Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan1
Author for correspondence: Nobuyuki Yoshikawa. Fax +81 19 621 6177. e-mail Yoshikawa{at}iwate-u.ac.jp
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Abstract |
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Introduction |
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Plant virus MPs are reported to be multifunctional. Generally, the MP is localized to the plasmodesmata in infected and transgenic plant cells and induces a significant increase in plasmodesmatal permeability (Atkins et al., 1991 ; Ding et al., 1992
; Tomenius et al., 1987
; Wolf et al., 1989
). The protein binds single-stranded nucleic acids and can traffic from cell to cell itself (Citovsky et al., 1990
; Fujiwara et al., 1993
; Waigmann & Zambryski, 1995
). Recent studies with MP fused to green fluorescent protein (GFP) showed that MP is associated with microtubules and the endoplasmic reticulum (ER), suggesting the involvement of the cytoskeleton and ER in the intracellular trafficking of MP from the site of synthesis in the cytoplasm to the plasmodesmata (Heinlein et al., 1995
, 1998
; McLean et al., 1995
; Huang & Zhang, 1999
). MPGFP fusions of Alfalfa mosaic virus (AlMV) and Cucumber mosaic virus (CMV) are reported to be capable of cell-to-cell trafficking in the leaf epidermis (Itaya et al., 1997
; Huang & Zhang, 1999
).
Apple chlorotic leaf spot virus (ACLSV), the type species of the genus Trichovirus, has very flexuous filamentous particles, approximately 600700 nm in length, and contains a polyadenylated, plus-sense ssRNA with a molecular mass of 2·48x106 Da and a single coat protein of 22 kDa (Yoshikawa & Takahashi, 1988 ). The genome of an apple isolate of ACLSV (P-209) consists of 7552 nt and contains three open reading frames (ORFs 1, 2 and 3) (Sato et al., 1993
). The 216 kDa protein (KP) encoded by ORF1 is a replication-associated protein and a coat protein is encoded by ORF3. The 50 kDa protein (50KP) encoded by ORF2 is thought to be an MP, based on the following evidence. (i) The amino acid sequence of 50KP has some similarity to MPs of other plant viruses and the protein was detected in the cell wall fraction from infected tissues (Sato et al., 1993
, 1995
). (ii) Immunoelectron microscopy with an antiserum against 50KP showed that the protein is localized to plasmodesmata in infected Chenopodium quinoa cells (Yoshikawa et al., 1999
). (iii) In transgenic plants expressing 50KP fused to GFP, the fluorescence was associated with plasmodesmata and accumulated in sieve elements (Yoshikawa et al., 1999
). (iv) Transgenic Nicotiana occidentalis plants producing 50KP can complement the systemic spread of movement-defective ACLSV (Yoshikawa et al., 2000
).
In this study, we transiently expressed ACLSV 50KP fused to GFP in leaf epidermal cells and in leaf mesophyll protoplasts of N. occidentalis and C. quinoa and analysed its subcellular distribution, intercellular trafficking in epidermal cells and tubule formation on the surface of protoplasts. The results indicate that 50KPGFP is associated with a network, thought to be cortical ER, on the periphery of epidermal cells and protoplasts. 50KPGFP moved into adjacent cells from the cells that produced it in the leaf epidermis. The protein also induced formation of tubular structures on the surface of protoplasts. Mutational analysis suggested that these activities are related to each other.
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Methods |
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Particle bombardment.
In transient GFP expression experiments, leaves were detached from N. occidentalis (12 true-leaf stage) or C. quinoa (8 true-leaf stage) plants and placed in a Petri dish containing wet filter paper. The lower epidermis was bombarded with microparticles coated with DNA constructs by using the PDS-1000/He particle delivery system (Bio-Rad) as described before (Satoh et al., 1999 ). Leaves were kept under moist conditions at 25 °C until used for observation.
To examine the complementation of cell-to-cell movement of 50KP-deficient virus by 50KPGFP, the fifth true leaf of a plant of C. quinoa (7 true-leaf stage) was bombarded with a mixture (1:1) of pStuNhe and p35S50KPGFP. The plants were maintained in a glass chamber for 5 days. Total RNA was extracted from bombarded leaves and then subjected to Northern hybridization analysis with an RNA probe, as described previously (Yoshikawa et al., 2000
).
Isolation and transfection of protoplasts.
Leaf mesophyll protoplasts were isolated from C. quinoa and N. occidentalis leaves as follows. Leaves were sliced into strips and then soaked for 3 h in an enzyme solution containing 2% cellulase Onozuka R-10 (Yakult Pharmaceutical), 0·1% pectolyase Y-23 (Seishin Pharmaceutical), 10 mM CaCl2 and 0·5 M mannitol, pH 5·6. The resulting protoplast suspension was washed twice with MC solution (0·5 M mannitol, 10 mM CaCl2) and centrifuged in MC solution containing 20% sucrose for 3 min at 700 r.p.m. Protoplasts were recovered from the middle layer and washed in MC solution.
To the protoplasts (about 3x105 cells), 20 µg plasmid DNA and 500 µl inoculation buffer (10 mM MES, 40 mM CaCl2, 0·5 M mannitol, pH 5·8) were added and then the suspension was mixed gently. Next, 900 µl PEG solution (40% PEG 4000, 40 mM CaCl2, 0·5 M mannitol) was added, followed by incubation on ice for 30 min. After washing with 50 mM glycine, 50 mM CaCl2, 0·5 M mannitol, pH 8·5, 10 ml inoculation buffer was added to the protoplasts, which were then incubated on ice for 30 min. The protoplasts were then suspended in a medium containing 0·5 M mannitol, 0·2 mM KH2PO4, 1 mM KNO3, 1 mM MgSO4, 10 mM CaCl2, 1 µM KI, 0·01 µM CuSO4, pH 6·5, and incubated at 25 °C.
Fluorescence and confocal laser scanning microscopy (CLSM).
Observation of GFP fluorescence in epidermal cells and protoplasts was conducted by using a Leica DMLB fluorescence microscope as described previously (Yoshikawa et al., 1999 ). A laser scanning microscope (Leica DMIRB equipped with Yokogawa CLSM unit CSU10) was also used with excitation at 488 nm and emission at 516700 nm. Digital images were acquired with a Yokogawa DNS10 CCD camera and processed by IPLab (Scananalytics).
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Results |
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In observations of leaf epidermis of C. quinoa plants bombarded with p35SGFP, non-specific trafficking of GFP was found in young, developing leaves, similar to the situation in N. occidentalis. However, in epidermal cells of both young and mature leaves of C. quinoa bombarded with p35S50KPGFP, fluorescent spots in cell walls were detected in only two or three cells around an originally transfected cell, rather fewer than in the case of N. occidentalis (data not shown).
Increased cell-to-cell trafficking of GFP co-expressed with 50KP in leaf epidermis
Plant virus MPs are reported to modify the plasmodesmata and to increase their size-exclusion limit (Derrick et al., 1992 ; Vaquero et al., 1994
; Poirson et al., 1993
; Wolf et al., 1989
). As mentioned above, GFP was restricted to single cells in most cases in mature leaves (Fig. 3
). In order to investigate whether GFP can spread from cell to cell in mature leaves when 50KP is co-expressed in the same cells, p35SGFP was bombarded into one half-leaf and p35S50KP plus p35SGFP were bombarded into the other half-leaf of N. occidentalis. When GFP was co-expressed with 50KP, the fluorescence spread more widely from the cells that initially produced it than when GFP was expressed alone (Table 2
; Fig. 2M
, N
). The result suggests that the 50KP expressed in cells may modify the plasmodesmata and facilitate cell-to-cell trafficking of GFP that is expressed in the same cells.
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Tubular structures protruding from the surface of the protoplasts were also observed in a few protoplasts expressing 50KPGFP (Fig. 2OQ). The tubules were of variable lengths and appeared to be very fragile and to fragment easily during observation. The percentages of protoplasts expressing 50KPGFP with tubules were 0·6 (1/165), 2 (3/145) and 3% (5/178) at 4, 12 and 24 h after transfection. The percentage did not increase at 48 h after transfection. The tubules were formed in protoplasts transfected with mutants
A,
B or
C but not
D,
E,
F or
G (Table 1
). Interestingly, small, irregular spots and fibrous network structures were never found in protoplasts containing tubular structures.
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Discussion |
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Cell-to-cell trafficking of MPGFP expressed transiently in leaf epidermis has been reported in CMV and AlMV, in which the protein moves into neighbouring cells from an originally transfected cell and, in contrast, free GFP remains in single cells (Itaya et al., 1997 ; Huang & Zhang, 1999
). Recently, Oparka et al. (1999)
reported that the capacity of plasmodesmata to traffic macromolecules depends on the physiological conditions of the leaves; in sink leaves, proteins of up to 50 kDa could move freely through plasmodesmata. Our results presented here show that free GFP (27 kDa) could move from cell to cell in young leaves of N. occidentalis and C. quinoa, but was mostly restricted to single cells in mature leaves (Fig. 3
). This result is in good agreement with the reported non-specific trafficking of GFP in developing tobacco leaves (Oparka et al., 1999
). In contrast, 50KPGFP (77 kDa) spread into neighbouring cells from cells that produced it, even in mature leaves, indicating that 50KP has a specific activity for cell-to-cell trafficking.
It was unexpected that cell-to-cell trafficking of 50KPGFP was restricted to only a few cells in leaf epidermis of C. quinoa plants. There may be structural and/or functional differences between the plasmodesmata that interconnect epidermal cells of N. occidentalis and C. quinoa plants. 50KPGFP expressed in epidermal cells of C. quinoa leaves was able to complement local spread of the movement-defective virus (Fig. 4). Thus, 50KPGFP may move from the epidermal cells to underlying mesophyll cells in C. quinoa leaves. It has been reported that a CMV mutant (M8) 3aGFP fusion protein was unable to traffic through plasmodesmata that interconnect epidermal cells, as the wild-type 3aGFP did, in tobacco (Nicotiana tabacum) and Nicotiana benthamiana (Canto & Palukaitis, 1999
). Because M8 CMV infects tobacco systemically, the virus spread via plasmodesmata to and within mesophyll tissue (Canto & Palukaitis, 1999
).
In addition to the cell-to-cell trafficking of 50KP itself, we showed that 50KP can facilitate the cell-to-cell trafficking of GFP when both proteins are co-expressed transiently in epidermal cells of N. occidentalis leaves (Table 2). However, there was no facilitation of cell-to-cell movement of GFP when the protein was expressed in leaf epidermis of transgenic N. occidentalis constitutively expressing a functional 50KP (data not shown).
Transgenic N. occidentalis plants expressing 50KP are known to complement 50KP-deficient ACLSV for movement (Yoshikawa et al., 2000 ). As shown in Fig. 3
, 50KP expressed transiently in leaf epidermis also complemented local spread of 50KP-deficient virus. It has been reported that transgenic plants expressing CMV 3a protein could complement 3a-deficient CMV, but plants expressing 3a protein fused to GFP could not, showing that CMV 3a protein fused to GFP is not biologically functional (Canto & Palukaitis, 1999
; Kaplan et al., 1995
). We also found that there was no complementation of the movement of 50KP-deficient virus in N. occidentalis expressing 50KPGFP (unpublished results). However, 50KPGFP could complement local movement of a 50KP-deficient virus when pStuStop was co-bombarded with p35S50KPGFP. These results suggest that complementation could occur when both p35S50KPGFP and pStuStop were introduced in cells at the same time. On the other hand, 50KP expressed transiently in cells may be functionally different from that in transgenic plant cells.
We do not know the biological significance of the tubules on the surface of protoplasts induced by 50KPGFP. No tubular structures spanning cell walls were found in ultrathin sections of tissues infected with ACLSV (Yoshikawa et al., 1997 , 1999
) and the tubules were observed in only a small portion of protoplasts that expressed 50KPGFP. This is similar to what has been described for TMV, where fluorescent protrusions were formed in a small percentage of protoplasts infected with TMV MPGFP (Heinlein et al., 1998
). In contrast, CMV 3aGFP generated tubules on the surface of 1485% of infected protoplasts (Canto & Palukaitis, 1999
). So far, tubule formation on protoplasts has been reported for spherical viruses in the Comoviridae, Bromoviridae and Caulimoviridae and for Tomato spotted wilt virus but not for TMV (Kasteel et al., 1996
, 1997
; Storms et al., 1995
; van Lent et al., 1991
; Zheng et al., 1997
).
As summarized in Table 1, mutational analysis has shown that the C-terminal region (between aa 287 and 457) of 50KP is not essential for localization to plasmodesmata, cell-to-cell trafficking through plasmodesmata, complementation of local movement of 50KP-deficient virus or tubule formation on the surface of protoplasts. In contrast, deletions in the N-terminal region of 50KP resulted in the complete disruption of all these activities, suggesting that there must be a close correlation between these functions. The expressed proteins (
D to
G) all formed large aggregates in the cytoplasm. It is probable that these proteins are not folded properly and that this makes the protein incapable of interacting with a subcellular structure(s) or undergoing intracellular and intercellular trafficking.
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Acknowledgments |
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References |
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Received 28 February 2000;
accepted 26 April 2000.