The First Report of a DNA Satellite (Geminivirus͞tomato Leaf Curl Virus͞dna Satellite)
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Proc. Natl. Acad. Sci. USA Vol. 94, pp. 7088–7093, June 1997 Plant Biology A novel subviral agent associated with a geminivirus: The first report of a DNA satellite (geminivirusytomato leaf curl virusyDNA satellite) IAN B. DRY*, LESLIE R. KRAKE,JUSTIN E. RIGDEN†, AND M. ALI REZAIAN Division of Horticulture, Commonwealth Scientific and Industrial Research Organization, Hartley Grove, Urrbrae, South Australia 5064, Australia Communicated by George Bruening, University of California, Davis, CA, March 31, 1997 (received for review November 4, 1996) ABSTRACT Numerous plant RNA viruses have associ- associated with a geminivirus. Geminiviruses are a group of ated with them satellite (sat) RNAs that have little or no plant viruses characterized by their twin-shaped isometric nucleotide sequence similarity to either the viral or host particles; these viruses are responsible for major crop losses genomes but are completely dependent on the helper virus for worldwide. Their genomes consist of either one or two circular replication. We report here on the discovery of a 682-nt single-stranded DNA components of approximately 2.5–3.0 kb circular DNA satellite associated with tomato leaf curl gemi- in size (6). Tomato leaf curl virus (TLCV) belongs to gemi- nivirus (TLCV) infection in northern Australia. This is the nivirus subgroup III, the members of which are characterized first demonstration that satellite molecules are not limited to by either monopartite or bipartite genomes, transmission by RNA viral systems. The DNA satellite (TLCV sat-DNA) is the whitefly Bemisia tabaci, and by the infection of dicotyle- strictly dependent for replication on the helper virus replica- donous hosts. tion-associated protein and is encapsidated by TLCV coat Small subgenomic DNA species are often associated with protein. It has no significant open reading frames, and it geminivirus infection (7). These DNAs are derived by a partial shows no significant sequence similarity to the 2766-nt helper- deletion of the wild-type viral genome and, as such, show a virus genome except for two short motifs present in separate high degree of sequence homology to the helper virus. In putative stem–loop structures: TAATATTAC, which is uni- contrast, the small circular DNA molecule found to be asso- versally conserved in all geminiviruses, and AATCGGTGTC, ciated with TLCV infection (which we describe here) is not which is identical to a putative replication-associated protein derived from the helper virus genome, and it displays charac- binding motif in TLCV. Replication of TLCV sat-DNA is also teristics analogous to the viral RNA satellites. This type of supported by other taxonomically distinct geminiviruses, in- molecule has not been found to be associated with any other cluding tomato yellow leaf curl virus, African cassava mosaic geminivirus or, indeed, any other plant, animal, or bacterial virus, and beet curly top virus. Therefore, this unique DNA DNA virus. satellite does not appear to strictly conform with the require- ments that dictate the specificity of interaction of geminiviral MATERIALS AND METHODS replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication. Construction of Virus Clones. Construction of infectious clones of the geminiviruses TLCV, TLCV-D1 strain, tomato Subviral agents, viroids, and satellites are well known in yellow leaf curl virus (TYLCV)-Sardinian strain, African infectious RNA systems (1–4). Apart from viroids, which are cassava mosaic virus (ACMV) (A and B), and beet curly top autonomously replicating, numerous plant RNA viruses (1, 2) virus (BCTV) have been described previously (8–12). TLCV have associated with them RNA satellites that show little or no mutant constructs were produced as described in Rigden et al. nucleotide sequence similarity to the viral or host genomes, but (13). which are completely dependent on the helper virus for Cloning and Manipulation of TLCV sat-DNA (DNA-2). replication. Unlike satellite viruses, which encode the genetic DNA-2 was purified from TLCV-infected tomato material as information for their own coat protein, RNA satellites are described previously for TLCV (8) but included a treatment packaged in coat protein encoded by the helper virus. Thus, with RNase A. Purified DNA-2 was treated with Klenow particles containing RNA satellites are antigenically indistin- fragment in the presence of random decamers (Bresatec, guishable from those of the helper virus. Adelaide, Australia) to convert any single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA) and digested with Both linear and circular satellites are found to be associated RsaI. The two resultant fragments (150 and 532 nt) were with plant RNA viruses (4). Some satellites have been found cloned into EcoRV-cut Bluescript SK1 (Strategene) and se- to exacerbate viral symptoms or induce symptoms quite dis- quenced. A unique NcoI site was identified within the se- tinct from the those induced by the helper virus alone (2). quence and was used to reclone the same DNA. Sequence However, a number of them interfere with helper virus comparison of the NcoI clone with the RsaI clones confirmed replication and ameliorate disease expression, which has led to it to be a full length circular monomer of DNA-2. considerable interest into the investigation of their potential as Site-directed mutagenesis of TLCV sat-DNA was carried sources of viral resistance (5). out using an Altered Sites kit (Promega). Wild-type and More than 30 RNA viruses are currently recognized as having one or more satellites associated with them (2). How- ever, to date, similar DNA species associated with DNA Abbreviations: TLCV, tomato leaf curl virus; TYLCV, tomato yellow leaf curl virus: ACMV, African cassava mosaic virus; BCTV, beet curly viruses have not been recorded. We now report on the top virus; ds, double-stranded; ss, single-stranded; Rep, replication- isolation and characterization of a small, circular DNA satellite associated protein; ORF, open reading frame. Data deposition: The sequence reported in this paper has been The publication costs of this article were defrayed in part by page charge deposited in the GenBank data base (accession no: U74627). *To whom reprint requests should be addressed. e-mail: ian.dry@adl. payment. This article must therefore be hereby marked ‘‘advertisement’’ in hort.csiro.au. accordance with 18 U.S.C. §1734 solely to indicate this fact. †Present address: R.W. Johnson Pharmaceutical Research Institute, © 1997 by The National Academy of Sciences 0027-8424y97y947088-6$2.00y0 PO Box 3331, Sydney 2001, Australia. 7088 Downloaded by guest on September 29, 2021 Plant Biology: Dry et al. Proc. Natl. Acad. Sci. USA 94 (1997) 7089 mutated TLCV sat-DNA constructs were cloned into pBin19 as either NcoI dimers or NcoIySpeI 1.3-mers and then intro- duced into Agrobacterium tumefaciens (strain C58) by electro- poration. Whole Plant Infectivity Assays. Agroinoculation was per- formed with an overnight culture of A. tumefaciens containing tandem–repeat constructs in pBin19 (8). Bacterial cultures bearing TLCV- and TLCV sat-DNA- encoding plasmids were mixed in equal proportions for coagroinoculation experiments. New developing leaves were sampled 21–28 days postinocula- tion, and the presence of replicative DNA forms was detected either by dot–blot analysis (14) or Southern blot analysis as described below. Leaf Strip Transient Assay. The transient replication assay was modified as described in ref. 15. Leaf strips (1–2 mm) cut from in vitro grown tobacco plants (Nicotiana tabacum cv. Samsun) were cocultivated with A. tumefaciens on 0.7% bacto- FIG. 1. Isolation and characterization of a circular 682-nt DNA agar plates containing culture medium (0.43% Murashige and molecule associated with TLCV infection. Ethidium bromide-stained Skoog basal salt mixture, 3% sucrose, 0.01% Gamborg vita- 1.2% agarose gel showing the presence of two DNA species (arrows) in TLCV-infected plant material. mins, 0.0001% benzyladenine, 0.00001% naphthaleneacetic acid) in the dark at 25°C. After 48 h, strips were transferred to molecule of 682 nt. This clone detected replicative forms of 25 ml of culture medium supplemented with 0.025% kanamy- DNA-2, but not TLCV, in TLCV-infected tomato tissues (Fig. cin and 0.05% cefotaxime and incubated for 6 days at 25°C in 2, lane 2). No hybridization was observed with this clone normal room light with gentle agitation at 50 rpm. Strips (150 against total nucleic acid extracted from healthy plants (Fig. 2, mg) were blotted dry and frozen in liquid N2 prior to DNA extraction. lane 1). Both double-stranded (ds) and single-stranded (ss) Extraction and Analysis of DNA. Total nucleic acid was forms of DNA-2 were found to be present in infected tissues extracted as described previously (8) but included a treatment (Fig. 2 compare lanes 2–4). with RNase A. Inoculation sites were excised from the stem as The complete sequence of the 682-nt DNA-2 molecule is described (16). DNA species were separated (5 mgylane) in shown in Fig. 3. Global sequence alignment of DNA-2 with 1.8% agarose gels containing 0.5 mgzml21 ethidium bromide to TLCV using the program GAP did not reveal any extensive facilitate separation of the ssDNA and dsDNA forms. Unless similarity, confirming that it is not a subgenomic DNA species otherwise stated, DNA was blotted and hybridized with 32P- of TLCV. Examination of the DNA-2 sequence did, however, labeled DNA probes as described (13). reveal the presence of a putative stem–loop structure formed Immunocapture PCR. Datura (Datura stramonium L.) by 11-nt GC-rich inverted repeats between nt 670-680 and nt leaves were extracted in 10 vol of 50 mM TriszHCl (pH 8.0), 5 10-20 containing the loop sequence TAATATTAC. This mM EDTA, 2% polyvinylpyrrolidone, and 0.05% Tween 20 by nonanucleotide sequence element, within a putative stem–loop grinding and centrifugation at 13,000 3 g for 15 min at 4°C.