Departamento de Biotecnología, ETSI Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain
Correspondence
F. García-Arenal
fga{at}bit.etsia.upm.es
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ABSTRACT |
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Present address: Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA.
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INTRODUCTION |
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While it is assumed that most viruses move systemically through the phloem, xylem transport has been proposed for some viruses, most notably for members of the beetle-transmitted Sobemovirus genus. Although not a clear indicator of a role for xylem in systemic movement, localization of virus particles in the xylem has been reported in many instances (e.g. Dubois et al., 1994; Fribourg et al., 1987
; Khan et al., 1994
; Urban et al., 1989
; Verchot et al., 2001
). Further evidence supporting xylem transport has been reported in a few instances (Dubois et al., 1994
; Opalka et al., 2000
; Schneider & Worley, 1959a
, b
). As well as the growing number of reported cases, one aspect of virus systemic movement via the xylem that still needs to be addressed is exactly how viruses translocate from the non-living tracheary elements to living cells (i.e. from the apoplast to the symplast) for the establishment of systemic infection. To explain this, a model by which the virus chelates calcium in the pit membrane between tracheids, relaxing the membrane's structure, and then moves into living immature tracheids has been proposed for Rice yellow mottle virus (Opalka et al., 2000
). Hence, systemic movement of plant viruses through the xylem is a relatively unexplored process. Further studies are needed for a better knowledge of the occurrence of xylem transport in different viral taxa and to analyse the mechanism of xylem transport of plant viruses and its relevance compared with systemic movement through the phloem.
Cucumber green mottle mosaic virus (CGMMV) is a Tobamovirus that shares the same genetic organization and similar particle morphology as Tobacco mosaic virus (TMV) (Fukuda et al., 1981; Namba et al., 1989
; Ugaki et al., 1991
; Wang & Stubbs, 1994
). The systemic transport of CGMMV has been analysed in our laboratory and was shown to circulate as virus particles in the phloem of infected cucumber plants (Cucumis sativus L.) (Simón-Buela & García-Arenal, 1999
). In this work, we report on the infection progress and cellular localization of CGMMV during the systemic colonization of cucumber plants. The data support previous results on the role of phloem in the systemic transport of CGMMV and show that CGMMV can also move systemically through the xylem. Both types of vascular tissue were used by CGMMV for systemic infection of cucumber plants, but systemic movement through the phloem was shown to be a more efficient process.
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METHODS |
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Microscopy.
Samples of plant material taken from CGMMV-inoculated plants at 4, 8, 12, 16, 20 and 24 days post-inoculation (p.i.) were cut to the approximate dimensions of 1x1x4 mm and fixed and embedded as described by Thompson & García-Arenal (1998). This material consisted of the following plant parts: the inoculated cotyledon, every leaf in the plant [one (1st) at 4 days p.i. and five (1st5th) by 2024 days p.i.] and stem internodes [one (1st) at 8 days p.i. and four (1st4th) by 24 days p.i.] (Table 1
). The youngest analysed leaf of the plant was at least 4 mm in size. The most apical internode analysed was the one below the youngest analysed leaf (for example, at 20 days p.i. the 4th internode, below the 5th leaf; Table 1
). Samples were taken from the central region of the leaf longitudinally following the path of veins of order I, II and III. Internode samples were taken longitudinally from a portion equidistant from each node. Similar samples were taken from mock-inoculated controls at 12 and 24 days p.i. Immunolocalization in semi- (1 µm) and ultrathin (100140 nm) sections using light and electron microscopy, respectively, was carried out according to the protocols described by Thompson & García-Arenal (1998)
. All sections were cut with glass knives with a Reichert ultramicrotome. Light microscopy was carried out with a Zeiss Axiophot and electron microscopy with a JEOL model JEM-1200EX II.
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Nucleic acid extraction and analyses.
Nucleic acids were extracted from 0·2 g fresh weight cotyledon samples, 0·1 g fresh weight leaf and root samples, 10 µl phloem exudate and 100 µl xylem washes. These samples were homogenized in 100 µl 0·2 M Tris/HCl, pH 8·2, 2 mM EDTA, 0·1 % SDS, incubated for 10 min at 70 °C and extracted in 2 : 1 vols of phenol : chloroform (1 : 1 by volume). Nucleic acids were precipitated at 4 °C in 2 M LiCl and concentrated by centrifugation at 10 000 g for 15 min. The nucleic acid pellets were washed with 200 µl 70 % ethanol and resuspended in 100 µl (leaf or root extracts) or 10 µl (phloem or xylem extracts) of double-distilled sterile water.
For Northern blot hybridization analyses, nucleic acid extracts corresponded to 20 mg fresh leaf or root tissue, 10 µl phloem exudate or 100 µl total xylem wash. The extracts were denatured as described by Sambrook et al. (1989) and blotted onto Hybond-N membranes (Amersham) using a manifold apparatus (Bio-Rad). CGMMV RNA was detected with a 32P-labelled RNA probe complementary to nt 43224681 of CGMMV-SH RNA. The lower limit for RNA detection by this procedure was about 1·5 ng as estimated from known amounts of CGMMV RNA diluted in a fivefold series in nucleic acid extract from uninfected plants (not shown). For CMV-Fny RNA detection, a 32P-labelled RNA probe complementary to the CP gene (Fraile et al., 1997
) was used.
For RT-PCR detection of CGMMV RNA, nucleic acid extracts corresponding to 4 mg fresh leaf or root tissue, 1 µl phloem sap or 10 µl total xylem wash were used. A 359 nt fragment representing nt 43224681 of CGMMV-SH was amplified using oligonucleotides LS1 (5'-GTTTCGCCTCAAAATTCC-3') and LS2 (5'-TCTAAATATGACAAGTCGC-3') as described by Simón-Buela & García-Arenal (1999). The lower limit for CGMMV RNA detection was about 8 pg as estimated from known amounts of CGMMV RNA diluted in a fivefold series in nucleic acid extracts from uninfected plants (not shown).
Protoplast isolation and FITC-labelling.
Mesophyll protoplasts from cucumber cotyledons were obtained and isolated as described by Taliansky & García-Arenal (1995). Each sample consisted of a pool of six cotyledon halves excised from inoculated plants or from uninoculated controls. After isolation, mesophyll protoplasts were fixed for 15 min in 10 ml 95 % ethanol and washed three times in 10 ml 10 mM Tris/HCl, pH 7·5, 100 mM NaCl. Protoplasts were then incubated for 2 h at 37 °C in 500 µl 10 mM Tris/HCl, pH 7·5, 100 mM NaCl, 1 % BSA with a 1 : 100 dilution of CGMMV antiserum (Simón-Buela & García-Arenal, 1999
). Protoplasts were washed as described before and incubated for 2 h at 37 °C in 500 µl 10 mM Tris/HCl, pH 7·5, 100 mM NaCl, 1 % BSA, with a 1 : 160 dilution of FITC-conjugate anti-rabbit IgG (Sigma F9887). After three additional washes, protoplasts were counted under a light microscope using a haemocytometer grid. FITC-labelled protoplasts were counted using a 450490 nm filter in a Zeiss Axiophot.
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RESULTS |
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Tissue and cellular localization of CGMMV
The progress of CGMMV infection in cucumber plants was analysed at the tissue and cellular level by immunosilver CP detection in semi-thin sections and immunogold CP detection in ultrathin sections. In the minor veins (order VII to V) of inoculated cotyledons, both the bundle sheath (BS) and the vascular parenchyma (VP) cells were shown to be infected at 8 days p.i. in 9 out of 17 veins scored, as exemplified in Fig. 1 (A) and (B). From 8 to 20 days p.i., high accumulation in xylem-associated VP was noteworthy (Fig. 1A
). CGMMV was detected in the phloem intermediary cells (ICs) in only one out of nine minor veins showing infection of cells of the BS and VP (not shown). The VPIC interface was observed as a boundary for CGMMV localization, as immunogold labelling of the CP was intense in the VP and completely lacking in the ICs (Fig. 2
A and B). CGMMV was not detected in the sieve elements (SEs) of minor or major veins in the inoculated cotyledons. In the major veins (order III and IV) of cotyledons, CGMMV was localized in the BS (six out of seven observations) and the VP (seven out of seven observations), but not in the phloem. The high frequency of CGMMV infection in major veins suggests that virus loading may occur in major veins as well as in minor veins.
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In the major veins (order I to III) of systemically infected young expanding leaves, CGMMV was first detected in the xylem cells, including the circulating elements, and in abaxial phloem CCs (arrows in Fig. 1F and G). Later on, high virus accumulation was observed in leaf parenchyma cells (Fig. 1G
). Remarkably, CGMMV accumulated to high levels in xylem cells (Fig. 1F
). Electron microscopy analysis showed that differentiating tracheary elements at different developmental stages were heavily labelled with immunogold (Fig. 2F
H), and large aggregates of immunogold-labelled virus-like particles were observed in some developing tracheary elements (T2 in Fig. 2F
, magnified in Fig. 2G
). Also, labelled virus-like particles were abundant on both sides of the middle lamella between tracheids (Fig. 2G
). In mature systemically infected leaves, CGMMV was detected in all vascular cell types of minor veins, e.g. of 14 observed veins from the 2nd leaf at 16 days p.i., CGMMV was detected within the BS, VP and ICs in seven veins and in the SEs in five veins (not shown).
All the results presented in this section indicate long-distance transport of CGMMV through the phloem. In addition, the results show a marked tropism of CGMMV for the VP and xylem, suggestive of a role for this tissue in the long-distance transport of the virus.
Assay of xylem and phloem transport of CGMMV
The role of phloem or xylem in the systemic transport of CGMMV was examined by analysing the progress of infection in plants in which cell death was induced in a portion of the 1st internode with a jet of steam (Schneider & Worley, 1959a). Plants with three fully expanded leaves and a well-developed 1st internode (1015 cm long) were inoculated in the cotyledons and 1st leaf. Thereafter, a jet of steam was applied to the central portion of the 1st internode for about 15 s, until water soaking became apparent. One hour after steaming, a segment about 2 cm long was reduced to a very thin strand and showed dry necrosis by 12 days p.i. (Fig. 3
). CMV, which has been used in the past as a negative control for xylem transport (Caldwell, 1930
), was included in some experiments to test how efficiently phloem transport was prevented in steamed plants. Most steamed plants developed axillary shoots below the steamed internode 7 days p.i. From 7 days p.i. onwards, occasional death of plant parts above the steamed portion was observed, reducing the number of plants available for systemic movement analysis. Typically, more than 50 % of the steamed plants survived until 14 days p.i., although less than 20 % survived until 21 days p.i.
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DISCUSSION |
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Little is known about how viruses circulate in the transport phloem and unload in systemically infected tissues (Oparka & Santa Cruz, 2000). CGMMV localized initially in the CCs of the external phloem and thereafter colonized the stem cortex, indicating that CGMMV unloads and probably reloads during its transport along the stem. Although graft experiments have shown that systemic movement in the internode does not require virus multiplication (Wisniewski et al., 1990
), a similar leaky pattern of transport has been described for photoassimilates (Nelson & Van Bel, 1998
) and for TMV in tobacco (Susi et al., 1999
) or Nicotiana benthamiana (Cheng et al., 2000
). Virus multiplication in the CC and SE reloading could increase the efficiency of transport. It has been shown that CGMMV RNA circulates in the transport phloem in the form of virus particles (Simón-Buela & García-Arenal, 1999
). Whether loading and reloading in the transport phloem involves uncoating and assembly of virions remains to be established. CGMMV localization in the external phloem contrasts with observations made for Pepper mottle virus or for TMV in the stem internal phloem of Capsicum anuum L. (Andrianifahanana et al., 1997
) or N. benthamiana L. (Cheng et al., 2000
), respectively. Virus and/or host factors determining differences in the stem transport route are yet to be identified but may depend on the plant vascular architecture and on the virus loading routes.
Symplastic unloading of Potato virus X in the major veins of N. benthamiana and of Barley stripe mosaic virus in barley longitudinal veins paralleled phloem unloading of carboxyfluoresceine (Haupt et al., 2001; Roberts et al., 1997
). Symplastic unloading along major veins in source leaves has also been reported for other virus species and host plants (Cheng et al., 2000
; Ding et al., 1998
; Silva et al., 2002
). Our data also indicate that unloading of CGMMV in young developing leaves involves the major veins, where it was first detected. Our data showed two noticeable features. First, in systemically infected developing leaves, CGMMV was first detected in the abaxial phloem and in xylem cells (Fig. 1F
). CGMMV accumulation was high in differentiating tracheids and immunogold-labelled particles were abundant in the lumen, the secondary cell wall and the middle lamella of intervascular pits (arrowheads in Fig. 2G
), indicating a xylem tropism. Secondly, the temporal pattern of CGMMV immunodetection in different organs (Table 1
) suggested that CGMMV could be unloaded in fully expanding, mature leaves, which may have undergone the sink to source transition. These two features suggest a function for the xylem in systemic movement of CGMMV, as reported for other viruses (Dubois et al., 1994
; Jones, 1975
; Schneider & Worley, 1959a
).
The involvement of phloem and xylem in the systemic movement was analysed using plants in which live cells in a portion of the 1st internode had been killed by steam treatment. Results indicated that CGMMV moved through the phloem of untreated controls, as was also the case for CMV, a virus previously reported to move exclusively through the phloem (Caldwell, 1930). However, systemic infection of upper leaves in steamed plants occurred at 29 °C, indicating xylem transport. Xylem transport in steamed plants was temperature dependent and inefficient, as deduced from the low percentage of steamed plants that became systemically infected (about 11 %) and from the delay in infection compared with untreated controls (Table 3
). Long-distance movement through the xylem has been proposed for some viruses, but few reports have addressed this issue directly (Chambers & Francki, 1966
; Dubois et al., 1994
; Jones, 1975
; Opalka et al., 1998
; Schneider & Worley, 1959a
, b
; Verchot et al., 2001
). Xylem localization of virus particles has been reported for a number of viruses and hosts (e.g. Fribourg et al., 1987
; Khan et al., 1994
; Robertson & Carroll, 1989
; Russo et al., 1967
; Urban et al., 1989
). Also, virus particles were recovered from guttation fluid in cucumber plants infected with 12 different viruses including CGMMV (French & Maureen, 1999
). Since guttate originates from xylem exudate, this was considered evidence of xylem transport for these viruses. Interestingly, the presence or absence of Brome mosaic virus particles in guttation fluid of different hosts correlated with virus-induced damage of cells mainly xylem cells in the veins suggesting a mechanism for virus exit to the apoplast (Ding et al., 2001
). Nevertheless, xylem localization need not result in infection via the xylem. Indeed, our data show that CGMMV RNA was present in the xylem of steamed plants at 24 and 29 °C, but systemic infection via the xylem only occurred at 29 °C.
The mechanisms of xylem transport of plant viruses remain largely unexplored. It could be that entry into the xylem occurs in differentiating xylem elements. Thus, it has been proposed that infection of the root tips before xylem elements differentiate is required for systemic movement of Beet necrotic yellow vein virus through the xylem (Dubois et al., 1994). This should not be a limiting factor in our system, as CGMMV was detected by Western blot and Northern hybridization (Fig. 4
) in root tips. A common feature of viruses reported to translocate through the xylem is the high stability of the virus particles and their resistance to proteases (Gergerich, 2002
), a feature shared by tobamoviral particles (Klug, 1999
), which would be a necessary characteristic to withstand the action of proteases during programmed cell death during tracheary element development (Kozela & Regan, 2003
). It is more difficult to envision how viruses would exit the xylem after transport and infect the protoplasts of living cells. A current hypothesis is that calcium binding by virus particles will disrupt pit membranes between mature and differentiating xylem vessels or tracheids, so enabling the virus to traffic through the membrane. This hypothesis was proposed on the basis of immunochemical analyses of the colonization of rice plants by Rice yellow mottle virus (Opalka et al., 1998
) and on the well-known role of calcium in the stabilization of Sobemovirus isometric capsids (Hsu et al., 1976
; Opalka et al., 2000
). It is known that calcium binding stabilizes TMV particles (Gallagher & Lauffer, 1983
; Namba et al., 1989
), and putative calcium binding sites have been described for CGMMV, watermelon strain (Wang & Stubbs, 1994
), which has the same amino acid sequence in the CP as the SH strain (Ugaki et al., 1991
). We could speculate that the higher efficiency of xylem transport of CGMMV at 29 versus 24 °C could be related to a greater virus accumulation and, hence, higher calcium binding in the xylem and/or to relaxation of the pit membrane at the higher temperature.
Thus, our data show that systemic movement of CGMMV in cucumber plants occurs through the phloem and, less efficiently, through the xylem. How general this pattern of virus infection is remains to be explored.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Caldwell, J. (1930). The physiology of virus diseases in plants. I. The movement of mosaic in the tomato plant. Ann Biol 17, 429443.
Chambers, T. C. & Francki, R. I. B. (1966). Localization and recovery of lettuce necrotic yellows virus from xylem tissues of Nicotiana glutinosa. Virology 29, 673676.
Cheng, N.-H., Su, C.-L., Carter, S. A. & Nelson, R. S. (2000). Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana. Plant J 23, 349362.[CrossRef][Medline]
Dalmay, T., Rubino, L., Burgyan, J. & Russo, M. (1992). Replication and movement of a coat protein mutant of cymbidium ringspot tombusvirus. Mol Plant Microbe Interact 5, 379383.[Medline]
Ding, X. S., Shintaku, M. H., Carter, S. A. & Nelson, R. S. (1996). Invasion of minor veins in tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement. Proc Natl Acad Sci U S A 93, 1115511160.
Ding, X. S., Carter, S. A., Deom, C. M. & Nelson, R. S. (1998). Tobamovirus and potyvirus accumulation in minor veins of inoculated leaves from representatives of Solanaceae and Fabaceae. Plant Physiol 116, 125136.
Ding, X. S., Boydston, C. M. & Nelson, R. S. (2001). Presence of Brome mosaic virus in barley guttation fluid and its association with localized cell death response. Phytopathology 91, 440448.
Dubois, F., Sangwan, R. S. & Sangwan-Norreel, B. S. (1994). Spread of Beet necrotic yellow vein virus in infected seedlings and plants of sugar beet (Beta vulgaris). Protoplasma 179, 7282.
Fraile, A., Alonso-Prados, J. L., Aranda, M. A., Bernal, J. J., Malpica, J. M. & García-Arenal, F. (1997). Genetic exchange by recombination or reassortment is infrequent in natural populations of a tripartite RNA plant virus. J Virol 71, 934940.[Abstract]
French, C. J. & Maureen, E. (1999). Virus particles in guttate and xylem of infected cucumber (Cucumis sativus L.). Ann Appl Biol 134, 8187.
Fribourg, C. E., Koenig, R. & Lesemann, D. E. (1987). A new tbamovirus from Passiflora edulis in Peru. Phytopathology 77, 486491.
Fukuda, M., Meshi, T., Okada, Y., Otsuki, Y. & Takebe, I. (1981). Correlation between particle multiplicity and location on virion RNA of the assembly initiation site for viruses of the tobacco mosaic virus group. Proc Natl Acad Sci U S A 78, 42314235.[Abstract]
Gallagher, W. H. & Lauffer, M. A. (1983). Calcium ion binding by tobacco mosaic virus. J Mol Biol 170, 905919.[Medline]
Gergerich, R. C. (2002). Beetles. Adv Bot Res 36, 101112.
Gross, K. C. & Pharr, D. M. (1982). A potential pathway for galactose metabolism in Cucumis sativus L., a stachyose transporting species. Plant Physiol 69, 117121.
Haritatos, E., Keller, F. & Turgeon, R. (1996). Raffinose oligosaccharide concentrations measured in individual cell and tissue types in Cucumis melo L. leaves: implications for phloem loading. Planta 198, 614622.
Haupt, S., Duncan, G. H., Holzberg, S. & Oparka, K. J. (2001). Evidence for symplastic phloem unloading in sink leaves of barley. Plant Physiol 125, 209218.
Haywood, V., Kragler, F. & Lucas, W. J. (2002). Plasmodesmata: pathways for protein and ribonucleoprotein signaling. Plant Cell 14, S303325.
Holthaus, U. & Schmitz, K. (1991). Distribution and immunolocalization of stachyose synthase in Cucumis melo L. Planta 185, 479486.
Hsu, C. H., Sehgal, O. P. & Pickett, E. E. (1976). Stabilizing effect of divalent metal ions on virions of Southern bean mosaic virus. Virology 69, 587595.[Medline]
Jones, R. A. C. (1975). Systemic movement of potato mop-top virus in tobacco may occur through the xylem. Phytopath Z 82, 352355.
Khan, J. A., Lohuis, H., Goldbach, R. W. & Dijkstra, J. (1994). Distribution and localization of bean common mosaic virus and bean black root virus in stems of doubly infected bean plants. Arch Virol 138, 95104.[Medline]
Klug, A. (1999). Tobacco mosaic virus particles: structure and assembly. Philos Trans R Soc Lond Ser B Biol Sci 354, 531535.[CrossRef][Medline]
Kozela, C. & Regan, S. (2003). How plants make tubes. Trends Plant Sci 4, 159164.[CrossRef]
Leisner, S. M., Turgeon, R. & Howell, S. H. (1992). Long-distance movement of cauliflower mosaic virus in infected turnip plants. Mol Plant Microbe Interact 5, 4147.
Lucas, W. J. & Wolf, S. (1999). Connections between virus movement, macromolecular signaling and assimilate allocation. Curr Opin Plant Biol 2, 192197.[CrossRef][Medline]
McGeachy, K. D. & Barker, H. (2000). Potato mop-top virus RNA can move long distance in the absence of coat protein: evidence from resistant, transgenic plants. Mol Plant Microbe Interact 13, 125128.[Medline]
Mitchell, D. E., Gadus, M. V. & Madore, M. A. (1992). Patterns of assimilate production and translocation in muskmelon (Cucumis melo L.). Plant Physiol 99, 959965.
Moreno, I. M., Bernal, J. J., García de Blas, B., Rodríguez-Cerezo, E. & García-Arenal, F. (1997). The expression level of the 3a movement protein determines differences in severity of symptoms between two strains of tomato aspermy cucumovirus. Mol Plant Microbe Interact 10, 171179.[Medline]
Namba, K., Pattanayek, R. & Stubbs, G. (1989). Visualization of proteinnucleic acid interactions in a virus. J Mol Biol 208, 307325.[Medline]
Nelson, R. S. & van Bel, A. J. E. (1998). The mystery of virus trafficking into, through and out of the vascular tissue. Prog Bot 59, 476533.
Opalka, N., Brugidou, C., Bonneau, C., Nicole, M., Beachy, R. N., Yeager, M. & Fauquet, C. (1998). Movement of rice yellow mottle virus between xylem cells through pit membranes. Proc Natl Acad Sci U S A 95, 33233328.
Opalka, N., Tihova, M., Brugidou, C., Kumar, A., Beachy, R. N., Fauquet, C. M. & Yeager, M. (2000). Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction. J Mol Biol 303, 197211.[CrossRef][Medline]
Oparka, K. J. & Santa Cruz, S. (2000). The great escape: phloem transport and loading of macromolecules. Annu Rev Plant Physiol Plant Mol Biol 51, 323347.[CrossRef]
Oparka, K. J. & Turgeon, R. (1999). Sieve elements and companion cells traffic control centers of the phloem. Plant Cell 11, 739750.
Petty, I. T. & Jackson, A. J. (1990). Mutational analysis of barley stripe mosaic virus RNA beta. Virology 179, 712718.[Medline]
Rizzo, T. M. & Palukaitis, P. (1990). Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: generation of infectious RNA transcripts. Mol Gen Genet 222, 249256.[Medline]
Roberts, A. G., Santa-Cruz, S., Roberts, I. M., Prior, D. A. M., Turgeon, R. & Oparka, K. J. (1997). Phloem unloading in sink leaves of Nicotiana benthamiana: comparison of a fluorescent solute with a fluorescent virus. Plant Cell 9, 13811396.
Robertson, N. L. & Carroll, T. W. (1989). Electron microscopy of the novel barley yellow streak mosaic virus. J Ultrastruct Mol Struct Res 102, 139146.[Medline]
Russo, M., Martelli, G. P. & Quacquarelli, A. (1967). Occurrence of artichoke mottle crinkle virus in leaf vein xylem. Virology 33, 555558.[Medline]
Ryabov, E. V., Robinson, D. J. & Taliansky, M. (2001). Umbravirus-encoded proteins both stabilize heterologous viral RNA and mediate its systemic movement in some plant species. Virology 288, 391400.[CrossRef][Medline]
Sambrook, J., Frisch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Santa Cruz, S. (1999). Perspective: phloem transport of viruses and macromolecules what goes in must come out. Trends Microbiol 7, 237241.[CrossRef][Medline]
Santa Cruz, S., Roberts, A. G., Prior, D. A. M., Chapman, S. & Oparka, K. J. (1998). Cell-to-cell and phloem-mediated transport of Potato virus X: the role of virions. Plant Cell 10, 495510.
Schneider, I. R. & Worley, J. F. (1959a). Upward and downward transport of infectious particles of southern bean mosaic virus though steamed portions of bean stems. Virology 8, 230242.[Medline]
Schneider, I. R. & Worley, J. F. (1959b). Rapid entry of infectious particles of southern bean mosaic virus into living cells following transport of the particles in the water stream. Virology 8, 243249.[Medline]
Silva, M. S., Wellink, J., Goldbach, R. W. & van Lent, J. W. M. (2002). Phloem loading and unloading of Cowpea mosaic virus in Vigna ungiculata. J Gen Virol 83, 14931504.
Simón-Buela, L. & García-Arenal, F. (1999). Virus particles of Cucumber green mottle mosaic tobamovirus move systemically in the phloem of infected cucumber plants. Mol Plant Microbe Interact 12, 112118.[Medline]
Susi, P., Pehu, E. & Lehto, K. (1999). Replication in the phloem is not necessary for efficient vascular transport of tobacco mosaic tobamovirus. FEBS Lett 447, 121123.[CrossRef][Medline]
Taliansky, M. E. & García-Arenal, F. (1995). Role of cucumovirus capsid protein in long-distance movement within the infected plant. J Virol 69, 916922.[Abstract]
Thompson, J. R. & García-Arenal, F. (1998). The bundle sheathphloem interface of Cucumis sativus is a boundary to systemic infection by tomato aspermy virus. Mol Plant Microbe Interact 11, 109114.
Thompson, G. A. & Schulz, A. (1999). Macromolecular trafficking in the phloem. Trends Plant Sci 4, 354360.[CrossRef][Medline]
Turgeon, R. (1996). Phloem loading and plasmodesmata. Trends Plant Sci 1, 418423.[CrossRef]
Ugaki, M., Tomiyama, M., Kakutani, T., Hidaka, S., Kiguchi, T., Nagata, R., Sato, T., Motoyoshi, F. & Nishiguchi, M. (1991). The complete nucleotide sequence of cucumber green mottle mosaic virus (SH strain) genomic RNA. J Gen Virol 72, 14871495.[Abstract]
Urban, L. A., Ramsdell, D. C., Klonparens, K. L., Lynch, T. & Hancock, J. F. (1989). Detection of blueberry shoestring virus in xylem and phloem tissues of highbush blueberry. Phytopathology 79, 488493.
Van Bel, A. J. E. & Gamalei, Y. V. (1992). Ecophysiology of phloem loading in source leaves. Plant Cell Environ 15, 265270.
Verchot, J., Driskel, B. A., Zhu, Y., Hunger, R. M. & Littlefield, L. J. (2001). Evidence that soilborne wheat mosaic virus moves long distance through the xylem in wheat. Protoplasma 218, 5766.[Medline]
Wang, H. & Stubbs, G. (1994). Structure determination of cucumber green mottle mosaic virus by X-ray fiber diffraction. J Mol Biol 239, 371384.[CrossRef][Medline]
Wisniewski, L. A., Powell, P. A., Nelson, R. S. & Beachy, R. N. (1990). Local and systemic spread of tobacco mosaic virus in transgenic tobacco. Plant Cell 2, 559567.
Received 31 July 2003;
accepted 13 November 2003.