Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Correspondence
Kazuyuki Mise
kmise{at}kais.kyoto-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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BMV is a well-studied positive-sense single-stranded RNA plant virus (Ahlquist, 1999). It is an established model for the study of plant RNA viruses and has been used for in-depth studies of viral gene expression, recombination and replication (Kao & Sivakumaran, 2000
). However, the cell-to-cell and long-distance movements of BMV are not as well characterized as those of some other plant viruses. The genome of BMV is divided into three RNA components. RNA1 and RNA2 encode the proteins, 1a and 2a, respectively, which are required for viral RNA replication. Dicistronic RNA3 encodes the 3a protein, which is an MP of BMV (Rao, 1997
), and CP. CP is translated from subgenomic RNA4, which is transcribed from minus-strand RNA3 (Miller et al., 1985
). BMV MP binds to single-stranded nucleic acids (Fujita et al., 1998
; Jansen et al., 1998
), localizes in the plasmodesmata (Fujita et al., 1998
), co-localizes with replication proteins in the BMV infection-inducing inclusion bodies (Dohi et al., 2001
) and induces tubular structures on the surfaces of protoplasts (Sanchez-Navarro & Bol, 2001
). Moreover, BMV MP is involved in host specificity (De Jong et al., 1995
; Fujita et al., 1996
, 2000
; Mise et al., 1993
; Mise & Ahlquist, 1995
; Sasaki et al., 2001
) and modulates symptom expression in susceptible host plants (Fujita et al., 1996
; Rao & Grantham, 1995a
). As mentioned, BMV MP is multifunctional and requires CP for cell-to-cell movement (Rao, 1997
; Rao & Grantham, 1995b
; Sasaki et al., 2003
; Schmitz & Rao, 1996
).
To gain further insight into the function of BMV MP, we used alanine-scanning (AS) mutagenesis (Cunningham & Wells, 1989) to map the BMV MP sequences that influence virus infectivity and symptom development. We then developed a BMV vector carrying the green fluorescent protein (GFP) gene to visualize BMV movement. We identified two BMV mutants with cell-to-cell movement more efficient than that of wild-type BMV. Furthermore, we showed that three BMV mutants can move from cell to cell independently of CP. An encapsidation analysis suggested that the capacities for virion formation and local spread are not sufficient to allow the long-distance movement of BMV, and that the C terminus of BMV MP plays a role in long-distance movement. We discuss the roles of MP and CP in BMV movement.
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METHODS |
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The AS mutation was introduced into pB3GAC2 to create pB3ASn-GAC2. The mutations in the MP gene within pB3AS262 and pB3AS267 were introduced into pB3CP/GFP2 to create pB3AS262-
CP/GFP2 and pB3AS267-
CP/GFP2, respectively. The nucleotides GAA encoding Glu-262 in the BMV MP gene of pB3TP8 were substituted with TAG, creating pB3C
42. This mutation was introduced into pB3
CP/GFP2 and pB3GAC2 to create pB3C
42-
CP/GFP2 and pB3C
42-GAC2, respectively.
The cDNAs generated by PCR were sequenced using a DNA sequencer (Applied Biosystems, model 310). All cDNA plasmids were linearized with EcoRI and capped viral RNA transcripts were synthesized as described previously (Kroner & Ahlquist, 1992). The transcript from the plasmid containing the RNA3 variant is referred to by its plasmid name without the prefix p and in parentheses after B3. For example, RNA3 produced from pB3AS12 is designated B3(AS12). In all experiments, RNA3 derivatives were inoculated together with wild-type RNA1 and RNA2.
Analysis of viral RNAs and proteins.
Isolation, inoculation and incubation of barley protoplasts (Hordeum vulgare cv. Hinodehadaka) were performed as described previously (Damayanti et al., 1999), except for a modification in the protoplast medium which was buffered by the addition of 5 mM MES, pH 6·5. Total and virion fraction RNAs were extracted from infected protoplasts and Northern blot analysis was performed as described previously (Damayanti et al., 1999
) with a DIG-labelled probe for the detection of positive-strand RNAs (Sasaki et al., 2001
). Total proteins were separated by electrophoresis on 12·5 or 15 % polyacrylamide gels containing 0·1 % SDS (Laemmli, 1970
). MP and CP were detected using a mouse monoclonal antibody raised against MP (Fujita et al., 1998
) and rabbit anti-BMV antiserum (PVAS178, ATCC), respectively.
Inoculation experiments in plants.
BMV RNA3-derivatives were inoculated onto Chenopodium quinoa plants as described previously (Nagano et al., 1999). Inoculated and upper uninoculated leaves were harvested at 14 days post-inoculation (p.i.). Viral RNAs were detected by tissue printing analysis, as described previously (Mise et al., 1993
). The leaves were then ground with 7 ml sample buffer (71·4 mM Tris/HCl, pH 6·8, 2·3 % SDS, 11·4 % glycerol, 0·01 % bromophenol blue, 5·7 % 2-mercaptoethanol) per 1 g of harvested leaves. GFP fluorescence in the inoculated leaves of harvested C. quinoa plants was observed under an epifluorescence microscope (Axioscope, Carl Zeiss) equipped with an FITC filter set (Carl Zeiss).
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RESULTS |
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Six AS mutants (AS46, AS69, AS201, AS208, AS240 and AS301) induced symptoms only in the inoculated leaves of C. quinoa (Table 2), suggesting that these AS mutants move from cell to cell, but not systemically. To confirm our visual observations, we examined the accumulation of CP. As expected, CP was detected only in the leaves inoculated with the six AS mutants (Fig. 1b
). We have never detected CP in leaves inoculated with an MP-frameshift mutant (see FS in Fig. 1b
), which suggests that the six AS mutants do move from cell to cell, but do not move systemically. In contrast, eight AS mutants (AS57, AS82, AS83, AS107, AS135, AS169, AS221 and AS227) did not induce symptoms (Table 2
) and no viral RNAs (data not shown) or CP (Fig. 1b
) were detected, even in the inoculated leaves, suggesting that these eight AS mutants do not move from cell to cell.
Visualization of the cell-to-cell movement of BMV using GFP
To further analyse the movement behaviour of the AS mutants, we attempted to visualize BMV movement using the GFP gene. In the family Bromoviridae, several RNA3 derivatives which express GFP have been used to visualize cell-to-cell movement in CMV (Canto et al., 1997) and Alfalfa mosaic virus (AMV) (Sanchez-Navarro & Bol, 2001
; Sanchez-Navarro et al., 2001
). Similarly, three RNA3-based BMV vectors, B3(
3a/GFP), B3(
CP/GFP2) and B3(TGF32), were inoculated onto C. quinoa together with wild-type RNA1 and RNA2. However, unlike the CMV system, co-inoculation with B3(
3a/GFP) plus B3(
CP/GFP2) (trans-complementation of MP and CP from two different RNA3 constructs) did not show multicellular GFP fluorescence on the inoculated leaves of C. quinoa at 5 days p.i. (data not shown). Furthermore, unlike the AMV system, inoculation with B3(TGF32), which contains the GFP gene inserted adjacent to the 5' leader sequence and a duplicated subgenomic promoter sequence, also showed no multicellular GFP fluorescence (data not shown). We then tested the other derivative, B3(GAC2), which encodes a GFPFMDV 2ACP fusion protein (Fig. 2
a) and detected foci with multiple fluorescent cells on the leaves inoculated with B3(GAC2) at 5 days p.i. (Table 3
). However, we failed to observe systemic spread of GFP fluorescence until 14 days p.i. (data not shown). The GFP fluorescence induced by B3(GAC2-FS), which expresses a dysfunctional BMV MP, was confined to single cells (see FS in Table 3
), suggesting that the cell-to-cell movement of B3(GAC2) depends on the function of MP. Passive diffusion of the GFP fusion proteins (GFP2ACP and GFP2A) was not observed under our experimental conditions using fully expanded source leaves of C. quinoa. Therefore, we used B3(GAC2) to examine the capacity of the MP mutants for cell-to-cell movement.
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Virion RNAs were not detected in protoplasts inoculated with B3(GAC2)
To elucidate the mechanism that increases the efficiency of cell-to-cell movement in B3(AS262-GAC2) and B3(AS267-GAC2), we characterized B3(GAC2). First, we examined the accumulation of viral proteins and RNAs in protoplasts inoculated with B3(GAC2). Western blot analysis for B3(GAC2)-inoculated protoplasts detected the uncleaved GFP2ACP fusion protein as well as 2A-cleaved CP with a slightly low electrophoretic mobility, probably due to the attachment of a proline residue at the N terminus of CP (Fig. 2d, lane 1). Thus, cleavage by FMDV 2A was incomplete. In the total RNA fraction, the accumulation of viral RNAs (RNA1RNA4) in B3(GAC2)-inoculated protoplasts was considerably lower than their accumulation in wild-type BMV-inoculated protoplasts (Fig. 2b
; compare lane 1 with lane 5). Surprisingly, in the virion RNA fraction, the accumulation of all four BMV RNAs was below the limit of detection in the B3(GAC2)-inoculated protoplasts (Fig. 2c
, lane 1), although wild-type RNA1 and RNA2 were easily detected in the total RNA fraction (Fig. 2b
, lane 1). These results suggest that a reduction in packaging in B3(GAC2)-infected protoplasts is caused by CP, namely a proline-attached CP cleaved by FMDV 2A or uncleaved GFP2ACP fusion protein.
To examine these possibilities, B3(GC2), which encodes a GFPCP fusion protein (Fig. 2a), and B3(AC2), which encodes an FMDV 2ACP fusion protein (Fig. 2a
), were inoculated into protoplasts and onto C. quinoa plants. In B3(GC2)-inoculated protoplasts, virion RNAs were not detected (Fig. 2c
, lane 2). In leaves inoculated with B3(GC2), GFP fluorescence was confined to single cells in most of the infection foci at 5 days p.i. (Table 4
). These results suggest that the GFPCP fusion protein does not encapsidate BMV RNAs nor function in cell-to-cell movement, suggesting that the uncleaved GFP2ACP fusion protein in B3(GAC2)-infected cells also does not perform these functions. Furthermore, these results suggest that the CP cleaved by FMDV 2A functions in the cell-to-cell movement of B3(GAC2). On the other hand, virion RNAs were detected in protoplasts inoculated with B3(AC2), which encodes an FMDV 2ACP fusion protein (Fig. 2c
, lane 3). B3(AC2) systemically infected C. quinoa plants with severe leaf distortions, like wild-type BMV (data not shown). These results indicate that CP cleaved by FMDV 2A has the ability to encapsidate viral RNAs and to mediate the cell-to-cell movement and even the long-distance movement of BMV. From these results, it appears that the uncleaved GFP2ACP fusion protein inhibits efficient virion formation in B3(GAC2) inoculation.
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The C terminus of BMV MP plays a role in long-distance movement
As shown above, we identified four amino acid residues at positions 262, 263, 267 and 268 within the C terminus of BMV MP, which are involved in the requirement for CP in cell-to-cell movement. On the other hand, our recent study with CMV MP showed that deletion of the C-terminal 33 amino acids alters the requirement for CP in virus cell-to-cell movement (Nagano et al., 2001). Taken together, these data suggest that deletion of the C terminus of BMV MP also alters the requirement for CP in cell-to-cell movement. To test this, we changed the codon for the amino acid at position 262 of BMV MP into a stop codon and investigated its effect on virus movement. Protoplast experiments showed that the accumulation of viral proteins and RNAs was similar in B3(C
42) and wild-type BMV inoculations (Fig. 3
ac). We then tested the effect of a C terminus truncation on cell-to-cell movement using the constructs B3(C
42-GAC2) and B3(C
42-
CP/GFP2). In C. quinoa leaves inoculated with B3(C
42-GAC2) or B3(C
42-
CP/GFP2), multiple GFP-fluorescent cells were observed at 5 days p.i. (Table 4
). These results demonstrate that a C-terminally-truncated BMV MP has the ability to mediate cell-to-cell movement independently of CP.
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DISCUSSION |
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We have shown that the C terminus of BMV MP plays a role in long-distance movement (Fig. 4). In this study, to investigate the role of the C terminus of BMV MP in virus movement, we constructed B3(C
42) by introducing a premature termination codon into the MP gene rather than a deletion mutation, because RNA sequences encoding the C terminus of BMV MP are required for efficient encapsidation of RNA3 (Choi & Rao, 2003
; Damayanti et al., 2002
, 2003
). As expected, the point mutations introduced into B3(C
42) did not affect the efficiency of encapsidation (Fig. 3
), although B3(C
42) did not move systemically (Fig. 4
). These results suggest that efficient virion formation is not sufficient to allow the long-distance movement of BMV, and that the C terminus of BMV MP plays a crucial role in long-distance movement. The inability of B3(C
42) to move over long distances may be explained by the putative interaction between MP and CP. BMV MP requires CP for virus movement (Rao, 1997
; Sasaki et al., 2003
) and this suggests that BMV MP interacts with BMV CP. Furthermore, a chimeric analysis of BMV and CMV suggested that BMV MP and its CP interact specifically (Nagano et al., 1999
). From these data, we infer that the C terminus of BMV MP plays a role in the putative interaction with its CP. The lack or reduction of interaction between BMV CP and a BMV MP after truncation of the C-terminal 42 amino acids may cause the defect in long-distance movement of B3(C
42). This scenario is further supported by recent studies of CMV (Nagano et al., 2001
), AMV (Sanchez-Navarro & Bol, 2001
) and CPMV (Carvalho et al., 2003
), which showed or suggested that the C termini of their MPs interact with their cognate CPs.
Alanine-scanning mutagenesis of BMV MP in this study gave novel information, as discussed below. First, the amino acid residues in the central region of BMV MP are important for cell-to-cell movement (Fig. 1b and Table 2
). The central region of BMV MP is highly conserved among the four bromoviruses (Table 1
) and shares sequence similarity with the MPs of viruses belonging to other genera (Koonin et al., 1991
). In Red clover necrotic mosaic virus, CMV and AMV, the regions in the MPs corresponding to the central region of BMV MP are also important for infectivity (Giesman-Cookmeyer & Lommel, 1993
; Li et al., 2001
; Huang et al., 2001
). Residues 189242 in the central region of BMV MP constitute the RNA-binding domain (Fujita et al., 1998
). The loss of infectivity of AS mutants with mutations in this domain might be due to a lack or reduction of RNA-binding activity.
Second, BMV MP plays a role in long-distance movement (Table 2, Figs 1b and 4
). In inoculated leaves, viruses initially infect epidermal cells and then move into mesophyll and bundle sheath cells and into sequential vascular systems to infect the plants systemically (Carrington et al., 1996
). Although six AS mutants and one C-terminally-truncated MP mutant moved locally, they did not move systemically in C. quinoa (Table 2
, Figs 1b and 4
). The accumulation of CP in leaves inoculated with AS201 and AS208 was significantly low (Fig. 1b
). These results suggest that these two AS mutants do not reach the vasculature in the inoculated leaves, due to quite low efficiency in cell-to-cell movement. The other four AS mutants and one C-terminally-truncated MP mutant may have reached the vasculature, but may have specifically failed to move through the vascular system of C. quinoa because the accumulation of CP from the five MP mutants was equal to that of AS244 in inoculated leaves (Figs 1b and 4
). AS244 moved systemically, although the accumulation of CP in the inoculated leaves was approximately 100-fold lower than that of wild-type BMV (Fig. 1b
). These results suggest that the five BMV MP mutants may not have the ability to overcome a later intercellular barrier(s) (e.g. epidermalmesophyll, mesophyllvascular cells and so on) as reported for other virus MPs (Fujita et al., 2000
; Wang et al., 1998
).
Third, BMV MP could play a role in the unloading process in upper uninoculated leaves. After moving into upper uninoculated leaves, plant viruses are sequentially unloaded into companion, phloem parenchyma, bundle sheath and mesophyll cells, and then move into the epidermal cells (Carrington et al., 1996). The lower accumulation of CP in the upper uninoculated leaves of plants infected with AS12, AS27 or AS244 (Fig. 1b
) suggests that the MPs of these AS mutants may have a defect in the unloading process. Furthermore, the upper uninoculated leaves of plants infected with these AS mutants displayed mild or no leaf distortions (Table 2
). These results suggest that the induction of severe leaf distortions in BMV-infected C. quinoa requires efficient cell-to-cell movement in upper uninoculated leaves.
As shown in this study, C-terminal truncation and point mutations in the C terminus of BMV MP alter the CP requirement in cell-to-cell movement. These findings suggest novel interpretations of two previous studies. First, Sanchez-Navarro & Bol (2001) showed that a chimeric AMV containing wild-type BMV MP (pGFP/B1-303/CP) did not move from cell to cell, whereas a chimeric AMV encoding BMV MP with a deletion of the C-terminal 48 amino acids (pGFP/B1-255/CP) did. We speculate that the BMV MP in pGFP/B1-255/CP has the ability to mediate cell-to-cell movement independently of CP. Second, Flasinski et al. (1995)
has exceptionally reported that BMV has the ability to move from cell to cell in the absence of CP. In that report they may not have observed the ability for cell-to-cell movement of a CP-less mutant of BMV but may, instead, have observed that of its progeny viruses, in which point mutations that alter the CP-requirement in cell-to-cell movement had been occasionally introduced into the MP gene during virus replication. This possibility is supported by our recent findings that BMV MP mutants with the ability to move from cell to cell in the absence of CP were isolated in the leaves inoculated with a CP-defective BMV variant (W. Nakamura, A. Takeda, N. Sasaki, and K. Mise, unpublished results).
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ACKNOWLEDGEMENTS |
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Received 15 January 2004;
accepted 6 February 2004.