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Short Communication |
1 CSIRO Plant Industry, PO Box 350, Glen Osmond, SA 5064, Australia
2 School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, SA 5064, Australia
3 Plant Protection Department, College of Agriculture, Shiraz University, Shiraz, Iran
4 Department of Plant Protection, School of Agriculture, Zanjan University, Zanjan, Iran
Correspondence
M. Ali Rezaian
ali.rezaian{at}adelaide.edu.au
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ABSTRACT |
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Present address: CSIRO Molecular & Health Technologies, 11 Julius Avenue, Riverside Corporate Park, Delhi Rd, NSW 2113, Australia. 
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MAIN TEXT |
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). The majority of geminiviruses fall into the genus Begomovirus. Members of this genus are transmitted by the whitefly Bemisia tabaci, infect dicotyledonous plants and have either monopartite or bipartite genomes. Tomato leaf curl virus (TLCV) is a monopartite begomovirus (Dry et al., 1993) containing a covalently closed, single-stranded DNA genome of 2766 nt that encodes six open reading frames (ORFs), two on the virion-sense strand (V1 and V2) and four on the complementary-sense strand (C1, C2, C3 and C4), interspersed by an intergenic region. The C1 ORF encodes the replication-associated protein (Rep), the only viral gene product absolutely required for virus replication.
The first viral satellite associated with a DNA virus (sat-DNA) was found in TLCV-infected plants from Australia (Dry et al., 1997). This 682 nt, circular, single-stranded DNA depends on TLCV for its replication and encapsidation, but can also be supported by some other geminiviruses (Dry et al., 1997). TLCV sat-DNA does not encode any proteins and has no discernible effect on the helper virus replication or on symptom development in the hosts studied. TLCV and its sat-DNA share two copies of the sequence motif (GGTGTCT) essential for binding the viral Rep in vitro. However, mutants of both TLCV and sat-DNA impaired in Rep binding were infectious (Lin et al., 2003). These results suggested that, in contrast to other geminiviruses studied (Fontes et al., 1994; Orozco et al., 1998), TLCV and sat-DNA replication is independent of high-affinity in vitro Rep binding (Lin et al., 2003). More recently, we have observed that TLCV Rep-binding-site mutants could support the replication of both wild-type and TLCV sat-DNA mutants (D. Li and M. A. Rezaian, unpublished). In these experiments, sat-DNA replication did not have an adverse effect on accumulation of TLCV Rep-binding-site mutants. This supports our earlier conclusion (Lin et al., 2003) that high-affinity Rep binding is not required for either TLCV or sat-DNA replication in the hosts studied.
We have previously observed that integrated TLCV promoter : 
-glucuronidase (GUS) fusion transgenes are transcriptionally silenced following TLCV infection (Seemanpillai et al., 2003). The silencing was transmitted vertically to progeny plants, whilst the virus itself was not seed transmitted (Seemanpillai et al., 2003). These results indicated that it might be possible to silence traits epigenetically, in plant progenies lacking TLCV. However, the single-stranded DNA genome of TLCV does not lend itself to insertion of additional sequences, as the viral origin of DNA replication and the six overlapping ORFs are all required for replication and movement (Rigden et al., 1993). On the other hand, TLCV sat-DNA does not encode any known proteins and its replication is supported by multiple geminiviruses that infect a variety of hosts (Dry et al., 1997). Thus, its potential to act as a vector for foreign DNA sequences into these hosts was investigated.
To test the hypothesis that TLCV sat-DNA has the potential to act as a gene expression/silencing vector, we first analysed the regions within the sat-DNA essential for replication by mutagenesis. Initially, six restriction sites (ApaI, SpeI, BglII, KpnI, StuI and SmaI) were introduced into the TLCV sat-DNA at nt 35, 146, 296, 420, 492 and 540, respectively, to allow subsequent sequence manipulation. Tandem-repeat mutant sat-DNA constructs were cloned into pBIN19 and agroinoculated into six Nicotiana benthamiana plants together with the helper TLCV as described previously (Lin et al., 2003). Each of the resulting mutant constructs was found to be as infectious as wild-type sat-DNA (data not shown). Using these introduced restriction sites, a series of seven sat-DNA deletion constructs (Fig. 1a) was produced representing deletions in all regions of the sat-DNA except for a 77 nt region (nt 641 through nt 682 to nt 35) flanking stem–loop I, which contains the conserved nonanucleotide sequence TAATATTAC essential for sat-DNA replication (Dry et al., 1997). These sat-DNA deletion constructs were co-agroinoculated, with TLCV, into whole plants (six) or leaf strips of N. benthamiana as described previously (Dry et al., 1997; Rigden et al., 1996).
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shows that, compared with the wild-type satellite, the deletion mutants either lacked the ability to replicate (constructs 4–7) or replicated poorly (constructs 1–3). The sequence from nt 296 to nt 35 (through nt 682) was found to be essential for sat-DNA replication. However, deletion of a 112 nt region downstream of the stem–loop I from nt 35 to 146 (construct 1) was tolerated, although sat-DNA replication was reduced significantly. Similarly, the deletion of 151 nt from nt 146 to 296 (construct 2) was tolerated, but the level of replication was reduced further. The combined deletion of these two regions (construct 3) diminished sat-DNA replication in leaf strips below that observed with either deletion on its own and further decreased the infectivity of the sat-DNA construct in whole plants (Fig. 1a).
To assess whether the effect of these deletions on sat-DNA replication was due to the loss of genomic sequences required for replication or due to changes in genome size, another set of constructs was produced in which the seven deleted regions were replaced with seven heterologous non-viral DNA fragments to restore the wild-type 682 nt sat-DNA size except for construct number 10, which was 60 nt larger than the original sat-DNA (Fig. 1b). The results of replication assays with these sat-DNA constructs showed that they were only capable of replication when the replacement occurred in the region between nt 35 and 296. These results resembled the relative infectivity of the deletion mutants (Fig. 1a). However, the sequence replacements in the region nt 35 to 296 of the sat-DNA (constructs 8–10) improved the accumulation of sat-DNA considerably relative to the deleted constructs (constructs 1–3). Taken together, these results demonstrated that sequence elements distributed within the entire sat-DNA molecule contribute to replication activity, but that sequence elements within the region from nt 35 to 296 are dispensable for replication. Furthermore, the size of the mutated satellite DNA molecule significantly influences replication efficiency.
Since TLCV sat-DNA was first described (Dry et al., 1997), a number of geminivirus satellite DNAs, known as DNA , have been discovered and characterized (Briddon et al., 2003). They share a satellite common region (nt 436–20 through nt 682) and an adenine-rich region (nt 152–307) with TLCV sat-DNA. The DNA satellites contain a complementary-sense ORF (C1), which is a pathogenicity determinant (Cui et al., 2004; Saeed et al., 2005; Saunders et al., 2004). The region of TLCV sat-DNA identified here as essential for replication (nt 296–35 through nt 682) includes the adenine-rich region and the satellite common region.
To analyse the size of DNA insert tolerated for replication, fragments of varying sizes from a 50 bp DNA ladder preparation were randomly introduced into the sat-DNA at nt 119 by shotgun cloning. Screening of the clones allowed the selection of sat-DNA constructs containing inserts of 100, 200, 600 or 700 nt, but other insert sizes were not obtained. All of these constructs were found to replicate in N. benthamiana leaf strips when co-inoculated with TLCV (Fig. 2, upper panel). PCR analysis indicated that the sizes of the recombinant DNAs were maintained in the progeny molecules (Fig. 2, lower panel, lanes 2, 4, 6, 8 and 10). Attempts to insert larger heterologous DNA fragments (i.e. 760 nt) within the region of sat-DNA shown to tolerate DNA insertions resulted in sequence truncations of the replicating mutant sat-DNA fragments (data not shown). These results demonstrated that heterologous DNA sequences can be inserted into sat-DNA, but that the size of the insert is a limiting factor.
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) may make it suitable as a gene vector. To assess this possibility, specific insertion constructs were produced by the introduction of fragments of the cauliflower mosaic virus (CaMV) 35S promoter. Constructs sat-35S-141 and sat-35S-320 were made by insertion of 141 bp (nt –133 to +8) and 320 bp (nt –312 to +8) fragments of the CaMV 35S promoter (Guilley et al., 1982; Odell et al., 1985) into the BglII site (nt 296) of the sat-DNA, respectively. Both of these 35S promoter fragments are sufficient to drive transcription of downstream sequences (Odell et al., 1985) and these constructs were used to study sat-DNA-mediated gene silencing.
Transgenic tobacco plants containing a functional 35S–GUS expression cassette (Dry et al., 2000) were inoculated with sat-35S-141 and sat-35S-320 in the presence of TLCV. Southern blot analysis of the inoculated plants at 21 days post-inoculation (p.i.) showed levels of mutant sat-DNA similar to the wild-type control (Fig. 3a, upper panel). The progeny sat-DNA molecules did not show sequence truncations based on the sizes of the PCR DNA fragments amplified (Fig. 3a, lower panel).
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and Dry et al. (2000). All three plants inoculated with either of the sat-35S promoter constructs showed significant GUS silencing at 21 days p.i., whereas GUS expression was unaffected in plants co-inoculated with TLCV and wild-type sat-DNA (Fig. 3b). Silencing of GUS expression at 21 days p.i. was found to be stronger in plants inoculated with the sat-DNA containing the longer (320 bp) 35S promoter sequence compared with the plants inoculated with sat-DNA containing the shorter (141 bp) 35S promoter (Fig. 3b). However, there was no discernible difference in silencing efficiency between the different sat-DNA constructs at 50 days p.i. (Fig. 3b). Southern blot analysis of DNA from leaves of the silenced plants at 50 days p.i. confirmed that both TLCV DNA and recombinant sat-DNAs were present in these plants (data not shown). Northern blot analysis also confirmed that GUS transcript was not detectable in the leaves of GUS silenced plants compared with the control plants infected with TLCV and wild-type sat-DNA (data not shown).
The results of this study demonstrate that TLCV sat-DNA has the potential to act as a gene expression/silencing vector in host plants co-infected with a helper geminivirus. We have also shown previously that the replication of sat-DNA is supported by multiple geminiviruses that infect a variety of hosts (Dry et al., 1997), offering the possibility of introducing epigenetic traits into a variety of plants via short DNA inserts.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Briddon, R. W., Bull, S. E. & Amin, I. Idris, A. M., Mansoor, S., Bedford, I. D., Dhawan, P., Rishi, N., Siwatch, S. S. & other authors (2003). Diversity of DNA , a satellite molecule associated with some monopartite begomoviruses. Virology 312, 106–121.[CrossRef][Medline]
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Saeed, M., Behjatnia, S. A. A., Mansoor, S., Zafar, Y., Hasnain, S. & Rezaian, M. A. (2005). A single complementary-sense transcript of a geminiviral DNA satellite is determinant of pathogenicity. Mol Plant Microbe Interact 18, 7–14.[CrossRef][Medline]
Saunders, K., Norman, A., Gucciardo, S. & Stanley, J. (2004). The DNA satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein C1. Virology 324, 37–47.[CrossRef][Medline]
Seemanpillai, M., Dry, I. B., Randles, J. & Rezaian, M. A. (2003). Transcriptional silencing of geminiviral promoter-driven transgenes following homologous virus infection. Mol Plant Microbe Interact 16, 429–438.[Medline]
Stanley, J., Bisaro, D. M., Briddon, R. W., Brown, J. K., Fauquet, C. M., Harrison, B. D., Rybicki, E. P. & Stenger, D. C. (2005). Geminiviridae. In Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp 301–326. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press.
Received 17 January 2007;
accepted 1 March 2007.
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