USDA, Agriculture Research Service, Fruit Laboratory, Bldg 010A, BARC-West, 10300 Baltimore Avenue, Beltsville, MD 20705, USA1
Author for correspondence: John Hartung. Fax +1 301 504 5062. e-mail hartungj{at}ba.ars.usda.gov
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Abstract |
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Introduction |
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Ahlawat et al. (1996a) provided a partial characterization of Citrus yellow mosaic virus (CYMV). The virus was transmitted by grafting and dodder to 14 citrus species and cultivars, including sweet orange, pummelo, Rangpur lime, Volkamer lemon and sour orange, but was not transmitted to Mexican lime. It was also mechanically transmitted to Citrus decumana, a native of India, Satgudi sweet orange and pummelo (Ahlawat et al., 1996b
). The authors were unable to transmit CYMV using either aphids or mealybugs (Ahlawat et al., 1996a
, b
). CYMV is a proposed member of the newly established family Caulimoviridae, genus Badnavirus (Pringle, 1998
), based on its serological relationship with other badnaviruses and PCR amplification using degenerate primers designed from conserved badnavirus sequences (Ahlawat et al., 1996b
).
Badnaviruses affect a wide range of tropical plant species, including economically important crops, such as banana, citrus, cacao, sugarcane, rice and yam. They are characterized by non-enveloped bacilliform particles (30x120150 nm), which contain a circular dsDNA genome of 7·17·6 kb (Lockhart & Olszewski, 1999 ). The genomes of five badnaviruses, including Commelina yellow mottle virus (ComYMV) (Medberry et al., 1990
), the type species of the genus, Sugarcane bacilliform virus (SCBV) (Bouhida et al., 1993
), Cacao swollen shoot virus (CSSV) (Hagen et al., 1993
), Banana streak virus (BSV) (Harper & Hull, 1998
) and Dioscorea bacilliform virus (DBV) (Briddon et al., 1999
) have been cloned and sequenced, but only the cloned genomes of ComYMV, SCBV and CSSV were shown to be infectious (Bouhida et al., 1993
; Jacquot et al., 1999
; Medberry et al., 1990
). The sequence of Rice tungro bacilliform virus (RTBV), a former member of the genus Badnavirus but now the type species of the new RTBV-like viruses genus within the family Caulimoviridae (Pringle, 1998
), has also been published (Hay et al., 1991
; Qu et al., 1991
). The genomes of all badnaviruses have similar genome organization and contain three open reading frames (ORFs), ORFs 13, capable of encoding proteins with a molecule mass greater than 10 kDa (except CSSV, which has two extra ORFs, designated X and Y, overlapping ORF 3). All of the ORFs are present on the plus-strand of the genome. ORFs 1 and 2 encode putative proteins of unknown function, although the C terminus of the ORF 2 product of CSSV has nucleic acid-binding activities for both dsDNA and ssRNA (Jacquot et al., 1996
). ORF 3 encodes a polyprotein that is cleaved post-translationally by the viral aspartic protease to produce a virus movement protein, a coat protein, the aspartic protease itself and a replicase comprising reverse transcriptase and ribonuclease H (RNase H) (Bouhida et al., 1993
; Hohn & Futterer, 1997
).
Like caulimoviruses, RTBV, soybean chlorotic mottle-like viruses, Cassava vein mosaic virus (CsVMV) and Petunia vein clearing virus (PVCV), badnaviruses are considered to be plant pararetroviruses. The replication model proposed for them involves an initial synthesis of a larger than unit length terminally redundant transcript of the viral genomic DNA by the host DNA-dependent RNA polymerase (Hohn et al., 1985 ; Medberry et al., 1990
; Pfeiffer & Hohn, 1983
; Qu et al., 1991
). This transcript serves both as a polycistronic mRNA for translation to produce viral proteins and as a template for reverse transcription to replicate the viral genome by the virus-encoded replicase (Gowda et al., 1989
; Joshi, 1987
). The host cytosolic initiator methionine tRNA (tRNAmet) is thought to serve as the primer for the reverse transcriptase in the synthesis of minus-strand DNA (Hohn et al., 1985
; Pfeiffer & Hohn, 1983
). RNase H digests the RNA in the RNA/DNA hybrid, leaving one or more specific RNA fragments, possibly a purine-rich region, as might be the case in ComYMV (Medberry et al., 1990
). The reverse transcriptase uses such fragments to prime the synthesis of plus-strand DNA by acting as a DNA-dependent DNA polymerase (Hohn & Futterer, 1991
; Howell & Hull, 1978
).
Citrus is the most widely grown fruit crop in the world and generates approximately $10 billion of economic activity in the United States alone. Therefore, a better understanding of CYMV is of great importance in order to control and prevent the disease that it causes in India from becoming established in other parts of the world. As a first step in this direction, we cloned an isolate of CYMV and sequenced its genome. We analysed its nucleotide (nt) and deduced amino acid (aa) sequences and determined its phylogenetic relationship to other badnaviruses and RTBV. We found that CYMV is most closely related to CSSV. We also demonstrated that the cloned CYMV genome is infectious in sweet orange via Agrobacterium-mediated inoculation.
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Methods |
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Virus purification and nucleic acid extraction.
CYMV was partially purified from infected sweet orange leaves by the method of B. Lockhart (University of Minnesota, MN, USA; personal communication) with modifications. A sample of 100 g of symptomatic leaves were ground in liquid nitrogen and extracted with 2 vol. of 500 mM potassium phosphate buffer, pH 7·5, containing 1 M urea, 5% polyvinylpyrrolidone (Mr 40000) and 0·5 % Na2SO3. The extract was filtered through eight layers of cheesecloth and Triton X-100 was added to the filtrate to a final concentration of 2% (v/v) and mixed thoroughly. The mixture was centrifuged at 12000 g for 15 min. The supernatant was then layered over a 5 ml cushion of 30% sucrose in 100 mM potassium phosphate, pH 7·2, and centrifuged for 70 min at 148000 g in a Beckman 50·2 Ti rotor. The resulting pellet was resuspended in 100 mM potassium phosphate, pH 7·2, centrifuged twice in a microcentrifuge to remove particulate matter and passed through a second 5 ml cushion of 30% sucrose at 148000 g for 70 min. The pellet resuspended in 100 mM potassium phosphate, pH 7·2, constituted the partially purified virus preparation. To isolate virion DNA, the partially purified virus sample was first treated with RNase A (100 µg/ml) and DNase (30 U/ml) in 10 mM phosphate buffer, pH 7·2, for 30 min at 37 °C. The reaction was stopped by the addition of EDTA to a final concentration of 1 mM. Virions were disrupted by adding Proteinase K to a final concentration of 0·5 mg/ml with 0·5% SDS and incubating for 30 min at 37 °C. DNA was purified by phenolchloroform extraction followed by ethanol precipitation.
Cloning, subcloning and DNA sequencing.
We determined by endonuclease digestion and agarose gel electrophoresis that there was apparently only one KpnI site within the CYMV genome. Thus, we ligated CYMV DNA, digested with KpnI, into the KpnI site of pBluescript KS+ (pBS) (Stratagene) to construct the genomic clones. Genomic subclones were produced by cloning the desired restriction fragments taken directly from the full-length CYMV clone into pBS. The subclones were then sequenced at the Interdisciplinary Center for Biotechnology Research (University of Florida, FL, USA).
Analysis of sequence data.
The web-based software available at eBioinformatics (http://www.bionavigator.com) was used to analyse the DNA sequence, as well as to deduce and analyse amino acid sequence data. Sequence similarities between CYMV and other badnaviruses were evaluated using the Gap program in the GCG package (Genetics Computer Group) (Devereux et al., 1984 ). Database searches were carried out using FastA (Pearson & Lipman, 1988
), tBlastn and BlastP (Altschul et al., 1997
). Phylogenetic relationships among badnaviruses were estimated using the Phylogeny Analysis program, a macro created by eBioinformatics. Nucleotide or amino acid sequences were first aligned using CLUSTAL W (fast) (Thompson et al., 1994
). The phylogenies were then estimated using the neighbour-joining method from a distance matrix generated by DNAdist or Protdist and unrooted phylogenetic trees were plotted by DrawTree using the PHYLIP software package (Felsenstein, 1989
). To estimate the confidence placed in the phylogenetic trees, bootstrap sets of resampled alignments (1000 replicates) were generated by Seqboot and a consensus tree was obtained by Consense in the PHYLIP package of programs.
Construction of an infectious clone.
A 1·4 genome length copy of CYMV was cloned into the binary vector pBI101.2 (Clontech). The BamHI/KpnI fragment of pCYMV, the full-length CYMV clone, was first cloned into pUC18 to create pBK. The SalI/KpnI fragment of pBK and the KpnI/XbaI fragment of pCYMV were then ligated together with pBI101.2 to create pBICYMV, which was then transformed into Agrobacterium tumefaciens C58C1 by the freezethaw method (An et al., 1988 ). A. tumefaciens C58C1 transformants containing pBICYMV and pBI101.2 only were selected on LB plates containing kanamycin (50 µg/ml).
Agrobacterium-assisted inoculation.
To introduce pBICYMV into plants, stems of 2-year-old sweet orange seedlings were wounded by three sets of 20 stem slashes using a disposable scalpel. Ten leaves of each plant were also wounded with a needle press inoculation tool. Both the scalpel and the needle press had been dipped into a saturated culture of A. tumefaciens grown at 28 °C in LB medium for 16 h. A total of nine plants was inoculated with C58C1(pBICYMV) and eight plants with C58C1(pBI101.2).
Confirmation of CYMV infection after Agrobacterium-assisted inoculation.
(i) PCR-based detection.
We used PCR to determine if the CYMV sequence was present in inoculated plants. Total DNA from 0·2 g of inoculated plant leaves was extracted using a QIAamp Tissue kit (Qiagen) and one-fifth of the DNA preparation was used as a template. The two primers used were 5' CGCAGGCGAAAAGACAAA and 5' CCAGATGGCAAACAACTT, corresponding to CYMV nt 45044521 and 52305213, respectively. PCR conditions used were 1 cycle of 95 °C for 5 min, 40 cycles of 95 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min and 1 cycle of 72 °C for 10 min. PCR products were analysed by electrophoresis in 1% agarose gels stained with ethidium bromide.
(ii) Detection of virus particles.
Immunosorbent electron microscopy was used to detect virus particles in leaves of inoculated plants by the method of Lockhart et al. (1992) , modified as follows. Carbon-coated Formvar grids (SPI) were coated for 15 min with 25 µl of SCBV antiserum (0·1 mg/ml) and then rinsed with 30 drops of 10 mM sodium phosphate, pH 7·2. The grids were then floated for 2 h on 50 µl of a partially purified virus sample prepared from 1·25 g of leaves, as described above. Finally, the grids were rinsed with 20 drops of the same buffer and incubated for 5 min in 25 µl of 2% sodium phosphotungstate, pH 7·2.
(iii) Symptoms.
Inoculated plants were observed at regular intervals for yellow mosaic symptoms in the leaves. Symptoms were compared to those occurring on plants infected by graft-inoculation in the same greenhouse as well as to healthy plants and plants inoculated with the binary vector only.
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Results |
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Discussion |
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CYMV differs from other badnaviruses in that it contains three additional, relatively small ORFs (46 with predicted translation products of 21, 11 and 15 kDa, respectively). ORF 4 of CYMV and ORF X of CSSV are similarly located within their respective ORF 3 and both encode very basic proteins (pI=11·2 and 11·1, respectively), but no sequence homology was detected between the predicted translation products. ORF 5 of CYMV overlaps the middle of ORF 3 and encodes a highly basic protein with a pI of 11·1. It has no counterpart ORF in either other badnaviruses or RTBV and has no sequence similarity to any proteins by BlastP and tBlastn searches. Direct sequence comparison using the Gap program, however, revealed that it has limited similarity to ORFs 4 and 5 of CsVMV and ORF 8 of SbCMV. ORF 6 of CYMV overlaps the C-terminal region of ORF 3, as does ORF Y of CSSV, and they share 36% amino acid similarity. It remains unclear, without the construction of mutants and a test of their effects, whether the three additional ORFs are fortuitous or correspond to real viral genes that maybe unique to CYMV. This is especially the case with ORF 4. Without any experimental evidence, it is also hard to predict how these three additional ORFs are translated, if they ever are. Leaky scanning by the ribosome, splicing of the primary transcript, as found for the expression of RTBV ORFs (Futterer et al., 1994 , 1997
), or the use of subgenomic RNAs are among the possible mechanisms for the expression of ORFs 46 of CYMV.
Previously, Ahlawat et al. (1996a ) reported that CYMV was related serologically to CSSV, BSV, ComYMV, DBV and SCBV, but had the closest serological relationship to SCBV. Our phylogenetic analysis based on complete nucleotide sequences, however, revealed that CYMV is most closely related to CSSV and DBV rather than to SCBV. Our result is supported by a separate analysis using the deduced amino acid sequences from ORF 3, which is the most conserved ORF among all of the badnaviruses and which includes the viral coat protein sequence. A recent phylogenetic study by Geering et al. (2000)
based on the conserved region of the RNase H domain also showed that CSSV is more similar to DBV than to SCBV, indirectly supporting our conclusion. We do not know why there is a discrepancy between the phylogenetic relationship determined by serological methods and the one based both on the total genome sequence and on the amino acid sequence of ORF 3. It is possible that the viral coat protein or some other regions, such as an epitope of the coat protein, evolve at a different rate (Hearne et al., 1990
). The coat protein sequence is not very well conserved among badnaviruses, except for the cysteine-rich, zinc finger-like RNA-binding region with the consensus sequence CXCX2CX4HX4C. It is also unclear where the coat protein sequence begins and ends in ORF 3, so it is difficult to determine whether the phylogenetic relationship based on the coat protein sequence agrees with the serological results. On the other hand, the serological study was carried out non-reciprocally and was based solely on immunosorbent electron microscopy. Because this method is more qualitative than quantitative in nature, additional quantitative serological tests are needed to determine precisely the serological relationship between CYMV and other badnaviruses. We also found that CYMV is more similar to other badnaviruses than to RTBV, reflecting the fact that badnaviruses and RTBV are also biologically distinct (Harper & Hull, 1998
; Hibino, 1983
; Hull, 1996
).
Agrobacterium-mediated inoculation has been used successfully to deliver cloned viral genomes into mono- and dicotyledons and to induce virus infection (Grimsley & Bisaro, 1987 ; Grimsley et al., 1986
, 1987
). For plant pararetroviruses, clones used for such inoculations require a greater than genome length insert that gives rise to a larger than unit length terminally redundant transcript. This is important for virus infection (Bouhida et al., 1993
; Dasgupta et al., 1991
; Jacquot et al., 1999
; Medberry et al., 1990
). Using a similar approach, we demonstrated that the cloned CYMV genome was infectious in sweet orange. However, we noticed that symptom development is somewhat slower in our plants, as it took 5 months or longer for the symptoms to develop. When CYMV is transmitted by grafting, disease symptoms become apparent 4 months after transmission. This may reflect high virus titres in infected buds used for graft-inoculation. When CYMV was transmitted by mealybugs, approximately 6 months were required for disease symptoms to develop (J. Hartung, unpublished data). Disease symptoms also developed slowly when ComYMV was inoculated to its host plant Commelina diffusa via A. tumefaciens (Medberry et al., 1990
). Our successful delivery of an infectious CYMV clone to sweet orange will facilitate further molecular studies of this virus, by making possible the introduction of mutated viral genes into host plants to test the effect of mutations in planta.
The sequence information obtained from this study will be used to design primers specific to CYMV for quarantine purposes. It may also be useful to develop this dsDNA virus into a practical tool for plant transformation and to control the expression of foreign genes in citrus and/or other dicotyledonous woody plants.
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Acknowledgments |
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References |
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Received 17 April 2001;
accepted 29 June 2001.
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