Tomato Leaf Curl Geminivirus Research Articles

Variation of field symptoms and molecular diversity of the virus isolates associated with chilli leaf curl complex in different agroecological regions of Sri Lanka

The present study was conducted to detect and identify the virus isolates and to determine the molecular diversity associated with CLCC-infected chilli plants grown in a wide range of agroecological zones in Sri Lanka over two consecutive cultivation seasons. Randomly-selected chilli plants showing virus-like symptoms were collected from five experimental sites representing different agroecological zones and symptoms were recorded. DNA extracted from the above chilli leaf samples were subjected to PCR amplification using JS35 F and JS36 R primers, which are specific to Chili leaf curl Sri Lanka virus (ChiLCSLV). Amplified PCR products which targeted a region of DNA-A genome of begomovirus were subjected to DNA sequencing. Subsequent DNA homology search identified virus isolates associated with the CLCC-infected chilli plants which gave the highest homology to chilli leaf curl Salem virus-India, chilli leaf curl-Bhavansagar-India, Pepper Leaf curl Bangladesh virus, ChiLCSLV-isolate 14, ChiLCSLV-isolate 15 and Tomato leaf curl geminivirus, but having a variation of percentage identity. Phylogenetic analysis confirmed the genetic divergence of the CL-14 and CL-15 isolates, being them more genetically closer to chilli leaf curl-Bhavansagar-India and chilli leaf curl Salem virus- India, respectively. Eventhough single nucleotide differences were found in different clades of virus isolates, there was no strong relationship with clade separation and the location or season from which the samples were collected. Clade separation also did not show a relationship with a particular type of symptom. Findings of the study clearly revealed the presence of a diverse number of begomovirus isolates associated with the CLCC-infected plants and the genomic variations of them.

Molecular Detection and Partial Characterization of Tomato Yellow Leaf Curl Virus in Sri Lanka

Tomato Yellow Leaf Curl Virus (TYLCV) is an important plant virus on one of the economically most important vegetable crops; tomato (Lycopersicon esculentum Mill.). This had not been molecularly detected before, in Sri Lanka. TYLCV-GN-SL was isolated from apparently infected tomato plants using modified Cetyltrimethyl Ammonium Bromide (CTAB) method in Gannoruwa. Associated Begomoviruses were detected using Deng 541/Deng 540 and AV 494/AC 1048 primer pairs. TYLCV was detected for the first time in tomato in Sri Lanka using P1V/P4C, TYLCV specific primer pair. Nucleotide sequence of coat protein of isolated TYLCV-GN-SL proved that the Indian strain of ToLC virus was closely related to Tomato Leaf Curl Sri Lanka Virus (TLCV-SL: 97%) and Tomato leaf curl Geminivirus (TLCGV: 93%) through direct sequencing data. TLCV-SL was confirmed as TYLCV isolate. TYLCV was molecularly detected from major tomato growing districts like Badulla, Nuwara-Eliya, Kandy and Matale in Sri Lanka.

Tomato leaf curl geminivirus in Australia: occurrence, detection, sequence diversity and host range

The occurrence of whitefly transmitted geminiviruses in Australia was studied using a mixed DNA probe capable of detecting a range of distinct geminiviruses. The only geminivirus species detected was Tomato leaf curl virus (TLCV), which is spread across a vast geographical region of far‐northern coastal Australia, an area inhabited by the Australasian‐Oceania biotype of Bemisia tabaci. The newly introduced silverleaf whitefly, B. tabaci biotype B, forms high population densities in the eastern coastal region of Queensland and is currently located approximately 150 km from the nearest known TLCV‐infected area. The viral host range appeared to be narrow and of 58 species of crop plants and weeds inoculated using the B biotype, only 11 became infected with the virus, including five that did not show foliar symptoms. A DNA fragment of 694 nt, including the complete C4 open reading frame (ORF), the overlapping N‐terminal part of the C1 ORF and the viral iterons involved in replication, was amplified from 11 TLCV field isolates and sequenced. Sequence analysis revealed an overall sequence variation of up to 14% in this region, as well as the presence of distinct viral iterons.

Identification of sources of resistance in Lycopersicon species to Tomato leaf curl geminivirus (ToLCV) by agroinoculation

A total of 90 genotypes of Lycopersicon species were tested forresistance to the Tomato leaf curlgeminivirus (ToLCV) by agroinoculation andthe vector whitefly (Bemisia tabaciGenn.) inoculation techniques underinsect-proof glasshouse conditions. Therate of infection in the inoculated plantswas determined by detection of the viralDNA in individual plants by the nucleicacid spot hybridization (NASH). Of the 38cultivars and 11 breeding lines of L.esculentum Mill. tested, none was highlyresistant or resistant while three andseven were moderately resistant whenexposed to the cloned virus DNAs byagroinoculation and whitefly inoculationrespectively. On the other hand, among the38 commercial cultivars screened, 16(42.1%) were highly susceptible in vectorinoculations and 31 (81.6%) inagroinoculation. Among the exoticcollection (EC) accessions six were highlyresistant, eleven resistant to whiteflyinoculation and none was highly susceptiblein either of the two tests, indicating thepresence of resistance among the ECaccessions. A higher degree of resistancewas observed in other species of Lycopersicon. While only one accession ofL. cheesmanii Riley was tested, itcould not be infected by either of the twomethods. L. pimpinellifolium (Jusl.)Mill. genotype EC 251580 was similarlyresistant. In L. peruvianum (L.)Mill., five EC accessions could not beinfected by whitefly inoculation, withthree of these being resistant and twomoderately resistant in agroinoculation.This study demonstrates the importance ofthe agroinoculation technique in the virusresistance screening programs andidentifies several good sources ofresistance to the Tomato leaf curlvirus in Lycopersicon species.

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types

We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using β-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.

Tomato leaf curl geminivirus associated with cantaloupe yellow leaf disease in Thailand

Geminivirus associated with yellow leaf disease of cantaloupe plants was detected using polymerase chain reaction (PCR) with geminivirus-specific degenerate primers which anneal within the AC1 ORF (replication initiator protein gene) and the AV1 ORF (coat protein gene). A DNA fragment of 1.2 kbp was amplified, cloned and sequenced. The 32-base stem loop region was found in the amplified fragment. This included the conserved nonanucleotide sequence TAATATTAC present in all geminiviruses. The nucleotide sequence of the intergenic region (IR) was compared with 28 whitefly-transmitted geminiviruses. The geminivirus associated with yellow leaf disease of cantaloupe plants showed 96.2% sequence identity with DNA A of tomato leaf curl geminivirus from India (ToLCV-In2). These data suggest that cantaloupe yellow leaf disease was caused by ToLCV.

First Report of Sinaloa Tomato Leaf Curl Geminivirus in Costa Rica

In October 1998, geminivirus-like symptoms were widespread in tomato plantings near Turrialba, Costa Rica. Isolates from several fields were experimentally transmitted to tomato seedlings with whiteflies from a Bemisia tabaci (Genn.) colony maintained at CATIE, which resulted in interveinal chlorosis and leaf curling symptoms indistinguishable from those observed in the field. Total DNA was extracted from leaves of 16 of these experimentally inoculated plants and assayed by polymerase chain reaction (PCR) for the presence of begomovirus DNA with the degenerate primers AV324 and AC889 (2) to amplify the core region of the coat protein gene (core Cp). PCR yielded the expected size core Cp fragment (576 bp) from 16 of 16 samples. The core Cp fragments of six samples were cloned and sequenced. A comparison of the core Cp sequences with reference begomovirus sequences indicated all Costa Rican isolates were >95% identical to Sinaloa tomato leaf curl geminivirus reported in 1994 from Sinaloa, Mexico (STLCV-SINALOA). Virus identity was confirmed by multiple sequence alignments of the viral coat protein gene (Cp) and the common region (CR) sequences of A and B components (CR-A and CR-B), respectively, with analogous reference begomovirus sequences. Cp and CRs were obtained by PCR, and amplicons were cloned and sequenced as described (1). The Cp open reading frame (ORF; 756 nucleotides) (AF110515) identified within the A component amplicon shared 92.9% sequence identity with STLCV-SINALOA Cp (AF040635). The CR sequences of the A (AF1150516) and B (AF110517) components (163 nucleotides) shared 98.2% sequence identity with each other, suggesting that they were amplified from the cognate A and B components of the same virus. Further, the CR-A and CR-B components contained the same putative Rep binding site, TGGGGT-AA-TGGGGT, which was also identical to that of STLCV-SINALOA. The mean percent divergences between viral Cp and CR amplicons (n = 6+) ranged from 98 to 100%. Collectively, STLCV-like symptoms in tomato, >92% identity between viral Cp sequences, and identical CR iterons indicate that the Costa Rican tomato virus is STLCV, or a closely related strain. This is the first report of an STLCV-like begomovirus in tomato in Costa Rica (STLCV-CR).

A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement.

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.

Expression of the Tomato Leaf Curl Geminivirus C4 Gene Produces Viruslike Symptoms in Transgenic Plants

Transgenic tobacco and tomato plants expressing the C4 gene from tomato leaf curl geminivirus (TLCV), under the control of the cauliflower mosaic virus 35S promoter, developed virus-disease-like phenotypes. Plants transformed with a frame-shift version of the C4 gene construct appeared normal but produced levels of transcript similar to those of plants expressing the C4 gene. Thus, the abnormal plant phenotypes result from translation of the C4 gene. These results support the hypothesis that the C4 open reading frame of TLCV encodes a protein involved in the development of disease symptoms during viral infection.

Identification of the replication-associated protein binding domain within the intergenic region of tomato leaf curl geminivirus.

The geminiviral replication-associated protein (Rep) is the only viral protein required for viral DNA replication. Tomato leaf curl virus (TLCV) Rep was expressed in Escherichia coli as a histidine-tagged fusion protein and purified to homogeneity in non-denaturing form. The fusion protein was used in in vitro binding experiments to identify the Rep-binding elements within the origin of replication of TLCV. Electrophoretic mobility shift assays demonstrated that the Rep binds specifically to a 120 bp fragment within the TLCV intergenic region. Fine resolution of the binding regions within the 120 bp fragment, using DNase I footprinting, demonstrated two footprints covering the sequences GCAATTGGTGTCTCTCAA and TGAATCGGTGTCTGGGG containing a direct repeat of the motif GGTGTCT (underlined). Our results suggest that the repeated motif is involved in virus-specific Rep-binding, but may not constitute the entire binding element. This is the first demonstration of geminivirus sequence elements involved in Rep-binding by direct protein-DNA interaction assays.

A novel subviral agent associated with a geminivirus: the first report of a DNA satellite.

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem-loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication.

The Role of AV2 (“Precoat”) and Coat Protein in Viral Replication and Movement in Tomato Leaf Curl Geminivirus

We analyzed various mutants of tomato leaf curl virus-India to investigate the role of ORFs AV3, AV2, and coat protein (CP) in viral replication, movement, and symptom development. The results of these studies indicate that ORF AV3 does not encode a protein. Plants inoculated with infectious DNA which contained deletions in AV2 developed very mild symptoms and accumulated only low levels of both single-stranded (ss) and double-stranded (ds) viral DNA, whereas inoculated protoplasts accumulated both ss and dsDNA to wild-type levels, showing that AV2 is required for efficient viral movement. However, both plants and protoplasts inoculated with substitution, frameshift, and other similar mutations in AV2 accumulated low levels of viral DNA. The low levels of accumulation of DNA of these mutants were apparently not due to a defect in AV2 synthesis. Mutations in the CP caused a marked decrease in ssDNA accumulation in plants and protoplasts while increasing dsDNA accumulation in protoplasts. Mutations in both AV2 and CP behaved like AV2 mutants in plants and like CP mutants in protoplasts. The results demonstrated that multiple functions provided by AV2, BV1, and BC1 are essential for viral movement, and that changes in A-component virion-sense mRNA structure or translation affect viral replication.

Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?

Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.

New Potato Spindle Tuber Viroid and Tomato Leaf Curl Geminivirus Strains from a WildSolanumsp.

Three distinct viruslike agents were isolated from a wild, symptomless Solanum sp. suspected of being a natural reservoir of tomato leaf curl geminivirus. Two of these contained circular DNA genomes of approximately 2.7 kb and were identified as geminiviruses. Restriction analysis and partial sequencing of viral DNA indicated that the two geminiviruses are related to, but distinct from, the Australian strain of tomato leaf curl virus. These viruses were not mechanically transmissible; however, a mechanically transmissible disease agent was also present, which caused stunting of infected tomato plants followed by shoot elongation and hairlessness. Analysis of nucleic acids from sap-inoculated tomato plants revealed the presence of a circular RNA, which was isolated and shown to be the causal agent of the transmissible disease. The complete nucleotide sequence of this 356-nucleotide RNA revealed that it was a sequence variant of potato spindle tuber viroid.

Nucleotide sequences from tomato leaf curl viruses from different countries: evidence for three geographically separate branches in evolution of the coat protein of whitefly-transmitted geminiviruses

The coat protein (CP) gene-containing circular DNA molecule of an isolate of tomato leaf curl geminivirus (ITmLCV; 2749 nt) obtained from southern India, and the CP genes of tomato yellow leaf curl geminivirus isolates from Nigeria and two regions of Saudia Arabia were sequenced. ITmLCV DNA had the same arrangement of ORFs, and the same pattern of repeats in the large intergenic region as is found in in DNA-A of other whitefly-transmitted geminivirus (WTGs) from the Old World. However, the sequence of ITmLCV DNA and the sequences of its predicted translation products differed substantially from those of other WTGs, including one isolate obtained from a tomato plant in northern India. Comparison of the four CP sequences deduced here with those of 18 WTGs previously studied indicated that their relationships can be represented by a tree with three branches that are unrelated to plant host species but which contains viruses from the Americas, Africa to the Middle East, and Asia to Australia, respectively. It is suggested that WTG CP evolution has proceeded along different paths in these three main regions, and that WTGs have adapted freely to new hosts in each region. Indeed, the virus isolates causing similar diseases of tomato plants in the different continents are, with few exceptions, not closely related and warrant recognition as separate species.

Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity

Genomes of two isolates of tomato leaf curl geminivirus from India (ToLCV-India) have been sequenced. ToLCV-India contains A and B components, both of which are required for systemic movement and symptom development. The two isolates have 94% sequence identify but one isolate gave mild symptoms in Nicotiana benthamiana and tomato. The genome organization of ToLCV-India is similar to other whitefly-transmitted geminiviruses (WTGs) with bipartite genomes. However, it contains an additional ORF, AV3, that has not been reported for other WTGs. Its coat protein (CP) sequence is highly homologous to that of Indian cassava mosaic virus (90%). Two mutations that truncated the CP after amino acids 65 or 172 did not affect systemic movement and symptom development in either N. benthamiana or tomato. However, the symptoms caused by mutant viruses were different from those in plants infected with unmodified viruses, and plants infected with the mutants had markedly reduced amounts of single-stranded viral DNA. Comparison of sequences and other biological features of ToLCV-India with other geminiviruses showed that ToLCV-India is a distinct virus and is related to the WTGs from the Old World. It is similar to African cassava mosaic virus in its requirement for B component and dispensability of coat protein for symptom development, unlike other geminiviruses that infect tomato in the Old World. It is proposed that ToLCV-India evolved more recently as compared to other geminiviruses that infect tomato in the Old World.

Mapping of the Polycistronic RNAs of Tomato Leaf Curl Geminivirus

Four major transcripts from infected tomato were identified and mapped onto the monopartite genome of the geminivirus tomato leaf curl virus (TLCV). The C1, C2, and C3 ORFs are spanned by one transcript and a second internal RNA covered C2 and C3. Both these RNAs have their counterparts in the DNA A components of the bipartite subgroup of geminiviruses. The 5′ ends of the virion-sense RNAs map either side of the first in-frame AUG of the V1 ORF, The 3′ ends of the virion-sense RNAs are coterminal and overlap with the 3′ ends of the complementary-sense RNAs. All of the RNAs have transcription regulatory sequences close to their mapped termini and the presence of overlapping transcripts suggests that temporal regulation of their synthesis may occur. The translation of these polycistronic RNAs is discussed in the light of the RNA mapping data.

Nucleotide sequence and genome organization of tomato leaf curl geminivirus.

The genome of tomato leaf curl virus (TLCV) from Australia was cloned and its complete nucleotide sequence determined. It is a single circular ssDNA of 2766 nucleotides containing the consensus nonanucleotide sequence present in all geminiviruses. It has six open reading frames with an organization resembling that of certain other dicotyledonous plant-infecting monopartite geminiviruses, i.e. tomato yellow leaf curl and beet curly top viruses. The regulatory sequences present indicate a bidirectional mode of transcription. A dimeric TLCV DNA clone was constructed in a binary vector and used to agroinoculate three different host species. Typical virus infections were produced, confirming that the single DNA component is sufficient for infectivity.

Particle purification, properties and epitope variability of Indian tomato leaf curl geminivirus

SUMMARYA whitefly‐transmissible stock isolate of Indian tomato leaf curl geminivirus (ITmLCV) was cultured in graft‐inoculated tomato plants and its particles purified from chloroform‐clarified extracts in citrate buffer by precipitation with 70 g/litre polyethylene glycol, ultracentrifugation and sucrose density gradient centrifugation. Contaminating helical filaments were eliminated by banding in caesium sulphate gradients. ITmLCV particles had the shape typical for geminiviruses, measured c. 30 × 20 nm and contained a single major protein of estimated mol. wt c. 32 000. They reacted in immunosorbent electron microscopy with antisera to four other whitefly‐transmitted geminiviruses. ITmLCV reacted with one out of 17 monoclonal antibodies specific for different epitopes in the particle protein of African cassava mosaic geminivirus and five or six out of 10 monoclonal antibodies to the particle protein of Indian cassava mosaic geminivirus. Virus isolates from tomato at nine locations in Karnataka State showed only slight differences in epitope profile, and isolates from four weed species in tomato fields were similar or identical to those from tomato.