School of Molecular and Cell Biology, University of the Witwatersrand, PO Wits 2050, Johannesburg, South Africa1
Author for correspondence: Maria Paximadis. Fax +27 11 321 4234. e-mail paxim{at}niv.ac.za
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Abstract |
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Introduction |
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A geminivirus was first reported to be the causative agent of tobacco leaf curl disease in Japan in 1981 (Osaki & Inouye, 1981 ). In Southern Africa, at least three symptom phenotypes in tobacco have been identified that could be classified as tobacco leaf curl (Paximadis & Rey, 1997
). One of these symptom phenotypes has been attributed to a phytoreovirus (Paximadis et al., 1997
; Rey et al., 1999
) and the other to a begomovirus, Tobacco leaf curl Zimbabwe virus (TbLCZWV) (Paximadis & Rey, 1997
; Paximadis et al., 1999
). Four plants, showing leaf curl symptom severity variation, associated with begomovirus infections in Zimbabwe (Paximadis & Rey, 1997
), were phylogenetically compared using coat protein gene (CP) and the common region (CR) nucleotide sequences. Isolates formed a distinct cluster and were
98% similar in their CP and CR sequences (Paximadis et al., 1999
). The present research characterizes TbLCZWV at a molecular level and reports, for the first time, defective DNA molecules associated with TbLCZWV.
Three pairs of overlapping degenerate primers [AV494 and AC1048 (Wyatt & Brown, 1996 ); PAL1c1960 and PAR1v722 (Paximadis & Rey, 1997
); prAV1134 (Idris & Brown, 1988) and PAR1c715 (5' GAT TTC TGC AGT TDA TRT TYT CRT CCA TCC A 3'); D. P. Maxwell, Univ. of Wisconsin, Madison, USA], designed to amplify regions of the DNA A components of most begomoviruses, were employed in PCR to amplify the DNA A from total DNA extracted (Doyle & Doyle, 1987
) from TbLCZWV-infected tobacco. PCR fragments were blunt-end cloned into the pBluescript (KS) vector (Stratagene) and three clones of each were sequenced automatically (ABI Prism 310) to obtain the complete TbLCZWV DNA A. TbLCZWV DNA A was phylogenetically compared to the DNA A sequences of other geminiviruses (GenBank) using the DNAMAN version 4.0 (Lynnon Biosoft, Quebec, Canada) full optimal alignment and neighbour-joining method options with 1000 bootstrap replications. Open reading frames (ORFs) of TbLCZWV DNA A were identified using DNAMAN, and percentage DNA A nucleotide and ORF amino acid sequence identities between virus isolates were calculated using the distances between all pairs of sequences in the multiple alignments.
Sequencing of cloned PCR fragments spanning the complete TbLCZWV DNA A revealed it to be 2767 nucleotides in size, exhibiting the typical genome organization of begomoviruses originating from the Old World, with six conserved ORFs (Fig. 1a, b
). Phylogenetic analysis of TbLCZWV DNA A placed TbLCZWV in the Old World African and Mediterranean cluster (Fig. 1c
), with the highest DNA A sequence identity (78·4%) being shared with Chayote mosaic virus (ChaMV), a cucurbit-infecting begomovirus from Nigeria, reported in GenBank as having a single genomic component. TbLCZWV also clustered with ChaMV in the DNA A relationship dendrogram (Fig. 1c
), sharing high amino acid sequence identity with this virus in the CP (95·3%) and C3 (77·4%) ORFs. The high CP amino acid sequence identity shared between ChaMV and TbLCZWV might suggest that these are strains of the same virus; however, the unique intergenic region (IR) putative iterons of TbLCZWV (Paximadis et al., 1999
) and <90% DNA A sequence identity shared with ChaMV demonstrate that TbLCZWV is a distinct species within the genus Begomovirus. Begomoviruses sharing <90% DNA A sequence identity are considered to be distinct species (Rybicki et al., 2000
). In both the DNA A and ORF amino acid comparisons, TbLCZWV generally showed high sequence identity with the monopartite Mediterranean tomato yellow leaf curl begomoviruses and with South African cassava mosaic virus (Berrie et al., 2001
).
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Although the failure to detect a DNA B component strongly suggested that TbLCZWV is a monopartite begomovirus, the ability of the single genomic component to move and cause disease symptoms in its original host (Nicotiana tabacum) had to be shown to unequivocally prove the monopartite nature of this virus. A full-length DNA A genomic clone (pBSZW-A) was prepared by linearizing TbLCZWV scDNA with BamHI, and cloning into the BamHI site of pBluescript (KS). A full-length head-to-tail dimer of the genomic DNA A was constructed in pBluescript (KS) and subcloned into plant transformation vectors pBin19 (Bevan, 1984 ) and pBI121 (Clontech), yielding clones pBinZW-DA and pBIZW-DA respectively. Agrobacterium tumefaciens C58C1 RifR (pMP90 GmR) (Koncz & Schell, 1986
), transformed with pBinZW-DA and pBIZW-DA using the freezethaw method of Holsters et al. (1978)
, was used to agroinoculate Nicotiana benthamiana and two cultivars of N. tabacum, namely Samsun and HG (a Zimbabwean cultivar). Lycopersicon esculentum cv. Moneymaker was also agroinoculated with A. tumefaciens C58C1 RifR (pMP90 GmR) transformed with pBIZW-DA. Exponential-phase recombinant Agrobacterium was pelleted, washed with sterile water and resuspended in 200 µl of Luria broth per ml of original culture. A 30 gauge needle was used to transfer A. tumefaciens cultures to plants by injecting the stems and pricking the leaves of seedlings at the four to six leaf stage. Plants were monitored for symptoms. All four hosts developed leaf curl symptoms similar to those noted in the field samples (Fig. 3af
), and both plant transformation vectors used were equally infectious. Southern hybridizations (using a DIG-labelled full-length TbLCZWV DNA A probe) verified the presence of both double-stranded and single-stranded forms of TbLCZWV-specific DNA in the agroinoculated plants, and PCR, using TbLCZWV-specific abutting primers, amplified expected full-length fragments, as well as ca.1300 bp fragments (only in tobacco), thought to be defective DNA molecules.
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In conclusion, TbLCZWV is a distinct, previously uncharacterized new member of the monopartite begomoviruses, associated with subgenomic defective DNA molecules that originate from its single genomic component. A monopartite genome organization has also been reported for Tobacco leaf curl Japan virus (Shimizu & Ikegami, 1999 ) and Tobacco leaf curl China virus (GenBank); however, these viruses are phylogenetically distantly related to TbLCZWV (Fig. 1c
) and furthermore, the infectious nature of their single genomic components has not been reported.
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Received 18 June 2001;
accepted 24 August 2001.