CURRENT MICROBIOLOGY Vol. 43 (2001), pp. 457– 458 DOI: 10.1007/s002840010339
Current Microbiology An International Journal © Springer-Verlag New York Inc. 2001
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Genetic Characterization of a Putative Densovirus from the Mealybug Planococcus citri MyLo L. Thao,1 Susan Wineriter,2,3 Gary Buckingham,3 Paul Baumann1 1
Microbiology Section, University of California, Davis, CA 95616-8665, USA University of Florida, IFAS, Gainsville, Florida 32614, USA 3 Agricultural Research Service, USDA, Florida Biological Control Laboratory, P.O. Box 14700, Gainsville, Florida 32614-7100, USA 2
Received: 25 April 2001 / Accepted: 3 May 2001
Abstract. Total genomic DNA preparations from the citrus mealybug, Planococcus citri, contained a DNA band corresponding to 5.5 kilobases. This DNA was a linear molecule and was cloned into pUC18. Nucleotide sequence determination indicated that it was the replicative form of a densovirus, most closely related to the virus from Periplaneta fuliginosa (smokybrown cockroach).
In the course of a study of mealybug endosymbionts, DNA was prepared from Planococcus citri (citrus mealybug) reared in Florida on Cucurbita sp. (a Japanese squash) [6]. Agarose gel electrophoresis of this DNA preparation indicated that it was unusual in containing a prominent 5.5 kilobase (kb) DNA band (Fig. 1, lane b). This band was electroeluted from an agarose gel [1, 2] (Fig. 1, lane c) and subjected to restriction enzyme analysis. A 0.7 kb BamHI-KpnI DNA fragement (Fig. 2) was cloned into pBCKS (Stratagene, LaJolla, CA, USA) and sequenced. Comparisons of the open reading frame (ORF) with those deposited in databases indicated a relationship to the nonstructural protein of Densovirus. Members of this viral genus are found in a variety of insect orders but have not been reported in the Hemiptera [7]. The Densovirus genome consists of single stranded DNA of about 5– 6 kb in size, containing an inverted terminal repeat. The replicative form found in the host consists of double stranded linear DNA [7]. Potential frayed ends of the purified P. citri densovirus (PcDNV) DNA (Fig. 1, lane c) were filed in with the Klenow fragment [1] and the DNA ligated into SmaI digested and phosphatase pUC18 [1]. Digestion of the recombinant plasmid with SacI and HindIII (which are in the multiple cloning site on each side of the vector) gave a fragment of 5.5 kb (Fig. 1, lane d). The recombinant Correspondence to: Paul Baumann; email:
[email protected]
plasmid was digested with KpnI or BamHI and the fragments shown in Fig. 2, were cloned into KpnI or BamHI digested pBCKS and the DNA sequence obtained by means of synthetic DNA primers and dideoxynucleotide
Fig. 1. Agarose gel electrophoresis of different preparations containing PcDNV. Lanes a and e, size markers 10, 8, 6, 4, 3, 2, 1.6, 1.4, .1, 0.8 kb; lane b, P. citri total DNA; lane c, purified PcDNV; lane d, PcDNV cloned into pUC18 and digested with SacI and HindIII.
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related to nonstructural genes [3, 7]. No function can be postulated for ORF-4. In the organization of the reading frames and their amino acid sequence similarity, PcDNV most resembles the densovirus (PfDNV) from Periplaneta fuliginosa (smokybrown cockroach) [5]. This is illustrated in Fig. 2 which also presents the percent amino acid identity between the major ORFs from the two viruses [4]. The present report constitutes the first case of a putative densovirus isolated from a mealybug. Fig. 2. Comparisons of the genetic maps of PcDNV and PfDNV. Double arrows, PcDNV fragments subcloned and sequenced; single arrows, inverted repeats; filled arrows, ORFs corresponding to genes coding for putative proteins for which a function can be suggested; open arrows, ORFs with no suggested functions; S, viral structural proteins; NS, viral nonstructural protein; percent between arrows, amino acid identity between ORFs; ORF designations for PfDNV from [5].
ACKNOWLEDGMENTS This material is based on work supported by National Science Foundation Award DEB-9978518 and the University of California Experiment Station. We thank Dr. A.B. Hamon, Florida Division of Plant Industry, Gainesville, Florida, for the identification of the citrus mealybug.
Literature Cited chain termination method [1, 2]. The 5380 bp nucleotide sequence was deposited in GenBank under the accession number AY032882. Figure 2 presents a genetic map of the 5.4 kb DNA fragment of PcDNV. There is a 122 bp inverted repeat at the ends of the fragment. The complete PcDNV is approximately 5.5 kb (Fig. 1, lane b) slightly larger than the 5.4 kb, sequenced, KpnI fragment. We were not able to obtain the sequence of the missing portion by the dideoxynucleotide chain termination method. This problem has also been encountered in other densoviruses due to extensive secondary structure at the beginning and end of the molecule [3, 7]. Four major ORFs were detected; from comparisons of sequences in the data banks ORF-1 and ORF-2 are related to densovirus structural genes, while ORF-3, transcribed in the opposite orientation, is
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