Agricultural Biotechnology Center, Plant Biology Institute, P.O. Box 411, H-2101, Gödöll, Hungary
Correspondence
Dániel Silhavy
silhavy{at}abc.hu
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ABSTRACT |
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Published ahead of print on 22 January 2003 as DOI 10.1099/vir.0.18987-0.
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MAIN TEXT |
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In vertebrate cells, dsRNAs also activate RNA-dependent protein kinase (PKR)-mediated, non-specific antiviral responses, including inhibition of translation and induction of cell death. As a counterdefence strategy, many vertebrate viruses express dsRNA-binding proteins (dsRBPs) that prevent PKR activation by sequestering dsRNAs (Kaufman, 1999). As dsRNAs play a role in RNAi and since many non-vertebrate RNA viruses also express dsRBPs, it is possible that virus-encoded dsRBPs could operate as inhibitors of RNAi. To address this issue, we tested to see if dsRBPs could suppress RNAi in plants.
Transgene expression can also trigger RNAi. Since virus- and transgene-induced RNAi operate in overlapping pathways, virus-encoded RNAi suppressors inhibit transgene-triggered RNAi. As the mechanism of plant and animal RNAi is conserved, the Agrobacterium tumefaciens infiltration assay has been used to identify silencing suppressors encoded by both plant and animal viruses (Li et al., 2002; Voinnet et al., 1999
). The infiltration of green fluorescent protein (GFP) transgenic Nicotiana benthamiana plants with A. tumefaciens carrying a vector in which the transcription of GFP is controlled by the 35S promoter (35S-GFP) not only results in transient GFP expression but also leads to the induction of GFP silencing. Cell-autonomous GFP silencing manifests as a weakening of green fluorescence, a decline in the level of GFP mRNA and an accumulation of GFP-specific siRNAs in the infiltrated patches (Brigneti et al., 1998
). siRNAs accumulate in two functionally different size classes. The 2123 nt siRNA fraction guides RISC, while the 2426 nt siRNA fraction is associated with systemic silencing (Hamilton et al., 2002
). If 35S-GFP is co-infiltrated with another A. tumefaciens expressing an RNAi suppressor, the levels of green fluorescence remain high, GFP mRNA levels do not decrease and siRNA accumulation is reduced in the infiltrated leaves (Voinnet et al., 2000
). Escherichia coli RNase III and the mammalian reovirus outer shell polypeptide
3 are among the best-characterized dsRBPs; therefore, we tested the RNAi suppressor capacity of these proteins and their mutants. Both proteins carry conservative dsRNA-binding motifs and bind dsRNAs in vitro and in vivo (Dasgupta et al., 1998
; Denzler & Jacobs, 1994
; Fierro-Monti & Mathews, 2000
; Huismans & Joklik, 1976
; Kharrat et al., 1995
; Nicholson, 1999
; Yue & Shatkin, 1997
). The postulated silencing suppressor capacity of E. coli RNase III, a mutant RNase III that binds dsRNA but lacks RNA cleavage activity (Rnc70) (Dasgupta et al., 1998
) and reovirus
3 proteins were tested in the Agrobacterium co-infiltration assay. The rnc+ (encodes RNase III) and rnc70 (encodes Rnc70) genes were amplified by PCR from plasmids pACS21 and pSDF70 (Dasgupta et al., 1998
) with primers RNC START (5'-ATGAACCCCATCGTAAT-3') and RNC STOP (5'-TCATTCCAGCTCCAGTT-3'). The PCR products were then cloned into the SmaI-digested Agrobacterium binary vector BIN61S (Silhavy et al., 2002
) to create the constructs 35S-rnc+ and 35S-rnc70 (Fig. 1
a). The S4 segment (encodes
3) was amplified by PCR with primers S4START (5'-ATGGAGTGTTGCTTGCC-3') and S4STOP (5'-TTAGCCAAGAATCATCGG-3') from plasmid pBC12BI (Giantini & Shatkin, 1989
) and cloned into the SmaI site of BIN61S to create the construct 35S-
3. As a negative control, a 35S-
3 clone was constructed by PCR, amplifying the 5' first 846 nt segment of the S4 gene with primers S4START (5'-ATGGAGTGTTGCTTGCC-3') and
S4STOP (5'-TTACATTTTACAGTTCCCAG-3'). Then, the PCR fragment was cloned into the SmaI-digested BIN61S plasmid. 35S-
3 encodes a truncated protein that fails to bind dsRNAs (Miller & Samuel, 1992
).
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dsRBPs inactivate PKR by depleting dsRNAs. If RNAi suppression of dsRBPs is also based on dsRNA sequestering, RNase III, Rnc70 and 3 should effectively bind dsRNAs in plant cells, thereby preventing the silencing-mediated degradation of dsRNAs. To test this hypothesis, silencing-mediated degradation of dsRNA was analysed in the presence and absence of dsRBPs. GFP transgenic N. benthamiana leaves were infiltrated with Agrobacteria carrying a GFP inverted repeat (Fig. 1a
); thus, the expressed mRNAs formed hairpin structures with a long stem (35S-IR) and could be digested by DICER. In line with previous reports (Johansen & Carrington, 2001
) at 3 days p.i., siRNAs were very abundant in 35S-IR-infiltrated cells of GFP transgenic N. benthamiana (Fig. 3
a, bottom panel), indicating that 35S-IR induced strong RNAi. As shown in Fig. 3(a)
, co-infiltration of 35S-
3 with 35S-IR did not influence RNAi-mediated dsRNA degradation, while dsRBPs inhibited 35S-IR-induced RNA silencing. siRNAs were not detected (35S-IR+35S-rnc+) or they accumulated to low levels (35S-IR+35S-rnc70 and 35S-IR+35S-
3) in 35S-IR and dsRBP co-infiltrated tissues (Fig. 3a
). The accumulation of a higher molecular mass mRNA fraction that corresponds to IR mRNA in samples taken from 35S-IR+35S-rnc70 and 35S-IR+35S-
3 co-infiltrated leaves (Fig. 3a
, top panel) suggests that dsRBPs prevented the degradation of IR dsRNA. The lack of this RNA fraction in control samples (Fig. 3a
, top panel) could reflect the activity of DICER and other dsRNases. IR mRNAs were also absent in 35S-IR+35S-rnc+ co-infiltrated samples, even though siRNAs were not detected (Fig. 3a
). These data suggest that E. coli RNase III degraded the co-expressed IR mRNAs.
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It is likely that certain virus suppressors target conserved elements of the RNAi machinery. Tombusvirus p19 RNAi suppressor binds ds siRNAs, thus inhibiting virus-induced systemic silencing in plants (Silhavy et al., 2002). Other RNAi suppressors might target another conserved elements of RNAi, long dsRNAs. Indeed, we showed that heterologous dsRBPs could effectively suppress RNAi, presumably by sequestering dsRNAs. We propose that many virus-encoded dsRBPs play important roles in pathogenicity by interfering with RNAi-mediated cell-autonomous and systemic host defences. As effective silencing suppression likely requires early, abundant cytoplasmic expression of virus-encoded dsRBPs, we think that only a subset of virus-encoded dsRBPs could operate as natural RNAi suppressors. For instance, in reovirus- or vaccinia virus-infected mammalian cells, the expression of
3 or E3L might lead to inactivation of RNAi-mediated defences in addition to inhibition of PKR-mediated responses (Kaufman, 1999
).
To confer broad-spectrum virus resistance, dsRNA-specific ribonucleases were expressed in transgenic plants (Sano et al., 1997; Watanabe et al., 1995
). RNase III- and Rnc70-expressing transgenic plants have shown virus resistance against viruses with segmented genomes (Langenberg et al., 1997
; Zhang et al., 2001
). However, finding that both RNase III and Rnc70 suppress RNA silencing suggests that the RNAi defence system of these transgenic plants could be compromised; therefore, these transgenic plants might be more susceptible to certain viruses.
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ACKNOWLEDGEMENTS |
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Received 18 November 2002;
accepted 9 January 2003.