Tomato Yellow Leaf Curl Virus Genome Research Articles

Establish an efficient inoculation system of tomato yellow leaf curl virus to assist tomato resistance breeding

Tomato yellow leaf curl virus (TYLCV) endangers the production of tomato in the world, which is largely overcome by cultivating resistant tomato cultivars. Since virus evolves fast and may disrupt the resistance in resistant cultivars, tomato resistance breeding against emerging TYLCV isolates is a long-term task, which requires an efficient inoculation system of TYLCV to screen and identify resistant tomato plants. Previous Agrobacterium-mediated TYLCV infectious clone is to construct the viral genome in the binary vector, and does not have high infection efficiency. In this study, we integrate TYLCV genome in pTRV2GW vector containing partial genome of Tobacco Rattle virus, and generate an Agrobacterium-mediated infectious clone pTRV2GW-TYLCV. Tomato plants are infected by vacuum infiltration with the Agrobacterium culture of infectious clone. PCR analysis with all TYLCV ORFs in infected plants reveals distinct detection of each ORF at different infection stages and a final 100 % infection rate of this inoculation method. Infected tomato plants have typical TYLCV disease symptoms including yellowing, curling, and smaller leaves. Plant growth and leaf photosynthesis are repressed in infected plants compared to non-infected plants. Moreover, the inoculation system is successfully applied for differentiating TYLCV resistant and susceptible cultivars, which can assist tomato resistance breeding.

Plant resistance to tomato yellow leaf curl virus is enhanced by Bacillus amyloliquefaciens Ba13 through modulation of RNA interference.

Tomato yellow leaf curl virus (TYLCV), which is a typical member of the genus Begomovirus, causes severe crop yield losses worldwide. RNA interference (RNAi) is an important antiviral defense mechanism in plants, but whether plant beneficial microbes used as biocontrol agents would modulate RNAi in defense against TYLCV remains unclear. Here, we employed whole-transcriptome, bisulfite, and small RNA sequencing to decipher the possible role of Bacillus amyloliquefaciens Ba13 as a bacterial biocontrol agent against TYLCV in RNAi modulation. Potted tomato plants were exposed to whiteflies for natural viral infection 14 days after bacterial inoculation. Compared with non-inoculated controls, the abundance of TYLCV gene in the leaves of inoculated plants decreased by 70.1% at 28 days post-infection, which mirrored the pattern observed for plant disease index. The expression of the ARGONAUTE family genes (e.g., AGO3, AGO4, AGO5, and AGO7) involved in antiviral defense markedly increased by 2.44-6.73-fold following bacterial inoculation. The methylation level at CpG site 228 (in the open reading frame region of the RNA interference suppressing gene AV2) and site 461 (in the open reading frame regions of AV1 and AV2) was 183.1 and 63.0% higher in inoculated plants than in non-inoculated controls, respectively. The abundances of 10 small interfering RNAs matched to the TYLCV genome were all reduced in inoculated plants, accompanied by enhancement of photosystem and auxin response pathways. The results indicate that the application of Ba. amyloliquefaciens Ba13 enhances plant resistance to TYLCV through RNAi modulation by upregulating RNAi-related gene expression and enhancing viral genome methylation.

Phylogenomic and population genetics analyses of extant tomato yellow leaf curl virus strains on a global scale

Tomato yellow leaf curl virus (TYLCV) is a monopartite DNA virus with a genome size of ~ 2,800 base pairs. The virus belongs to the genus Begomovirus within the family Geminiviridae. Extant TYLCV strains are differentiated based on an established threshold of 94% genome-wide pairwise nucleotide identity. The phylogenetic relationships, diversification mechanisms, including recombination, and extent of spread within and from the center of origin for TYLCV have been reported in previous studies. However, the evolutionary relationships among strains, strains’ distribution and genomic diversification, and genetic mechanisms shaping TYLCV strains’ evolution have not been re-evaluated to consider globally representative genome sequences in publicly available sequence database, including herein newly sequenced genomes from the U.S. and Middle East, respectively. In this study, full-length genome sequences for the extant strains and isolates of TYLCV (n=818) were downloaded from the GenBank database. All previously published genome sequences, and newly sequenced TYLCV genomes of TYLCV isolates from Kuwait and USA, determined herein (n=834), were subjected to recombination analysis. To remove the ‘phylogenetic noise’ imparted by interspecific recombination, the recombinant genomes were removed from the data set, and the remaining non-recombinant genome sequences (n=423) were subjected to population genetics and Bayesian analyses. Results of the phylogeographical analysis indicated that the type strain, TYLCV-Israel, and TYLCV-Mild strain, were globally distributed, spanning Africa, America, Asia, Australia/Oceania, Europe, and New Caledonia, while the other TYLCV strains were prevalent only throughout the Middle East. The results of Bayesian evolutionary (ancestral) analysis predicted that TYLCV-Israel represents the oldest, most recent common ancestor (MRCA) (41,795 years), followed by TYLCV-Mild at 39,808 years. These were closely followed by two Iranian strains viz., TYLCV-Kerman and TYLCV-Iran at 37,529 and 36,420 years, respectively. In contrast, the most recently evolving strains were TYLCV-Kuwait and TYLCV-Kahnooj at 12,445 and 298 years, respectively. Results of the neutrality test indicated that TYLCV-Israel and TYLCV-Mild populations are undergoing purifying selection and/or population expansion, although statistically significant selection was documented for only TYLCV-Israel, based on positive selection acting on five codons.

Intracellular bottlenecking permits no more than three tomato yellow leaf curl virus genomes to initiate replication in a single cell

Viruses are constantly subject to natural selection to enrich beneficial mutations and weed out deleterious ones. However, it remains unresolved as to how the phenotypic gains or losses brought about by these mutations cause the viral genomes carrying the very mutations to become more or less numerous. Previous investigations by us and others suggest that viruses with plus strand (+) RNA genomes may compel such selection by bottlenecking the replicating genome copies in each cell to low single digits. Nevertheless, it is unclear if similarly stringent reproductive bottlenecks also occur in cells invaded by DNA viruses. Here we investigated whether tomato yellow leaf curl virus (TYLCV), a small virus with a single-stranded DNA genome, underwent population bottlenecking in cells of its host plants. We engineered a TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that among the cells entered by both replicons, less than 65% replicated both, whereas at least 35% replicated either of them alone. Further probability computation concluded that replication in an average cell was unlikely to have been initiated with more than three replicon genome copies. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons at the intracellular level. In conclusion, the intracellular population of the small DNA virus TYLCV is actively bottlenecked, and such bottlenecking may be a virus-encoded, evolutionarily conserved trait that assures timely selection of new mutations emerging through error-prone replication.

Tomato Yellow Leaf Curl Virus-Resistant and -Susceptible Tomato Genotypes Similarly Impact the Virus Population Genetics.

Tomato yellow leaf curl virus is a species in the genus Begomovirus and family Geminiviridae. Tomato yellow leaf curl virus (TYLCV) infection induces severe symptoms on tomato plants and causes serious yield losses worldwide. TYLCV is persistently transmitted by the sweetpotato whitefly, Bemisia tabaci (Gennadius). Cultivars and hybrids with a single or few genes conferring resistance against TYLCV are often planted to mitigate TYLCV-induced losses. These resistant genotypes (cultivars or hybrids) are not immune to TYLCV. They typically develop systemic infection, display mild symptoms, and produce more marketable tomatoes than susceptible genotypes under TYLCV pressure. In several pathosystems, extensive use of resistant cultivars with single dominant resistance-conferring gene has led to intense selection pressure on the virus, development of highly virulent strains, and resistance breakdown. This study assessed differences in TYLCV genomes isolated from susceptible and resistant genotypes in Florida and Georgia. Phylogenetic analyses indicated that Florida and Georgia isolates were distinct from each other. Population genetics analyses with genomes field-collected from resistant and susceptible genotypes from Florida and/or Georgia provided no evidence of a genetic structure between the resistant and susceptible genotypes. No codons in TYLCV genomes from TYLCV-resistant or susceptible genotypes were under positive selection, suggesting that highly virulent or resistance-breaking TYLCV strains might not be common in tomato farmscapes in Florida and Georgia. With TYLCV-resistant genotypes usage increasing recently and multiple tomato crops being planted during a calendar year, host resistance-induced selection pressure on the virus remains a critical issue. To address the same, a greenhouse selection experiment with one TYLCV-resistant and susceptible genotype was conducted. Each genotype was challenged with TYLCV through whitefly-mediated transmission serially 10 times (T1-T10). Population genetics parameters at the genome level were assessed at T1, T5, and T10. Results indicated that genomes from resistant and susceptible genotypes did not differentiate with increasing transmission number, no specific mutations were repeatedly observed, and no positive selection was detected. These results reiterate that resistance in tomato might not be exerting selection pressure against TYLCV to facilitate development of resistance-breaking strains. TYLCV populations rather seem to be shaped by purifying selection and/or population expansion.

Tomato Yellow Leaf Curl Virus: Impact, Challenges, and Management.

Tomato yellow leaf curl virus (TYLCV) is one of the most studied plant viral pathogens because it is the most damaging virus for global tomato production. In order to combat this global threat, it is important that we understand the biology of TYLCV and devise management approaches. The prime objective of this review is to highlight management strategies for efficiently tackling TYLCV epidemics and global spread. For that purpose, we focus on the impact TYLCV has on worldwide agriculture and the role of recent advances for our understanding of TYLCV interaction with its host and vector. Another important focus is the role of recombination and mutations in shaping the evolution of TYLCV genome and geographical distribution.

Localized surface plasmon resonance biosensing of tomato yellow leaf curl virus

Current techniques for plant virus detection, such as RT- PCR and ELISA, require multistep procedures and rely on sophisticated equipment. Due to the global spread of plant viruses, the development of simpler, faster and cheaper assay methods is inevitable. Gold nanoparticles (AuNPs) had raised much interest during recent years due to their novel optical properties or diagnostic purposes. The localized surface plasmon resonance (LSPR11LSPR: localized surface plasmon resonance.) of AuNPs had been used in the development of novel colorimetric nano-biosensing systems. The frequency and intensity of the LSPR peak generally depend on the shape, size and the surrounding medium of the AuNPs. In this study, unmodified AuNPs had been used to detect the unamplified Tomato yellow leaf curl virus (TYLCV) genome in infected plants. A specific DNA probe complementary to the coat protein region of virus genome was designed. The extracted total DNA of uninfected and infected plants was mixed with hybridization buffer and the designed probe. The mixture was denatured, annealed and then cooled to room temperature and was followed by AuNPs addition. The color changes in the samples indicating the presence of target virus infections were assessed visually after the addition of salt and confirmed by UV–Vis spectroscopy. The results showed that this strategy allowed for fast and sensitive detection of TYLCV genome and eliminated the need for PCR amplification and detection equipment.

Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

ABSTRACTCRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

Molecular variation of tomato yellow leaf curl virus in the insect vector Bemisia tabaci

Insect vectors play significant roles in geminivirus spread and evolution in nature. To date little is known about the population dynamics of begomoviruses in their insect vector Bemisia tabaci. In this study we analyzed the genetic variation of tomato yellow leaf curl virus (TYLCV) in its host plant, Solanum lycopersicum, in its transmission vector B. tabaci raised on TYLCV-infected S. lycopersicum plants, and in B. tabaci after being transferred from S. lycopersicum to Gossypium hirsutum. We found that the levels of variability of TYLCV remained stable in S. lycopersicum plants, but increased significantly in both invasive and indigenous species of B. tabaci. We also presented evidence that the elevated mutation frequencies in TYLCV populations from vector whiteflies were caused mainly by mutations that occurred at several distinct sites within the TYLCV genome. Simultaneous introduction of mutations in the hot spots did not affect the ability of TYLCV to be transmitted by B. tabaci, but reduced its pathogenicity in both S. lycopersicum and Nicotiana benthamiana. Our findings provide new information on population variability of TYLCV in its insect vector, extending the knowledge of the influence of insect vector on plant virus population dynamics.

Tomato Yellow Leaf Curl Virus: A Serious Threat to Tomato Plants World Wide

Tomato (Solanum lycopersicum) is one of the widely grown crops worldwide. It is consumed in various forms and has excellent nutritional values. Presently, this crop is facing a serious threat to its yield and survival because of Geminivirus infection. One of the Geminivirus species hampering tomato productions world-wide is Tomato yellow leaf curl virus (TYLCV). Tomato yellow leaf curl disease is one of the most destructive plant diseases destroying the tomato crops globally. It has spread to many countries worldwide including Southern, Central and Northern parts of America, Southern Asia, Africa and Mediterranean basin. TYLCV genome consists of a circular single stranded DNA of 2.7 kb in size and occasionally contains beta satellite of 1.3 kb. TYLCV genome encodes six open reading frames, of which two are in the viral orientation and four are in the complementary orientation. TYLCV is transmitted by the insect vector Bemisia tabaci, commonly known as the silverleaf white fly. This review provides an overview of Geminiviruses with special emphasis on TYLCV, virus vector relationship between TYLCV and white fly, different strains of TYLCV, its genome organization and replication, present status of its spread in different parts of the world and strategies employed for controlling it. The knowledge about the recent progress in the study of TYLCV would help develop novel strategies for its control in agriculture.

Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates

Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted by the whitefly Bemisia tabaci to tomato and other crops. TYLCV proteins are endangered by the host defenses. We have analyzed the capacity of the tomato plant and of the whitefly insect vector to degrade the six proteins encoded by the TYLCV genome. Tomato and whitefly demonstrated the highest proteolytic activity in the fractions containing soluble proteins, less—in large protein aggregates; a significant decrease of TYLCV proteolysis was detected in the intermediate-sized aggregates. All the six TYLCV proteins were differently targeted by the cytoplasmic and nuclear degradation machineries (proteases, ubiquitin 26S proteasome, autophagy). TYLCV could confront host degradation by sheltering in small/midsized aggregates, where viral proteins are less exposed to proteolysis. Indeed, TYLCV proteins were localized in aggregates of various sizes in both host organisms. This is the first study comparing degradation machinery in plant and insect hosts targeting all TYLCV proteins.

PCR–RFLP-based typing for differentiation of Tomato yellow leaf curl virus (TYLCV) genotypes from infected host plants in Korea

A polymerase chain reaction (PCR) using two sets of primers designed from published Tomato yellow leaf curl virus (TYLCV) genomes was developed to distinguish from the TYLCV-IL groups. The specificity of the two sets of primers was proven by testing against control TYLCV genomes and the symptomatic leaves of 34 different tomato cultivars naturally infected with TYLCV in greenhouses. One set for TYLCV-IL strain-specific primers (TYLCV-UNI-F and TYLCV-UNI-R) amplified full-length genome fragments from all the 34 tomato cultivars. Another set for TYLCV-IL group-II strain-specific primers (TYLCV-GPII-F and TYLCV-GPII-R) amplified target DNA fragments from only 9 tomato cultivars. Digestion by BglII and EcoRV of the PCR amplicons produced restriction fragment length polymorphism pattern that distinguished the TYLCV-IL group-I with two fragments from the TYLCV-IL group-II with no digested fragment. PCR coupled with BglII and EcoRV digestion confirmed that the 9 tomato cultivars were infected with the TYLCV-IL group-II and the remained 25 tomato cultivars were infected with the TYLCV-IL group-I.

Two Distinct Isolates of Tomato yellow leaf curl virus Threaten Tomato Production in Arizona and Sonora, Mexico.

Severe yellow leaf curl and plant stunting symptoms were observed in tomato plants from two home gardens in central Arizona (Phoenix area) and a tomato field in Sonora, Mexico during the fall of 2006. Disease symptoms were reminiscent of those reported in Florida during 1994 (4) and more recently in tomato fields in the Pacific Coast state of Sinaloa, Mexico found to be infected with the exotic Tomato yellow leaf curl virus (TYLCV) (2). Total DNA was extracted from two symptomatic tomato plants from Arizona and Sonora and used as a template in PCR. PCR products of the core region of the begomovirus coat protein gene (Cp) were cloned (n = 3) and the DNA sequence was determined. BLAST analysis of the 579 bases with sequences available in the NCBI GenBank database indicated the closest match was to an isolate of the monopartite begomovirus TYLCV from Israel, which was known to have been introduced into the Caribbean region, including Puerto Rico, the southeastern United States, and Mexico from 1990 to 1996 (1,4). The full-length TYLCV genome (approximately 2,800 bases) was amplified for a field isolate from each location by rolling circle amplification (RCA) using TempliPhi (Amersham Biosciences, Piscataway, NJ). RCA products were cloned into the plasmid vector pGEM7 (Promega, Madison, WI) that had been previously digested with SacI endonuclease. The complete TYLCV genome sequence was determined for six clones from each RCA product. Nucleotide analysis indicated that the complete TYLCV genome sequences from Sonora and Arizona, respectively, shared 97.6 and 97.7% nt identity. The comparative sequence analysis indicated that TYLCV-Sonora (TYLCV-Son) (GenBank Accession No. EF210555) was 99.1% nt identical to TYLCV reported recently from Culiacan, Mexico (GenBank Accession No. DQ631892). In contrast, TYLCV-AZ (GenBank Accession No. EF210554) shared 99.3% identity with an isolate from Texas, TYLCV-TX (GenBank Accession No. EF110890) (3). Interestingly, the TX and AZ TYLCV isolates contained a unique 29-nt deletion in the intergenic region (IR) between the TATA-box and the nonanucleotide, initiating at nt coordinate 2696. Except for the deletion in the IR region of the AZ and TX isolates, these viruses shared 97.6 to 99.1% nt identity to other TYLCV isolates reported in the Western Hemisphere. The genome sequence for TYLCV-Son shares high nt identity with TYLCV isolates identified in the Yucatan Peninsula and Pacific Coast of Mexico (2), the Caribbean region, and the southeastern United States, suggesting that a single TYLCV species was introduced and has spread throughout North America and the Caribbean (4). The absence of other TYLCV isolates in the Western Hemisphere with the novel 29-nt deletion noted for the TX and AZ isolates suggests that the latter two isolates originated from the same U.S. source. In Mexico, TYLCV was first introduced in the east coast and Yucatan region approximately in 1996. From there, this isolate has spread to the western part of the country (Sinaloa and Sonora) from 2004 to 2006 (2). Similarly, in the United States, TYLCV was introduced and spread in the eastern U.S. states beginning in 1994 (4), where it had been confined until it was discovered in Texas (3) and now Arizona during 2006.

Simultaneous and rapid differentiation of members of the tomato yellow leaf curl virus complex by multiplex PCR.

A multiplex PCR method was developed to identify simultaneously either single or multiple infections with members of the Tomato yellow leaf curl virus (TYLCV) complex. Several sets of species-specific primers, designed from sequenced genomes of TYLCV from the Mediterranean region (Sicily, Israel and Spain), were tested using plants infected with Egyptian and Tunisian isolates. The results show that this multiplex PCR is rapid, simple and a useful alternative to conventional tools such as PCR-RFLP or PCR-sequencing for typing virus isolates. Furthermore, this approach revealed evidence of recombination among Tunisian TYLCV genomes.

Complete nucleotide sequence of Iranian tomato yellow leaf curl virus isolate: further evidence for natural recombination amongst begomoviruses. Brief Report.

Complete nucleotide sequence of the Iranian strain of tomato yellow leaf curl virus (TYLCV-IR) was determined and compared with some begomoviruses. The complete sequence of TYLCV-IR clustered together with TYLCV and TYLCV-MId from Israel. A similar relationship holds when the deduced amino acid sequences of V1, V2, C2 and C3 and nucleotide sequences of IR, and RIR were compared. In contrast, phylogenetic analyses of amino acid sequences of C4, C1, and nucleotide sequences of LIR revealed that TYLCV-IR clustered with TLCIRV and two Indian species: ToLCBV- [Ban4], and ToLCKV. The phylogenetic analyses, Recombination Detection Program analyses, and sequence alignment survey provided evidence of the occurrence of recombination between an Israeli TYLCV-MId, as major parent, and TLCIRV, as minor parent. In this recombination event, a region (from nt 2149 to 2766) of TYLCV-MId genome were replaced with corresponding genome sequences of TLCIRV (RDP P-value = 5.976 x 10(-72)), which include LIR, C4, and N-terminal of C1. Infectivity of the cloned TYLCV-IR genome was demonstrated by successful agroinoculation of tomato ( Lycopersicon esculentum) and other plant species. The disease was transmitted by the natural vector Bemisia tabaci from agroinoculated plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-IR consists of only one circular single-stranded DNA molecule.

Appearance of Tomato yellow leaf curl virus in North Carolina.

In the summers of 2000 and 2001, tomato plants (Lycopersicon esculentum) with symptoms of stunting, curling, and marginal chlorosis of leaves, reduced leaf size, and marked reduction in fruit number, similar to those caused by Tomato yellow leaf curl virus (TYLCV), were seen in Henderson County, NC. In 2001, symptomatic plants appeared in a 40-A (18.2 ha) field in 12 foci of ≈12 plants each, at a total incidence of less than 1%. In August 2001, DNA was extracted from leaf samples from four symptomatic plants and tested by polymerase chain reaction (PCR) amplification for the presence of one or more geminiviruses. Two sets of primers were used to test for begomoviruses, AC1048 and PCRv181 (3,4), which amplify a 1,020-bp DNA product from a wide range of monopartite and bipartite (A component only) begomoviruses, and C473 and PTYC1v2406, which preferentially amplifies a 859-bp DNA product from the monopartite TYLCV (1,2). Fragments of the expected size were obtained from all four samples, and all PCR products were sequenced. The sequences of the 1,020-bp PCR product from each of the four samples were compared and found to be 100% identical. The same was found for the 859-bp products. These sequences were compared with equivalent regions of begomoviruses and were identical to sequences of TYLCV. Since the two primer sets amplify overlapping regions of the TYLCV genome, the 1,020 and 859-bp products generated by the two primer sets from one plant were combined to create a 1,464-bp sequence that represented approximately half of the TYLCV genome and encompasses the C4 ORF, the intergenic region, and most of the coat protein gene. This 1,464-bp sequence from North Carolina was 99.2 to 99.6% identical to TYLCV sequences reported from Cuba (GenBank Accession No. AJ223505), the Dominican Republic (GenBank Accession No. AF024715), and Florida, and 96.9 to 98.2% identical to TYLCV sequences reported from the Bahamas, Israel (GenBank Accession No. X15656), Jamaica (GenBank Accession No. U84146), Mexico (GenBank Accession No. AF168709), and Spain (GenBank Accession No. AF071228). Symptomatic plants appeared to be infected with an isolate of TYLCV that is most similar to TYLCV isolates reported from Florida and the northeastern Caribbean. To our knowledge, this is the first report of TYLCV in North Carolina. TYLCV may have been introduced on transplants since the infected plants showed symptoms at an early growth stage. The appearance of infected plants in clusters of limited size suggests no spread or very limited spread in the field. Reports of populations of the whitefly (Bemisia tabaci) vector in the field were not available since whiteflies are not normally a problem in this area due to the higher altitude and relatively cool temperatures characteristic of Henderson County. It is not clear at this time what threat TYLCV poses to tomato production in the county, though its appearance indicates that the geographic range of TYLCV is continuing to expand in the southeastern United States.

Characterization of a tomato karyopherin that interacts with the Tomato Yellow Leaf Curl Virus (TYLCV) capsid protein

cDNA from poly(A)+ RNA which has been extracted from A karyopherin a (LeKAPa1) cDNA was isolated from tomato Lycopersicon esculentum cv. MoneyMaker leaves and used as plants. The deduced LeKAPa1 protein sequence of 527 amino template for reverse transcription. The cDNA preparation was acids showed similarity to other plant karyopherin a proteins. then subcloned using Eco RI adapters into the Eco RI site of When LeKAPa1 was expressed in a yeast two-hybrid system the lgt10 phage vector (Stratagene). The recombinant l phage together with the gene coding for the capsid protein (CP) of the DNA was packaged in vitro using the Stratagene lambda packtomato yellow curl leaf virus (TYLCV), it interacted directly with aging extract Gigapack II according to the manufacturer’s CP. Thus, LeKAPa1 may be involved in the nuclear import of instructions, and used to infect the Escherichia coli C600 hfl’ TYLCV CP and, potentially, the TYLCV genomes during viral strain. The resulting cDNA library (2.0◊106 plaques) was screinfection of the host tomato cells. ened with the AtKAPa probe as described (Ausubel et al., 1994). Briefly, a 32P-labelled 2 kb fragment of AtKAPa cDNA containing the complete open reading frame was hybridized for 16 h

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes.

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid of Agrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2-5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation, L. chilense LA1969 and L. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.

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.