Frequent occurrence of recombinants in mixed infections of tomato yellow leaf curl disease-associated begomoviruses.
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Begomoviruses are plant DNA viruses for which recombination plays a key role in driving evolution. However, little is known about how frequently begomovirus recombinants arise in mixed infected plants. To tackle this issue, co-infections of tomato with monopartite begomoviruses associated with the tomato yellow leaf curl disease, Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus, have been studied as a model system. The frequency of recombinant genotypes in the progeny populations was evaluated at several times post inoculation. Recombinants constituted a significant proportion of the viral population. Interestingly, not all regions of the genome contributed equally to genetic exchange. In addition to the intergenic region, a known hot spot for recombination, a second hot spot region was found. Implication of secondary structure sequence features in cross-over sites is suggested, which might favor discontinuous DNA replication with the replication complex switching between homologous regions of DNA templates. (+info)
A conserved binding site within the Tomato golden mosaic virus AL-1629 promoter is necessary for expression of viral genes important for pathogenesis.
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We have identified a nine base pair sequence in Tomato golden mosaic virus that is required for binding of nuclear proteins from tobacco and Arabidopsis to viral DNA. The sequence is located within the promoter for a 0.7 kb complementary sense mRNA (AL-1629). Mutation of the binding site results in a two- to six-fold reduction in the accumulation of AL-1629 mRNA, leading to reduced AL2 and AL3 gene expression. Viral sequences located immediately adjacent to the core binding site appear to influence AL2 and AL3 expression, but retain some binding affinity to a soluble host protein(s). The ability of a nuclear protein(s) to bind sequences within the AL-1629 promoter correlates with efficient viral DNA replication, as mutation of these sequences results in reduced viral DNA levels. Analysis of begomo- and curtoviruses indicates extensive conservation of this binding site, which suggests a common mechanism regulating expression of two viral genes involved in replication and suppression of host defense responses. (+info)
Genomic regions of tomato leaf curl virus DNA satellite required for replication and for satellite-mediated delivery of heterologous DNAs.
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Tomato leaf curl virus (TLCV) satellite DNA (sat-DNA) is a 682 nt, circular, single-stranded molecule that lacks an open reading frame (ORF) or an apparent promoter. It contains binding motifs for the TLCV replication-associated protein, but these are dispensable for replication. To identify the regions of the sat-DNA critical for replication, the entire sequence was scanned by deletion/replacement mutagenesis. Transient assays using Nicotiana benthamiana revealed that sequences within nt 296-35 (through nt 682) are essential for replication. Sequence deletions and replacements between nt 35 and 296 were tolerated but with a significant loss of infectivity, indicating that genome size strongly influences replication efficiency. Within the permissible region, inserts of 100-700 nt were retained in transient assays although with a slight reduction in replication. In addition, sat-DNA constructs containing short non-viral DNAs replicated and spread in tobacco plants, indicating their potential as gene-delivery vectors. (+info)
RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris).
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Bean golden mosaic virus (BGMV) is transmitted by the whitefly Bemisia tabaci in a persistent, circulative manner, causing the golden mosaic of common bean (Phaseolus vulgaris L.). The characteristic symptoms are yellow-green mosaic of leaves, stunted growth, or distorted pods. The disease is the largest constraint to bean production in Latin America and causes severe yield losses (40 to 100%). Here, we explored the concept of using an RNA interference construct to silence the sequence region of the AC1 viral gene and generate highly resistant transgenic common bean plants. Eighteen transgenic common bean lines were obtained with an intron-hairpin construction to induce post-transcriptional gene silencing against the AC1 gene. One line (named 5.1) presented high resistance (approximately 93% of the plants were free of symptoms) upon inoculation at high pressure (more than 300 viruliferous whiteflies per plant during the whole plant life cycle) and at a very early stage of plant development. Transgene-specific small interfering RNAs were detected in both inoculated and non-inoculated transgenic plants. A semiquantitative polymerase chain reaction analysis revealed the presence of viral DNA in transgenic plants exposed to viruliferous whiteflies for a period of 6 days. However, when insects were removed, no virus DNA could be detected after an additional period of 6 days. (+info)
Multiple introductions of the Old World begomovirus Tomato yellow leaf curl virus into the New World.
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A phylogenetic analysis of three genomic regions revealed that Tomato yellow leaf curl virus (TYLCV) from western North America is distinct from TYLCV isolated in eastern North America and the Caribbean. This analysis supports a second introduction of this Old World begomovirus into the New World, most likely from Asia. (+info)
A monopartite begomovirus-associated DNA beta satellite substitutes for the DNA B of a bipartite begomovirus to permit systemic infection.
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DNA beta is a circular single-stranded satellite DNA which co-infects with certain monopartite helper begomoviruses to cause economically important diseases, such as cotton leaf curl disease (CLCuD). DNA beta encodes a single protein, betaC1. Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus in which both DNA A and DNA B are required for systemic infection. Inoculation of tomato plants with ToLCNDV DNA A alone induced local but not systemic infection, whereas co-inoculation with DNA A and the DNA beta associated with CLCuD resulted in systemic infection. DNA beta containing a disrupted betaC1 open reading frame (ORF) did not mobilize DNA A systemically. Co-inoculation of plants with DNA A and a construct of the betaC1 ORF, under the control of the cauliflower mosaic virus 35S promoter, resulted in the systemic movement of DNA A. In inoculated tobacco and onion epidermal cells, betaC1 fused to GFP was localized at the cell periphery in association with punctate bodies, around and within the cell nucleus and with the endoplasmic reticulum. It is concluded that heterologous betaC1 protein can replace the movement function of the DNA B of a bipartite begomovirus. Evidence is also provided that tomato leaf curl virus-encoded C4 protein confers the same movement function to ToLCNDV DNA A. The intracellular distribution of betaC1 is consistent with the hypothesis that it has a role in transporting the DNA A from the nuclear site of replication to the plasmodesmatal exit sites of the infected cell. (+info)
Early and late gene expression in pepper huasteco yellow vein virus.
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Viral infections usually take place in an orderly manner and can be divided into at least two phases: an early and a late stage. In geminiviruses, plant viruses with a circular, single-stranded DNA genome, expression of viral genes involves complex regulation strategies that suggest the existence of a pattern of temporal gene expression. In this work, the transcription of pepper huasteco yellow vein virus (PHYVV) genes was studied. Green fluorescent protein replacements and RT-PCR analyses were used to monitor PHYVV gene expression chronologically in suspension cells and plant tissue. A model is proposed to describe the order of geminivirus gene expression, where the genes that encode Rep, TrAP and REn are expressed during an early stage of infection. The genes that encode the coat protein and the nuclear shuttle protein are expressed during the late stage of infection. (+info)
Detection and frequency of recombination in tomato-infecting begomoviruses of South and Southeast Asia.
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BACKGROUND: Tomato-infecting begomoviruses are widely distributed across the world and cause diseases of high economic impact on wide range of agriculturally important crops. Though recombination plays a pivotal role in diversification and evolution of these viruses, it is currently unknown whether there are differences in the number and quality of recombination events amongst different tomato-infecting begomovirus species. To examine this we sought to characterize the recombination events, estimate the frequency of recombination, and map recombination hotspots in tomato-infecting begomoviruses of South and Southeast Asia. RESULTS: Different methods used for recombination breakpoint analysis provided strong evidence for presence of recombination events in majority of the sequences analyzed. However, there was a clear evidence for absence or low Recombination events in viruses reported from North India. In addition, we provide evidence for non-random distribution of recombination events with the highest frequency of recombination being mapped in the portion of the N-terminal portion of Rep. CONCLUSION: The variable recombination observed in these viruses signified that all begomoviruses are not equally prone to recombination. Distribution of recombination hotspots was found to be reliant on the relatedness of the genomic region involved in the exchange. Overall the frequency of phylogenetic violations and number of recombination events decreased with increasing parental sequence diversity. These findings provide valuable new information for understanding the diversity and evolution of tomato-infecting begomoviruses in Asia. (+info)