Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, Via Amendola 165/A, 70126 Bari, Italy
Correspondence
Luisa Rubino
l.rubino{at}area.ba.cnr.it
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ABSTRACT |
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INTRODUCTION |
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The molecular biology of tombusvirus sat RNAs has been much less studied. So far, the best characterized sat RNA is the 621 nt sat RNA of Cymbidium ringspot virus (CymRSV) whose cis-acting sequence requirements essential for replication and the significance and nature of multimeric forms found in infected tissues have been studied (Rubino et al., 1990; Dalmay & Rubino, 1994
, 1995
). Two additional satellites, associated with natural infections by Tomato bushy stunt virus (TBSV) (Celix et al., 1997
), have been studied in relation to helper virus replication and symptom modulation in infected plants (Celix et al., 1999
). Sat RNAs contain little sequence homology with the viral genome other than a region of approximately 50 nt present both in the 5' non-coding region of the corresponding helper genomes and in all DI RNAs of tombusviruses (Rubino et al., 1990
; Russo et al., 1994
; Celix et al., 1997
). The replication of sat RNA originally associated with CymRSV infection can also be supported in plants by the related species Artichoke mottled crinkle virus and Carnation Italian ringspot virus (CIRV) (M. Russo, unpublished information).
The availability of a yeast-based system to analyse the replication of tombusviruses prompted us to reinvestigate the flexibility of the system using several CIRV or CymRSV DI RNA species and CymRSV sat RNA. It was found that the yeast system allowing the replication of homologous and heterologous DI RNAs did not support the replication of sat RNA.
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METHODS |
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RNA and protein analysis.
Total yeast RNA was extracted and analysed as described previously (Pantaleo et al., 2003). Riboprobes for positive- and negative-strand DI or sat RNAs were obtained by cloning approximately 300 nt of the 3'-terminal regions of DI or sat RNAs in the vector pTL7SN (Oh & Carrington, 1989
) in the appropriate orientation.
Protein extraction, electrophoresis and Western blot analysis were done as previously described (Rubino et al., 2000), except that the replicase proteins were detected using anti-c-Myc or anti-HA antibodies (Santa Cruz Biotechnology) (Pantaleo et al., 2004
).
Plant growth, agroinfiltration and protoplasts.
Transgenic Nicotiana benthamiana plant line 92KA11, expressing the full-length CymRSV replicase gene (Rubino et al., 1993), and non-transgenic plants were grown in growth chambers at 25 °C with a 14 h photoperiod. For agroinfiltration, sequences encoding the proteins p92 and p95 were cloned into the expression vector pRTL2 (Carrington et al., 1990
) and DI-3 and sat RNA sequences were cloned into the vector pRT101 (Töpfer et al., 1987
). Expression cassettes were excised from the corresponding constructs and inserted into the binary vector pGA482. The resulting plasmids were electroporated into Agrobacterium tumefaciens strain C58C1. Recombinant clones were cultured and bacterial suspensions of each clone were infiltrated alone or in combination into N. benthamiana leaves as described previously (Rubino et al., 2001
). For co-infiltration, equal volumes of the designated cultures were mixed before infiltration, i.e. p92 and DI-3 or sat RNA, p95 and DI-3 or sat RNA. Several small tissue pieces showing clear signs of infiltration were excised after 2448 h and nucleic acids were extracted as described previously (Rubino et al., 2001
). Preparation of protoplasts from fully expanded leaves, transfection and RNA analysis were done as previously described (Dalmay et al., 1993a
; Rubino et al., 2001
). Protoplasts were transfected with full-length CIRV genomic RNA (Burgyan et al., 1996
) or deletion mutants
B/H (see above) or
B/N (obtained by restriction of the CIRV full-length clone with BamHI and NcoI at nt 3870, at the beginning of ORF4), together with DI (Burgyan et al., 1992
) or sat (Rubino et al., 1990
) RNA in vitro transcripts.
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RESULTS |
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The above results indicated that the p36+p95+ transformants were indeed fully competent for the replication of subviral RNAs, regardless of whether these were originally associated with CIRV (DI-7 RNA) or CymRSV (DI-3 and DI-13 RNAs).
Since previous analysis has shown that the replication of sat RNA in plants is inhibited by temperatures above 25 °C (Burgyan & Russo, 1988), p36+p95+ yeast cells expressing either DI-3 or sat RNA were cultured at 26 and 20 °C. At the mid-exponential phase, cells were extracted and examined by Northern blotting. While DI RNA replication still took place in cells grown at 20 °C although yielding fewer progeny than at 26 °C, no sat RNA was synthesized at either temperature (not shown).
The replication of DI RNA does not transactivate the replication of sat RNA
Knowing that both DI and sat RNA can replicate in the same plant cell (Rubino et al., 1992), yeast cells were co-transformed with plasmids expressing CIRV replicase proteins and plasmids pBsat and pBDI-3 to see whether sat RNA transcripts could replicate in yeast cells in which an active replication complex was formed by another template RNA, such as DI RNA. However, since these plasmids must contain different nutritional markers to permit simultaneous selection, pBsat was first engineered to have the marker URA3 in place of TRP1. Cell growth and GAL1 induction and repression were performed as before, and Northern blot analysis was carried out in duplicate using DI RNA- and sat RNA-specific probes. Robust DI RNA replication took place either in galactose-containing (Fig. 3a
, lanes 1 and 2) or glucose-containing (Fig. 3a
, lanes 4 and 5) medium, regardless of whether yeast cells expressed sat RNA (Fig. 3a
, lanes 1 and 4) or not (Fig. 3a
, lanes 2 and 5). By contrast, there was no evidence of sat RNA replication, regardless of the presence (Fig. 3b
, lanes 1 and 4) or absence (Fig. 3b
, lanes 3 and 6) of DI RNA, notwithstanding the active plasmid DNA-dependent transcription (Fig. 3b
, lanes 1 and 3), which was not affected by the concomitant transcription of DI RNA (Fig. 3b
, lane 1). These results suggested that a replication complex was correctly formed in transformed yeast cells, but that it was unsuitable for the replication of sat RNA, even if already activated by the replicating DI RNA.
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Protoplasts were extracted from fully expanded leaves of the transgenic N. benthamiana line 92KA11, which expresses the active CymRSV replicase under the control of the cauliflower mosaic virus 35S promoter and terminator (Rubino & Russo, 1995). Approximately 106 cells were transfected with 50 ng of in vitro-synthesized DI or sat RNA together or not with the helper genome (200 ng) or with water, as described previously (Dalmay et al., 1993a
). Protoplasts were incubated for 24 h at room temperature (2225 °C) under continuous fluorescent light, then disrupted and extracted with phenol. RNA extracts were analysed by Northern blotting. Whereas transgenic protoplasts supported the replication of DI RNA in the absence or presence of the helper (Fig. 4a
, lanes 1 and 3, respectively), sat RNA replication was not detected when it was transfected alone, but occurred when there was co-transfection with genomic RNA (Fig. 4b
, lane 1 and 3, respectively). No signal was detected in extracts of mock-inoculated protoplasts (Fig. 4a and b
, lane 2).
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It was next decided to test whether the competence of the helper genome to sustain the replication of sat RNA was due to expression of genes other than the viral replicase. To do this, two deletion mutants of the full-length infectious clone of CIRV (Burgyan et al., 1996) were constructed (Fig. 5a
). The first mutant clone (
B/N) contained a deletion from nt 20 of the CP gene to the start codon of ORF5, while the second mutant clone (
B/H) had a longer deletion as it lacked not only the entire CP gene, but also most of the ORF4 and -5 sequences. Protoplasts were obtained from non-transgenic N. benthamiana plants and co-transfected with in vitro-synthesized wild-type genomic or deletion mutant
B/N or
B/H RNAs and DI or sat RNA. Both DI (Fig. 5b
, upper panel, lanes 2, 4 and 6) and sat RNA (Fig. 5b
, lower panel, lanes 1, 3 and 5) replication was supported by the different helpers. It was concluded that sat RNA can be replicated only in the presence of a cis-replicating helper molecule.
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DISCUSSION |
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In particular, the replication of sat RNA requires the replication of the helper genome, thus suggesting an interaction with the self-replicating helper molecule. The nature of this interaction is unknown. It could be hypothesized that the replicating genome, besides supplying the replicase, acts as a trans-replication enhancer. Long-distance RNARNA interactions have been identified for several viruses for the regulation of subgenomic RNAs, as in the case of Red clover necrotic mosaic virus (Sit et al., 1998) and TBSV (Zhang et al., 1999
). With FHV, the replication of subgenomic RNA3, which is synthesized by transcription from FHV RNA1, is necessary for the replication of RNA2 (Eckerle & Ball, 2002
; Eckerle et al., 2003
). We speculate that a similar interaction regulates the replication of CymRSV sat RNA.
In yeast cells, genomic B/H RNA could only be replicated in trans, although it is fully competent for autonomous replication in plant cells. Contrary to our results, Brome mosaic virus (BMV) RNA2 was shown to act both as mRNA for the synthesis of the polymerase protein 2a and as replication template in protein 1a-expressing yeast cells (Chen et al., 2001
). A major difference between BMV, a tripartite-genome virus, and CIRV, a monopartite-genome virus, lies in the strategy of expression of replication-associated proteins. In particular, because the presence of an amber stop codon in CIRV may be a stumbling block to the efficiency of our replication system in yeast, overcoming this impairment is currently being addressed.
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ACKNOWLEDGEMENTS |
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Received 20 May 2004;
accepted 23 June 2004.
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