1 Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Gakuen-cho, Sakai, Osaka 599-8531, Japan
2 Kyoto Prefectural Institute of Agricultural Biotechnology, Seika-cho, Soraku-gun, Kyoto 619-0244, Japan
Correspondence
Satoshi T. Ohki
ohki{at}plant.osakafu-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Almost all eukaryotic organisms possess a sequence-specific RNA-degradation system, referred to as RNA silencing (Voinnet, 2001; Waterhouse et al., 2001
). Observations that plant viruses encode proteins to suppress RNA silencing provide the most compelling evidence that RNA silencing functions as an inducible, host RNA-surveillance system (Marathe et al., 2000
; Li & Ding, 2001
; Voinnet, 2001
; Waterhouse et al., 2001
). The coat protein (CP) is often, but not invariably, essential for systemic spread of plant viruses (Gilbertson & Lucas, 1996
; Séron & Haenni, 1996
; Nelson & van Bel, 1998
; Kobori et al., 2002
). The helper component proteinase (HC-Pro) of potyviruses, the 2b protein of cucumoviruses, the p19 protein of tombusviruses and the P1 protein of sobemoviruses are also involved in systemic spread (Cronin et al., 1995
; Ding et al., 1995
; Scholthof et al., 1995
; Bonneau et al., 1998
; Kasschau & Carrington, 2001
; Soards et al., 2002
). These proteins, which have been characterized as important pathogenicity determinants, have been identified as suppressors of RNA silencing (Anandalakshmi et al., 1998
; Brigneti et al., 1998
; Voinnet et al., 1999
).
Mixed virus infections can result in the complementation of a movement defect in heterologous viruses in non-host plants (Malyshenko et al., 1989; Taliansky & García-Arenal, 1995
; Hacker & Fowler, 2000
; Takeshita & Takanami, 2000
; Choi et al., 2002
). Several studies have shown that different viral proteins expressed from transgenes (Giesman-Cookmeyer et al., 1995
; Kaplan et al., 1995
; Cooper et al., 1996
), heterologous sequences cloned into defective genomes (De Jong & Ahlquist, 1992
; Ryabov et al., 1999
; Spitsin et al., 1999
) or cotransfected plasmids (Agranovsky et al., 1998
; Fedorkin et al., 2001
) can functionally replace non-homologous proteins from other viruses.
In this report, we have shown that Potato virus Y (PVY, T01 isolate) accumulated in inoculated leaves (the oldest pair of leaves along the stem) of tobacco plants and spread systemically up the stem to sequential pairs of leaves but, atypically, did not move to young, developing tissues. However, when tobacco plants were doubly infected with PVY and Cucumber mosaic virus (CMV, strain Pepo), PVY moved to and accumulated in young, developing tissues and the resulting infection resulted in systemic symptoms distinct from those caused by each virus alone. Systemic spread of PVY in tobacco appeared to be regulated in phloem tissues.
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METHODS |
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Inoculated plants were grown in a growth chamber (NK systems) at 24 °C with a 14 h light/10 h dark cycle. Virus accumulation was assessed at different times post-inoculation (p.i.) by double-antibody sandwich (DAS)-ELISA (Clark & Adams, 1977). Four 6 mm diameter discs per leaf were collected from six plants for each sample. Samples were ground in 400 µl PBS containing 0·05 % Tween 20 (PBST), and 100 µl samples were tested. Coating antibodies were diluted 1 : 1000 in sodium carbonate buffer (pH 9·6) and alkaline phosphatase-conjugated IgG secondary antibody was diluted 1 : 1000 in PBST. A405 was measured with a SPECTRAmax 250 (Molecular Devices) ELISA plate reader. Experiments were done in triplicate. Tissue immunoblot analysis was carried out independently on two plants as described by Andrianifahanana et al. (1997)
. Cuts were made across the axes of petioles or stems. The cut surface of the tissue was then pressed directly on to nitrocellulose membranes (Bio-Rad) that had been treated with 0·2 M CaCl2 prior to blotting. PVY or CMV CP was detected in tissue blots by using specific antibodies, diluted 1 : 2000, as the primary antibody, and alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma), diluted 1 : 4000, as the secondary antibody. Alkaline phosphatase was detected by using a BCIP/NBT liquid substrate system (Sigma). Experiments were done in triplicate.
Immunostaining of infected tissues.
Sampled pieces were immersed immediately in fixative (50 % ethanol, 5 % acetic acid, 3·7 % formalin) and left overnight at 4 °C. After dehydration and infiltration in a graded series of ethanol solutions (50, 70, 90 and 100 %), each for 30 min, samples were embedded in paraffin (Paraplast-plus; Sigma). Sections (12 µm thick) of stem tissue were cut with a rotary microtome (Yamato Kohki) and placed on glass slides (Matsunami Glass). Sections were dewaxed in xylene and washed in 100 % ethanol. After hydration in a graded series of ethanol solutions (70, 50 and 30 %) and distilled water for 10 min each, sections were incubated in PBST and 1 % BSA for 1 h and then incubated with PVY CP-specific antibody, diluted 1 : 200 in PBST/BSA, for 2 h at 37 °C. After washing in PBST, sections were incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma), diluted 1 : 200 in PBST/BSA, for 2 h at 37 °C and then washed three times for 10 min in PBST. After washing, samples were stained by using the BCIP/NBT liquid substrate system. Stained sections were then washed in distilled water and observed with a BX-50 microscope (Olympus).
Infectious cDNA plasmids.
Plasmids pCP1TP1, pCP2TP1 and pCP3TP2, containing full-length cDNA copies of CMV strain Pepo genomic RNAs 1, 2 and 3, respectively (Saitoh et al., 1999), were used to generate CMV inocula. To prepare a modified CMV containing RNA 2 that was unable to translate the 2b protein (Pepo
2b), nucleotide changes were introduced into pCP2TP1. As the 2b protein of CMV strain Pepo contains three methionines, at amino acid positions 1, 8 and 18, we generated two translational termination codons at the beginning of the 2b gene, one after the first AUG codon and another after the third, by site-directed mutagenesis (Kunkel, 1985
). A pair of complementary mutagenic primers (5'-GCGAAAGAAATATGGAATAGAACGTAGGTGCAATGAC-3' and its complement) was used. An additional translational termination codon was then introduced into this plasmid by using a pair of complementary mutagenic primers (5'-GCTGGCTCACATGTAGGAGGCGAAGAAGC-3' and its complement). Neither mutated sequence induced C-terminal amino acid changes in the 2a protein, which overlaps the 2b protein. In the PCR, 12 cycles of amplification followed incubation at 95 °C for 30 s under the following conditions, using 0·05 U Pfu Turbo DNA polymerase µl1 (Stratagene): denaturation at 95 °C for 30 s, annealing at 55 °C for 1 min and extension at 68 °C for 12 min. After treatment with DpnI (Stratagene), the products were transformed into Escherichia coli DH5
. The nucleotide sequences for the cDNA clone (pCP2
2bM3stp) constructed were determined by using a Beckman Coulter DNA sequencer, model CEQ 8000. In vitro transcripts of Pepo
2b by T7 RNA polymerase (TaKaRa Bio) were generated by combining pCP2
2bM3stp with infectious clones of pCP1TP1 and pCP3TP2.
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RESULTS |
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Immunohistochemistry revealed details of the distribution of PVY in the stem tissues (Fig. 3a, position E) in singly and doubly infected plants. PVY antigen was confined to external phloem cells and was not detected in internal phloem cells in singly infected plants (Fig. 4b
). However, it was distributed uniformly in whole tissues, including the external phloem, xylem parenchyma and internal phloem cells in doubly infected plants (Fig. 4c
). Therefore, CMV assisted the accumulation of PVY in stem tissues, which could be associated with enhanced spread of PVY into younger tissues.
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CMV 2b protein assists PVY systemic spread in doubly infected plants
The CMV 2b protein is a known suppressor of RNA silencing (Brigneti et al., 1998). To examine its possible contribution to the successful systemic spread of PVY, we inoculated N. tabacum plants simultaneously with PVY and Pepo
2b, a modified CMV in which the 2b protein has been rendered untranslatable (Fig. 5a
). Systemic spread of PVY was then traced by tissue immunoblot of stem sections and symptoms were observed. Plants inoculated with Pepo
2b had very mild mosaic symptoms (Fig. 1e
). Plants inoculated with PVY and Pepo
2b developed mild chlorotic spots in older leaves without any PVY-induced symptoms in the younger leaves, as in the case of plants inoculated with PVY only (Fig. 1f
). PVY was detected in the external phloem only of systemically infected stem tissues, but Pepo
2b was detected in external and internal phloem tissues (Fig. 5a
). Similar results were obtained for plants inoculated with Pepo
2b and then with PVY (data not shown).
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DISCUSSION |
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Members of the plant family Solanaceae have two phloem tissues external and internal. The structural differences of these have functional consequences for photoassimilate transport: metabolites descend in the external phloem and ascend in the internal phloem (Turgeon, 1989). Previous studies have indicated that Pepper mottle virus (PepMoV) in Capsicum annuum and Tobacco mosaic virus (TMV) in N. benthamiana accumulate first in the external phloem in inoculated leaves and stem tissues below the inoculated leaves, and then accumulate in the internal phloem tissues in systemically infected stem sections or leaves (Andrianifahanana et al., 1997
; Cheng et al., 2000
). Our results indicate that PVY was transported readily from the inoculated leaves to the external phloem in stem tissues, but had no or little ability to gain access to the internal phloem tissues in singly infected plants. In plants doubly infected with PVY and CMV, invasion of stem tissues by PVY was not restricted to the external phloem and the virus gained access to the xylem parenchyma and internal phloem tissues. Although the nature of the connecting cells between the external and internal phloem is not fully understood, these cells may have an important role in regulating movement of plant viruses. Guerini & Murphy (1999)
observed a similar restriction of PepMoV in the external phloem of resistant C. annuum cv. Avelar plants and resistance was overcome by dual infection with CMV. In Tetragonia expansa, which possesses temperature-sensitive systemic resistance against CMV, systemic transport of virus is considered to be inhibited in the tissue between the external and internal phloem (Kobori et al., 2002
, 2003
). We also consider that the internal phloem is the pathway for rapid transport of PVY up the stem to the younger tissues in doubly infected plants. As PVY cannot use this pathway fully, it does not move up the stem to young tissues in singly infected plants.
The mechanism by which CMV alleviates the limitation of PVY systemic spread can be explained by the ability of the CMV 2b protein to suppress host factors that are involved in the defence system, as the 2b protein suppresses RNA silencing (Brigneti et al., 1998). It has already been shown to be important in overcoming the phloem restriction of a luteovirus, which could also be related to RNA silencing (Ryabov et al., 2001
). We showed that double infection with Pepo
2b, a CMV mutant that does not translate the 2b protein, did not assist systemic spread of PVY (Fig. 5b
). Another possible mechanism is that PVY may lack a factor for systemic spread in N. tabacum and that this is complemented by CMV in doubly infected plants. Pepo
2b would possibly not complement this defect, as it might not produce a sufficient amount of complementing factor, i.e. protein 1a, 2a, 3a or CP (Taliansky & García-Arenal, 1995
); this is because Pepo
2b accumulates at lower levels than CMV (Fig. 5c
). However, this is unlikely. PVY accumulation in plants doubly infected with Pepo
2b was significantly lower than that in singly infected plants (Fig. 5b
). In the absence of functional 2b protein, other CMV factor(s) may impede PVY multiplication in plants doubly infected with PVY and Pepo
2b.
RNA silencing is a natural regulatory mechanism in which particular RNAs are targeted and destroyed in a sequence-specific manner (Ratcliff et al., 1997, 1999
; Voinnet et al., 1999
; Voinnet, 2001
; Waterhouse et al., 2001
). Ding et al. (2003)
suggested that plants have evolved a powerful mechanism to traffic selected macromolecules in the symplasmic pathway: systemic transport of a protein or RNA would be regulated at multiple checkpoints, including phloem entry, transport and exit. RNA silencing may be hyperactivated in cells that control access to the phloem (Marathe et al., 2000
), especially near and in the internal phloem cells, and may be activated to suppress rapid systemic spread of a virus as an adaptive defence mechanism. The ability of a virus to move within infected tissues has been suggested to depend on its ability to block the systemic signalling that is generated by RNA silencing (Vance & Vaucheret, 2001
; Voinnet, 2001
; Baulcombe, 2002
). CMV 2b protein autonomously enters, and probably translocates through, the phloem tissues (Guo & Ding, 2002
). Consistent with these suggestions, we consider that CMV 2b protein acts as a suppressor protein, blocking the systemic, mobile signals that are associated with silencing against PVY. PVY then accumulates in the internal phloem tissue and is subsequently transported rapidly and systemically in doubly infected plants.
PVY isolate T01 readily infected N. benthamiana systemically, but not N. tabacum cv. Xanthi-nc. One possible mechanism is that the movement factor of PVY T01 may interact with a host factor that is different in N. benthamiana and N. tabacum cv. Xanthi-nc. Several viruses spread systemically in N. benthamiana, but are limited to the inoculated leaves of other hosts. For example, TMV with a mutation in the 30 kDa movement protein and Odontoglossum ringspot virus spread systemically in N. benthamiana, but not in N. tabacum (Hilf & Dawson, 1993; Waigmann et al., 2000
). Another possibility is that HC-Pro of PVY T01 may manipulate the suppressor function of systemic spread in N. benthamiana, but not in N. tabacum. HC-Pro of Plum pox virus (PPV) can strongly enhance the pathogenicity of Potato virus X in the systemic host of PPV, but it does not intensify the symptoms in N. tabacum that is not infected systemically by PPV (Sáenz et al., 2002
). HC-Pro is also involved in systemic spread and virulence of potyviruses. This function could be due to the silencing of suppressor activity (Kasschau & Carrington, 2001
; Voinnet, 2001
).
We conclude that the compensation by CMV strain Pepo of the inhibition of PVY T01 transport in the phloem is related to a restriction of the host defence mechanism by the 2b protein of CMV. However, we did not assess systemic spread of other PVY strains or combinations with other CMV strains. Our present results and more such data should provide important insights to help further exploration of the defence mechanism(s) against plant viruses in phloem tissue.
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ACKNOWLEDGEMENTS |
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Received 7 April 2004;
accepted 27 July 2004.
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