Trapping of Tomato yellow leaf curl virus (TYLCV) and other plant viruses with a GroEL homologue from the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Summary. To avoid destruction in the haemolymph of their vector, many plant circulative viruses interact with GroEL homologues produced by insect endosymbiotic bacteria. We have exploited this phenomenon to devise tools allowing trapping of plant viruses by either GroEL purified from the whitefly Bemisia tabaci or by whitefly GroEL over-expressed in E. coli. PCR tubes or 96-well plates coated with a GroEL preparation were incubated with cleared sap of virus infected plant leaves or insect vectors. GroEL-bound viruses were then identified by PCR or RT-PCR using virus-specific primers or by ELISA with virus specific antibodies. In this way Tomato yellow leaf curl virus (TYLCV) – a whitefly-transmitted geminivirus – was detected in plant sap, in extracts of leaf squashes and in homogenates of individual viruliferous whiteflies. Anti-GroEL antibody prevented TYLCV binding to GroEL. GroEL-bound virus was also detected by ELISA. GroEL was much more potent in binding TYLCV than commercial anti-TYLCV antibodies. In addition to several other geminiviruses, these procedures allowed detecting a variety of RNA viruses such as Cucumber mosaic virus (CMV), Prune dwarf virus (PDV) and Tomato spotted wilt (TSWV), but not Potato virus X and Potato virus Y (PVX and PVY), Grapevine leafroll-associated viruses (GLRV) and Tobacco mosaic virus (TMV). Predictions pertaining to viruses that do, or do not bind to GroEL, and applications in plant virus diagnosis, are presented.     

Tomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: Implications for the delimitation of <Emphasis Type="Italic">cis</Emphasis>- and <Emphasis Type="Italic">trans</Emphasis>-acting replication specificity determinants

Summary. Over the last decade, the tomato production in Cuba has been affected by new whitefly-associated diseases. In addition to the well-documented presence of Tomato yellow leaf curl virus (TYLCV) along the island, the occurrence of bipartite begomoviruses has also been reported. One of them, tentatively named Tomato mottle Taino virus (ToMoTV), has now been cloned and characterized at the molecular level. Its genomic organization is similar to other bipartite geminiviruses. Phylogenetic analyses placed ToMoTV in a subcluster with other geminiviruses isolated in the Caribbean Basin: Tomato mottle virus (ToMoV), Bean dwarf mosaic virus, Abutilon mosaic virus, Sida golden mosaic virus and Potato yellow mosaic virus (PYMV). Biolistic inoculation of tobacco and tomato plants with cloned viral DNA showed that ToMoTV pseudorecombines with PYMV-GP as predicted by the identity of their iterative elements, whereas it does not show the same ability with ToMoV, even when their replication-associated proteins (Rep and REn) show the highest percentage of similarity. A comparative analysis of Rep proteins from begomoviruses that are able to produce viable reassortants suggests that some key elements for virus replication specificity are located in the first ten amino acids of this protein.Received October 18, 2002; accepted April 22, 2003 Published online July 2, 2003     

Making a friend from a foe: expressing a GroEL gene from the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> in the phloem of tomato plants confers resistance to tomato yellow leaf curl virus

Summary Some (perhaps all) plant viruses transmitted in a circulative manner by their insect vectors avoid destruction in the haemolymph by interacting with GroEL homologues, ensuring transmission. We have previously shown that the phloem-limited begomovirus tomato yellow leaf curl virus (TYLCV) interacts in vivo and in vitro with GroEL produced by the whitefly vector Bemisia tabaci. In this study, we have exploited this phenomenon to generate transgenic tomato plants expressing the whitefly GroEL in their phloem. We postulated that following inoculation, TYLCV particles will be trapped by GroEL in the plant phloem, thereby inhibiting virus replication and movement, thereby rendering the plants resistant. A whitefly GroEL gene was cloned in an Agrobacterium vector under the control of an Arabidopsis phloem-specific promoter, which was used to transform two tomato genotypes. During three consecutive generations, plants expressing GroEL exhibited mild or no disease symptoms upon whitefly-mediated inoculation of TYLCV. In vitro assays indicated that the sap of resistant plants contained GroEL-TYLCV complexes. Infected resistant plants served as virus source for whitefly-mediated transmission as effectively as infected non-transgenic tomato. Non-transgenic susceptible tomato plants grafted on resistant GroEL-transgenic scions remained susceptible, although GroEL translocated into the grafted plant and GroEL-TYLCV complexes were detected in the grafted tissues.     

Evidence for Transovarial Transmission of Tomato Yellow Leaf Curl Virus by Its Vector, the WhiteflyBemisia tabaci

The whiteflyBemisia tabaciis the only vector of the tomato yellow leaf curl geminivirus (TYLCV). The insect transmits the virus in a persistent-circulative manner. TYLCV DNA was detected by polymerase chain reaction and by Southern blot hybridization in progeny (eggs, first and second instars, adults) of single viruliferous whiteflies that developed on eggplant or on cotton (two TYLCV nonhost plants). Furthermore, TYLCV DNA was present in the progeny of insects that had acquired the virus through the egg. The adult progeny of the viruliferous insects and their own progeny were able to infect tomato test plants, producing typical disease symptoms. Ovaries and maturing eggs of viruliferous insects contained viral DNA, as did eggs laid by viruliferous insects maintained on an artificial diet. Eggs laid by nonviruliferous whiteflies on cotton plants previously caged with viruliferous insects did not acquire viral DNA from the plant. Hence, TYLCV can be transmitted through the egg for at least two generations. In the absence of an available plant host, the whitefly may serve as a reservoir of the virus between growing seasons.     

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 characterization of two sweepoviruses from China and evaluation of the infectivity of cloned SPLCV-JS in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Sweepoviruses are important begomoviruses that infect Ipomoea plants worldwide and cause sweet potato yield losses and cultivar decline. Two sweepoviruses, sweet potato leaf curl virus-Jiangsu (SPLCV-JS) and sweet potato leaf curl China virus-Zhejiang (SPLCCNV-ZJ), were cloned from diseased sweet potato plants collected in the Jiangsu and Zhejiang provinces of China. Sequence characterization and phylogenetic analysis demonstrated that both are typical monopartite begomoviruses and have close relationships to several reported SPLCV and SPLCCNV isolates, respectively, from Asian countries. Analysis of the protein alignments and subcellular localizations of the six SPLCV-JS proteins was also conducted to verify their putative functions. In Nicotiana benthamiana, an infectivity assay of the infectious SPLCV-JS clone resulted in mild symptoms and weak viral DNA accumulation. Interestingly, SPLCV-JS, together with a heterologous betasatellite DNA (tomato yellow leaf curl China virus isolate Y10 [TYLCCNV-Y10] DNA-β), showed a synergistic effect on enhanced symptom severity and viral DNA accumulation. This is the first reported infectious SPLCV clone.     

Molecular characterization and experimental host range of an isolate of <Emphasis Type="Italic">Wissadula golden mosaic St. Thomas virus</Emphasis>

Partial genome segments of a begomovirus were previously amplified from Wissadula amplissima exhibiting yellow-mosaic and leaf-curl symptoms in the parish of St. Thomas, Jamaica and this isolate assigned to a tentative begomovirus species, Wissadula golden mosaic St. Thomas virus. To clone the complete genome of this isolate of Wissadula golden mosaic St. Thomas virus, abutting primers were designed to PCR amplify its full-length DNA-A and DNA-B components. Sequence analysis of the complete begomovirus genome obtained, confirmed that it belongs to a distinct begomovirus species and this isolate was named Wissadula golden mosaic St. Thomas virus-[Jamaica:Albion:2005] (WGMSTV-[JM:Alb:05]). The genome of WGMSTV-[JM:Alb:05] is organized similar to that of other bipartite Western Hemisphere begomoviruses. Phylogenetic analyses placed the genome components of WGMSTV-[JM:Alb:05] in the Abutilon mosaic virus clade and showed that the DNA-A component is most closely related to four begomovirus species from Cuba, Tobacco leaf curl Cuba virus, Tobacco leaf rugose virus, Tobacco mottle leaf curl virus, and Tomato yellow distortion leaf virus. The putative Rep-binding-site motif in the common region of WGMSTV-[JM:Alb:05] was observed to be identical to that of Chino del tomate virus-Tomato [Mexico:Sinaloa:1983], Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005], and Tomato leaf curl Sinaloa virus-[Nicaragua:Santa Lucia], suggesting that WGMSTV-[JM:Alb:05] is capable of forming viable pseudo-recombinants with these begomoviruses, but not with other members of the Abutilon mosaic virus clade. Biolistic inoculation of test plant species with partial dimers of the WGMSTV-[JM:Alb:05] DNA-A and DNA-B components showed that the virus was infectious to Nicotiana benthamiana and W. amplissima and the cultivated species Phaseolus vulgaris (kidney bean) and Lycopersicon esculentum (tomato). Infected W. amplissima plants developed symptoms similar to symptoms observed under field conditions, confirming that this virus is a causal agent of Wissadula yellow mosaic disease in W. amplissima.     

Complete genomic sequences of <Emphasis Type="Italic">Tomato yellow leaf curl Mali virus</Emphasis> isolates infecting tomato and pepper from the North Province of Cameroon

Complete genomic sequences of Tomato yellow leaf curl Mali virus isolates infecting tomato and pepper from the North Province of Cameroon. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Infectivity analysis of a blackgram isolate of <Emphasis Type="Italic">Mungbean yellow mosaic virus</Emphasis> and genetic assortment with MYMIV in selective hosts

Yellow mosaic disease in grain legumes in Indian subcontinent is caused by two important virus species viz. Mungbean yellow mosaic virus (MYMV) and Mungbean yellow mosaic India virus (MYMIV), belonging to the genus Begomovirus of the family Geminiviridae. The genomic components of a begomovirus causing yellow mosaic disease in blackgram in southern India were cloned and sequenced. Nucleotide sequence comparison of DNA A component shows the virus isolate to be a variant of Mungbean yellow mosaic virus:–(MYMV-[IN:Vam:05]). However, DNA B component of the present virus isolate has greater similarity (92%) to Mungbean yellow mosaic India virus. Agroinoculations of the viral clones produced typical yellow mosaic symptoms in blackgram and mungbean, severe leaf curl and stunting in French bean, similar to blackgram isolate of MYMIV. Blackgram isolates of both the virus species were only mildly infectious on cowpea, produced atypical leaf curl symptoms and not yellow or golden mosaic. In agroinoculations done by exchanging genomic components, symptom expression was seen only in French bean. In cowpea, blackgram and mungbean there was no visible symptoms though viral DNA could be detected by PCR.     

Evidence for recombination among isolates of <Emphasis Type="Italic">Tobacco leaf curl Japan virus</Emphasis> and <Emphasis Type="Italic">Honeysuckle yellow vein mosaic virus</Emphasis>

Summary Complete nucleotide sequences of Tobacco leaf curl Japan virus (TbLCJV) isolates from infected tomato (Lycopersicon esculentum) plants in Nara (-[Jp2], 2764nt; -[Jp3], 2761nt), Kochi (-[Koc], 2760nt) and Yamaguchi (-[Yam], 2758nt) Prefectures, of Japan were determined. These sequences were compared with each other and the sequences of further begomoviruses from Japan. TbLCJV, TbLCJV-[Jp2], TbLCJV-[Jp3], TbLCJV-[Koc], TbLCJV-[Yam], Honeysuckle yellow vein mosaic virus (HYVMV), Eupatorium yellow vein virus (EpYVV), EpYVV-[MNS2], EpYVV-[SOJ3], EpYVV-[Yam] and EpYVV-[Tob] are monophyletic. The intergenic region (IR) of TbLCJV has highest nucleotide sequence identity with that of HYVMV (93%) whereas the rest of the genomic DNA had higher identity with that of TbLCJV-[Jp2] or -[Jp3] (91100%) than with that of HYVMV. In conclusion, TbLCJV has a chimeric genome which may have arisen by recombination between TbLCV-[Jp2] or -[Jp3]-like and HYVMV-like ancestors. Similarly, TbLCJV-[Yam] DNA has a hybrid genome, with a major parent HYVMV and minor parent TbLCJV-[Koc].     

Absence of interaction of genomic components and complementation between <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> isolates in cowpea

Summary. Agroinoculations were performed with DNA A and DNA B components of Mungbean yellow mosaic India virus (MYMIV) isolates differing in their infectivity on cowpea. Exchange of genomic components of the MYMIV isolates occurred in all the leguminous species but not in cowpea. Extremely low viral DNA accumulation and atypical leaf curl symptoms produced by reassortants in cowpea suggest barriers both for replication and systemic movement despite genetic similarity.     

Tomato leaf curl virus from Bangalore (ToLCV-Ban4): sequence comparison with Indian ToLCV isolates, detection in plants and insects, and vector relationships}

Summary.  Tomato leaf curl virus (ToLCV) is a whitefly (Bemisia tabaci) transmitted geminivirus (family Geminiviridae, genus Begomovirus) causing a destructive disease of tomato in many regions of India, East Asia and Australia. While ToLCV isolates from Australia and Taiwan have a single genomic component (designated DNA-A), those from Northern India have two components (DNA-A and DNA-B). The ToLCV isolates from Southern India (Bangalore) previously cloned seem to have a DNA-A-like monopartite genome. We have used degenerate DNA-A-specific PCR primers to clone the genome of a ToLCV isolate (named ToLCV-Ban4) from field-infected tomato plants growing in Bangalore, India, in 1997. Degenerate DNA-B-specific PCR primers have not allowed to amplify a putative DNA-B from infected tomato, at the time when DNA-B fragments were amplified from plants infected by known bipartite begomoviruses. The full-length 2759 nucleotide-long DNA-A-like viral genome was sequenced. Similarly to other monopartite ToLCV and TYLCV isolates, ToLCV-Ban4 contains six open reading frames, two on the virion strand and four on the complementary strand. Sequence comparisons indicated that ToLCV-Ban4 is similar to the other three isolates from Bangalore previously sequenced, and is closely related to ToLCV-Ban2 (approximately 91\% nucleotide sequence identity). Phylogenetic analysis showed that the ToLCV isolates from Bangalore constitute a group of viruses separated from those of Northern India. ToLCV-Ban4 was detected in tomato and in its whitefly vector Bemisia tabaci by one or by a combination of ELISA, Southern blot hybridization and PCR. Parameters of virus acquisition, retention and transmission by the whitefly vector were investigated in the laboratory. Single whiteflies were able to acquire ToLCV-Ban4 from infected tomato and to transmit the virus to tomato test plants, but five insects were necessary to achieve 100% transmission. Minimum acquisition access and inoculation access periods were 10 min and 20 min, respectively. A latent period of 6 h was required for B. tabaci to efficiently infect tomato test plants. Following a 24 h acquisition access period the insect retained its ability to infect tomato test plants for 12 days, but not for its entire life. In one insect/one plant inoculation tests, female whiteflies were more efficient (∼95%) than males (∼25%) in transmitting the virus. Received July 5, 1999 Accepted March 2, 2000     

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.     

A simple and efficient method for agroinfection of <Emphasis Type="Italic">Vernonia cinerea</Emphasis> with infectious clones of <Emphasis Type="Italic">Vernonia yellow vein virus</Emphasis>

Vernonia yellow vein virus (VeYVV) is a distinct monopartite begomovirus associated with a satellite DNA β. After constructing dimers of both DNA A and DNA β in binary vectors, a number of infection methods were attempted. However, only a modified stem-prick method produced up to 83% infection in the natural host Vernonia cinerea, thus, fulfilling the Koch’s postulate. The presence of the viral DNA in the agroinfected plants was confirmed by rolling circle amplification (RCA), followed by Southern hybridization. DNA β induces typical symptoms of Vernonia yellow vein disease (VeYVD) when co-agroinoculated with the begomovirus to Vernonia and also leads to the accumulation of DNA A systemically. VeYVV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction.     

Characterization and genetic diversity of <Emphasis Type="Italic">Potato yellow mosaic virus</Emphasis> from the Caribbean

Summary. The begomovirus Potato yellow mosaic virus (PYMV) is responsible of significant yield losses in tomato in Guadeloupe. Four field isolates from Guadeloupe were analyzed in term of their host range using three inoculation methods (mechanical, grafting and insect vector), sequences analysis of PCR fragments and phylogenetic analysis of an infectious clone, PYMV-[GP]. Capsicum annuum, Datura stramonium, Nicotiana benthamiana, N. tabacum Xanthi NC, Petunia hybrida, and Solanum tuberosum were found to be hosts. All isolates from Guadeloupe, Martinique, Puerto Rico and the Dominican Republic were closely related to PYMV-[GP]. Sequence identity between PYMV-[GP] and PYMV-Ve from Venezuela and PYMTV from Trinidad and Tobago clearly confirmed that it is a new strain of PYMV.     

PCR-based detection and partial genome sequencing indicate high genetic diversity in Bangladeshi begomoviruses and their whitefly vector, <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

The population diversity of Bangladeshi begomoviruses and their vector, Bemisia tabaci was analysed by PCR-based detection and partial genome sequencing. B. tabaci adults and plants expressing symptoms of virus infection were collected from locations representing diverse agro-ecological regions of the country. Universal and species-specific primers were used to detect begomoviruses in seven crops (chilli, okra, papaya, pumpkin, sponge gourd, tomato and yardlong bean) and two common weeds (Ageratum conyzoides and Croton bonplandianum). At least five distinct species of tomato leaf curl viruses infected tomato and other host-plants. Phylogenetic analyses of their nucleotide sequences (∼530 bases) from the intergenic region and capsid protein of DNA-A indicated the existence of five distinct clusters of begomoviruses. Begomoviruses infecting tomato, chilli and dolichos have been reported previously, and those infecting Ageratum, Croton, okra, papaya, pumpkin and yardlong bean are described for the first time. Phylogenetic analyses based on mitochondrial cytochrome oxidase I gene sequences of 21 B. tabaci from Bangladesh and other reference sequences grouped them into at least two independent clusters. Some sequences from different countries, e.g., Bangladesh, China, India, Nepal, Pakistan and Thailand were almost identical while others collected from plants within the same field diverged by as much as 15%, indicating high diversity even at the local level. None of the B. tabaci from Bangladesh grouped with the reference B- and Q-biotype sequences, thus these two aggressive biotypes were apparently absent from Bangladesh.     

Sequence Analysis of the Glycoproteins of <Emphasis Type="Italic">Tomato Chlorotic Spot Virus</Emphasis> and <Emphasis Type="Italic">Groundnut Ringspot virus</Emphasis> and Comparison with other Tospoviruses

The tospoviruses Tomato chlorotic spot virus (TCSV) and Groundnut ringspot virus (GRSV) cause high economic losses in several vegetable crops in Brazil. The glycoprotein precursor coding sequence was still not available for these two viruses. In this study, the 3' 4kb M RNA of TCSV and GRSV genome was cloned and sequenced. The sequences were compiled with the available 5' region sequence (NS_M gene and 5' UTR) of the same isolates. The M RNA of TCSV was deduced as formed by 4,882 nucleotides, while of GRSV by 4,855 nucleotides. Both M RNA comprised two ORFs in an ambisense arrangement. The vcRNA ORF coded for viral glycoprotein (G1/G2) precursor of TCSV (128.46kDa) and for glycoprotein precursor of GRSV (128.16kDa). Comparison of the TCSV and GRSV glycoprotein precursor proteins with those of other tospoviruses showed the highest identity with Tomato spotted wilt virus (81 and 79%, respectively). The amino acid sequence comparison of glycoprotein precursor between TCSV and GRSV revealed a high identity of 92%. However, the nucleotide sequence of the M RNA intergenie region showed only 78%. Phylogenetic analysis was done based on glycoprotein precursor and on M RNA intergenic region of tospoviruses and parameters on tospovirus taxonomic classification were discussed.     

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.     

The complete sequence of the genomic RNA of an isolate of <Emphasis Type="Italic">Lily virus X</Emphasis> (genus <Emphasis Type="Italic">Potexvirus</Emphasis>)

Summary. The complete sequence of the genomic RNA of an isolate of Lily virus X (LVX) has been determined for the first time. The isolate from the Netherlands was 5823 nucleotide (nt) long excluding the 3-poly(A) tail, making it the shortest reported potexvirus sequence. The 5-non-coding region begins with GGAAAA like that of Scallion virus X (ScaVX) and some isolates of Cymbidium mosaic virus (CymMV), whereas those of other sequenced potexviruses probably all begin with GAAAA. The genome organisation was similar to that of other members of the genus except that a TGBp3-like region lacked a normal AUG start codon. A phylogenetic analysis based on the entire coding sequence showed that LVX was most closely related to Strawberry mild yellow edge virus and belonged in a subgroup of the genus that also contains CymMV, Narcissus mosaic virus, ScaVX, Pepino mosaic virus, Potato aucuba mosaic virus and White clover mosaic virus.