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1 The United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan
2 The 21st Century Center of Excellence Program, Iwate University, Morioka 020-8550, Japan
3 Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
4 Kyoto Prefectural Institute of Agricultural Biotechnology, Soraku-gun, Kyoto 619-0244, Japan
5 Graduate School of Life and Environmental Sciences, University of Osaka Prefecture, Sakai 599-8531, Japan
Correspondence
Nobu Yoshikawa
Yoshikawa{at}iwate-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hannon, 2002; Zamore, 2002). The pathway is triggered initially by double-stranded RNAs, which are processed into small interfering RNAs (siRNAs) of 21–25 nt by an RNase III-like enzyme called Dicer (Hamilton & Baulcombe, 1999). These siRNAs are incorporated into a protein complex called RNA-induced silencing complex (RISC) and guide the RISC to degrade target RNAs that have sequences identical to those of the siRNAs (Hammond et al., 2000).
In plants, RNA silencing functions as an immune system against viruses and transposons (Vance & Vaucheret, 2001; Baulcombe, 2004; Ding et al., 2004; Voinnet, 2005a; Wang & Metzlaff, 2005). During virus infection, long double-stranded (ds) RNAs generated from the replication intermediates of virus RNA trigger RNA silencing. When RNA silencing is induced at one site, silencing signals then move both from cell to cell and long distance (Palauqui et al., 1997; Voinnet & Baulcombe, 1997; Guo & Ding, 2002; Himber et al., 2003) and trigger systemic silencing of target RNA in distant tissues of plants. If the silencing signals induced by virus replication spread in advance of virus movement, sequence-specific virus resistance may be established in whole plants and the virus cannot infect systemically. Although the exact nature of silencing signals remains to be elucidated, RNA is probably a key component to confer sequence specificity in RNA silencing (Mlotshwa et al., 2002; Voinnet, 2005b).
To counteract RNA silencing, viruses have evolved RNA-silencing suppressors. Over 30 viral suppressors have been identified among plant, animal and insect viruses. However, these suppressors have no obvious sequence similarity to each other and they interfere with the RNA-silencing pathway at different points (Roth et al., 2004; Voinnet, 2005a). For instance, the helper component–proteinase (HC-Pro) of potyviruses suppresses intracellular local silencing by interfering with the upstream production of siRNA, and it also affects the biogenesis and function of microRNA (Chapman et al., 2004; Dunoyer et al., 2004). On the other hand, the 2b protein encoded by Cucumber mosaic virus (CMV) and the P25 protein of Potato virus X (PVX) prevent the spread of RNA-silencing signals by inhibiting their movement and/or production (Brigneti et al., 1998; Voinnet et al., 2000; Guo & Ding, 2002). The P21 protein of Beet yellows virus and P19 protein of tombusviruses bind to and presumably inactivate siRNA (Chapman et al., 2004; Lakatos et al., 2004). Interestingly, Citrus tristeza virus (CTV) encodes three distinct suppressors that interfere with multiple steps of RNA silencing (Lu et al., 2004). Identification and analysis of viral silencing suppressors are important both for understanding the survival strategy of viruses in host plants and for dissecting the RNA-silencing pathway.
Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus. It has flexuous, filamentous particles of approximately 600–700 nm in length, which contain a polyadenylated, single-stranded, plus-sense RNA and multiple copies of a single coat protein (CP) of 21 kDa (Yoshikawa & Takahashi, 1988). ACLSV is distributed worldwide and is known to infect Rosaceae fruit-tree species, including apple, peach, pear, plum, cherry and apricot (Lister, 1970; Martelli et al., 1994). In Japan, ACLSV is one of the causative agents of apple topworking disease and induces lethal decline in apple trees grown on Maruba kaido (Malus prunifolia Ringo) rootstocks (Yanase, 1974). Complete nucleotide sequences have been reported for the P863 and PBM isolates from plum, for BAL1 from cherry and for P-205 from apple (German et al., 1990; German-Retana et al., 1997; Sato et al., 1993). The genome of an apple isolate of ACLSV (P-205) consists of 7552 nt excluding the 3' poly(A) tail and contains three open reading frames (ORFs 1, 2 and 3) that encode a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and CP, respectively (Sato et al., 1993). Both P50 and CP are expressed from 3'-coterminal subgenomic RNA. At present, it has not been elucidated which of these proteins might act as a suppressor of RNA silencing.
In this study, we have used an Agrobacterium-mediated transient assay in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c to show that ACLSV P50 is an unusual suppressor of RNA silencing that interferes with the spread of systemic silencing induced by both single-stranded (ss) RNA and dsRNA, but not with local silencing in infiltrated leaves. The suppression of systemic silencing by P50 is probably due to inhibition of the movement of silencing signals into upper leaves, not suppression of the production of signals.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. For expression and induction of RNA silencing of GFP, the binary plasmids pBI-GFP and pBI-dsGFP (both kindly provided by Dr T. Kon and Professor M. Ikegami, Touhoku University, Miyagi, Japan) were used. These plasmids consist of the cauliflower mosaic virus (CaMV) 35S promoter, the mGFP5 sequence (inserted in the sense orientation or as an inverted repeat) and nopaline synthase (nos) terminator. The binary vector pBE2113 (Mitsuhara et al., 1996), consisting of enhanced CaMV 35S promoter, tobacco mosaic virus 5' untranslated region (
) and nos terminator, was also used. The plasmid pBE2113-P35T was constructed from pBE2113-EGFP (Yoshikawa et al., 1999) by eliminating the enhanced GFP (EGFP) sequence. The ORF1 sequence encoding P216 was amplified from pCLSF (Satoh et al., 1999) by PCR with the Expand Long Template PCR system (Roche) and two primers: ACORF1Xba (+) [5'-GCTCTAGAATGGCTTTCTCTCTTATAGA-3', corresponding to nt 152–169 (underlined) of the ACLSV (P-205) genome (GenBank accession no. D14996
[GenBank]
; Sato et al., 1993) and containing an XbaI site], and ACORF1Sac (–) [5'-TACATGAGCTCTCAGAATAAATTCTGGAGCT-3', corresponding to nt 5790–5809 (underlined) of the virus genome and containing a SacI site]. The PCR product was double-digested with XbaI and SacI and ligated to pBE2113-EGFP previously restricted with the same enzymes. The resulting plasmid was denoted pBE2113-P216. pBE2113-P50 and pBE2113-CP, which contain the P50 and CP sequences, respectively, have been reported previously (Yoshikawa et al., 2000). The frame-shift mutant of the P50 sequence, which has been deleted of 5 nt containing the initiation codon, was amplified by PCR from pCLSF using two primers: AC50KFSBam (+) [5'-CGCGGATCCGATAAGGGGTCACAAATTGA-3', corresponding to nt 5732–5751 (underlined) of the virus genome and containing a BamHI site], and AC50Ksac (–) [5'-TACATGAGCTCTCACACACTTGGCGGAAGGT-3', corresponding to nt 7081–7100 (underlined) of the virus genome and containing a SacI site]. The PCR product was double-digested with BamHI and SacI and ligated to pBE2113-EGFP restricted with the same enzymes. The resulting plasmid was denoted pBE2113-FSP50. The HC-Pro gene of Clover yellow vein virus (ClYVV) (Riechmann et al., 1992; Takahashi et al., 1997) was amplified from infected Chenopodium quinoa leaves by RT-PCR with two primers: ClHCProBam (+) [5'-CGCGGATCCATGTCTGCAGGAGATTTGTTT-3', corresponding to nt 1–19 (underlined) of the HC-Pro gene and containing a BamHI site], and ClHCProsac (–) [5'-TACATGAGCTCTCAACCAACTCTGTAAAA-3', corresponding to nt 1357–1371 (underlined) of the HC-Pro gene and containing a SacI site]. The 2b gene of Cucumber mosaic virus (strain pepo) was amplified by PCR from pBKP2b (Saiga et al., 1998; Kobori et al., 2003; Ryang et al., 2004) by using two primers: Pepo2bBam (+) [5'-CGCGGATCCATGGAATTGAACGTAGGT-3', corresponding to nt 1–18 (underlined) of the 2b gene and containing a BamHI site], and Pepo2bSac (–) [5'-TACATGAGCTCTCAGAAAGCACCTTCCG-3', corresponding to nt 317–333 (underlined) of the 2b gene and containing a SacI site]. These PCR products were ligated to the pBE2113 binary plasmid by using the BamHI and SacI sites as described above. The resulting plasmids were designated pBE2113-HCPro and pBE2113-2b, respectively.
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Agroinfiltration and GFP imaging.
A. tumefaciens carrying each construct was cultured on Luria–Bertani (LB) agar containing kanamycin, rifampicin and tetracycline at 28 °C for 48 h. Bacterial cells were harvested and resuspended in an infiltration buffer [10 mM MES (pH 5.6), 10 mM MgCl2 and 150 µM acetosyringone] to a final OD600 of 1.0 for general infiltration and incubated for 2–4 h at room temperature. In co-infiltration, equal volumes of each suspension were mixed prior to infiltration. Five-week-old GFP-expressing transgenic N. benthamiana line 16c plants (kindly provided by Professor David Baulcombe, Sainsbury Laboratory, Norwich, UK) and non-transgenic Nicotiana occidentalis 37B plants were infiltrated on the fourth, fifth and sixth true leaves with a 1 ml syringe without a needle. The infiltrated plants were kept at 25 °C in a growth chamber. GFP fluorescence was observed under long-wavelength UV light (Black Ray model B 100A; UV Products) and photographed by using a FinePix S1 Pro digital camera (Fujifilm) with a yellow filter.
Western blot analysis.
Total proteins extracted from co-infiltrated N. benthamiana line 16c leaf tissues were electrophoresed in an SDS/polyacrylamide (12.5 %) gel and transferred electrophoretically to a PVDF membrane (Millipore). The membrane was incubated with polyclonal antibodies against P50 or ACLSV particles, followed by anti-rabbit IgG (H&L) alkaline phosphatase-linked antibody (Cell Signaling Technology), and then immersed in development solution containing Fast Red TR salt (Sigma) and naphthol AS-MX phosphate (Sigma).
RNA analysis.
Total RNAs were extracted from 200–300 mg leaf tissue by using TRI reagent (Sigma) according to the manufacturer's protocol. For Northern blot analysis of GFP mRNA, the same amount of total RNA was separated on a 1 % agarose gel containing 6 % formaldehyde and transferred to Hybond-N+ membrane (Amersham Biosciences). For analysis of siRNAs, low-molecular-weight RNAs (LMW-RNAs) were enriched from total RNAs by removing high-molecular-mass RNAs using 10 % polyethylene glycol (PEG8000) and 1 M NaCl. The LMW-RNA (4 µg) in each sample was separated on a 7 M urea/15 % polyacrylamide gel and then transferred to Hybond-N+ membrane. After UV cross-linking, the membranes were hybridized with digoxigenin (DIG)-labelled antisense RNA probes complementary to the full-length GFP sequence. The hybridized membranes were immunodetected with anti-DIG Fab fragments coupled to alkaline phosphatase (Roche) and visualized with a chemiluminescent substrate (CSPD; Amersham Biosciences) on X-ray films.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The constructs used in the analysis are shown diagramatically in Fig. 1
. pBE2113-P35T (vector) and pBE2113-HCPro that expresses the HC-Pro protein of ClYVV (Riechmann et al., 1992; Takahashi et al., 1997) were used as negative and positive controls for silencing-suppressor activity, respectively.
When leaves of N. benthamiana line 16c were infiltrated with a mixture of agrobacteria carrying pBI-GFP and pBE2113-P35T, GFP fluorescence derived from pBI-GFP reached the highest level in infiltrated regions 2–3 days post-infiltration (p.i.), followed by decrease or disappearance of fluorescence at 4–5 days p.i. [Fig. 2a(i)]. In contrast, leaves infiltrated with pBI-GFP plus pBE2113-HCPro showed a marked increase in GFP fluorescence at 2–3 days p.i. and the fluorescence remained at a high level for at least 2 weeks [Fig. 2a(ii)], indicating that ClYVV HC-Pro has strong suppressor activity, as described previously for other potyviruses (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau & Carrington, 1998). When a mixture of agrobacteria carrying pBI-GFP and pBE2113-P216, pBE2113-P50 or pBE2113-CP was infiltrated, none of P216, P50 or CP showed any effects on the intensity of GFP fluorescence, and the fluorescence in infiltrated regions disappeared at 5 days p.i. [Fig. 2a(iii–v)].
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), compared with leaves infiltrated with buffer only (Fig. 2b, mock). An approximate estimation of the level of GFP mRNA using serially diluted RNA samples indicated that GFP mRNA levels in leaves infiltrated with GFP plus empty vector or vector expressing P216, P50 or CP were at least eightfold lower than in mock-infiltrated leaves. In contrast, the accumulation of GFP mRNA in leaves infiltrated with GFP plus HC-Pro was at least sixfold higher than in leaves infiltrated with buffer (Fig. 2b).
As the presence of siRNA is a hallmark of RNA silencing (Hamilton & Baulcombe, 1999; Hamilton et al., 2002), the accumulation of siRNA specific for GFP mRNA was analysed. Fig. 2(b) showed that the accumulation of siRNAs of 21–25 nt length was detected readily in leaves infiltrated with GFP plus empty vector or vector expressing P216, P50 or CP, but not in samples infiltrated with buffer only (mock) or GFP plus vector expressing HC-Pro. From these results, it is concluded that none of the three known proteins of ACLSV, P216, P50 or CP, has a suppressor activity that interferes with local silencing in N. benthamiana line 16c.
To demonstrate that P50 and CP accumulate in infiltrated leaves, leaves infiltrated with GFP plus vector expressing P50 or CP were collected at 3 and 5 days p.i. and Western blot analysis using polyclonal antibodies against P50 (anti-P50) and ACLSV particles (anti-CP) was carried out. As shown in Fig. 2(c), both P50 and CP were expressed and detected readily at 3 days p.i., but the accumulation level of both proteins had decreased at 5 days p.i. (Fig. 2c). These results support the hypothesis that neither P50 nor CP could suppress local silencing. However, we cannot strictly rule out the possibility that the failure of P216 to suppress local silencing could be due to instability of the protein.
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P50 interferes with systemic silencing in N. benthamiana
After the onset of RNA silencing, a mobile silencing signal is thought to spread systemically and induce systemic silencing of homologous sequences in upper leaves (Guo & Ding, 2002; Mlotshwa et al., 2002; Palauqui et al., 1997; Voinnet, 2005b; Voinnet & Baulcombe, 1997). To investigate whether P216, P50 or CP can interfere with the induction of systemic silencing, GFP fluorescence was monitored in upper leaves of N. benthamiana infiltrated with a mixture of agrobacteria carrying pBI-GFP plus pBE2113-P216, pBE2113-P50 or pBE2113-CP as described above. In this analysis, pBE2113-2b, which expresses 2b protein of CMV-pepo (Saiga et al., 1998; Kobori et al., 2003; Ryang et al., 2004), was used as a positive control for a protein with systemic silencing-suppressor activity (Brigneti et al., 1998; Guo & Ding, 2002).
When 16c plants were infiltrated with GFP plus empty vector, fluorescence in upper uninfiltrated leaves started to fade out at 8 days p.i. and disappeared extensively after 14 days p.i. [Fig. 3a(ii)]. The disappearance of GFP fluorescence in upper leaves was found on 96.7 % of plants (29 of 30 infiltrated plants) (Table 1). In contrast, upper leaves of all plants infiltrated with GFP plus CMV 2b showed green fluorescence similar to that in mock-infiltrated plants at 14 days p.i. [Fig. 3a(i, iii); Table 1], indicating that CMV 2b functioned as a suppressor of systemic silencing as reported before (Brigneti et al., 1998; Guo & Ding, 2002). When 16c plants were infiltrated with GFP plus P50, upper leaves of 73.3 % (22 of 30) plants remained brightly green fluorescent at 14 days p.i. [Fig. 3a(iv-a)], whilst the remaining plants exhibited partial disappearance of GFP fluorescence mainly on and around the veins of a few leaves [Fig. 3a(iv-b)]. Thus, P50 appears to be almost as efficient as CMV 2b in suppression of systemic silencing. On most of the plants infiltrated with GFP plus a vector expressing P216, CP or FSP50 (a frame-shift mutant of P50), GFP fluorescence in all upper leaves had mostly disappeared at 14 days p.i. [Fig. 3a(v); Table 1].
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). In contrast, the accumulation of GFP mRNA in upper leaves of plants infiltrated with GFP plus empty vector or vector expressing P216, CP or FSP50 was about eightfold lower than that of a mock-infiltrated plant (Fig. 3b). Conversely, siRNA derived from GFP mRNA was found to have accumulated in upper leaves of plants infiltrated with GFP plus empty vector or vector expressing P216, CP or FSP50, but not in those infiltrated with GFP plus vector expressing CMV 2b or P50 (Fig. 3b).
We next investigated the suppressor activity of P50 when systemic silencing was induced in plants by infiltration with dsRNA. The 16c plants were infiltrated with GFP plus dsGFP (pBI-dsGFP) plus empty vector or vector expressing CMV 2b or P50, and the resulting systemic silencing in upper leaves was monitored. Most plants infiltrated with GFP plus dsGFP plus CMV 2b (13 of 15 plants) and GFP plus dsGFP plus empty vector (14 of 15 plants) developed systemic silencing in upper uninfiltrated leaves (Table 1). On the other hand, 60 % of plants (nine of 15 plants) infiltrated with GFP plus dsGFP plus vector expressing P50 did not show systemic silencing in upper leaves (Table 1), indicating that P50 interferes with systemic silencing induced by both ssRNA and dsRNA. Our finding that CMV 2b suppresses systemic silencing triggered by ssRNA, but not by dsRNA, is consistent with the result reported for 2b of Tomato aspermy virus (Cao et al., 2005).
It has been reported that short (21–22 nt) and long (25 nt) siRNAs may be involved in cell-to-cell and long-distance movement of silencing signals, respectively (Hamilton et al., 2002; Himber et al., 2003). We analysed the accumulation level of these two classes of siRNA in leaves infiltrated with GFP plus empty vector or vector expressing CMV 2b or P50. Fig. 3(c) shows that both short (21–22 nt) and long (25–26 nt) siRNAs from GFP mRNA accumulated to a similar level in the P50- and empty vector-infiltrated leaves. From these results, it appears that P50 does not inhibit the production of long siRNA, a candidate component of the signal for systemic silencing. In CMV 2b-infiltrated leaves, on the other hand, local silencing in infiltrated regions was suppressed and only a trace of siRNA was detected by Northern blot hybridization (Fig. 3c).
P50 interferes with the movement of silencing signals
To test whether P50 suppress systemic silencing by inhibiting the movement of silencing signals, we conducted an assay described by Guo & Ding (2002), in which CMV 2b was expressed locally along the presumed path of movement of silencing signals. The agrobacteria carrying pBI-GFP and pBE2113-P50 were infiltrated simultaneously but separately into tip (T) or base (B) portion of two basal leaves of N. benthamiana line 16c and systemic silencing of GFP in upper leaves was monitored. In this assay, empty vector and vector expressing CMV 2b were used as a negative and a positive control for a protein with systemic silencing-suppressor activity, respectively (Guo & Ding, 2002).
The results are summarized in Table 2. Most of the 16c plants infiltrated with GFP (T) and empty vector (B) (14 of 15 infiltrated plants) showed systemic silencing of GFP in upper leaves at 14 days p.i. In contrast, 73.3 % of 16c plants (11 of 15 plants) infiltrated with GFP (T) and CMV 2b (B) showed green fluorescence similar to that in mock-infiltrated plants at 14 days p.i., in agreement with the results for CMV 2b reported previously (Guo & Ding, 2002). When 16c plants were infiltrated with GFP (T) and P50 (B), uninfiltrated upper leaves of 50 % of infiltrated plants (10 of 20 plants) did not show systemic silencing of GFP at 14 days p.i. Because P50 could spread into neighbouring cells from cells that produce it (Satoh et al., 2000), 16c plants were infiltrated with both constructs in the opposite orientation to exclude the possibility that P50 moves into silenced cells and prevents the production of silencing signals in it. All 16c plants infiltrated with GFP (B) and P50 (T) exhibited systemic silencing of GFP in upper leaves. These results suggest that P50 suppressed systemic silencing by inhibiting the movement of silencing signals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2006); (ii) it can spread from cells that initially produce it into neighbouring cells (Satoh et al., 2000); (iii) it enables cell-to-cell trafficking of GFP when P50 and GFP are co-expressed in leaf epidermis (Satoh et al., 2000); (iv) it induces the production of tubular structures protruding from the surface of protoplasts (Satoh et al., 2000; Isogai et al., 2003); (v) it has two independently active, single-stranded nucleic acid-binding domains (Isogai & Yoshikawa, 2005); and (vi) it interferes specifically with the functions of the MP encoded by Grapevine berry inner necrosis virus (Isogai et al., 2003; Yoshikawa et al., 2000, 2006). In addition, the present study indicates that P50 functions as a suppressor of RNA silencing. P50 is an MP assigned to the ‘30K’ superfamily (Mushegian & Koonin, 1993) and is the first silencing suppressor reported to be a member of this superfamily.
The suppressor activity of P50 is relatively unusual among suppressors encoded by plant viruses reported so far, in that P50 suppresses systemic silencing in upper leaves without interfering with local silencing in infiltrated leaves. Similar behaviour for suppressors encoded by plant viruses have been reported for P1 protein of Rice yellow mottle virus (RYMV) and a coat protein (CP) of Citrus tristeza virus (CTV) (Himber et al., 2003; Lu et al., 2004). Of the two, P50 appears to be closer to CTV CP than to RYMV P1, because RYMV P1 reduces accumulation specifically of the long species of siRNA (Hamilton et al., 2002; Himber et al., 2003). On the other hand, neither P50 (Fig. 3c) nor CTV CP (Lu et al., 2004) affected the accumulation of long or short siRNAs. Both ACLSV and CTV are fruit-tree viruses and a suppressor activity that only suppresses systemic silencing might be favourable for persistent infection in fruit trees.
The suppression of systemic silencing by P50 was not complete and the percentages of silenced plants out of infiltrated plants were 73.3 and 60 % in systemic silencing induced by ssRNA and dsRNA, respectively (Table 1). This incomplete suppression might be due to the lack of suppressor activity for local silencing in P50, because transient expression of a candidate protein in the agroinfiltration assay ceases at 2–3 days p.i. unless it can suppress local silencing (Voinnet et al., 2003). In fact, P50 in agroinfiltrated regions was detected readily by Western blot analysis at 3 days p.i., but was difficult to detect at 5 days p.i. (Fig. 2c, left).
In this study, we found that CMV 2b protein could suppress both local and systemic silencing induced by ssRNA (pBI-GFP), but it did not prevent systemic silencing by dsRNA (pBI-dsGFP). The result is similar to findings with 2b of Tomato aspermy virus (Cao et al., 2005). On the other hand, P50 suppressed systemic silencing induced by both ssRNA and dsRNA without affecting the accumulation of either short (21–22 nt) or long (25 nt) siRNA (Table 1; Fig. 3c). This suggests that the step in the silencing pathways at which P50 works is different from that of CMV 2b. Moreover, the result from concurrent infiltration at the tip and the base of a leaf suggested strongly that P50 suppressed systemic silencing by inhibiting the movement of silencing signals (Table 2). Although the nature of the systemic silencing signals remains to be determined, the signals move from cell to cell through plasmodesmata and systemically through phloem (Palauqui et al., 1997; Voinnet et al., 1998). P50 could spread from cells that initially produce it into neighbouring cells (Satoh et al., 2000) and accumulate in sieve elements in P50-plant (P50-expressing transgenic N. occidentalis) and ACLSV-infected plant leaves (Yoshikawa et al., 1999, 2006). At these sites, P50 might be positioned to prevent the movement of silencing signals rather than blocking the production of the signals in cells. Alternatively, P50 would interfere with host protein(s) required for movement of the silencing signals. Screening for plant factor(s) interacting with P50 might help to elucidate the nature of the silencing signals.
Because silencing signals move from cell to cell through plasmodesmata and systemically through phloem (Palauqui et al., 1997; Voinnet et al., 1998) in a manner similar to that of virus movement, localization of P50 in plasmodesmata and its accumulation on the parietal layer of sieve elements and on sieve plates (Yoshikawa et al., 1999, 2006) may correlate with the ability of P50 to prevent systemic silencing.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 12 July 2006;
accepted 22 September 2006.
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