1 Pathogen Plant Interactions Group, Plant Breeding Science, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2 Plant Genetic Engineering Laboratory, Biotechnology Institute, Akita Prefectural University, Ogata, Akita 010-0444, Japan
Correspondence
Ichiro Uyeda
uyeda{at}res.agr.hokudai.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Most potyviral proteins are multifunctional. For example, HCPro is involved in aphid transmission (Atreya et al., 1990), genome replication (Klein et al., 1994
; Kasschau et al., 1997
), long-distance movement (Cronin et al., 1995
; Kasschau et al., 1997
; Saenz et al., 2002
) and autoproteolysis (Carrington et al., 1989
). In addition, it has been demonstrated that HCPro has plasmodesmatal gating (Rojas et al., 1997
) and nucleic acid binding properties (Maia & Bernardi, 1996
). Recently HCPro has been identified as a suppressor of post-transcriptional gene silencing (reviewed by Marathe et al., 2000
; Anandalakshmi et al., 1998
; Brigneti et al., 1998
; Kasschau & Carrington, 1998
).
VPg is also multifunctional and needed for virus replication (Shahabuddin et al., 1988; Murphy et al., 1990
, 1991
, 1996
). It is also needed for virus cell-to-cell and long-distance movement (Nicolas et al., 1997
; Schaad et al., 1997
; Keller et al., 1998
; Rajamäki & Valkonen, 2002
). It contains a nuclear localization signal that is important for virus replication (Schaad et al., 1996
) and a sequence-non-specific RNA-binding domain (Merits et al., 1998
). Furthermore, the plant cap-binding proteins eIF4E and eIF(iso)4E have been found to bind the VPg of potyviruses (Wittmann et al., 1997
; Leonard et al., 2000
; Schaad et al., 2000
). This interaction was found to be important for the replication of the Turnip mosaic virus (TuMV), Lettuce mosaic virus (LMV), Potato virus Y (PVY) and Tobacco etch virus (TEV) in Arabidopsis thaliana and pepper (Duprat et al., 2002
; Lellis et al., 2002
; Ruffel et al., 2002
).
Proteinprotein interactions play important roles in the virus infection cycle. Several interactions between potyviral proteins have been studied using the yeast two-hybrid system (YTHS) (Guo et al., 2001). However, the reported interactions between major proteins are not necessarily common to all potyviruses, and there are many inconsistent observations among the potyviruses that have been analysed by YTHS (Hong et al., 1995
; Li et al., 1997
; Merits et al., 1999
; Urcuqui-Inchima et al., 1999
; Choi et al., 2000
; Guo et al., 2001
; Lopez et al., 2001
). In this report, we describe a novel proteinprotein interaction between HCPro and VPg in ClYVV that has not been reported in any other potyvirus, suggesting that the HCProVPg interaction participates in some function unique to ClYVV. Since the reported results for proteinprotein interactions of other potyviruses are not always applicable to ClYVV, the present work will serve as a molecular basis for further characterization of ClYVV proteins.
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METHODS |
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Yeast two-hybrid system and plasmid construction.
Yeast two-hybrid assays were carried out according to the MATCHMAKER Two-Hybrid System 3 protocol (Clontech). The yeast (Saccharomyces cerevisiae) strain AH109 was used to determine proteinprotein interactions. The DNA binding domain (BD) vector pAS2-1 and the activation domain (AD) vector pACT2 were used throughout. HCPro cDNA was inserted in-frame into the BD between the SmaI and SalI sites of pAS2-1 and in-frame into the AD between the BamHI and SacI sites of pACT2. Wild-type VPg cDNA and its deletion clones were inserted in-frame into the BD between the EcoRI and BamHI sites of pAS2-1, or into the BamHI and EcoRI sites of pACT2. PCR primers were designed to add the required restriction endonuclease recognition sequences so that the PCR products could be cloned in-frame into the AD or BD gene. Recombinant plasmids were transformed into E. coli strain JM109. Plasmid DNA was isolated by Quantum Prep Plasmid Miniprep Kit (Bio-Rad). Competent cells of S. cerevisiae strain AH109 were transformed simultaneously with pACT2 and pAS2-1 recombinant DNAs by the lithium acetate method (Gietz et al., 1995). Yeast cells were plated on a high-stringency selective medium without tryptophan, leucine, histidine and adenine, and their
-galactosidase activities were tested at 30 °C on the same selective media containing X-
-Gal. Yeast cells co-transformed with pAS2-1 and pACT2 without inserts were used as a negative control, whereas those co-transformed with pTD1-1 [AD simian virus 40 (SV40) large T antigen] and pVA3-1 (BD mouse p53) were used as a positive control.
Anti-VPg polyclonal antibody production.
Due to the insolubility of the full-length VPg expressed in E. coli, the 5' (aa 1100) and 3' (aa 153191) halves of the VPg gene were separately cloned into the pGEX vector (Amersham) to produce glutathione S-transferase (GST)VPg fusion proteins in E. coli JM109. The former protein was designated VPgN and the latter VPgC. Each purified fusion protein was used to immunize BALB/c mice.
Anti-HCPro monoclonal antibody production.
The entire HCPro gene was cloned into pMAL-c2 (New England Biolabs) to express the maltose-binding protein (MBP)HCPro fusion protein in E. coli JM109. The fusion protein was purified with an MBP affinity column and used to produce a monoclonal antibody against HCPro, essentially as described by Inoue et al. (1984).
Western blot analysis.
Total proteins were prepared from either the leaves or stem tissues of infected plants. Tissues were homogenized in denaturing buffer containing 2 % SDS and 5 % -mercaptoethanol (Laemmli, 1970
) or in PBS. The extracts were boiled for 5 min and centrifuged to collect the supernatants. Equal amounts of protein (
10 µg) were separated by SDS-PAGE. Proteins were then transferred to nitrocellulose membranes (Amersham Pharmacia Biotech) and the blots were probed with anti-HCPro monoclonal antibody. Proteins were visualized using anti-mouse secondary antibodies conjugated to alkaline phosphatase, followed by treatment with NBT/BCIP.
Extraction of yeast proteins.
Yeast cells were grown in appropriate selective synthetic medium SD (Difco) to reach the mid-exponential phase (OD600=0·60·8). Total proteins from yeast were prepared as described in the Yeast Protocols Handbook (Clontech). Yeast cells were lysed with glass beads (425600 µm) in cracking buffer (8 M urea, 5 % SDS, 40 mM Tris/HCl, pH 8·8, 0·1 mM EDTA and 0·4 mg bromophenol blue ml-1) supplemented with -mercaptoethanol and protease inhibitors. Protein samples (1020 µg) were fractionated by SDS-PAGE through a 12·5 % gel, transferred to nitrocellulose membranes and subjected to immunoblot analysis. The membranes were probed with antibodies against GAL4 AD or BD (1 : 200 dilution; Santa Cruz Biotechnology) and then with alkaline phosphatase-conjugated goat anti-rabbit IgG (1 : 250 dilution; Bio-Rad).
Far-Western blot analysis.
The VPgHCPro interaction was further examined in vitro by far-Western blotting. Two different probes, GSTVPg (VPgN+VPgC) and MBPHCPro, were used. Both probes were produced in E. coli JM109. Bacterially expressed proteins and total protein extracts from ClYVV-infected or healthy stem tissues were separated by SDS-PAGE and the fractionated proteins were then transferred on to nitrocellulose membranes. Alternatively, bacterially expressed proteins were directly dot-blotted on to membranes. The blots were first incubated in binding buffer (0·2 M PBS) containing E. coli-produced probe proteins (GSTVPg or GST only and MBPHCPro or MBP only). After being probed with these proteins, the membranes were extensively washed in PBS and treated with specific antibodies (anti-GSTVPg polyclonal antibodies or anti-HCPro monoclonal antibody). All the membranes were treated with alkaline phosphatase-conjugated anti-mouse IgG (Bio-Rad) and finally immersed in the substrate solution containing BCIP/NBT as described above.
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RESULTS |
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Identification of the domain of VPg involved in its self-interaction
We also analysed the VPg domain(s) involved in its self-interaction. A series of VPg deletion mutants from both the BD and AD vectors were made (Fig. 5). When the C terminus of VPg was deleted, no growth was observed on the selective medium. To eliminate the possibility that the lack of interaction was due to instability of the proteins expressed in the yeast cells, Western blot analysis was performed (Fig. 2
). Comparison of the results obtained from BD 51191 and BD 81191 with BD 77153 and BD 50153 suggested that the 38 C-terminal amino acids (residues 153191) are important for VPg self-interaction (Fig. 5
). Furthermore, the combination of BD 81191 and AD 81191 showed a positive interaction (Fig. 5
). The interaction was stronger in both directions when the combination of the region from residues 51 to 191 and the full-length VPg was used. The structural requirement for VPg self-interaction probably favours the entire VPg, although the C-terminal region is indispensable.
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DISCUSSION |
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The interaction found between VPg and HCPro in ClYVV is a novel feature, so far not shown for any other Potyvirus. Whether this interaction is required for ClYVV replication, protein synthesis or movement is unclear. Potyviruses encode several proteins with common functions in their infection cycle. At least seven proteins (P1, HCPro, P3, CI, 6K2, VPg and NIb) have been found to be involved in replication (reviewed by Revers et al., 1999), and five (HCPro, CI, 6K2, VPg and CP) are involved in virus movement (Dolja et al., 1994
, 1995
; Klein et al., 1994
; Cronin et al., 1995
; Kasschau et al., 1997
; Schaad et al., 1997
; Andersen & Johansen, 1998
; Carrington et al., 1998
; Lopez-Moya & Pirone, 1998
; Rajamäki & Valkonen, 1999
). Although HCPro and VPg have common functions, especially in replication and virus movement, it is conceivable that VPg and HCPro act only cooperatively and not independently in replication and movement.
The involvement of VPg in potyviral movement has been reported in TEV, TVMV and PVA (Schaad et al., 1997; Nicolas et al., 1997
; Rajamäki & Valkonen, 1999
, 2002
). In these systems, these authors indicated that VPg formed a movement complex with another viral protein and/or a host factor to function in virus movement. Furthermore, in a separate study VPg was found in an early stage of infection of PVA in the companion cells of infected Solanum commersonii, emphasizing its role in the movement of potyviruses (Rajamäki & Valkonen, 2003
). Since our observations indicate that the central domain of VPg is important for its interaction with HCPro and the same domain was reported to be important for viral movement in other potyviruses (Rajamäki & Valkonen, 2002
), we speculate that ClYVV VPg and HCPro may play an important role in the virus movement process. In view of the fact that VPg is attached to the 5' end of viral RNA and that HCPro has been identified as a movement protein that increases the size exclusion limit of plasmodesmata (Rojas et al., 1997
), the association of VPg and HCPro might facilitate the movement process in a host plant.
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ACKNOWLEDGEMENTS |
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Received 23 April 2003;
accepted 9 June 2003.