1 Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, 10135 Torino, Italy
2 ENEA CR Casaccia, Settore Biotec, Via Anguillarese 301, 00060 Rome, Italy
Correspondence
Mario Tavazza
tavazza_m{at}casaccia.enea.it
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ABSTRACT |
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These authors contributed equally to this work.
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MAIN TEXT |
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To counterattack the RNA silencing defence mechanism, most plant viruses have evolved RNA silencing suppressors (Voinnet et al., 1999). Although viral suppressors can target different steps of the RNA silencing pathway (Guo & Ding, 2002
; Llave et al., 2000
; Mallory et al., 2001
; Silhavy et al., 2002
; Voinnet et al., 2000
), transgene RNA silencing has been successfully exploited to confer resistance to RNA viruses (Baulcombe, 1996
). Little is known on the possible use of RNA silencing as an antiviral strategy to control geminivirus DNA infection (Pooggin et al., 2003
; Vanitharani et al., 2003
). We have shown that transgenic expression of a truncated form of TYLCSV Rep protein (Rep-210) confers resistance to viral infection (Brunetti et al., 1997
) and that resistance is lost when TYLCSV shuts off transgene expression by post-transcriptional gene silencing (PTGS) (Lucioli et al., 2003
). This suggests that TYLCSV can somehow evade transgene-mediated RNA silencing of two essential viral genes, rep and the nested C4.
To evaluate directly the impact of RNA silencing on TYLCSV infection, we used two classes of post-transcriptionally silenced (PTS) transgenic tomato plants, a sensexantisense hybrid and multicopy sense lines. The sensexantisense hybrid 10x47 has been described previously (Brunetti et al., 1997). The concomitant expression of sense and antisense sequences leads to the production of dsRNAs that activate RNA silencing (Waterhouse et al., 1998
). We examined whether 10x47 hybrids accumulated transgene-specific siRNAs by Northern blotting, as described by Lucioli et al. (2003)
. Plants containing only the sense gene and accumulating large amounts of Rep-210 protein (Fig. 1
a, lanes 25) did not contain transgene-specific siRNAs. However, Rep-210-specific siRNAs were readily detected in plants with both antisense and sense genes (Fig. 1a
, lanes 611), indicating that the reduction of Rep-210 mRNA (Brunetti et al., 1997
) and protein were due to RNA silencing.
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Since virus infection can trigger silencing of homologous transgenes (Kumagai et al., 1995; Ruiz et al., 1998
), we analysed whether infection by TYLCSV would further reduce the level of Rep-210 mRNA, i.e. enhance silencing. At the same time, such a PTS enhancement may in turn also reduce the homologous rep gene expression of the infecting TYLCSV, eventually leading to plant recovery. Total RNA was extracted from samples of PTS plants before inoculation and several weeks following virus infection (i.e. 7 weeks p.i. for line 200 and 19 weeks p.i. for hybrids 10x47) and analysed by Northern blotting for the accumulation of transgene-specific siRNAs (Fig. 2
a) and Rep-210 mRNA (Fig. 2b
). As reported previously, wt tomato plants naturally infected with TYLCSV accumulate Rep-specific siRNAs (Lucioli et al., 2003
). Therefore, a probe homologous to Rep-210-derived transgene sequences cannot discriminate between transgene- and virus-derived Rep-210 siRNAs. Thus, the blot containing siRNAs was first hybridized with a cauliflower mosaic virus terminator (CaMV-T) RNA probe specifically recognizing the 3' untranslated region of the Rep-210 mRNA and then rehybridized with the pGEM103 probe (Lucioli et al., 2003
) that recognizes Rep-210-specific siRNAs (Fig. 2a
). The CaMV-T probe, spanning nt 796910 of pJIT60 (Guerineau & Mullineaux, 1993
), was hybridized at 33 °C without prior hydrolysis. Hybridization signals were evaluated by Phosphorimager (Typhoon; Molecular Dynamics). siRNAs homologous to the CaMV-T were readily detected at 0 weeks p.i. in all plants of line 200 but not in 10x47 hybrids (Fig. 2a
). However, in accordance with the results shown in Fig. 1
, when the same filter was rehybridized with the pGEM103 probe, siRNAs were readily detected at 0 weeks p.i. in all PTS plants. In 10x47 plants, the ratio between Rep-210- and CaMV-T-derived siRNA sequences was five times higher than in line 200 plants. This was not surprising considering that in 10x47 plants expression of the sense and antisense rep-210 transgenes did not directly produce negative- and positive-sense RNAs homologous to the CaMV-T region (Brunetti et al., 1997
). At 7 weeks p.i. (4 weeks after establishment of systemic infection), a two- to fivefold increase in transgene-derived siRNAs was observed in plants of line 200 using both probes (Fig. 2a
). A similar increase in Rep-210-specific siRNAs was also observed in the two 10x47 plants at 19 weeks p.i., which correlated with a further 40 % decrease in the steady-state level of Rep-210 mRNA (Fig. 2b
). The very low steady-state level of Rep-210 mRNA in plants of line 200, already seen at 0 weeks p.i., did not allow precise evaluation. Nevertheless, collectively our results suggest that TYLCSV infection further reduces the steady-state mRNA of an already silenced rep-210 transgene. Notably, TYLCV is limited to phloem tissue (Rojas et al., 2001
), so a diffusible signal molecule should be involved in the observed phenomenon. TYLCSV-infected PTS plants showed characteristic TYLCSV symptoms until the end of the experiment (20 weeks p.i. for 10x47 plants and 23 weeks p.i. for line 200), indicating that further reduction in rep-210 transgene expression (Fig. 2b
) did not lead to plant recovery.
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Agroinoculation is commonly used to transmit TYLCSV and other mechanically non-transmissible geminiviruses. However, it could be envisaged that due to the long persistence of Agrobacterium within the inoculated plant and the integration of the T-DNA into the plant genome agroinoculation may result in a prolonged or continuous source of virus inoculum. Therefore, R1 progeny of line 201 were challenged with Bemisia tabaci, the natural vector of TYLCSV, under low (LP) or high (HP) inoculation pressure, obtained by modifying the acquisition access period, the number of insects per plant and the inoculation access period (Table 1). All plants were analysed at 0 weeks p.i. for transgene expression and screened for virus infection by tissue print at 2, 3, 6 and 10 weeks p.i. (Table 1
). Under LP conditions, when less than 100 % of the non-transgenic control plants were infected, PTGS was able to prevent infection in some plants, whereas under HP conditions all PTS plants were susceptible. Interestingly, delayed infections were observed in both LP and HP mode. This suggests that if the virus reaches a certain level of expression/replication in the initially infected cells, then virus spread cannot be prevented. This is in accordance with the evidence that all the PTS plants challenged with TYLCSV either by agroinoculation or by high vector inoculum were susceptible to TYLCSV and with the recent evidence that siRNA targeting of the African cassava mosaic virus rep gene in cultured plant cells, although drastically reducing rep mRNA accumulation, has only a limited impact on virus replication (Vanitharani et al., 2003
). Our experimental system allowed us to compare directly two different strategies of conferring TYLCSV resistance, suggesting that a trans-dominant negative mutant approach may be more appropriate. Recently, it has been shown that RNA-mediated interference (RNAi) targeting of a geminivirus promoter is able to cure, in a transient assay, Vigna mungo plants of the viral disease (Pooggin et al., 2003
). The viral DNA genome seems the primary target of the RNAi, possibly through an RNA-dependent DNA methylase (RdDM). We do not know whether and to what extent Rep-210 RNA silencing can target TYLCSV DNA by a RdDM. However, it appears that RNAi targeting of a non-regulatory region of the TYLCSV genome does not confer virus resistance.
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ACKNOWLEDGEMENTS |
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Received 31 December 2003;
accepted 11 February 2004.
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