Predominance of tomato leaf curl Gujarat virus as a monopartite begomovirus: association with tomato yellow leaf curl Thailand betasatellite

Tomato leaf curl is a serious malady in the state of Maharashtra, India, causing nearly 100 % yield loss. An extensive survey was done in the affected fields of tomato in the year 2008, and members of three species of begomoviruses were identified as causing the disease. More than 60 % of the samples from diseased plants were infected with tomato leaf curl Gujarat virus (ToLCGuV). Isolates collected from these fields differed from the Varanasi isolate of ToLCGuV in not having a DNA B component. Instead, they were like typical Old World monopartite begomoviruses in that they were associated with only one betasatellite, tomato yellow leaf curl Thailand betasatellite (TYLCTHB). ToLCGuV alone is readily infectious, expressing systemic symptoms in Nicotiana benthamiana and tomato. Co-inoculation of ToLCGuV with TYLCTHB, increased symptom severity and reduced the incubation time required for symptom expression. ToLCGuV successfully interacted with heterologous DNA B component of ToLCNDV [IN:Pun:JID:08], and co-inoculation of these two resulted in yellow mottling symptoms that were typical of DNA B.     

Tomato leaf curl Joydebpur virus: a monopartite begomovirus causing severe leaf curl in tomato in West Bengal

A begomovirus was isolated from tomato plants showing leaf curl and stunting symptoms in farmers’ fields near the district of Kalyani, West Bengal, India. Viral genomic components amplified by rolling-circle amplification were cloned and sequenced. The genome organization of this virus was found to be similar to those of Old World monopartite begomovirus, with DNA A and a betasatellite component. Neither alphasatellite nor DNA B component was detected. The begomovirus showed highest sequence identity of 93.6% to tomato leaf curl Joydebpur virus (ToLCJoV-[IN:Kal:Chi:06]) and was thus identified to be an isolate of ToLCJoV. The betasatellite isolated from these samples was identified as tomato leaf curl Joydebpur betasatellite. ToLCJoV-[IN:Kal:Tom:08] alone induced severe symptoms in Solanum lycopersicum, N. benthamiana and N. glutinosa plants, and its severity was enhanced when co-inoculated with the cognate betasatellite. ToLCJoV-[IN:Kal:Tom:08] trans-replicated four more non-cognate betasatellites and induced severe symptoms in N. benthamiana and tomato. Since DNA A replicated efficiently and caused systemic symptom expression, it is hypothesized that ToLCJoV is essentially a monopartite virus, which could have acquired a betasatellite from an unknown source.     

Stachytarpheta leaf curl virus is a novel monopartite begomovirus species

Summary. Begomovirus isolates were obtained from Stachytarpheta jamaicensis plants showing leaf curl and chlorosis symptoms collected in the Hainan province of China. The complete sequences of isolates Hn5-4, Hn6-1, Hn30 and Hn34 were determined to be 2748, 2751, 2748 and 2748 nucleotides long, respectively. The complete sequences of the four isolates share more than 94.9% nucleotide sequence identity, but all of them have less than 86% nucleotide sequence identity with other reported begomoviruses. The molecular data show that Hn5-4, Hn6-1, Hn30 and Hn34 are isolates of a distinct begomovirus species, for which the name Stachytarpheta leaf curl virus (StaLCV) is proposed. PCR and Southern blot analyses demonstrate that all the collected field samples are not associated with DNAβ or DNA-B components. An infectious clone of StaLCV isolate Hn5-4 was constructed, and could efficiently infect Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Lycopersicon esculentum and Petunia hybrida plants, inducing upward leaf roll and vein swelling symptoms. In addition, we illustrate that StaLCV can functionally interact with distinct DNAβ molecules in plants. These authors contributed equally to this work.     

Suppressor of RNA silencing encoded by Beet yellows virus

Using an Agrobacterium-mediated transient assay, we screened the 15.5-kb genome of the Beet yellows virus for proteins with RNA silencing suppressor activity. Among eight proteins tested, only a 21-kDa protein (p21) was able to suppress double-stranded (ds) RNA-induced silencing of the green fluorescent protein (GFP) mRNA. Restoration of GFP expression by p21 under these conditions had no apparent effect on accumulation of the small interfering RNAs. In addition, p21 elevated the transient expression level of the GFP mRNA in the absence of dsRNA inducer. Similar activities were detected using homologs of p21 encoded by other members of the genus Closterovirus. Computer analysis indicated that p21-like proteins constitute a novel protein family that is unrelated to other recognized suppressors of RNA silencing. Examination of the subcellular distribution in BYV-infected plants revealed that p21 is partitioned between soluble cytoplasmic form and proteinaceous inclusion bodies at the cell periphery.     

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.     

Host range and genetic diversity of croton yellow vein mosaic virus, a weed-infecting monopartite begomovirus causing leaf curl disease in tomato

Croton yellow vein mosaic virus (CYVMV) is a widely occurring begomovirus in Croton bonplandianum, a common weed in the Indian subcontinent. In this study, CYVMV (genus Begomovirus, family Geminiviridae) was transmitted by whiteflies (Bemisia tabaci) to as many as 35 plant species belonging to 11 families, including many vegetables, tobacco varieties, ornamentals and weeds. CYVMV produced bright yellow vein symptoms in croton, whereas in all the other host species, the virus produced leaf curl symptoms. CYVMV produced leaf curl in 13 tobacco species and 22 cultivars of Nicotiana tabacum and resembled tobacco leaf curl virus (TobLCV) in host reactions. However, CYVMV was distinguished from TobLCV in four differential hosts, Ageratum conyzoides, C. bonplandianum, Euphorbia geniculata and Sonchus bracyotis. The complete genome sequences of four isolates originating from northern, eastern and southern India revealed that a single species of DNA-A and a betasatellite, croton yellow vein mosaic betasatellite (CroYVMB) were associated with the yellow vein mosaic disease of croton. The sequence identity among the isolates of CYVMV DNA-A and CroYVMB occurring in diverse plant species was 91.8-97.9 % and 83.3-100 %, respectively. The CYVMV DNA-A and CroYVMB generated through rolling-circle amplification of the cloned DNAs produced typical symptoms of yellow vein mosaic and leaf curling in croton and tomato, respectively. The progeny virus from both the croton and tomato plants was transmitted successfully by B. tabaci. The present study establishes the etiology of yellow vein mosaic disease of C. bonplandianum and provides molecular evidence that a weed-infecting monopartite begomovirus causes leaf curl in tomato.     

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.     

Characterization and distribution of tomato yellow margin leaf curl virus, a begomovirus from Venezuela

A begomovirus causing mottling and leaf deformation in tomato from the State of Mérida was cloned and sequenced. The virus has a bipartite genome comprised of a DNA-A (2,572 nucleotides) and a DNA-B (2,543 nucleotides) with a genome organization typical of New World begomoviruses. Both components share a common region of 115 nucleotides with 98 % sequence identity. Phylogenetic analysis indicated that while no virus sequences were closely related, the A component was distantly related to those of two other tomato-infecting viruses, tomato leaf deformation virus and Merremia mosaic virus; and the DNA-B, to those of pepper huasteco yellow vein virus and Rhynchosia golden mosaic Yucatan virus. The DNA-A and DNA-B sequences were submitted to GenBank (accession no. AY508993 and AY508994, respectively) and later accepted by the International Committee on Taxonomy of Viruses as the genome of a member of a unique virus species with the name Tomato yellow margin leaf curl virus (TYMLCV). Tomato (Solanum lycopersicum L. ‘Fl. Lanai’) plants inoculated with cloned TYMLCV DNA-A and DNA-B became systemically infected and showed chlorotic margins and leaf curling. The distribution of TYMLCV in tomato-producing states in Venezuela was determined by nucleic acid spot hybridization analysis of 334 tomato leaf samples collected from ten states using a TYMLCV-specific probe and confirmed by PCR and sequencing of the PCR fragment. TYMLCV was detected in samples from the states of Aragua, Guárico, and Mérida, suggesting that TYMLCV is widely distributed in Venezuela.     

Recent evolution of a novel begomovirus causing tomato leaf curl disease in the Al-Batinah region of Oman

For last two decades, begomoviruses (family Geminiviridae) have been a major constraint for tomato production in Oman, particularly in the Al-Batinah region, the major agricultural area of Oman. Farms in the Al-Batinah region were surveyed during January-March and November-December in 2012 and January-February in 2013. Leaf samples of tomato plants showing typical leaf curl disease symptoms were collected and analyzed for begomoviruses. Out of fifteen begomovirus clones sequenced, seven were shown to be tomato yellow leaf curl virus strain Oman (TYLCV-OM); three, chili leaf curl virus strain Oman (ChLCV-OM); and one, tomato leaf curl Oman virus (ToLCOMV) – viruses that have previously been shown to occur in Oman. Four sequences were shown to have relatively low percent identity values to known begomoviruses, with the highest (86 %) to isolates of pepper leaf curl Lahore virus, indicating that these should be included in a new species, for which the name “Tomato leaf curl Al Batinah virus” (ToLCABV) is proposed. Although the betasatellite tomato leaf curl betasatellite (ToLCB; 7 full-length sequences isolated) was identified with some isolates of ChLCV-OM, TYLCV-OM and ToLCOMV, it was not identified in association with any of the ToLCABV isolates. Analysis of the sequences of the TYLCV-OM and ToLCOMV isolates characterized here did not show them to differ significantly from previously characterized isolates of these viruses. The three isolates of ChLCV-OM characterized were shown to have a recombination pattern distinct from earlier characterized isolates. ToLCABV was shown to have resulted from recombination between ChLCV-OM and ToLCOMV. A clone of ToLCABV was infectious by Agrobacterium-mediated inoculation to Nicotiana benthamiana and tomato, inducing symptoms typical of those seen in tomato in the field. Additionally, ToLCABV was shown to be able to interact in planta with ToLCB, resulting in a change in symptom phenotype, although the betasatellite did not appear to affect viral DNA levels.     

Tomato yellow leaf curl Thailand virus-[Y72] from Yunnan is a monopartite begomovirus associated with DNAβ

Previous studies have shown that isolates of tomato yellow leaf curl Thailand virus (TYLCTHV) originated from Thailand are bipartite begomoviruses, while all the seven TYLCTHV isolates found in China are associated with DNAβ molecules. In this study, infectious clones of TYLCTHV isolate Y72 (TYLCTHV-[Y72]) and its DNAβ were constructed to verify the bipartite or monopartite nature of TYLCTHV. Agroinoculation showed that TYLCTHV-[Y72] alone was able to induce significant symptoms in Nicotiana benthamiana, Nicotiana glutinosa, and Solanum lycopersicum plants, but co-inoculation with its associated satellite DNAβ produced more severe symptoms, which is similar to tobacco curly shoot virus. Southern blot results showed that TYLCTHV DNAβ could increase the virus accumulation in systemically infected tissues. Thus, TYLCTHV-[Y72] is a monopartite begomovirus, which may represent an evolutionary intermediate between the begomoviruses requiring DNAβ and begomoviruses dispensable of DNAβ. Wei Guo and Xiuling Yang contributed equally to this paper.     

Evidence for interspecific-recombination for three monopartite begomoviral genomes associated with the tomato leaf curl disease from central Sudan

Summary. Two distinct viral genotypes were identified in the same tomato plant collected from Gezira, Sudan and are provisionally designated Tomato leaf curl Sudan virus (ToLCSDV-Gez) and Tomato yellow leaf curl virus-Sudan (TYLCV-SD). A third genotype was identified in tomato samples collected in Shambat, Sudan (ToLCSDV-Sha). The ToLCSDV-Gez and ToLCSDV-Sha isolates were 90% identical, TYLCV-SD from Gezira shared 93% identity with TYLCV-Mld. Recombination analyses identified two fragments in the ToLCSDV-Gez and TYLCV-SD genomes, providing evidence that these two genomes had undergone intermolecular recombination. A half unit size (737nt) single-stranded satellite DNA was associated with ToLCSDV-Gez and TYLCV-SD.     

A begomovirus associated with ageratum yellow vein disease in Indonesia: evidence for natural recombination between tomato leaf curl Java virus and Ageratum yellow vein virus-[Java]

Summary. A begomovirus (2747 nucleotides) and a satellite DNAβ component (1360 nucleotides) have been isolated from Ageratum conyzoides L. plants with yellow vein symptoms growing in Java, Indonesia. The begomovirus is most closely related to Tomato leaf curl Java virus (ToLCJV) (91 and 98% in the total nucleotide and coat protein amino acid sequences, respectively), although the products of ORFs C1 and C4 are more closely related to those of Ageratum yellow vein virus-[Java] (91 and 95% identity, respectively). For this reason, the begomovirus it is considered to be a strain of ToLCJV and is referred to as ToLCJV-Ageratum. The virus probably derives from a recombination event in which nucleotides 2389–2692 of ToLCJV have been replaced with the corresponding region of the AYVV-[Java] genome, which includes the 5′ part of the intergenic region and the C1 and C4 ORFs. Infection of A. conyzoides with ToLCJV-Ageratum alone produced no symptoms, but co-infection with DNAβ induced yellow vein symptoms. Symptoms induced in Nicotiana benthamiana by ToLCJV-Ageratum, ToLCJV and AYVV-[Java] are consistent with the exchange of pathogenicity determinant ORF C4 during recombination.     

Identification of a disease complex involving a novel monopartite begomovirus with beta- and alphasatellites associated with okra leaf curl disease in Oman

Okra leaf curl disease (OLCD) is an important viral disease of okra in tropical and subtropical areas. The disease is caused by begomovirus-satellite complexes. A begomovirus and associated betasatellite and alphasatellite were identified in symptomatic okra plants from Barka, in the Al-Batinah region of Oman. Analysis of the begomovirus sequences showed them to represent a new begomovirus most closely related to cotton leaf curl Gezira virus (CLCuGeV), a begomovirus of African origin. The sequences showed less than 85 % nucleotide sequence identity to CLCuGeV isolates. The name okra leaf curl Oman virus (OLCOMV) is proposed for the new virus. Further analysis revealed that the OLCOMV is a recombinant begomovirus that evolved by the recombination of CLCuGeV isolates with tomato yellow leaf curl virus-Oman (TYLCV-OM). An alpha- and a betasatellite were also identified from the same plant sample, which were also unique when compared to sequences available in the databases. However, although the betasatellite appeared to be of African origin, the alphasatellite was most closely related to alphasatellites originating from South Asia. This is the first report of a begomovirus-satellite complex infecting okra in Oman.     

Evidence of local evolution of tomato-infecting begomovirus species in West Africa: characterization of tomato leaf curl Mali virus and tomato yellow leaf crumple virus from Mali

Tomato yellow leaf curl (TYLC) and tomato leaf curl (ToLC) diseases are serious constraints to tomato production in Mali and other countries in West Africa. In 2003 and 2004, samples of tomato showing virus-like symptoms were collected during a survey of tomato virus diseases in Mali. Three predominant symptom phenotypes were observed: (1) TYLC/ToLC (stunted upright growth and upcurled leaves with interveinal yellowing and vein purpling), (2) yellow leaf crumple and (3) broccoli or bonsai (severe stunting and distorted growth). Squash blot (SB) hybridization with a general begomovirus probe and/or SB/PCR analyses revealed begomovirus infection in plants with each of these symptom phenotypes and no evidence of phytoplasma infection. Sequence analysis of PCR-amplified begomovirus fragments revealed two putative new begomovirus species associated with the TYLC/ToLC and yellow leaf crumple symptom phenotypes, respectively. Full-length clones of these begomoviruses were obtained using PCR and overlapping primers. When introduced into N. benthamiana and tomato plants, these clones induced upward leaf curling and crumpling (the TYLC/ToLC-associated begomovirus) or downward leaf curl/yellow mottle (yellow leaf crumple-associated begomovirus) symptoms. Thus, these begomoviruses were named tomato leaf curl Mali virus (ToLCMLV) and tomato yellow leaf crumple virus (ToYLCrV). The genome organization of both viruses was similar to those of other monopartite begomoviruses. ToLCMLV and ToYLCrV were most closely related to each other and to tobacco leaf curl Zimbabwe virus (TbLCZV-[ZW]) and tomato curly stunt virus from South Africa (ToCSV-ZA). Thus, these likely represent tomato-infecting begomoviruses that evolved from indigenous begomoviruses on the African continent. Mixed infections of ToLCMLV and ToYLCrV in N. benthamiana and tomato plants resulted in more severe symptoms than in plants infected with either virus alone, suggesting a synergistic interaction. Agroinoculation experiments indicated that both viruses induced symptomatic infections in tomato and tobacco, whereas neither virus induced disease symptoms in pepper, common bean, small sugar pumpkin, African eggplant, or Arabidopsis. Virus-specific PCR primers were developed for detection of ToLCMLV and ToYLCrV and will be used to further investigate the distribution and host range of these viruses.     

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.     

Molecular characterization of Tobacco leaf curl Pusa virus, a new monopartite Begomovirus associated with tobacco leaf curl disease in India

Leaf curl disease of tobacco (TbLCD) is endemic in India. A monopartite Begomovirus, a betasatellite and an alphasatellite were found associated with the disease in Pusa, Bihar. The DNA-A of the Begomovirus associated with TbLCD in Pusa, Bihar was found to comprise of 2707 nt with a typical Old World begomovirus-like genome organization. The full-length sequence of DNA-A [HQ180391] showed that the Pusa isolate is a newly described member of the genus Begomovirus, as it had <89% sequence homology with DNA-A of all the known begomoviruses. The isolate is tentatively named as Tobacco leaf curl Pusa virus [India:Pusa:2010]. The betasatellite (HQ180395) associated with TbLCD in Pusa was identified as a variant of Tomato leaf curl Bangladesh betasatellite [IN:Raj:03], with which it shared 90.4% sequence identity. The alphasatellite (HQ180392) associated with the disease had highest 87% nucleotide sequence identity with Tomato leaf curl alphasatellite. The Begomovirus, betasatellite, and alphasatellite associated with TbLCD in Pusa, Bihar, India were found to be recombinants of extant begomoviruses, betasatellites and alphasatellites spreading in the Indian sub-continent and South-East Asia.     

A new strain of tomato severe leaf curl virus and a unique variant of tomato yellow leaf curl virus from Mexico

The complete genome sequence of a distinct variant of tomato yellow leaf curl virus-Israel (TYLCV-IL) and the DNA-A sequence of a new strain of tomato severe leaf curl virus (ToSLCV) isolated in San Luis Potosi, Mexico, are described and analyzed. The TYLCV-IL[MX:SLP:11] variant differs from all TYLCV-IL isolates described so far by a unique 42-nt duplicated sequence comprising a part of the conserved stem-loop element of the virion-strand replication origin and adjacent regulatory sequences. TYLCV-IL[MX:SLP:11] was associated with tomato chino La Paz virus (ToChLPV-B[MX:SLP:11]) in a Solanum pimpinellifolium plant, and with pepper huasteco yellow vein virus (PHYVV-[MX:SLP:11]) and ToSLCV-GT[MX:SLP:11] in a Solanum lycopersicum plant. In addition, a distinct ToSLCV exhibiting low sequence identity (<89?%) to other ToSLCV isolates from Mexico was found in a tomato plant collected in the same field. Sequence analysis of this new ToSLCV strain indicates that it is a recombinant of close relatives of ToSLCV-GT[MX:SLP:11] and ToChLPV-B[MX:SLP:11] found in mixed infections with TYLCV-IL[MX:SLP:11].