem direct repeats of the A genome component. In the studies presented here, petunia plants transgenic for either both components (A×B hybrids) or the A component alone were examined for the presence of virus particles and encapsidated, single stranded viral DNA. The results of DNase protection experiments and direct observation of extracts from transgenic plants by electron microscopy indicate that single stranded TGMV DNA is in both cases packaged into paired particles identical to those obtained from virus-infected plants. DNase-treated virions isolated from A×B hybrid petunia are infectious when inoculated onto healthy Nicotiana benthamiana. Likewise, virions obtained from transgenic A petunia are infectious for plants transgenic for the B component.Our observations of TGMV replication in transgenic plants indicate that TGMV A DNA encodes all viral functions necessary for the replication and encapsidation of viral DNA. The possible role of the B component in TGMV replication is discussed.

Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants

Phenotypically normal petunia plants carrying chromosomal inserts of either the tomato golden mosaic virus (TGMV) A or the B component DNA, as single or tandem inserts, were obtained using an Agrobacterium tumefaciens Ti plasmid-based transformation system. Southern hybridization analysis revealed that the tandem, direct-repeat A plants contained free single and double stranded A component DNAs. No free B component DNA was detected in plants carrying tandem repeats of the B component. Progeny of self-fertilized plants appeared normal. In contrast, one-quarter of the progeny from tandem A by tandem B plant crosses showed chlorotic lesions on their leaves similar to virus symptoms. The significance of these results and the use of this method for the study of virus functions involved in TGMV replication and symptom production are discussed.