High-affinity Rep-binding is not required for the replication of a geminivirus DNA and its satellite

The 682-nt satellite DNA (sat-DNA) of Tomato leaf curl virus (TLCV) depends on the helper virus for its replication. In contrast to the strict specificity that exists in each geminivirus for its cognate replication associated protein (Rep), TLCV sat-DNA can utilize Rep encoded by distinct geminiviruses. We have used a combination of protein-binding assays and mutagenesis to show that repeat motifs in TLCV and sat-DNA are essential for Rep-binding in vitro. Surprisingly, mutants of TLCV and sat-DNA impaired in their ability to bind TLCV Rep in vitro were infectious in tomato. Thus, in contrast to other geminiviruses reported, TLCV and sat-DNA replication is independent of the high-affinity in vitro Rep binding. These results prompt a reassessment of the current model of geminivirus replication where Rep/DNA interaction is a highly specific step in the initiation of rolling circle replication.     

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.     

Characterization and transient replication of tomato leaf curl virus defective DNAs

Summary. Distinct subgenomic DNA species known as defective (df) DNA molecules were found in plants infected with tomato leaf curl virus (TLCV). Four df DNAs derived from TLCV Type and Darwin 1 strains were found to contain large deletions that disrupt all of the viral genes required for viral replication, encapsidation and spread. However, the viral origin of replication (ori), including the replication-associated protein (Rep) binding domains, was present in all four df DNAs. Co-agroinfection of leaf strips with tandem repeat constructs of the viral and df DNAs resulted in their replication in the presence of the respective TLCV strain. However, the df DNAs failed to move in whole plants when co-inoculated with TLCV. The df DNAs were shown to be associated with TLCV coat protein, which may indicate encapsidation. Mutational analysis showed the minimum sequence requirements for df DNA replication by TLCV to be the intergenic region containing the Rep-binding domains.     

Molecular characterization of a novel defective DNA isolated from tobacco tissues infected with tobacco leaf curl virus

Defective DNA of tobacco leaf curl virus (TLCV) was identified in TLCV-infected tobacco plants. The defective DNA was cloned and sequenced. The sequence showed it was about half the size of the TLCV DNA-A, and was derived from TLCV DNA-A by a large deletion. The defective DNA contained the intergenic region and part of the AC1 (Rep) gene of TLCV, and also novel open reading frames (ORFs). The immunotrapping tests showed the defective DNA was associated with geminate particles, suggesting it could be encapsidated in virus particles. It was transmitted, along with full-length DNA-A, to tobacco plants by grafting and whitefly (Bemisia tabaci).     

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.     

Replicative intermediates of Tomato leaf curl virus and its satellite DNAs

Several plant geminiviruses have been shown recently to utilize both rolling-circle replication (RCR) and recombination-dependent replication (RDR) strategies. A highly specific binding of the viral replication-associated protein (Rep) to its cognate DNA is essential for initiation of viral DNA replication and for the recognition of DNA components of the bipartite geminiviruses of the Begomovirus genus. We have extended the replication analysis to the monopartite Australian Tomato leaf curl virus (ToLCV), its Rep binding deficient mutants, and the satellite DNAs it supports. Analyses of viral DNA by two-dimensional agarose gel electrophoresis after fractionation by single-stranded (ss) DNA-selective cellulose chromatography revealed that DNA intermediates of ToLCV and its mutant were identical. Both RCR and RDR intermediates were identified. New ToLCV DNA forms were observed and characterized as subgenomic topoisomers, heterogeneous open circular double-stranded (ds) DNA, and degradation products. A 1350-nt DNA beta satellite associated with the unrelated Cotton leaf curl Multan virus (CLCuMV) was supported by ToLCV and produced intermediates of both RCR and RDR, suggesting that replication strategies of satellites are determined by the helper virus. Replicative intermediates of the 682 nt ToLCV satellite DNA could not be resolved; however, concatemers of up to octamer were detected, together with a field of hybridizing material suggestive of complementary strand replication on heterogeneous circular ssDNA templates.     

A single-tube PCR assay for detecting viruses and their recombinants that cause tomato yellow leaf curl disease in the Mediterranean basin

Tomato yellow leaf curl disease (TYLCD) is well known in Mediterranean countries, where it has been causing severe losses in tomato crops for decades. Until recently, two viruses (with several isolates) in the genus Begomovirus, family Geminiviridae, have been associated with the epidemics: Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV). However, recombinants between these, such as Tomato yellow leaf curl Malaga virus (TYLCMalV), are spreading, and new methods for detecting all viruses present in the region are needed. By considering all DNA sequences available of viruses causing TYLCD in the Mediterranean basin, a PCR/RFLP protocol was developed that amplifies the intergenic region in a multiplex reaction, followed by digestion with AclI (=Psp1406I) restriction enzyme. This procedure generates an easily recognizable pattern on gels, with DNA fragments of specific size for each virus species and each recombinant: 800 bp for TYLCSV, 410 bp for TYLCV, 570 bp for TYLCMalV and the other detected recombinants, 640 bp for hypothetical recombinants of different type. This new method gives, with a single reaction, an overview of the species present in the sample and will be useful for screening the causal agents of TYLCD, as well as in breeding programs for resistance.     

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.     

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.     

Founder effect, plant host, and recombination shape the emergent population of begomoviruses that cause the tomato yellow leaf curl disease in the Mediterranean basin

Tomato yellow leaf curl disease (TYLCD)-associated viruses present a highly structured population in the western Mediterranean basin, depending on host, geographical region and time. About 1,900 tomato and common bean samples were analyzed from which 111 isolates were characterized genetically based on a genome sequence that comprises coding and non-coding regions. Isolates of three distinct begomoviruses previously described were found (Tomato yellow leaf curl virus, TYLCV, Tomato yellow leaf curl Sardinia virus, TYLCSV, and Tomato yellow leaf curl Málaga virus, TYLCMalV), together with a novel recombinant virus. Mixed infections were detected in single plants, rationalizing the occurrence of recombinants. Except for TYLCV-type strain, single, undifferentiated subpopulations were present for each virus type, probably the result of founder effects. Limited genetic variation was observed in genomic regions, with selection against amino acid change in coding regions.     

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.     

TYLCSV DNA, but not infectivity, can be transovarially inherited by the progeny of the whitefly vector Bemisia tabaci (Gennadius)

The transovarial transmission of two species of begomovirus, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV), through generations of Bemisia tabaci of the B and Q biotypes has been investigated. Different life stages of the progeny of viruliferous female whiteflies have been analysed by PCR detection of viral DNA and infectivity tests. Our results indicate that TYLCSV DNA can be detected in eggs and nymphs, and to a lesser extent adults, of the first-generation progeny. Infectivity tests using a large number of adult progeny of the first, second, and third generation indicate that even when viral DNA is inherited, infectivity is not. For TYLCV, neither viral DNA nor infectivity were associated with the progeny of viruliferous female whiteflies. Because the inherited viral DNA is unable to give rise to infections, the transovarial transmission of TYLCSV DNA appears to have no epidemiological relevance.     

Begomovirus genetic diversity in the native plant reservoir Solanum nigrum: Evidence for the presence of a new virus species of recombinant nature

We examined the native plant host Solanum nigrum as reservoir of genetic diversity of begomoviruses that cause the tomato yellow leaf curl disease (TYLCD) emerging in southern Spain. Presence of isolates of all the species and strains found associated with TYLCD in this area was demonstrated. Mixed infections were common, which is a prerequisite for recombination to occur. In fact, presence of a novel recombinant begomovirus was demonstrated. Analysis of an infectious clone showed that it resulted from a genetic exchange between isolates of the ES strain of Tomato yellow leaf curl Sardinia virus and of the type strain of Tomato yellow leaf curl virus. The novel biological properties suggested that it is a step forward in the ecological adaptation to the invaded area. This recombinant represents an isolate of a new begomovirus species for which the name Tomato yellow leaf curl Axarquia virus is proposed. Spread into commercial tomatoes is shown.     

Differential pathogenicity among Tomato leaf curl Gujarat virus isolates from India

Tomato leaf curl Gujarat virus (ToLCGV) has been identified as one of the most destructive pathogens causing tomato leaf curl disease (ToLCD) in India. In the tomato growing regions of Dhanbad and Ramgarh, plants bearing severe symptoms of ToLCD such as leaf curling, leaf crinkling, yellowing and leaf rolling was observed in the farmer fields. The association of begomovirus in these samples was confirmed by PCR and the causal viruses were identified as the isolates of ToLCGV. However, association of cognate DNA B component could not be ascertained from these samples. Indeed, like other Old World begomoviruses, the present ToLCGV isolates were found to be associated with a particular betasatellite, Tomato yellow leaf curl Thailand betasatellite (TYLCTHB). Although DNA A of both ToLCGV isolates could alone infect tomato inducing systemic symptoms, the difference in virulence was observed. Co-inoculation of TYLCTHB reduced the incubation period without influencing the accumulation of helper virus DNA and hence, differential pathogenesis among ToLCGV isolates was governed by the helper component rather than betasatellite. ToLCGV infection with DNA B increases the accumulation of DNA A component of Dhanbad isolate but not of Ramgarh isolate. Results indicated that the begomovirus identified from Ramgarh sample was a mild strain of ToLCGV.     

A worldwide survey of tomato yellow leaf curl viruses

Summary.  The name tomato yellow leaf curl virus (TYLCV) has been given to several whitefly-transmitted geminiviruses affecting tomato cultures in many tropical and subtropical regions. Hybridization tests with two DNA probes derived from a cloned isolate of TYLCV from Israel (TYLCV-ISR) were used to assess the affinities of viruses in naturally infected tomato plants with yellow leaf curl or leaf curl symptoms from 25 countries. Probe A which included most of the intergenic region was expected to detect only isolates closely related to TYLCV-ISR, especially after high stringency washes. In contrast probe B, which included the full-length genome, was expected to detect a wide range of whitefly-transmitted geminiviruses. Tomato samples from six countries in the Middle East, from Cuba or the Dominican Republic proved to be closely related to TYLCV-ISR and probably were infected by strains of the same virus. Samples from Senegal and Cape Verde Islands were also related to the Middle Eastern virus. Samples from nine other countries in the western Mediterranean area, Africa, or South-East Asia were more distantly related and probably represent one or more additional geminivirus species. Samples from five countries in Africa, Central or South America gave hybridization signals with the full-length viral genome, only after low stringency wash, indicating that these samples were infected by remote viruses. These results were supported by DNA and protein sequence comparison, which indicate that tomato geminiviruses fall into three main clusters representing viruses from 1) the Mediterranean/Middle East/African region, 2) India, the Far East and Australia, and 3) the Americas. Within the first cluster, two sub-clusters of viruses from the western Mediterranean or from the Middle East/Caribbean Islands were distinguished. The incidence of tomato yellow leaf curl diseases has increased considerably between 1990 and 1996. Accepted January 28, 1997; Received April 19, 1996