Detection and Variability of Begomoviruses in Tomato from the Andean States of Venezuela.

The three Andean states of Mérida, Táchira, and Trujillo in Venezuela produce tomatoes (Lycopersicon esculentum) year-round with a high frequency of virus-like symptoms. Polymerase chain reaction amplification using degenerate primers detected begomoviruses in 18% of leaf samples collected from tomato plants showing virus-like symptoms in commercial fields in these states between 1993 and 1994. A comparison of the sequences of the amplified DNA products revealed a diversity of begomovirus sequences in tomato plants from this region. Partial A component sequences (approximately 1,100 bp) clustered in four groups based on BLAST, GAP, phylogenetic analyses of the nucleic acid sequences, and comparisons of iteron sequences with known begomoviruses. Two groups of sequences were closely related to Potato yellow mosaic virus-Venezuela strain tomato and Tomato Venezuela virus, respectively, begomoviruses previously reported from other Venezuelan states. The other two groups of sequences appear to belong to two new begomovirus species.

A Virus Related to Clover Yellow Mosaic Virus Found East of the Mississippi River in Verbena canadensis in Florida.

In September 2002, several unthrifty Verbena canadensis 'Homestead purple' plants were received at the Division of Plant Industry in Gainesville, FL. Symptoms included very subtle yellowing and distortion or stunting of the younger leaves, symptoms that could be overlooked as a nutritional problem. Symptomatic leaves were ground in phosphate buffer and mechanically inoculated to a variety of plants that included Antirrhinum majus, Chenopodium amaranticolor, Datura stramonium, Gomphrena globosa, Pisum sativum, Trifolium pratense, T. repens, Vicia faba, and Vigna unguiculata. These hosts showed symptoms similar to those described for infection by Clover yellow mosaic virus (ClYMV) (3). In addition, the virus systemically infected Arachis hypogaea, Catharanthus roseus, C. quinoa, Nicotiana benthamiana, and healthy seed-grown Verbena × hybrida. No symptoms were seen in inoculated Zinnia elegans, N. glutinosa, N. clevelandii, Lycopersicon esculentum, Cucumis sativus, or Capsicum annuum. However, back inoculations of Cucumis sativus to C. amaranticolor gave typical local and systemic symptoms. Microscopic examination of leaf strips of infected V. faba revealed banded inclusions typical of the genus Potexvirus (1). Reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers for the genus Potexvirus (2) produced a 750-bp product that is the expected product for the genus Potexvirus. The RT-PCR product was cloned in pGem-T easy (Promega, Madison, WI) and sequenced. BLAST ( http://www.ncbi.nlm.nih.gov/ BLAST/ ) analysis of the sequence showed an 82% identity at the nucleotide level with the RNA polymerase gene of ClYMV. Leaf extracts of the original verbena, several inoculated hosts, and a known sample of ClYMV in N. benthamiana were tested in sodium dodecyl sulfate immunodiffusion (4) against antiserum to the degraded capsid protein of ClYMV. A reaction of identity was seen with infected samples but not with samples for comparable virus-free plants. To our knowledge, this is the first time this virus has been found in the eastern United States. It is not known how or if the presence of this noninsect transmitted virus in an ornamental will affect the agricultural, forage, or ornamental industries in the east or how widely distributed Verbena sp. infected with this virus may be at this time. References: (1.) R. G. Christie and J. R. Edwardson. Fla. Agric. Exp. Stn. Monogr. 9, 1994. (2.) A. Gibbs et al. J. Virol. Methods 74:67,1998 (3.) M. J. Pratt. Can. J. Bot. 39:655. 1961. (4) D. E. Purcifull and D. L. Batchelor. Fla. Agric. Ext. Stn. Bull. 788, 1977.

Use of Tomato yellow leaf curl virus (TYLCV) Rep Gene Sequences to Engineer TYLCV Resistance in Tomato.

ABSTRACT Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus (family Geminiviridae), causes severe losses in tomato production in the tropics and subtropics. In order to generate engineered resistance, eight different constructs of the TYLCV replication-associated protein (Rep) and C4 gene sequences were tested in transformed tomato inbred lines. Transgenic plants were screened for resistance to TYLCV using viruliferous whiteflies. No symptoms were observed and no TYLCV genomic DNA was detected by both hybridization and polymerase chain reaction in progenies of plants transformed with three constructs. This resistance was observed in plants that contained one of the following transgenes: 2/5Rep (81 nucleotides [nt] of the intergenic region [IR] plus 426 nt of the 5' end of the TYLCV Rep gene), Delta2/5Rep (85 nt of the IR plus 595 nt of the 5' end of the TYLCV Rep gene in the antisense orientation), and RepDelta2/5Rep (81 nt of the IR, the entire Rep gene, and 41 nt 3' to the end of the Rep gene fused to Delta2/5Rep). Our study differs from other transgenic Geminivirus resistance reports involving the Rep gene in that viruliferous whiteflies were used for challenge inoculation instead of agroinoculation or biolistic inoculation, and TYLCV resistance was evaluated under field conditions.

Two Newly Described Begomoviruses of Macroptilium lathyroides and Common Bean.

ABSTRACT Macroptilium lathyroides, a perennial weed in the Caribbean region and Central America, is a host of Macroptilium yellow mosaic Florida virus (MaYMFV) and Macroptilium mosaic Puerto Rico virus (MaMPRV). The genomes of MaYMFV and MaMPRV were cloned from M. lathyroides and/or field-infected bean and the DNA sequences were determined. Cloned A and B components for both viruses were infectious when inoculated to M. lathyroides and common bean. Comparison of the DNA sequences for cloned A and B components with well-studied begomovirus indicated that MaMPRV (bean and M. lathyroides) and MaYMFV (M. lathyroides) are unique, previously undescribed begomo-viruses from the Western Hemisphere. Phylogenetic analysis of viral A components indicated that the closest relative of MaYMFV are members of the Bean golden yellow mosaic virus (BGYMV) group, at 76 to 78% nucleotide identity, whereas the closest relative for the A component of MaMPRV was Rhynchosia golden mosaic virus at 78% nucleotide identity. In contrast, BGYMV is the closest relative for the B component of both MaYMFV and MaMPRV, with which they share approximately 68.0 and approximately 72% identity, respectively. The incongruent taxonomic placement for the bipartite components for MaMPRV indicates that they did not evolve entirely along a common path. MaYMFV and MaMPRV caused distinctive symptoms in bean and M. lathyroides and were transmissible by the whitefly vector and by grafting; however, only MaYMFV was mechanically transmissible. The experimental host range for the two viruses was similar and included species within the families Fabaceae and Malvaceae, but only MaYMFV infected Malva parviflora and soybean. These results collectively indicate that MaMPRV and MaYMFV are new, previously undescribed species of the BGYMV group, a clade previously known to contain only strains and isolates of BGYMV from the Caribbean region that infect Phaseolus spp. Both MaYMFV and MaMPRV may pose an economic threat to bean production in the region.

Serological Studies Using Polyclonal Antisera Prepared Against the Viral Coat Protein of Four Begomoviruses Expressed in Escherichia coli.

Polyclonal rabbit antisera were produced to the coat protein of Bean golden mosaic virus Brazil isolate (BGMV), Cabbage leaf curl virus (CabLCV), Tomato yellow leaf curl virus (TYLCV), and Tomato mottle virus (ToMoV), all expressed in Escherichia coli by the pETh expression vector. The expressed coat protein of each virus was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for use as an immunogen. The antisera to BGMV, CabLCV, TYLCV, and ToMoV reacted in indirect (plate-trapping) enzyme-linked immunosorbent assay (ELISA) with extracts from begomovirus-infected tissue. The antisera to BGMV, CabLCV, TYLCV, and ToMoV also reacted specifically with the test begomovirus antigens in leaf imprint blots and Western blots. The CabLCV and TYLCV antisera were used to detect Bean golden yellow mosaic virus antigens by immunogold labeling of thin sections of infected bean tissues. In tissue blot immunoassays, the TYLCV antiserum reacted well with TYLCV antigens but not with ToMoV antigens, while CabLCV antiserum reacted well with ToMoV antigens and weakly with TYLCV antigens. The results indicate that polyclonal antisera prepared to expressed begomovirus coat proteins were useful for the detection of begomoviruses in an array of assays.

Molecular characterization and coat protein serology of watermelon leaf mottle virus (Potyvirus).

A cDNA library was generated from purified RNA of watermelon leaf mottle virus (WLMV) (Genus Potyvirus). Two overlapping clones totaling 2,316 nucleotides at the 3' terminus of the virus were identified by immunoscreening with coat protein antiserum. The sequence analyses of the clones indicated an open reading frame (ORF) of 2,050 nucleotides which encoded part of the replicase and the coat protein, a 243-nucleotide non-coding region (3'UTR), and 23 adenine residues of the poly (A) tail. The taxonomic status of WLMV was determined by comparisons of the sequence of the cloned coat protein gene and 3'UTR with potyvirus sequences obtained from GenBank. The nucleotide sequence identities of WLMV compared with 17 other potyviruses ranged from 55.6 to 63.5% for the coat protein, and from 37.2 to 48.3% for the 3'UTR. Phylogenetic analyses of the coat protein region and the 3'UTR indicated that WLMV did not cluster with other potyviruses in a clade with high bootstrap support. The coat protein gene was expressed in Escherichia coli and a polyclonal antiserum was prepared to the expressed coat protein. In immunodiffusion tests, WLMV was found to be serologically distinct from papaya ringspot virus type W, watermelon mosaic virus 2, zucchini yellow mosaic virus, and Moroccan watermelon mosaic virus. In Western blots and ELISA, serological cross-reactivity with other cucurbit potyviruses was observed. Serological and sequence comparisons indicated that watermelon leaf mottle virus is a distinct member of the Potyvirus genus.

Tobacco plants transformed with a modified coat protein of tomato mottle begomovirus show resistance to virus infection.

ABSTRACT Tobacco plants (Nicotiana tabacum 'Xanthi') were transformed with a binary vector containing the coat protein gene of tomato mottle begomo-virus (ToMoV) modified by the deletion of 30 nucleotides in the 5' end. The R(1) generation was screened for resistance to ToMoV by inoculation with viruliferous whiteflies. Fifteen days after inoculation, symptom development was recorded weekly for up to 120 days using a visual scale, and ToMoV infection was confirmed by polymerase chain reaction and enzyme-linked immunosorbent assay. The response to high inoculation levels of ToMoV varied and ranged from susceptibility to immunity. The transgene transcript was detected by northern blot analysis; however, the transgene product could not be detected by protein blot analysis using antisera reactive with ToMoV coat protein. The lack of detection of the transgene product in resistant plants suggests that it is not involved in eliciting the resistance response and that resistance may be mediated by the transgene transcript.

First Report of Tomato Yellow Leaf Curl Virus in Tomato in South Georgia.

In October 1998, symptoms characteristic of tomato yellow leaf curl virus (TYLCV) were observed on fresh market tomato (Lycopersicon esculentum Mill.) in four production fields, two in Decatur County, Georgia, and two in Gadsden County, Florida. Symptoms observed were plant stunting, reduced leaf size, yellow leaf margins, and mottling. The incidence of symptomatic plants was less than 1% in all fields examined. In most cases, symptoms were observed only on the upper portion of plants, suggesting these plants had been infected by secondary spread from an unknown source. Nuclear inclusions characteristic of geminiviruses were observed by light microscopy in leaf tissue from symptomatic plants (1). To identify the geminivirus, total DNA from infected plants was extracted from six symptomatic tomato plants (two from Georgia and four from Florida) for polymerase chain reaction (PCR; J. E. Polston, personal communication). DNA was amplified with geminivirus DNA A degenerate primer set PAL1v1978 and PAR1c496 (2) from these extracts in addition to extracts from a known TYLCV-infected, a tomato mottle virus (ToMoV)-infected, and a healthy tomato plant. A PCR product of 1.4 kb was obtained from plants with TYLCV-like symptoms, while a 1.4-kb product and a 1.1-kb product were obtained from extracts of the known TYLCV-infected and ToMoV-infected tomato plants, respectively. No PCR product was obtained from extracts of healthy tomato plants. The 1.4-kb PCR products from one Georgia sample and one Florida sample were compared with those of TYLCV and ToMoV by restriction enzyme (RE) digestion with EcoRI and ClaI. The RE pattern of the 1.4-kb fragment from both samples was identical to the RE pattern of TYLCV and different from that of ToMoV. Adult and immature whiteflies collected from the fields where TYLCV was found were identified as Bemisia tabaci, the vector of TYLCV, but the biotype was not established. This report of TYLCV in south Georgia and north Florida extends the geographic range of TYLCV in the U.S. northward approximately 100 km. Georgia is the second state in which TYLCV was found since its initial detection in south Florida in July 1997 (J. E. Polston, personal communication). Monitoring of silverleaf whitefly populations and detection of TYLCV on alternate hosts will continue in order to estimate the potential impact of this virus on south Georgia and north Florida agriculture. References: (1) R. G. Christie and J. R. Edwardson. Plant Dis. 70:273, 1986, (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Asymmetric infectivity of pseudorecombinants of cabbage leaf curl virus and squash leaf curl virus: implications for bipartite geminivirus evolution and movement.

The bipartite geminiviruses squash leaf curl virus (SqLCV) and cabbage leaf curl virus (CLCV) have distinct host ranges. SqLCV infects a broad range of plants within the Cucurbitaceae, including pumpkin and squash, and CLCV has a broad host range within Brassicaceae that includes cabbage and Arabidopsis thaliana. Despite this, the genomic A components of these viruses share a high degree of sequence identity, particularly in the gene encoding the replication protein AL1, and their common regions are 77% identical. However, there is unexpected sequence diversity in the common regions of the two CLCV genomic A and B components, these being only 80% identical. Based on these sequence similarities, we investigated the host range properties of pseudorecombinants of SqLCV and CLCV. We found that in a pseudorecombinant virus consisting of the A component of CLCV and the B component of SqLCV, both components replicated in tobacco protoplasts, and this pseudorecombinant was infectious and caused systemic disease in Nicotiana benthamiana, a common host to all bipartite geminiviruses. However, this pseudorecombinant did not move systemically in pumpkin or Arabidopsis, despite the demonstrated replication compatibility of the genome components. As a result of the greater sequence differences between the common regions, the pseudorecombinant of SqLCV A and CLCV B components neither replicated the CLCV B component nor systemically infected any of the hosts tested. These findings demonstrate that for different geminiviruses with distinct host ranges, the replication origins and AL1 proteins can be sufficiently similar to permit infectious pseudorecombinants, but replication alone is not sufficient to cause systemic disease, and host range may ultimately be limited at the level of movement. The results of this study further suggest that CLCV is an evolving virus that can provide insights into how new bipartite geminiviruses arise from mixed infections.

The nucleotide sequence of the 3'-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production.

A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA. The sequence of the 3'-terminal 3158 nucleotides, which consisted of the 3'-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57-68% similar at the nucleotide level and 72-82% similar at the amino acid level when compared with other potyviruses. Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus. A recombinant DsMV-Ch CP (approximately 39 kDa) expressed in E. coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA.

Partial Characterization of a Distinct Potyvirus Isolated from Watermelon in Florida.

Conspicuous, unusual nuclear inclusions in stained epidermal strips of leaves implicated a virus (designated isolate 2932) as the cause of foliar mosaic in a watermelon plant (Citrullus lanatus) received for analysis from South Florida in 1990. In greenhouse tests, mechanically inoculated plants of Cucurbita pepo (Small Sugar pumpkin and Early Prolific Straightneck squash) and watermelon (Crimson Sweet) developed mosaic or mottle symptoms. Isolate 2932 caused foliar symptoms in 16 cultivars of Cucurbita pepo, including Freedom II and Prelude II, and in six cultivars of watermelon. None of five cultivars of melon (Cucumis melo) or 11 cultivars of cucumber (Cucumis sativus) developed consistent, distinctive symptoms, but all of these cultivars were systemically infected based on back-inoculations to squash. No systemic infection of mechanically inoculated plants of 25 species representing 13 noncucurbitaceous plant families was detected. Crystalline nuclear inclusions, cytoplasmic amorphous inclusions, and cytoplasmic cylindrical inclusions were detected by light and electron microscopy in leaf tissues of infected squash and watermelon. Electron microscopy of squash leaf extracts revealed filamentous particles, and 86% of 159 particles measured ranged from 800 to 890 nm in length. The virus was transmitted in a nonpersistent manner by Myzus persicae from squash to squash in two of three trials. Immunodiffusion tests with polyclonal antisera prepared to partially purified 2932 or its capsid protein showed that the isolate was antigenically different from papaya ringspot virus type W, watermelon mosaic virus 2, and zucchini yellow mosaic virus. In limited testing of field samples of squash and watermelon since 1990, no additional isolates of the 2932 type have been found. The characteristics of isolate 2932 obtained thus far indicate that it is a distinct potyvirus. It is tentatively named watermelon leaf mottle virus to distinguish it from other potyviruses commonly isolated from cucurbits in Florida.

Location of Geminiviruses in the Whitefly Bemisia tabaci (Homoptera: Aleyrodidae).

The location of tomato mottle virus (ToMoV) and cabbage leaf curl virus (CabLCV) (Geminiviridae, genus Begomovirus) in the whitefly vector Bemisia tabaci B-biotype (Homoptera: Aleyrodidae) was elucidated using a novel technique incorporating indirect immunofluorescent labeling in freshly dissected whiteflies. Begomoviruses were visualized in the whitefly by indirect-fluorescent-microscopy. Polyclonal and monoclonal primary antibodies were used to successfully detect both ToMoV and CabLCV. Both begomoviruses were located in the anterior region of the midgut and filter-chamber of adult whiteflies, with ToMoV detected in the salivary glands. CabLCV was detected at a greater frequency than ToMoV, with a positive detection of 16% (89 out of 560) for CabLCV and 3% (25 out of 840) for ToMoV. Possible sites involved in geminivirus transport from the gut lumen of whiteflies into the hemocoel were located in the filter-chamber and anterior portion of the midgut. The location of these begomoviruses suggests a possible scenario of virus movement through the whitefly, which is discussed.

Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins.

Tobacco plants were transformed with the movement protein (pathogenicity) gene (BC1) from tomato mottle geminivirus (TMoV), using Agrobacterium-mediated transformation. Different transgenic tobacco lines that expressed high levels of the BC1 protein had phenotypes ranging from plants with severe stunting and leaf mottling (resembling geminivirus symptoms) to plants with no visible symptoms. The sequence data for the BC1 transgene from the transgenic plants with the different phenotypes indicated an association of spontaneously mutated forms of the BC1 gene in the transformed tobacco with phenotype variations. One mutated transgene associated with an asymptomatic phenotype had a major deletion at the C terminus of 119 amino acid residues with a recombination resulting in the addition of 26 amino acid residues of unidentified origin. This asymptomatic, mutated BC1 attenuated the phenotypic expression of the symptomatic BC1 in a tobacco line containing both copies of the BC1 gene. Another mutated form of the BC1 gene amplified from an asymptomatic, multicopy transgenic tobacco plant did not induce symptoms when transiently expressed in tobacco via a virus vector. The symptom attenuation in the transgenic tobacco by the asymptomatic BC1 may involve trans-dominant negative interference.

Characterization of the P1 protein and coding region of the zucchini yellow mosaic virus.

The nucleotide sequence of the 5'-terminal P1 coding region of an aphid-transmissible isolate of zucchini yellow mosaic virus (ZYMV; strain FL/AT), a mild isolate (strain MD) and a severe isolate (strain SV), all from Florida, were compared with two other ZYMV isolates. The ZYMV MD and SV isolates and an isolate from California (ZYMV CA) had 95-98% sequence similarities to FL/AT, whereas an isolate from Reunion Island (ZYMV RU) had a 60% sequence similarity to FL/AT. ZYMV MD had an 18 nucleotide insert following the start codon of the P1 coding region. The P1 proteins of all ZYMV isolates shared conserved amino acids in areas of the C terminus similar to those reported for other potyviruses. Polyclonal antisera were prepared to the P1 proteins of ZYMV FL/AT and RU expressed in Escherichia coli. The FL/AT and RU P1 antisera showed varying degrees of reactivity in Western blots with extracts of pumpkin (Cucurbita pepo L.) singly infected with a number of distinct ZYMV isolates. The reaction of the FL/AT P1 antiserum with isolate RU-infected tissue extracts was very weak compared to the homologous reaction. Neither antiserum reacted with extracts from plants singly infected with three other potyviruses, a potexvirus, or a cucumovirus. The P1 proteins of ZYMV isolates ranged in molecular mass from 33 kDa to 35 kDa. The P1 protein of strain MD was larger (35 kDa) than that of FL/AT (34 kDa). Indirect immunofluorescence tests with FL/AT P1 antiserum indicated that the P1 protein aggregates in ZYMV-infected tissues. The antisera to the ZYMV P1 proteins have potential as serological probes for identifying ZYMV and for distinguishing ZYMV isolates by immunoblotting.

The nucleotide sequence of tomato mottle virus, a new geminivirus isolated from tomatoes in Florida.

A new geminivirus, tomato mottle virus (TMoV), affecting tomato production in Florida has been cloned and sequenced. Sequence analysis of the cloned replicative forms of TMoV revealed four potential coding regions for the A component [2601 nucleotides (nt)] and two for the B component (2541 nt). Comparisons of the nucleotide sequence of the TMoV genome with those of other whitefly-transmitted geminiviruses indicate that TMoV is a typical bipartite geminivirus of the New World and is closely related to but distinct from abutilon mosaic virus.

A comparison of pepper mottle virus with potato virus Y and evidence for their distinction.

Pepper mottle virus (PepMOV) was identified as a distinct potyvirus infecting peppers in Arizona and Florida in the 1970's. The distinction of PepMoV from potato virus Y (PVY) has recently been challenged on the basis of sequence comparisons of the coat proteins and of the 3' nontranslated regions of the viral RNAs. We summarize the biological, cytological, serological, and in vitro translational studies which compare the apparent differences, and also similarities, between PepMoV and PVY. We conclude that although PepMoV may be more closely related to PVY than to other known potyvirus, PepMoV should be maintained as a separate virus on the basis of its distinctive characteristics.

Serological relationships involving potyviral nonstructural proteins.

This report represents a compilation of many of the publications on antigenic properties of potyviral-specified nonstructural proteins. Polyclonal antisera have been prepared for use in characterization of six nonstructural proteins. These include antisera to the cylindrical inclusion proteins of at least 28 potyviruses, to small nuclear inclusion protein (protease) of four potyviruses, to large nuclear inclusion protein (putative replicase) of three viruses, helper component-protease or amorphous inclusion protein of at least four viruses, to the P1 protein (located at the N-terminus of the polyprotein) of one virus, and to the P3 protein (located between helper component protease and cylindrical inclusion protein) of one virus. Monoclonal antibodies also have been prepared to several of these nonstructural proteins. The evidence thus far indicates that cylindrical inclusions of different potyviruses have both conserved and unique epitopes. Nuclear inclusion proteins and amorphous inclusion proteins also may have conserved and unique epitopes. Antigenic relationships of potyviral nonstructural proteins have potential for the identification and classification of potyviruses.

The nucleotide sequences of the 3'-terminal regions of papaya ringspot virus strains W and P.

The sequences of cDNA clones encoding most of the NIb protein, the coat protein and the 3' untranslated region of papaya ringspot virus (PRV) strains W and P have been determined. The open reading frame of P strain PRV was confirmed by amino acid analysis. Nucleotide sequence comparisons of these strains show that they share a 98.2% identity in their NIb gene regions and a 97.7% identity in their coat protein genes. The sequences of these two strains are distinct from other potyvirus types, confirming their classification as two strains of the same virus. The NIb amino acid sequence possesses conserved amino acids characteristic of RNA-dependent RNA polymerases. Comparison of the coat protein amino acid sequence with those of other potyviruses shows perfectly conserved amino acids which may have functional significance.

Complementary DNA cloning and expression of the papaya ringspot potyvirus sequences encoding capsid protein and a nuclear inclusion-like protein in Escherichia coli.

Three cDNA clones that express viral gene products in Escherichia coli JM83 were derived from a watermelon mosaic virus-1 strain of papaya ringspot virus (PRSV-W). DNAs complementary to portions of the viral RNA were inserted into the pUC8 and pUC9 plasmids, and the expressed polypeptides were fusion products with the amino terminus of beta-galactosidase. Clones W1-77 and W2-1 expressed fusion products with apparent molecular weights of 40,000 (40K) and 14K, respectively, which were serologically related to PRSV capsid protein. A 52K product serologically related to a 54K nuclear inclusion protein of tobacco etch virus was produced by clone W1-18. The sequences encoding the capsid and 57K nuclear inclusion-like proteins of PRSV were physically mapped to adjacent positions through Southern blot analyses of clones W1-77 and W1-18.