1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia
2 M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Str., Moscow 117997, Russia
3 Institute of Plant Virology, Microbiology and Biosafety, Federal Biological Research Centre for Agriculture and Forestry, Messeweg 11/12, D-38104 Braunschweig, Germany
Correspondence
S. Yu. Morozov
morozov{at}genebee.msu.su
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ABSTRACT |
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INTRODUCTION |
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Plant viruses have evolved an ability to counteract RNA silencing with silencing-suppressor proteins (Kasschau & Carrington, 1998; Voinnet et al., 1999
). By now, silencing suppressors have been identified in more than 12 genera of plant viruses. The structure of viral silencing suppressors is strikingly diverse. Moreover, the ability to suppress silencing is often found as an additional activity of proteins that were previously known to have another function in the virus life cycle, such as movement protein, coat protein (CP) or a component of the viral replicase (Moissiard & Voinnet, 2004
; Silhavy & Burgyán, 2004
).
The molecular mechanisms of RNA silencing suppression by viral proteins are poorly understood. A remarkable exception is the tombusviral suppressor P19, which has recently been demonstrated to block the silencing machinery by specifically sequestering siRNA molecules, thereby preventing their incorporation into RISC complexes (Silhavy et al., 2002; Vargason et al., 2003
; Ye et al., 2003
). Many viral silencing suppressors are RNA-binding proteins and, moreover, even silencing-unrelated, dsRNA-binding proteins have been shown to exert silencing-suppression activity in plants (Lichner et al., 2003
). However, siRNA binding is unlikely to be the general mechanism of suppression, as the unique structural fold formed by the P19 homodimer to selectively bind the 19 nt RNA duplex (Vargason et al., 2003
; Ye et al., 2003
) is apparently absent in many viral suppressors (Moissiard & Voinnet, 2004
). Due to the diversity of suppressors, it is generally believed that these proteins could target different components of the silencing machinery and, therefore, interfere with different steps of RNA silencing suppression (Moissiard & Voinnet, 2004
; Silhavy & Burgyán, 2004
). For instance, the potyviral suppressor HC-Pro inhibits RISC effector complexes that are involved in siRNA-guided degradation of target RNAs (Kasschau et al., 2003
) and interferes with dsRNA processing into siRNA (Dunoyer et al., 2004
), whereas the cucumoviral suppressor 2b efficiently blocks systemic spread of the RNA silencing signal (Guo & Ding, 2002
).
Recently, a silencing suppressor has been identified in the genus Hordeivirus (Yelina et al., 2002; Bragg & Jackson, 2004
). Viruses of this genus contain a tripartite, positive-stranded RNA genome consisting of RNA
, RNA
and RNA
. RNA
is monocistronic and encodes a component of viral replicase; RNA
encodes the CP and three movement proteins; RNA
is bicistronic and encodes the other component of viral replicase (
a protein) and a non-structural
b protein (Jackson et al., 1989
; Solovyev et al., 1996
; Savenkov et al., 1998
; Lawrence et al., 2000
). The N-terminal
b region is able to bind ssRNA and Zn2+ (Donald & Jackson, 1996
; Bragg et al., 2004
), whereas the C-terminal
b region contains a predicted coil-coiled structure that is responsible for protein self-interactions (Bragg & Jackson, 2004
). Heterologous complementation and cross-protection assays have revealed the silencing-suppressor function of
b encoded by Poa semilatent virus (PSLV) (Yelina et al., 2002
). For
b of Barley stripe mosaic virus (BSMV, the type species of the genus Hordeivirus), the ability to suppress RNA silencing was demonstrated in an agrobacterium-mediated transient-expression assay (Bragg & Jackson, 2004
). These findings are consistent with previous reports showing that
b is dispensable for virus replication, but influences hordeivirus genome amplification, determines viral long-distance movement and affects infection phenotype (Petty et al., 1990
, 1994
; Donald & Jackson, 1994
).
In this paper, we studied the subcellular localization of PSLV b protein and the possible role of its subcellular localization signal in the silencing-suppression function.
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METHODS |
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Monoclonal antibodies (mAbs).
For mouse immunization, 50 µg soluble PSLV GFPb fusion, mixed with an equal volume of Freund's complete adjuvant, was injected intraperitoneally and boosted twice with the same dose mixed with incomplete adjuvant at 2 week intervals. The final injection of 50 µg antigen was without adjuvant. Immunized spleen cells were fused 3 days later with the mouse myeloma cell line Sp2/0 by using 45 % polyethylene glycol. Cells were cultured under selective conditions on Dulbecco's HAT MEM (Amresco) supplemented with 15 % fetal calf serum (Loewe) in the presence of mouse peritoneal macrophages as feeder cells, as described previously (Erokhina et al., 2000
). Culture fluids were screened for specific antibody production by indirect ELISA. Hybridomas secreting specific mAbs were cloned twice under limiting-dilution conditions. mAbs were purified from ascitic fluids by affinity chromathography on protein ASepharose (Sigma).
Transient expression and plant analyses.
Inoculation of Nicotiana benthamiana plants with recombinant viruses and Western and Northern blot analyses were carried out as described by Yelina et al. (2002). Agrobacterium tumefaciens strain GV2260/C58C1 was used for the agroinfiltration assay. Bacteria carrying pLH7000-based vectors were cultured overnight at 28 °C with 10 mM MES and 20 µM acetosyringone, then resuspended in 10 mM MES (pH 5·5), 10 mM MgCl2 and 150 µM acetosyringone to a final density of OD600=2, incubated for 3 h at room temperature and infiltrated into N. benthamiana leaves. In silencing-suppression experiments, Agrobacterium cultures were mixed prior to all infiltrations. Green fluorescent protein (GFP) reporter-gene culture (1 vol.) was mixed with either (i) 2 vols culture containing empty vector, (ii) 1 vol. dsGF-containing culture and 1 vol. culture containing empty vector or (iii) 1 vol. dsGF-containing culture and 1 vol.
b- or
b
SKL-containing culture. Total RNA was isolated as described by Verwoerd et al. (1989)
. High-molecular-mass RNA was precipitated with an equal volume of 10 % PEG8000/1 M NaCl. For analysis of siRNAs, 5 µg low-molecular-mass RNA fraction was separated in a 15 % polyacrylamide gel containing 8 M urea, transferred to Hybond-N membranes (Amersham Biosciences) and hybridized with a radiolabelled, negative-sense T7 transcript of the GFP gene. For subcellular fractionation, leaf tissue was ground to a fine powder with liquid nitrogen in a buffer containing 400 mM sucrose, 100 mM Tris/HCl (pH 7·5), 10 mM
-mercaptoethanol, 2 mM PMSF, 1 mM EDTA, 1 mM EGTA and 2 µg aprotinin ml1. The slurry was filtered through two layers of Miracloth (Calbiochem). The filtrate was taken for Western blot analysis as a total fraction and the residual portion of the filtrate was centrifuged at 30 000 g for 30 min to yield pellet (P30) and supernatant (S30) fractions. GFPC3 fluorescence in infiltrated leaves was monitored under a hand-held long-wave UV lamp (UVL-56; UVP). Particle bombardment of N. benthamiana leaves was performed by using the flying-disc method with the PDS-1000 system (Bio-Rad), as described previously (Morozov et al., 1997
). Cells expressing fusions of fluorescent proteins were imaged with a Leica TCS SP2 system. GFP was visualized with an argon ion laser at 488 nm and an acquisition window of 500530 nm. For imaging of co-expressed yellow fluorescent protein (YFP) and GFP constructs, argon ion laser-excitation lines (488 nm for GFP and 514 nm for YFP) were used alternately. Accordingly, the fluorescence of GFP and YFP was detected alternately by using the switching between lines' option of the confocal system in the 496510 nm acquisition window for GFP and the 560615 nm window for YFP. In this way, any cross-talk between the GFP and YFP channels was eliminated (Brandizzi et al., 2002
).
Immunogold labelling and electron microscopy.
Sections of N. benthamiana leaves were embedded in Epon 812 resin after fixation with 2·5 % glutaraldehyde in 0·1 M phosphate buffer, pH 7·0 (Erokhina et al., 2001). Ultrathin sections (90110 nm) were cut with a diamond knife and placed on Formvarcarbon-coated grids. Grids were preblocked by incubation for 15 min in 0·1 M sodium cacodylate buffer, pH 7·4 (CB), with 1 % BSA, washed with CB and incubated overnight at room temperature with solution containing purified mAbs (5 µg ml1). After washing, the grids were incubated for 2 h at room temperature with goat anti-mouse IgG conjugated to 15 nm gold beads (Biocell) and washed with distilled water. Finally, grids were stained with 1 % uranyl acetate for 30 min and examined under a LEO EM-906 electron microscope.
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RESULTS |
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For protein immunodetection, mAbs against PSLV b were raised. In preliminary experiments, 11 mAbs showed a strong positive reaction with Escherichia coli-expressed
b, which was used for mice immunization (data not shown). To test the reaction of mAbs with PSLV
b that accumulated in plants during viral infection, we inoculated N. benthamiana plants with B
P, a previously described hybrid hordeivirus representing BSMV in which the native
b gene has been replaced by that of PSLV (Yelina et al., 2002
). Western blotting of B
P-infected tissues with five mAbs confirmed their ability to recognize
b in plant extracts (Fig. 1
a and data not shown); three mAbs (3D5, 2B2 and 1D3) were selected for further experiments. In plants, PSLV
b was detected not only as a monomer, but also as a dimer and as oligomers (Fig. 1a
), which are presumably due to a putative coiled-coil region that is found in the PSLV
b sequence (Bragg & Jackson, 2004
). Such a predicted coiled-coil region in BSMV
b was shown to be responsible for protein self-interactions (Bragg & Jackson, 2004
).
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Crude fractionation of tissues expressing b and
b
SKL demonstrated that
b was localized mostly in the P30 fraction, which contained cell membranes (Fig. 1b
); this was in agreement with the previously reported association of PCV P15 with the P30 fraction (Dunoyer et al., 2002
). On the other hand,
b
SKL was detected mainly in the S30 fraction (Fig. 1b
), in accordance with earlier reports showing that
b encoded by the ND18 strain of BSMV (which lacks the C-terminal tripeptide SKL) was found predominantly in the S30 fraction (Donald et al., 1993
; Donald & Jackson, 1994
).
Subcellular localization of GFP-fused PSLV b and its SKL mutant
To analyse the subcellular localization of PSLV b and to verify that its C-terminal tripeptide SKL could serve as a peroxisomal targeting signal, we used particle bombardment-mediated transient expression of GFP-tagged proteins. In these experiments, the expression vectors pRT-GFP-
b and pRT-GFP-
b
SKL were used. Confocal laser-scanning microscopy of bombarded N. benthamiana epidermal cells revealed that GFP-fused
b was localized to punctate structures (Fig. 2
a), resembling those in which GFP-fused PCV 15K was localized in virus-infected protoplasts (Dunoyer et al., 2002
). The latter structures were proposed to represent peroxisomes (Dunoyer et al., 2002
). Therefore, to resolve the nature of
b-containing structures, we used peroxisome-targeted fluorescent marker proteins.
|
In contrast to GFPb, GFP-fused
b
SKL was distributed diffusely in the cytoplasm and in the nucleus (Fig. 2b
), thus resembling the GFP distribution in plant cells (Reichel et al., 1996
). These data show that PSLV
b is directed to peroxisomes and that its C-terminal signal, SKL, is implicated in this targeting. Interestingly, a GFP fusion to the BSMV ND18 strain
b, which lacks the C-terminal tripeptide SKL, was also distributed diffusely in plant cells (Lawrence & Jackson, 2001a
, b
).
Immunogold studies of PSLV b in virus-infected cells
To verify whether PSLV b is localized to peroxisomes in virus-infected cells, we used immunogold electron microscopy of thin sections of N. benthamiana leaves collected 6 days post-inoculation (d.p.i.) with B
P. mAbs 3D5, 2B2 and 1D3 gave similar results, therefore only data obtained with mAb 3D5 will be presented below. In B
P-infected cells, immunogold labelling was found predominantly in oval structures (Fig. 3
) that were classified, according to their size, shape and electron density, as peroxisomes (Nishimura et al., 1996
; Pastori & del Río, 1997
). Gold particles were distributed uniformly over the matrix of a given labelled peroxisome; however, not all observed peroxisomes appeared to be labelled (Fig. 3
). Examination of other subcellular structures revealed that labelling of the chloroplasts was comparable to the background labelling in healthy cells (Table 1
). On the other hand, low but statistically significant labelling was found in the cytoplasm and nucleus of B
P-infected cells (Table 1
, Fig. 3
). However, this labelling did not exhibit the consistent localization that is characteristic of proteins associated with specific cytoplasmic or nuclear structures. Additionally, no specific immunolabelling was observed in control sections of B
P-infected leaves treated with mAbs specific to Beet yellows virus methyltransferase and leader papain-like proteinase (Erokhina et al., 2001
; Zinovkin et al., 2003
) (data not shown).
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Ability of the b protein and its SKL mutant to suppress RNA silencing in an agroinfiltration assay
Recently, BSMV b was reported to exhibit silencing-suppression activity in an agrobacterium-mediated transient-expression system (Bragg & Jackson, 2004
). To compare the abilities of
b and
b
SKL to suppress RNA silencing, we employed a similar assay. This approach involved simultaneous transient co-expression of a reporter gene with a dsRNA silencing inducer and a candidate silencing suppressor (Johansen & Carrington, 2001
). In this study, GFPC3 (Crameri et al., 1996
) was used as a reporter. As an inducer of GFPC3-targeted RNA silencing, we used a construct, referred to as dsGF, that contained two copies of the 5'-proximal part of the GFPC3 coding sequence in the sense and antisense orientations separated by a spacer, so that transcription of this construct in plant cells gave rise to an RNA that formed a long RNA duplex.
To test the silencing-suppression activity of PSLV b, A. tumefaciens harbouring binary vector pLH-
b was mixed with A. tumefaciens containing the binary vectors pLH-GFPC3 and pLH-dsGF. A similar mixture was prepared with agrobacteria harbouring binary vector pLH-
b
SKL. The mixtures were infiltrated into N. benthamiana leaves. In controls, infiltrations were carried out with either a bacterial culture carrying pLH-GFPC3 or a mixture of cultures carrying pLH-GFPC3 and pLH-dsGF. For simplicity, we shall refer below to each A. tumefaciens culture used in the infiltrations by the name of the gene that it harbours in expression cassettes of the binary vectors. Observations of leaves under a long-wave UV light revealed bright GFP fluorescence in leaf areas infiltrated with GFP, but not with GFP+dsGF (Fig. 5
a), confirming that dsRNA efficiently induced silencing of homologous target RNA (Johansen & Carrington, 2001
). In leaf areas infiltrated with GFP+dsGF+
b or GFP+dsGF+
b
SKL, GFP fluorescence was restored to a level similar to that in the areas expressing GFP only (Fig. 5a
), suggesting that both PSLV
b and
b
SKL efficiently suppressed RNA silencing in the agroinfiltration assay.
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DISCUSSION |
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In experiments with transient expression of GFP-fused b, we found that the protein was localized in punctate structures that were dispersed in transfected cells (Fig. 2a
). To resolve the nature of the
b-containing structures, we employed both fluorescent microscopy and immunoelectron microscopy. Transient co-expression of GFP
b with peroxisomal markers, either YFPPTS1 or PTS2YFP, revealed their precise colocalization in punctate bodies (Fig. 2c and d
). These data demonstrate that PSLV
b is localized in peroxisomes and are consistent with the proposed peroxisomal localization of a GFP fusion of PCV cysteine-rich protein P15 (Dunoyer et al., 2002
), which exhibits sequence similarity to hordeiviral
b genes (Savenkov et al., 1998
).
The occurrence of b in peroxisomes of virus-infected cells was not uniform. Only a subpopulation of these organelles observed by immunoelectron microscopy was found to be labelled by
b-specific mAbs (Fig. 3
). On the other hand, in transient-expression experiments, all peroxisomes that contained the peroxisomal marker proteins, either YFPPTS1 or PTS2YFP, also accumulated GFP-fused
b (Fig. 2c and d
), showing that GFP
b localized only to peroxisomes that actively import matrix proteins. Presumably, hordeivirus-infected cells contain both functional peroxisomes and import-inactive peroxisomes or peroxisome-like structures. It is not yet known whether healthy plant cells contain such structures in parallel with normal peroxisomes. In animal cells, accumulation of peroxisome-like structures, known as ghost peroxisomes, is associated with deficiencies in peroxisome import (Wilson, 1991
; Santos et al., 1992
). One can speculate that hordeiviral infections induce changes in peroxisomal metabolism, resulting in the appearance of subpopulations among leaf peroxisomes, as has been described for plant cells under stress conditions (Nishimura et al., 1996
; Pastori & del Río, 1997
).
In the patch agroinfiltration assay, PSLV b effectively suppressed silencing of GFP, in agreement with similar experiments with BSMV
b (Bragg & Jackson, 2004
), but had little effect on GFP-specific siRNA accumulation (Fig. 5
). The latter observation is consistent with the finding that PCV P15, which is structurally similar to
b, is unable to prevent processing of dsRNA into siRNAs (Dunoyer et al., 2004
). However, unlike PSLV
b, PCV P15 significantly reduced the siRNA level (Dunoyer et al., 2004
). PCV 15K has been proposed to exert its suppression activity at a step downstream of siRNA production, e.g. 15K may block incorporation of siRNA into RISC. This may cause an instability of RISC-excluded siRNA that is manifested by reduced siRNA levels (Dunoyer et al., 2004
). Our observations indicate that PSLV
b may also act as a silencing suppressor downstream of siRNA production. On the other hand, as siRNAs accumulate in the presence of
b, one can speculate that
b does not prevent incorporation of siRNA into RISC, but instead may block RISC activity.
To study whether b peroxisomal localization was related to its function as an RNA-silencing suppressor, we constructed the mutant
b
SKL, which has a deletion of five C-terminal amino acid residues that include the C-terminal tripeptide SKL. Localization of pecluviral P15 has been shown to depend on the C-terminal tripeptide SKL (Dunoyer et al., 2002
), which represents the canonical peroxisome targeting signal PTS1, directing transport of cellular proteins into the matrix of peroxisomes (Olsen, 1998
; Subramani et al., 2000
; Hayashi & Nishimura, 2003
; Reumann, 2004
). Therefore, we envisaged blockage of
b
SKL transport to peroxisomes. To verify this prediction, we carried out a crude subcellular fractionation of N. benthamiana leaf tissues expressing either wild-type
b or
b
SKL. Similarly to PCV 15K (Dunoyer et al., 2002
),
b was found predominantly in the P30 fraction containing cell membranes, whereas most
b
SKL was detected in the S30 fraction containing soluble cytoplasmic proteins (Fig. 1b
). Accordingly, GFP-fused
b
SKL showed no association with peroxisomes and was found to localize diffusely in the cytoplasm and nucleus (Fig. 2b
). Thus, as expected, localization of
b in cell peroxisomes depended on the C-terminal signal SKL and we used
b
SKL in further experiments to study the functional significance of such protein targeting.
Firstly, we analysed the effect of b
SKL deletion on the infection phenotype of recombinant hordeivirus B
P, which represented a previously described chimera of BSMV carrying the PSLV
b gene in place of the native
b gene (Yelina et al., 2002
). For this purpose, the mutant
b
SKL gene was introduced into B
P to replace the full-length
b gene. Inoculation of N. benthamiana plants revealed that timing and phenotype of B
P-
b
SKL infection (both local and systemic) were similar to those of B
P (Fig. 4e, f and h
). Secondly, we found that the dramatic effect of PSLV
b on the phenotype of PVX infection, manifested as extensive necrosis resulting in fast death of infected plants (Fig. 4c
; Yelina et al., 2002
), could also be induced by
b
SKL expressed in the PVX background (Fig. 4a
). These observations suggest that deletion of the
b subcellular targeting signal has little effect on the ability of the protein to influence viral infection phenotype.
Direct comparison of b
SKL silencing-suppression potential with that of
b revealed that, although the mutant lost its subcellular localization, it fully retained suppression activity in an agroinfiltration assay (Fig. 5
). Thus, localization of
b in peroxisomes is dispensable for its ability to suppress RNA silencing and is thus consistent with the general view that RNA silencing is a cytoplasmic process (Voinnet, 2001
; Moissiard & Voinnet, 2004
) and argues in favour of the suggestion that the cytoplasm, not peroxisomes, is the site of
b silencing-suppression activity. This suggestion is supported by an earlier finding showing that the peroxisome localization motif is not required for silencing suppression mediated by pecluviral P15 (Dunoyer et al., 2002
). However, when the SKL motif in P15 was deleted, local and systemic transport of PCV was suppressed dramatically (Dunoyer et al., 2002
), whereas
b
SKL supported hordeivirus movement as effectively as the native protein (Fig. 4e, f and h
).
Interestingly, the tobraviral cysteine-rich protein shows sequence similarity to hordeiviral b, but lacks the C-terminal SKL tripeptide (Savenkov et al., 1998
). However, hordeiviral
b can substitute functionally for the tobraviral protein (Liu et al., 2002
), supporting the hypothesis that peroxisomes are not the site of
b action. This conclusion is supported by earlier observations that, among four BSMV strains, only two have the SKL tripeptide at the
b C terminus (Gustafson et al., 1987
; Kozlov et al., 1989
; Edwards, 1995
). One can speculate that peroxisomes contain a sequestered excess of PSLV
b that is produced during viral infection, whereas the functional form of the protein can exist in cells in low amounts and/or temporarily. Depositions of presumably excessive amounts of a silencing suppressor have been reported for PVX p25, which is implicated in both silencing suppression and viral movement. Immunoelectron microscopy revealed that p25 localized in specific cytoplasmic inclusions consisting of filamentous material (Davies et al., 1993
), but never in the cytoplasm, plasmodesmata or other sites where it would be expected to perform its suppression and movement functions.
At least one viral silencing suppressor is known to accumulate only to very limited levels during infection. Indeed, high levels of the suppressor P0 were shown to be unfavourable for Beet western yellows virus (BWYV, genus Polerovirus) and P0 synthesis in BWYV infection is downregulated to undetectable amounts by conservation of the non-optimal context of the translation initiation codon of the P0 gene (Pfeffer et al., 2002). Sequestering of
b in peroxisomes might be another way to reduce the effective concentration of silencing suppressor at the sites of its action. It remains to be investigated whether small amounts of PSLV
b detected in the cytoplasm and nucleus (Table 1
, Fig. 4
) represent the functional form of the protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Bass, B. L. (2000). Double-stranded RNA as a template for gene silencing. Cell 101, 235238.[Medline]
Bragg, J. N. & Jackson, A. O. (2004). The C-terminal region of the Barley stripe mosaic virus b protein participates in homologous interactions and is required for suppression of RNA silencing. Mol Plant Pathol 5, 465481.[CrossRef]
Bragg, J. N., Lawrence, D. M. & Jackson, A. O. (2004). The N-terminal 85 amino acids of the Barley stripe mosaic virus b pathogenesis protein contain three zinc-binding motifs. J Virol 78, 73797391.
Brandizzi, F., Frangne, N., Marc-Martin, S., Hawes, C., Neuhaus, J.-M. & Paris, N. (2002). The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain. Plant Cell 14, 10771092.
Crameri, A., Whitehorn, E. A., Tate, E. & Stemmer, W. P. C. (1996). Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14, 315319.[Medline]
Davies, C., Hills, G. & Baulcombe, D. C. (1993). Sub-cellular localization of the 25-kDa protein encoded in the triple gene block of potato virus X. Virology 197, 166175.[CrossRef][Medline]
Donald, R. G. K. & Jackson, A. O. (1994). The barley stripe mosaic virus b gene encodes a multifunctional cysteine-rich protein that affects pathogenesis. Plant Cell 6, 15931606.
Donald, R. G. K. & Jackson, A. O. (1996). RNA-binding activities of barley stripe mosaic virus b fusion proteins. J Gen Virol 77, 879888.[Abstract]
Donald, R. G. K., Zhou, H. & Jackson, A. O. (1993). Serological analysis of barley stripe mosaic virus-encoded proteins in infected barley. Virology 195, 659668.[CrossRef][Medline]
Dunoyer, P., Pfeffer, S., Fritsch, C., Hemmer, O., Voinnet, O. & Richards, K. E. (2002). Identification, subcellular localization and some properties of a cysteine-rich suppressor of gene silencing encoded by peanut clump virus. Plant J 29, 555567.[CrossRef][Medline]
Dunoyer, P., Lecellier, C.-H., Parizotto, E. A., Himber, C. & Voinnet, O. (2004). Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16, 12351250.
Edwards, M. C. (1995). Mapping of the seed transmission determinants of barley stripe mosaic virus. Mol Plant Microbe Interact 8, 906915.[Medline]
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494498.[CrossRef][Medline]
Erokhina, T. N., Zinovkin, R. A., Vitushkina, M. V., Jelkmann, W. & Agranovsky, A. A. (2000). Detection of beet yellows closterovirus methyltransferase-like and helicase-like proteins in vivo using monoclonal antibodies. J Gen Virol 81, 597603.
Erokhina, T. N., Vitushkina, M. V., Zinovkin, R. A., Lesemann, D. E., Jelkmann, W., Koonin, E. V. & Agranovsky, A. A. (2001). Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus. J Gen Virol 82, 19831994.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806811.[CrossRef][Medline]
Guo, H. S. & Ding, S. W. (2002). A viral protein inhibits the long range signaling activity of the gene silencing signal. EMBO J 21, 398407.
Gustafson, G., Hunter, B., Hanau, R., Armour, S. L. & Jackson, A. O. (1987). Nucleotide sequence and genetic organization of barley stripe mosaic virus RNA-. Virology 158, 394406.[CrossRef][Medline]
Hamilton, A. J. & Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950952.
Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. (2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293296.[CrossRef][Medline]
Hausmann, L. & Töpfer, R. (1999). Entwicklung von Plasmid-Vektoren. Vortr Pflanzenzücht 45, 155172 (in German).
Hayashi, M. & Nishimura, M. (2003). Entering a new era of research on plant peroxisomes. Curr Opin Plant Biol 6, 577582.[CrossRef][Medline]
Hayashi, M., Aoki, M., Kondo, M. & Nishimura, M. (1997). Changes in targeting efficiencies of proteins to plant microbodies caused by amino acid substitutions in the carboxy-terminal tripeptide. Plant Cell Physiol 38, 759768.[Medline]
Himber, C., Dunoyer, P., Moissiard, G., Ritzenthaler, C. & Voinnet, O. (2003). Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J 22, 45234533.
Jackson, A. O., Hunter, B. G. & Gustafson, G. D. (1989). Hordeivirus relationships and genome organization. Annu Rev Phytopathol 27, 95121.[CrossRef]
Johansen, L. K. & Carrington, J. C. (2001). Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol 126, 930938.
Kasschau, K. D. & Carrington, J. C. (1998). A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95, 461470.[Medline]
Kasschau, K. D., Xie, Z., Allen, E., Llave, C., Chapman, E. J., Krizan, K. A. & Carrington, J. C. (2003). P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev Cell 4, 205217.[Medline]
Kato, A., Hayashi, M., Kondo, M. & Nishimura, M. (1996). Targeting and processing of a chimeric protein with the N-terminal presequence of the precusor to glyoxysomal citrate synthase. Plant Cell 8, 16011611.
Klahre, U., Crété, P., Leuenberger, S. A., Iglesias, V. A. & Meins, F., Jr (2002). High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants. Proc Natl Acad Sci U S A 99, 1198111986.
Kozlov, Yu. V., Afanas'ev, B. N., Rupasov, V. V., Golova, Iu. B. G., Kulaeva, O. I., Dolia, V. V., Atabekov, I. G. & Baev, A. A. (1989). Primary structure of RNA 3 of barley stripe mosaic virus and its variability. Mol Biol (Mosk) 23, 10801090 (in Russian).[Medline]
Lawrence, D. M. & Jackson, A. O. (2001a). Interactions of the TGB1 protein during cell-to-cell movement of Barley stripe mosaic virus. J Virol 75, 87128723.
Lawrence, D. M. & Jackson, A. O. (2001b). Requirements for cell-to-cell movement of Barley stripe mosaic virus in monocot and dicot hosts. Mol Plant Pathol 2, 6575.[CrossRef]
Lawrence, D. M., Solovyev, A. G., Morozov, S. Yu., Atabekov, J. G. & Jackson, A. O. (2000). Genus Hordeivirus. In Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses, pp. 899904. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. London: Academic Press.
Lichner, Z., Silhavy, D. & Burgyán, J. (2003). Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences. J Gen Virol 84, 975980.
Liu, H., Reavy, B., Swanson, M. & MacFarlane, S. A. (2002). Functional replacement of the Tobacco rattle virus cysteine-rich protein by pathogenicity proteins from unrelated plant viruses. Virology 298, 232239.[CrossRef][Medline]
Mano, S., Nakamori, C., Hayashi, M., Kato, A., Kondo, M. & Nishimura, M. (2002). Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP: dynamic morphology and actin-dependent movement. Plant Cell Physiol 43, 331341.
Moissiard, G. & Voinnet, O. (2004). Viral suppression of RNA silencing in plants. Mol Plant Pathol 5, 7182.
Morozov, S. Yu., Fedorkin, O. N., Jüttner, G., Schiemann, J., Baulcombe, D. C. & Atabekov, J. G. (1997). Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes. J Gen Virol 78, 20772083.[Abstract]
Nishimura, M., Hayashi, M., Kato, A., Yamaguchi, K. & Mano, S. (1996). Functional transformation of microbodies in higher plant cells. Cell Struct Funct 21, 387393.[Medline]
Olsen, L. J. (1998). The surprising complexity of peroxisome biogenesis. Plant Mol Biol 38, 163189.[CrossRef][Medline]
Pastori, G. M. & del Río, L. A. (1997). Natural senescence of pea leaves: an activated oxygen-mediated function for peroxisomes. Plant Physiol 113, 411418.
Petty, I. T. D., French, R., Jones, R. W. & Jackson, A. O. (1990). Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement. EMBO J 9, 34533457.[Abstract]
Petty, I. T. D., Donald, R. G. K. & Jackson, A. O. (1994). Multiple genetic determinants of barley stripe mosaic virus influence lesion phenotype on Chenopodium amaranticolor. Virology 198, 218226.[CrossRef][Medline]
Pfeffer, S., Dunoyer, P., Heim, F., Richards, K. E., Jonard, G. & Ziegler-Graff, V. (2002). P0 of beet western yellows virus is a suppressor of posttranscriptional gene silencing. J Virol 76, 68156824.
Reichel, C., Mathur, J., Eckes, P., Langenkemper, K., Koncz, C., Schell, J., Reiss, B. & Maas, C. (1996). Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells. Proc Natl Acad Sci U S A 93, 58885893.
Reumann, S. (2004). Specification of the peroxisome targeting signals type 1 and type 2 of plant peroxisomes by bioinformatics analyses. Plant Physiol 135, 783800.
Santos, M. J., Hoefler, S., Moser, A. B., Moser, H. W. & Lazarow, P. B. (1992). Peroxisome assembly mutations in humans: structural heterogeneity in Zellweger syndrome. J Cell Physiol 151, 103112.[Medline]
Savenkov, E. I., Solovyev, A. G. & Morozov, S. Yu. (1998). Genome sequences of poa semilatent and lychnis ringspot hordeiviruses. Arch Virol 143, 13791393.[CrossRef][Medline]
Shivprasad, S., Pogue, G. P., Lewandowski, D. J., Hidalgo, J., Donson, J., Grill, L. K. & Dawson, W. O. (1999). Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255, 312323.[CrossRef][Medline]
Silhavy, D. & Burgyán, J. (2004). Effects and side-effects of viral RNA silencing suppressors on short RNAs. Trends Plant Sci 9, 7683.[CrossRef][Medline]
Silhavy, D., Molnár, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M. & Burgyán, J. (2002). A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21, 30703080.
Solovyev, A. G., Savenkov, E. I., Agranovsky, A. A. & Morozov, S. Yu. (1996). Comparisons of the genomic cis-elements and coding regions in RNA components of the hordeiviruses barley stripe mosaic virus, lychnis ringspot virus, and poa semilatent virus. Virology 219, 918.[CrossRef][Medline]
Solovyev, A. G., Stroganova, T. A., Zamyatnin, A. A., Jr, Fedorkin, O. N., Schiemann, J. & Morozov, S. Yu. (2000). Subcellular sorting of small membrane-associated triple gene block proteins: TGBp3-assisted targeting of TGBp2. Virology 269, 113127.[CrossRef][Medline]
Subramani, S., Koller, A. & Snyder, W. B. (2000). Import of peroxisomal matrix and membrane proteins. Annu Rev Biochem 69, 399418.[CrossRef][Medline]
Vargason, J. M., Szittya, G., Burgyán, J. & Hall, T. M. T. (2003). Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115, 799811.[CrossRef][Medline]
Verwoerd, T. C., Dekker, B. M. M. & Hoekema, A. (1989). A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17, 2362.[Medline]
Voinnet, O. (2001). RNA silencing as a plant immune system against viruses. Trends Genet 17, 449459.[CrossRef][Medline]
Voinnet, O. & Baulcombe, D. C. (1997). Systemic signalling in gene silencing. Nature 389, 553.[CrossRef][Medline]
Voinnet, O., Pinto, Y. M. & Baulcombe, D. C. (1999). Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96, 1414714152.
Wilson, G. N. (1991). Structurefunction relationships in the peroxisome: implications for human disease. Biochem Med Metab Biol 46, 288298.[CrossRef][Medline]
Ye, K., Malinina, L. & Patel, D. J. (2003). Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426, 874878.[CrossRef][Medline]
Yelina, N. E., Savenkov, E. I., Solovyev, A. G., Morozov, S. Yu. & Valkonen, J. P. T. (2002). Long-distance movement, virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families. J Virol 76, 1298112991.
Zinovkin, R. A., Erokhina, T. N., Lesemann, D. E., Jelkmann, W. & Agranovsky, A. A. (2003). Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus. J Gen Virol 84, 22652270.
Received 8 September 2004;
accepted 1 November 2004.