Tomato yellow leaf curl Sardinia virus can overcome transgene-mediated RNA silencing of two essential viral genes

Emanuela Noris1,{dagger}, Alessandra Lucioli2,{dagger}, Raffaela Tavazza2, Piero Caciagli1, Gian Paolo Accotto1 and Mario Tavazza2

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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To evaluate RNA silencing for the control of geminivirus infection, two classes of post-transcriptionally silenced (PTS) plants were tested using Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep-210-transgenic plants, a sensexantisense hybrid and two multicopy sense lines. In both classes, PTS plants accumulated low or undetectable amounts of Rep-210 protein and mRNA but high amounts of Rep-210 small interfering RNAs. PTS plants were susceptible to TYLCSV when challenged by agroinoculation or using high viruliferous whitefly (Bemisia tabaci) pressure, although some plants were resistant at low whitefly pressure. Delayed infections were also observed, indicating that TYLCSV could overcome transgene silencing of rep and of the nested C4 gene. TYLCSV infection boosted transgene silencing but this did not lead to recovery. The data suggest that if the virus reaches a threshold level of expression/replication in the initially infected cells then virus spreading can no longer be prevented.

{dagger}These authors contributed equally to this work.


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RNA silencing represents a broad family of gene regulation mechanisms operating at the RNA level, implicated among others in defence against viruses (Voinnet, 2001; Waterhouse et al., 2001). Partially duplex viral RNAs or dsRNAs generated by virus- or host-encoded RNA polymerases are recognized and diced into small interfering RNAs (siRNAs) of 21–26 nt by a Dicer-like protein (Bass, 2000; Bernstein et al., 2001; Hamilton et al., 2002; Hamilton & Baulcombe, 1999; Tang et al., 2003). The siRNAs are recruited to an RNA-induced silencing complex, guiding a sequence-specific recognition of the RNAs to be degraded (Elbashir et al., 2001a, b; Hammond et al., 2000). The presence of siRNAs is a hallmark of the activated RNA silencing process. We have recently shown that plants infected by the geminivirus Tomato yellow leaf curl Sardinia virus (TYLCSV) accumulate siRNAs homologous to viral rep and C4 genes (Lucioli et al., 2003). This suggests that the host RNA silencing machinery targets mRNAs of two essential TYLCSV genes. Rep is a multifunctional protein absolutely required for virus replication (Laufs et al., 1995), while C4 is essential for establishing a systemic infection in tomato but not in Nicotiana benthamiana plants (Jupin et al., 1994).

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. 1a, lanes 2–5) 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 6–11), indicating that the reduction of Rep-210 mRNA (Brunetti et al., 1997) and protein were due to RNA silencing.



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Fig. 1. Molecular analysis of Rep-210-transgenic lines 10x47 (a), 200 (b) and 201 (c). Migration of Rep-210 protein, Rep-210 mRNA and Rep-210-specific siRNAs are indicated on the right. Equal loading of siRNAs is shown by ethidium bromide staining. The presence or absence of sense and antisense truncated C1 genes in each plant tested by PCR is indicated as + and –, respectively, at the top of each panel. For Western blots, 500 ng total protein was analysed by 12 % SDS-PAGE, blotted and incubated with Rep-specific rabbit polyclonal antiserum; secondary antibody was goat anti-rabbit IgG conjugated to horseradish peroxidase. For Northern blots, 10 and 25 µg of total RNA were loaded on gels for mRNA and siRNAs, respectively; detection was with specific 32P-labelled RNA probes.

 
PTGS of transgenes is often observed in plants carrying the transgene in multiple copies and/or in repeated and inverted orientations (Goodwin et al., 1996; Stam et al., 1997). Progeny of transgenic plants harbouring multiple insertions are often composed of both silenced and non-silenced plants. In particular, in the case of Rep-210-transgenic plants, this would allow comparison of, in the same line, the effect of the expression of Rep-210 protein or siRNAs on viral infection. Transgenic tomato plants were derived by transformation of cotyledons with an Agrobacterium strain carrying pTOM102C4(–), a plasmid derived from pTOM102 (Noris et al., 1996) where the nested C4 gene has been knocked out without altering the coding capacity of the rep-210 gene. pTOM102C4(–) was obtained by inserting the KpnI–BglII expression cassette from pTOM100C4(–) (Brunetti et al., 2001) into KpnI/BamHI-cut pBIN19. Two R0 transgenic plants, namely 200 and 201, both having three copies of the rep-210 transgene (data not shown), were selected and their progeny were analysed in detail. Most R1 plants inherited the transgene but only a few expressed the protein (Fig. 1b, lanes 1–3, and c, lanes 2–5). All R1 transgenic plants not expressing Rep-210 protein (Fig. 1b, lanes 4–14 and c, lanes 6–13) accumulated reduced amounts of transgene mRNA but large amounts of transgene-specific siRNAs, showing that the transgene was silenced at the post-transcriptional level. Plants of the above two classes were thus challenged with TYLCSV using agroinoculation (Noris et al., 1996). Before inoculation at 0 weeks post-infection (p.i.), all plants were analysed for transgene expression. Fig. 1 shows representative examples of each class. TYLCSV infection was monitored by tissue print hybridization using a coat-protein-specific probe as described previously (Lucioli et al., 2003). As already reported (Brunetti et al., 1997), when the 10x47 progeny were challenged with TYLCSV, all sensexantisense silenced plants became infected at 3 weeks p.i., as did wild-type (wt) plants, while sense plants expressing Rep-210 were resistant. Similarly, when 17 and 31 R1 plants of lines 201 and 200, respectively, were challenged with TYLCSV, the only plants that remained healthy at 4 weeks p.i. were the non-silenced ones, accumulating Rep-210 and showing the typical altered Rep-210-associated phenotype (Brunetti et al., 1997). These results suggested that siRNA targeting of two essential viral genes was not sufficient to prevent TYLCSV infection.

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. 2a) 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 796–910 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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Fig. 2. Molecular analysis of post-transcriptionally silenced transgenic lines 200 and 10x47, before and after TYLCSV infection, by siRNA (a) and mRNA (b) Northern blot analysis. Numbers at the top of the panels indicate the weeks post-inoculation (wpi) at which samples were collected; numbers below each panel indicate individual plants. M, mock-inoculated wt plants; I, infected wt plants. In (b), C is a control non-silenced Rep-210-transgenic plant of line 200. For the siRNA gel (a), equal loading is shown by ethidium bromide staining and the probes used are indicated on the left.

 
To evaluate the impact of silencing on viral DNA accumulation, total DNA was extracted from infected 10x47 and 200 PTS plants at 19 and 18 weeks p.i., respectively, and analysed by dot blotting (Noris et al., 1996) (data not shown). A slight reduction (up to threefold) in the amount of viral ssDNA was observed in PTS compared with wt plants. Therefore, siRNA targeting of two essential viral genes has only a limited impact on virus infection.

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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Table 1. Analysis of resistance of tomato line 201 challenged with TYLCSV-viruliferous B. tabaci

 
How can TYLCSV overcome transgene-mediated siRNA targeting of two essential viral genes, rep and C4? TYLCSV, unlike most viruses for which RNA-mediated resistance has been described, has a DNA genome and replicates through DNA intermediates. Therefore, TYLCSV could be less susceptible to RNA silencing of coding regions than viruses having an RNA genome or replicating through RNA intermediates. The viral C2 protein of Tomato yellow leaf curl virus-China has been reported as a silencing suppressor (van Wezel et al., 2002), probably acting at an early step (Dong et al., 2003). Although no direct evidence of the existence of a similar viral suppressor for TYLCSV is available, a protein with similar activity could contribute to silencing evasion. The ability of TYLCSV to infect PTS 10x47 plants suggests that if a silencing suppressor is involved in the evasion phenotype it should work downstream from the production of dsRNAs. However, the ability of TYLCSV to overcome Rep-210-mediated resistance by silencing the transgene (Lucioli et al., 2003) cannot easily be reconciled with a pivotal role for the viral suppressor in the evasion phenotype. It will be of interest to know whether this unusual behaviour is related to the phloem localization of TYLCSV or is a more general characteristic of members of the family Geminiviridae.


   ACKNOWLEDGEMENTS
 
We thank E. Vecchiati and D. Marian for excellent technical assistance. We are also grateful to Jozsef Burgyan and Bob Milne for critically reading the manuscript. This work was partially funded by the PNR Biotecnologie Avanzate II and by the PNR Programma Strategico Post-genoma.


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Received 31 December 2003; accepted 11 February 2004.



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