1 Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan
2 Graduate Institute of Biochemistry, National Chung Hsing University, Taichung 402, Taiwan
3 Institute of Botany, Academia Sinica, Taipei 115, Taiwan
Correspondence
Yau-Heiu Hsu
yhhsu{at}nchu.edu.tw
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ABSTRACT |
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INTRODUCTION |
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The three proteins encoded by the triple gene block (TGBps) of potexviruses are essential for the cell-to-cell movement of viruses of this group (Angell et al., 1996; Beck et al., 1991
). However, TGBps are structurally and functionally different from the MPs described above. Among the three TGB proteins, TGBp1 appears to exist in soluble and cytoplasmic inclusion forms. The soluble form of TGBp1 binds cooperatively to ssRNA, possesses ATPase (Rouleau et al., 1994
; Wung et al., 1999
) and RNA helicase activities (Kalinina et al., 2002
), increases the SEL of plasmodesmata (Angell et al., 1996
; Lough et al., 1998
) and forms a ribonucleoprotein complex (Lough et al., 1998
, 2000
). TGBp1 is also a gene silencing suppressor, which prevents spread of the gene silencing signal in Nicotiana benthamiana (Voinnet et al., 2000
). TGBp1 can move from cell to cell (Lough et al., 1998
; Yang et al., 2000
) and mediates cell-to-cell movement of TGBp2 and TGBp3 (Krishnamurthy et al., 2002
). Further analysis of the soluble TGBp1 of Bamboo mosaic virus (BaMV) has revealed that Arg-11, Arg-16 and Arg-21 in the N-terminal region of this protein are essential for its RNA-binding activity (Liou et al., 2000
). Although BaMV TGBp1 belongs to the RNA helicase superfamily I (Kadare & Haenni, 1997
; Morozov et al., 1999
; Liou et al., 2000
), Arg-16 and Arg-21, which are located outside the seven conserved motifs of the superfamily I RNA helicase, are also critical for the NTPase activity of TGBp1 (Liou et al., 2000
). TGBp1s of potexviruses are in fact mainly associated with cytoplasmic inclusions in infected tissues (Chang et al., 1997
; Davies et al., 1993
; Rouleau et al., 1994
). The TGBp1 obtained from the cytoplasmic inclusions also possesses NTP-binding and NTPase activities but lacks the RNA-binding activity (Liou et al., 2000
).
TGBp2 and TGBp3 both contain stretches of hydrophobic amino acids and are associated with cell wall and membrane fractions both in vivo and in vitro (Donald et al., 1993; Hefferon et al., 1997
; Morozov et al., 1990
, 1991
; Niesbach-Klosgen et al., 1990
; Solovyev et al., 2000
). Using a microinjection and transient expression strategies, TGBp2 was shown to assist the movement of the ribonucleoprotein complex of potexviruses (Lough et al., 1998
) and to be capable of increasing the SEL of the plasmodesmata (Tamai & Meshi, 2001
). However, contradictory results were obtained for TGBp3. It has been reported to be required for the movement of White clover mosaic virus (WClMV) (Lough et al., 1998
) but is not absolutely required for the movement of potato virus X (Tamai & Meshi, 2001
). Recently, Krishnamurthy et al. (2003)
and Mitra et al. (2003)
have demonstrated that TGBp2 and TGBp3 are endoplasmic reticulum (ER)-targeted proteins and that ER targeting is important for virus movement.
A satellite RNA (satBaMV) has been found to associate with the BaMV-V isolate (Lin & Hsu, 1994). The satBaMV is a linear RNA molecule 836 nucleotides in length, excluding the poly(A) tail, and encodes a 20 kDa non-structural protein (P20). The satBaMV can be used as a plant expression vector since P20 is not essential for satellite replication and can be replaced with other proteins such as chloramphenicol acetyltransferase (Lin et al., 1996
).
In this study, we analysed the relationship between the RNA-binding and NTPase activities of TGBp1 and the capability of BaMV to move from cell to cell. With the aid of the infectious cDNA clones of BaMV-S and a satellite replicon based on satBaMV (Lin et al., 1996), we have demonstrated that Arg-16 and Arg-21 in the N-terminal region of the BaMV TGBp1, residues that are essential for the RNA-binding and NTPase activities of TGBp1 (Wung et al., 1999
; Liou et al., 2000
), are also crucial for cell-to-cell movement of BaMV in host plants.
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METHODS |
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Construction of mutant BaMV and satellite replicons.
Each of the mutant BaMV with ArgAla substitution(s) in TGBp1 was derived from pCBG by site-directed mutagenesis (Sambrook et al., 1989
). Primers used for mutagenesis are shown in Table 1
. pR11A, pR16A and pR21A are plasmids that contain a single Arg
Ala substitution at amino acid positions 11, 16 and 21 of TGBp1, respectively. Plasmids pR11/16A, pR11/21A and pR16/21A containing double Arg
Ala substitutions were derived from pR11A or pR16A. To construct pSat28, the P20-coding sequence of the satBaMV in pCBSF4 was replaced with the TGBp1-coding sequence amplified from pCBG using primers P28N and P28C (Table 1
). The plasmid pSat28f was a mutant clone of pSat28, in which a single-base deletion was observed at position 7 of the TGBp1-coding sequence, resulting in a shift of reading frame from the third amino acid of TGBp1.
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Protoplast preparation, inoculation and Northern and Western blot analyses of viral products.
Methods for preparation and inoculation of protoplasts were as previously described (Cheng & Tsai, 1999) except that plasmid DNA, rather than RNA transcript, was used as the inoculum. In brief, the protoplasts (2x105) prepared from mesophyll cells of N. benthamiana were inoculated with 10 µg plasmid DNA. Methods for Northern and Western blot analyses of viral RNA and proteins in infected protoplasts were as previously described (Cheng & Tsai, 1999
).
FluorImage and confocal laser scanning microscopy of the BaMV-infected leaves.
Fluorescent images of leaves infected with the wild-type BaMV or the TGBp1 mutants were obtained with a FluorImager (Molecular Dynamics, model 595) with an excitation filter of 488 nm and an emission filter using calibration files. Fluorescent images for epidermal and mesophyll cells were obtained through a confocal laser-scanning microscope (Zeiss LSM410). Laser illumination at 488 nm (argon ion laser) was recorded through a bandpass filter normally used for the detection of fluorescence. Image analysis and display (adjustments in contrast, brightness, etc.) were all performed using Adobe PhotoShop.
RT-PCR and cDNA sequencing.
Total RNAs were extracted from the inoculated leaves of C. quinoa and N. benthamiana as previously described (Lin et al., 1996). Reverse transcription was then carried out to synthesize the first strand cDNA of TGBp1 using primer 5081 (Table 1
) complementary to the 3' end of the genomic or subgenomic RNA for TGBp1. Following that, 30 PCR cycles were performed using the forward primer (3572R) and the reverse primer (4818), which are complementary to the cDNA sequence of TGBp1. The PCR products were finally sequenced using primer 3759R with the IRDye 800 terminator sequencing protocol designed for the LI-COR IR2 automated DNA sequencer.
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RESULTS |
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BaMV mutants defective in cell-to-cell movement are able to replicate and synthesize TGBp1
Several explanations could account for the failure of mutant BaMVs with TGBp1 ArgAla substitutions at either position 16, 21 or both 16 and 21 to move from cell to cell. One possibility is that the Arg
Ala substitution(s) directly renders the mutant TGBp1 defective in movement function. Alternatively, it could be that these TGBp1 mutations interfere with virus replication. Finally, it is possible that the synthesis and accumulation of TGBp1 in leaves infected with the mutant BaMVs are blocked for as yet unknown reasons. To rule out the latter two possibilities, the replication of viral RNA and the synthesis of TGBp1 were analysed in protoplasts of N. benthamiana infected with each of the movement-defective BaMVs. As shown in Fig. 6
(A), similar patterns of genomic and subgenomic RNAs of BaMV were observed in protoplasts inoculated with the wild-type or mutant BaMVs, indicating that the wild-type and mutant BaMVs possess similar replication activities. In addition, similar amounts of TGBp1 were also detected in protoplasts infected with the wild-type and mutant BaMVs (Fig. 6B
). These results indicated that the movement-defective BaMVs are able to replicate and to synthesize TGBp1 in the plant cells. Taken together, these results demonstrate that the inability of the mutant BaMVs with Arg
Ala substitutions at position 16, 21 or both 16 and 21 of TGBp1 to move from cell to cell must be attributed to the loss of certain important TGBp1 functions.
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DISCUSSION |
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Complementation has long been used as a strategy to confirm the function of a specific gene. Strategies for complementation of a defective gene in a plant virus include co-bombardment of plants with the defective virus together with a plasmid expressing the target gene (Lough et al., 2001; Morozov et al., 1997
; Tamai & Meshi, 2001
), mixed infection of plants with the defective virus and a viral RNA replicon (Bleykasten-Grosshans et al., 1997
), and microinjection of the target protein into a transgenic plant lacking the corresponding gene (Lough et al., 1998
). The new strategy adopted in the present study involves the use of a novel satellite replicon, pSat28. The presence of TGBp1 and the spread of BaMV in N. benthamiana leaves co-infected with the mutant BaMVs and pSat28 (Fig. 7A
) demonstrated that the satellite replicon strategy works as well as other strategies for complementation analyses.
There are several possible explanations for how the loss of ATPase and RNA-binding activities of TGBp1 could interfere with the movement of the mutant BaMVs. It is highly possible that the loss of the ATPase activity of TGBp1 leads to inactivation of its helicase activity, which is essential for unwinding the duplex region of the viral RNA (Kalinina et al., 2002). In addition, loss of RNA-binding activity of a TGBp1 mutant would probably negatively affect the ability of the mutant TGBp1 to form a streamlined nucleoprotein complex with the viral RNA suitable for movement (Lough et al., 1998
, 2000
). Thus, the movement process of the virus, at least in the initial stage of transport, is probably hampered by the loss of each of these two activities. However, it is also possible that the defective TGBp1 is unable to carry out steps in the movement process subsequent to the streamlining of viral RNA. For example, the TGBp1 mutations could prevent the viral ribonucleoprotein complex from interacting with TGBp2, TGBp3 or some other host protein so that the movement of viral nucleic acid along the cytoskeleton and toward the plasmodesmata is blocked. Furthermore, loss of the ATPase activity may inhibit the ability of TGBp1 to increase the SEL of plasmodesmata (Lough et al., 1998
) and thus decrease the efficiency of viral transport across the cell wall. Further studies are required to clarify these possibilities.
Since the R11A TGBp1, while retaining its NTPase activity, has significantly reduced RNA-binding activity (Liou et al., 2000; Wung et al., 1999
), we originally predicted that this mutant would be unable to move from cell to cell. However, the R11A mutant was shown to be able to move in the present study (Figs 2, 4 and 5
). Because no reversion was detected in the progenies of the R11A mutant (Fig. 3
), we conclude that the R11A TGBp1 mutant is able to fulfil movement function. Probably, the residual RNA-binding activity of the R11A TGBp1 is enough to assist virus movement. Alternatively, there may be some other factors that are able to enhance the RNA-binding activity of the mutant TGBp1 in vivo. The ability of the TGBp1 protein of WClMV to form ribonucleoprotein complexes with the viral CP (Lough et al., 1998
, 2000
) raises the possibility that CP may be one such factor. Other possibilities are interactions with TGBp2 and TGBp3 (Krishnamurthy et al., 2002
).
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ACKNOWLEDGEMENTS |
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Received 19 June 2003;
accepted 2 October 2003.