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.     

Phylogenetic lineage of Tobacco leaf curl virus in Korea and estimation of recombination events implicated in their sequence variation

New strains of Tobacco leaf curl virus (TbLCV) were isolated from tomato plants in four different local communities of Korea, and hence were designated TbLCV-Kr. Phylogenetic analysis of the sequences of the whole genome and of individual ORFs of these viruses indicated that they are closely related to the Tobacco leaf curl Japan virus (TbLCJV) cluster, which includes Honeysuckle yellow vein virus (HYVV), Honeysuckle yellow vein mosaic virus (HYVMV), and TbLCJV isolates. Four putative recombination events were recognized within these virus sequences, suggesting that the sequence variations observed in these viruses may be attributable to intraspecific and interspecific recombination events involving some TbLCV-Kr isolates, Papaya leaf curl virus (PaLCV), and a local isolate of Tomato yellow leaf curl virus (TYLCV).     

A multiplex PCR method discriminating between the TYLCV and TYLCV-Mld clades of tomato yellow leaf curl virus

Tomato yellow leaf curl virus (TYLCV) is one of the causal agents of tomato yellow leaf curl disease (TYLCD) and can cause up to 100% yield losses in tomato fields. As TYLCV continues to spread, many isolates have been described in different parts of the world. Recently two closely related but distinct TYLCV clades, called TYLCV and TYLCV-Mld, have been identified. Isolates from those two clades differ mainly in the nucleotide sequences of their replication associated protein genes but do not display significantly different symptomatology. In order to improve monitoring of the rapidly expanding worldwide TYLCD epidemic, a multiplex polymerase chain reaction assay (mPCR) was developed. A set of three primers were designed to detect and characterize the TYLCV and TYLCV-Mld clade isolates. The specificity and sensitivity of the mPCR were validated on TYLCV infected tomato plants and Bemisia tabaci whiteflies. Being cheap, fast and highly sensitive this new diagnostic tool should greatly simplify efforts to trace the global spread of TYLCV.     

Pathogenicity and insect transmission of a begomovirus complex between tomato yellow leaf curl virus and Ageratum yellow vein betasatellite

Tomato yellow leaf curl virus (TYLCV) and Ageratum yellow vein betasatellite (AYVB) are members of the genus Begomovirus (family Geminiviridae). TYLCV and AYVB have been found in Japan over the last 15 years, and are associated with tomato leaf curl and the tomato yellow leaf curl diseases (TYLCD). AYVB is also associated with some monopartite begomoviruses. We have cloned both TYLCV and AYVB and demonstrated that TYLCV can trans-replicate with AYVB in Nicotiana benthamiana and tomato plants. A mixed infection of TYLCV and AYVB induced more severe symptoms of upward leaf curl, stunting, vein thickening, and swelling compared with TYLCV infection alone. The symptoms induced by infection of AYVB included a rise in abnormal cell proliferation, and pigmentation around leaf vein tissues. This is the first study to show that a complex of TYLCV and AYVB can be transmitted by vector insects among tomato plants. These results indicate that TYLCV possesses the potential to induce severe TYLCD by associating with AYVB.     

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 characterization of tomato-infecting begomoviruses in Thailand

Bipartite geminiviruses infecting tomatoes in Thailand were detected by polymerase chain reaction (PCR) using CPA5/CPA2 primers. Products derived from PCR-amplified full-length DNA-A and DNA-B of TYLCV collected from Chiang Mai, Nong Khai, and Sakon Nakhon were cloned and sequenced. DNA-A from Chiang Mai was 2747 nts long; Nong Khai, 2744 nts; and Sakon Nakhon, 2747 nts, and those of DNA-B from Chiang Mai were 2750 nts long; Nong Khai, 2749 nts; and Sakon Nakhon, 2749 nts. The genomes of these virus isolates were organized like those of other begomoviruses. The DNA-A had two ORFs in the virion sense and four ORFs in the complementary sense. The DNA-B had two ORFs in the virion sense and one ORF in the complementary sense. Nucleotide sequences of DNA-A of TYLCV from Chiang Mai, Nong Khai, and Sakon Nakhon were closely related to those of Tomato yellow leaf curl Thailand virus (TYLCTHV) and Tomato yellow leaf curl Thailand virus-[Myanmar] (TYLCTHV-[MM]) with nucleotide sequence identity ranging from 89% to 95%. Based on sequence comparisons and phylogenetic analyses, these three virus isolates studied were identified as new strains of TYLCTHV and named Tomato yellow leaf curl Thailand virus-Chiang Mai (TYLCTHV-[CM]The GenBank accession codes for DNA-A of TYLCTHV-[CM], -[NK], and -[SK] are , and , respectively. The GenBank accession codes for DNA-B of TYLCTHV-[CM], -[NK], and -[SK] are , , and , respectively.), Nong Khai (TYLCTHV-[NK] and Sakon Nakhon (TYLCTHV-[SK]).     

Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide

Tomato yellow leaf curl (TYLC) is one of the most devastating viral diseases of cultivated tomato (Lycopersicon esculentum) in tropical and subtropical regions worldwide, and losses of up to 100% are frequent. In many regions, TYLC is the main limiting factor in tomato production. The causal agents are a group of geminivirus species belonging to the genus Begomovirus of the family Geminiviridae, all of them named Tomato yellow leaf curl virus (TYLCV) (sensu lato). There has been almost 40 years of research on TYLCV epidemics and intensive research programmes have been conducted to find solutions to the severe problem caused by these viruses. This paper provides an overview of the most outstanding achievements in the research on the TYLCV complex that could lead to more effective control strategies.     

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     

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.     

Whiteflies (<Emphasis Type="BoldItalic">Bemisia tabaci</Emphasis>) issued from eggs bombarded with infectious DNA clones of <Emphasis Type="BoldItalic">Tomato yellow leaf curl virus</Emphasis> from Israel (TYLCV) are able to infect tomato plants

Summary.  We have reported previously that Tomato yellow leaf curl virus from Israel (TYLCV) penetrates the reproductive system of its vector, the whitefly Bemisia tabaci biotype B, and may be transmitted to progeny [9]. In order to mimic this phenomenon and to understand how TYLCV accompanies the development of the insect, we have bombarded B. tabaci eggs with an infectious DNA clone of TYLCV. After a linear full-length genomic copy of TYLCV DNA was delivered to eggs, the DpnI-sensitive DNA became circular and DpnI resistant. When a dimeric copy of TYLCV DNA was delivered to eggs, the viral DNA was detected in all the whitefly developmental stages. Adult insects that developed from the treated eggs were able to infect tomato test plants with variable frequency. Viral DNA was detected in the progeny of whiteflies that developed from eggs bombarded with TYLCV. Similarly, when insect eggs were bombarded with a dimeric copy of an infectious clone of the genome of Tomato yellow leaf curl virus from Sardinia, Italy (TYLCSV), adults that eclosed from the treated eggs were able to infect tomato test plants. Received February 19, 2001 Accepted September 20, 2001     

Sweet pepper confirmed as a reservoir host for tomato yellow leaf curl virus by both agro-inoculation and whitefly-mediated inoculation

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, has a single-stranded DNA genome. TYLCV can induce severe disease symptoms on tomato plants, but other hosts plants such as cucurbits and peppers are asymptomatic. A full-length DNA clone of a Korean TYLCV isolate was constructed by rolling-circle amplification from TYLCV-infected tomatoes in Korea. To assess relative susceptibility of sweet pepper varieties to TYLCV, 19 cultivars were inoculated with cloned TYLCV by agro-inoculation. All TYLCV-infected sweet peppers were asymptomatic, even though Southern hybridization and polymerase chain reaction analysis showed TYLCV genomic DNA accumulation in roots, stems, and newly produced shoots. Southern hybridization indicated that TYLCV replicated and moved systemically from agro-inoculated apical shoot tips to roots or newly produced shoots of sweet peppers. Whitefly-mediated inoculation experiments showed that TYLCV can be transmitted to tomatoes from TYLCV-infected sweet peppers. Taken together, these results indicate that sweet pepper can be a reservoir for TYLCV in nature.     

Biolistic inoculation of plants with tomato yellow leaf curl virus DNA

Tomato yellow leaf curl virus (TYLCV) full-length DNA was amplified by PCR and cloned into a bacterial plasmid. The cloned TYLCV DNA was excised from the plasmid, ligated and the resulting monomeric circular double-stranded TYLCV DNA was used to inoculate tomato (Solanum lycopersicom) and datura (Datura stramonium) plants by particle bombardment. The bombarded plants produced typical disease symptoms, similar to those produced following whitefly-mediated inoculation, albeit 5-7 days later than whitefly-inoculated plants. The success rate of inoculating tomato plants by particle bombardment averaged 37%, whereas with datura plants, it averaged 85%. With whitefly-mediated inoculation of TYLCV, the success rate of inoculation was also higher in datura plants than in tomato plants. Bombardment of datura plants with a linear form of TYLCV DNA also resulted in viral infection, with an inoculation success rate similar to that with the closed-circular TYLCV DNA. Bombarding datura plants with the bacterial plasmid containing the cloned TYLCV DNA did not result in viral infection, but bombardment with a bacterial plasmid containing a cloned dimer of TYLCV DNA yielded an infection rate of 50-100%. This is the first report of TYLCV inoculation of plants using particle bombardment of a cloned monomeric linear or closed-circular form of TYLCV double-stranded DNA.     

Recombinant-antibody-mediated resistance against <Emphasis Type="Italic">Tomato yellow leaf curl virus</Emphasis> in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Tomato yellow leaf curl virus (TYLCV) is a geminivirus species whose members cause severe crop losses in the tropics and subtropics. We report the expression of a single-chain variable fragment (scFv) antibody that protected Nicotiana benthamiana plants from a prevalent Iranian isolate of the virus (TYLCV-Ir). Two recombinant antibodies (scFv-ScRep1 and scFv-ScRep2) interacting with the multifunctional replication initiator protein (Rep) were obtained from phage display libraries and expressed in plants, both as stand-alone proteins and as N-terminal GFP fusions. Initial results indicated that both scFvs and both fusions accumulated to a detectable level in the cytosol and nucleus of plant cells. Transgenic plants challenged with TYLCV-Ir showed that the scFv-ScRep1, but more so the fusion proteins, were able to suppress TYLCV-Ir replication. These results show that expression of a scFv-ScRep1-GFP fusion protein can attenuate viral DNA replication and prevent the development of disease symptoms. The present article describes the first successful application of a recombinant antibody-mediated resistance approach against a plant DNA virus.     

Molecular identification and the complete nucleotide sequence of TYLCV isolate from Shanghai of China

Tomato yellow leaf curl virus (TYLCV) belongs to the genus Begomovirus, family Geminiviridae. It is transmitted by the whitefly Bemisia tabaci. A pair of primers was designed according to the specific sequence of the TYLCV gene and used in the PCR detection of the virus in the infected tissues of tomato grown in the Shanghai area of China. DNA was extracted from leaves, fruits, seeds, and roots of infected tomato plants separately. The results showed that the target fragment of about 570 bp could be isolated from the leaves, fruits, roots of infected tomato plants, but not from the seeds. Thus, the PCR-based detection technology for the Shanghai TYLCV (TYLCV-Sh10) has been established. Primers were then designed based on the sequence of the 570 bp fragment to obtain the complete DNA-A sequence of TYLCV-Sh10 by genome walking. Sequencing results indicated that the DNA-A sequence of TYLCV-Sh10 contained 2781 nt that included six ORFs. BLAST results showed that DNA-A of TYLCV-Sh10 had low homology with the characterized TYLCV in China (TYLCCNV) except TYLCV-ZJ8 (TYLCV isolated from Zhejiang China). But it was most closely related to TYLCV-USA (99.28% sequence identity). TYLCV-Sh10, TYLCV-ZJ8, TYLCV-USA, TYLCV-Mex, and TYLCV-Eg formed an independent branch by pairwise comparison and phylogenetic analysis. All these results strongly suggested that Sh10 was an isolate of America or Africa TYLCV.     

Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus

The functional properties of proteins [capsid protein (CP), V1, and C4] potentially involved with movement of the monopartite begomovirus, Tomato yellow leaf curl virus (TYLCV), were investigated using microinjection of Escherichia coli expressed proteins and transient expression of GFP fusion proteins. The TYLCV CP localized to the nucleus and nucleolus and acted as a nuclear shuttle, facilitating import and export of DNA. Thus, the CP serves as the functional homolog of the bipartite begomovirus BV1. The TYLCV V1 localized around the nucleus and at the cell periphery and colocalized with the endoplasmic reticulum, whereas C4 was localized to the cell periphery. Together, these patterns of localization were similar to that of the bipartite begomovirus BC1, known to mediate cell-to-cell movement. However, in contrast to BC1, V1 and C4, alone or in combination, had a limited capacity to move and mediate macromolecular trafficking through mesophyll or epidermal plasmodesmata. Immunolocalization and in situ PCR experiments, conducted with tomato plants at three stages of development, established that TYLCV infection was limited to phloem cells of shoot apical, leaf, stem, and floral tissues. Thus, the V1 and/or C4 may be analogs of the bipartite begomovirus BC1 that have evolved to mediate TYLCV movement within phloem tissue.     

A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus

Evidence for the involvement of a Bemisia tabaci GroEL homologue in the transmission of tomato yellow leaf curl geminivirus (TYLCV) is presented. A approximately 63-kDa protein was identified in B. tabaci whole-body extracts using an antiserum raised against aphid Buchnera GroEL. The GroEL homologue was immunolocalized to a coccoid-shaped whitefly endosymbiont. The 30 N-terminal amino acids of the whitefly GroEL homologue showed 80% homology with that from different aphid species and GroEL from Escherichia coli. Purified GroEL from B. tabaci exhibited ultrastructural similarities to that of the endosymbiont from aphids and E. coli. In vitro ligand assays showed that tomato yellow leaf curl virus (TYLCV) particles displayed a specific affinity for the B. tabaci 63-kDa GroEL homologue. Feeding whiteflies anti-Buchnera GroEL antiserum before the acquisition of virions reduced TYLCV transmission to tomato test plants by >80%. In the haemolymph of these whiteflies, TYLCV DNA was reduced to amounts below the threshold of detection by Southern blot hybridization. Active antibodies were recovered from the insect haemolymph suggesting that by complexing the GoEL homologue, the antibody disturbed interaction with TYLCV, leading to degradation of the virus. We propose that GroEL of B. tabaci protects the virus from destruction during its passage through the haemolymph.     

Transreplication of a Tomato yellow leaf curl Thailand virus DNA-B and replication of a DNAbeta component by Tomato leaf curl Vietnam virus and Tomato yellow leaf curl Vietnam virus

The genomes of two tomato-infecting begomoviruses from Vietnam were cloned and sequenced. A new variant of Tomato leaf curl Vietnam virus (ToLCVV) consisting of a DNA-A component and associated with a DNAbeta molecule as well as an additional begomovirus tentatively named Tomato yellow leaf curl Vietnam virus (TYLCVV) consisting also of a DNA-A component were identified. To verify if monopartite viruses occurring in Vietnam and Thailand are able to transreplicate the DNA-B component of Tomato yellow leaf curl Thailand virus-[Asian Institute of Technology] (TYLCTHV-[AIT]) infectivity assays were performed via agroinoculation and mechanically. As result, the DNA-B component of TYLCTHV-[AIT] was transreplicated by different DNA-A components of viruses from Vietnam and Thailand in Nicotiana benthamiana and Solanum lycopersicum. Moreover, the TYLCTHV-[AIT] DNA-B component facilitated the mechanical transmission of monopartite viruses by rub-inoculation as well as by particle bombardment in N. benthamiana and tomato plants. Finally, defective DNAs ranging from 735 to 1457 nucleotides were generated in N. benthamiana from those combinations containing TYLCTHV-[AIT] DNA-B component.     

V2 protein encoded by Tomato yellow leaf curl China virus is an RNA silencing suppressor

The V2 protein of Tomato yellow leaf curl China virus (TYLCCNV) was identified as an RNA silencing suppressor by Agrobacterium-mediated co-infiltration. The V2 protein could inhibit local RNA silencing, systemic RNA silencing of the green fluorescent protein (GFP) gene and the spread of a systemic GFP RNA silencing signal. However, the V2 could not interfere with the cell-to-cell spread of RNA silencing. Subcellular localization assay indicated that the V2 protein was distributed in the cytoplasm of Nicotiana benthamiana cells, and accumulated in irregular cytoplasmic bodies. The V2 bound 21 nt and 24 nt small interfering RNA (siRNA) duplexes and 24 nt single-stranded (ss)-siRNA but not 21 nt ss-siRNA in electrophoresis mobility shift assays. Expression of the V2 protein via the Potato virus X (PVX) vectors heterogenous system induced severe symptoms in N. benthamiana. In a yeast two-hybrid system, TYLCCNV V2 could interact with itself, but not with SlSGS3, which is known to been involved in RNA silencing pathway and to interact with a closely related Tomato yellow leaf curl virus (TYLCV) V2. These results indicate that TYLCCNV V2 is an RNA silencing suppressor, possibly through sequestering siRNA molecules.     

Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component

The genome of the tomato yellow leaf curl virus (TYLCV), a Bemisia tabaci-transmitted geminivirus, was cloned. All clones obtained were of one genomic molecule, analogous to DNA A of African cassava mosaic virus. Nucleotide sequence analysis of the TYLCV genome showed that it comprises 2787 nucleotides, encoding six open reading frames, two on the virion strand and four on the complementary strand. All of them have counterparts in other geminiviruses. Dimeric copies of the cloned viral genome were introduced into tomato plants by agroinoculation. Severe yellow leaf curl disease symptoms developed in all of them. Effective whitefly-mediated transmission of the virus from agroinoculated plants to test plants demonstrated that the cloned molecule carries all the information needed for virus replication, systemic infection, and transfer by whiteflies. Restriction and hybridization analyses of viral DNA forms in infected plants and viruliferous whiteflies did not support the presupposed existence of a second genomic component. This is the first report of a whitefly-transmitted geminivirus that possesses a single genomic molecule.