Laboratory of Virology, Department of Plant Sciences, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1
Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands2
Author for correspondence: Jan van Lent. Fax +31 317 484820. e-mail jan.vanlent{at}viro.dpw.wag-ur.nl
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Abstract |
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Introduction |
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Extensive studies over the past two decades have identified two major strategies for plant virus cell-to-cell movement through plasmodesmata. Tobacco mosaic virus (TMV) utilizes one strategy, wherein the virus moves in a non-virion form in the absence of coat protein (CP) through plasmodesmata modified by the viral MP (reviewed by Carrington et al., 1996 ). A second strategy is exemplified by Cowpea mosaic virus (CPMV), wherein mature virions are transported through virus-induced tubules that cross the walls of adjacent cells (Wellink & Van Kammen, 1989
; Van Lent et al., 1990
, 1991
; Kasteel et al., 1993
). Several viruses, e.g. potexviruses, need the CP for cell-to-cell movement, but its exact role has not yet been established (Chapman et al., 1992
; Foster et al., 1992
, Oparka et al., 1996
).
CPMV (reviewed by Goldbach & Wellink, 1996 ) represents a large group of different plant viruses, including comoviruses (Van Lent et al., 1990
, 1991
), nepoviruses (Wieczorek & Sanfaçon, 1993
; Ritzenthaler et al., 1995
), caulimoviruses (Perbal et al., 1993
) and tospoviruses (Storms et al., 1995
), that employ the tubule-guided movement mechanism of virions. No information, however, is available on how these viruses are loaded into and unloaded from the plant vascular tissue and which classes of veins are involved in these processes. Other relevant questions are whether entry into or exit from the sieve element by CPMV also involves a tubule-guided mechanism and in which form the virus is loaded or unloaded (i.e. virion or ribonucleoprotein).
The vascular loading and unloading of several plant viruses have been demonstrated to occur in different patterns in different host-virus systems (e.g. Cheng et al., 2000 ; Roberts et al., 1997
; Sudarshana et al., 1998
). Since CPMV represents a group of plant viruses with a different cell-to-cell movement strategy, we investigated its vascular loading and unloading characteristics in cowpea (Vigna unguiculata) at the macroscopic and microscopic levels. To facilitate this, the green fluorescent protein (GFP) gene was inserted in the CPMV RNA-2 coding region to act as a reporter for virus infection and spread. GFP-expressing recombinant viruses were used to determine the preferred sites (vein classes) for virus loading and unloading. Moreover, veins actively involved in CPMV loading and unloading were analysed for virus pathology at the cellular level.
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Methods |
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Viral recombinants and inoculations.
Two CPMV recombinant viruses, M19GFP7 and M19GFP2A, expressing the green fluorescent protein (GFP) through different strategies (Fig. 1) were described previously (Gopinath et al., 2000
). To obtain these recombinants, the GFP gene was inserted into the viral RNA-2 segment within in vitro transcription vectors containing the T7 polymerase promoter. Infectious RNA copies of these constructs or a construct containing no GFP sequence (i.e. wild-type viral RNA) were made by in vitro transcription of the DNA templates. Plasmid DNA templates were purified with Midiprep columns (Qiagen). In vitro transcription was carried out in 20 µl reactions using T7 RNA polymerase (Gibco BRL). Each reaction contained 400500 ng of template DNA, 20 units of RNase inhibitor (RNasin, Gibco BRL), 10 units of ClaI to linearize the DNA, 1·25 mM of each rNTP (Promega, 25 mM each), 25 units of T7 RNA polymerase and its buffer at appropriate final concentration as suggested by the manufacturer. The reactions were incubated at 37 °C for 11·5 h. RNA quantity and quality were checked on agarose gels. In vitro transcripts were kept at -20 °C until used as inoculum.
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Alternatively, recombinant virus inoculum was obtained by inoculation of protoplasts with the transcripts. For this, aliquots of 1x106 protoplasts were inoculated with 5 µg each of RNA-1 and RNA-2 in vitro transcripts using polyethylene glycol (PEG mol. mass 6000) as described by Van Bokhoven et al. (1993 ). The protoplasts were then incubated under continuous illumination at 25 °C for 48 h and observed for infection using a Zeiss LSM510 laser-scanning microscope. The protoplasts were pelleted and pellets were kept at -20 °C until their use as an inoculum or, for immediate inoculation, 150 µl of PBS was added to the pellets and the protoplasts were disrupted by repeated resuspension through a syringe with a large gauge needle.
Electron microscopy.
Plant tissues were fixed with 3% (w/v) glutaraldehyde2% (w/v) paraformaldehyde, 1% (w/v) osmium tetroxide and 1% (w/v) uranyl acetate, dehydrated in ethanol and embedded in London Resin White (LR White, Hard Grade; Electron Microscopy Sciences) essentially as described by Van Lent & Verduin (1987 ). Ultra-thin sections, 70 nm thick, were cut with a diamond knife (Diatome). Prior to gold labelling, sections were treated for 1 h with a saturated solution of sodium metaperiodate (Bendayan & Zollinger, 1983
) and washed with distilled water. Immunogold labelling with 10 nm protein Agold complexes was performed essentially as described by Van Lent & Verduin (1986
), using rabbit primary antibodies to CPMV particles (Van Lent et al., 1991
), the viral 24 kDa protease (Wellink et al., 1987a
) and MP (Wellink et al., 1987b
). The gold particles were then enlarged by a silver enhancement using R-GENT SE-LM reagents (Aurion) as suggested by the manufacturer. Finally, sections were stained for 5 min with 2% (w/v) uranyl acetate and for 1 min with lead citrate (Reynolds, 1963
). Specimens were observed with a Philips CM12 transmission electron microscope.
Surgical isolation procedure.
The surgical isolation of lamina flaps and midveins of cowpea primary leaves was done essentially as described by Cheng et al. (2000 ) (see also Fig. 3
). Plants were inoculated the day after surgical isolation of leaf flaps and midveins (Class I veins) in order to allow the isolated part to recover. The Carborundum-dusted lamina flaps and isolated midveins were pinpoint inoculated with infected leaf extract using a flamed Pasteur pipette as described by Wisniewski et al. (1990
) and Cheng et al. (2000
). Each inoculated leaf contained 510 surgically isolated flaps or one isolated midvein. Only plants with at least one successful pinpoint inoculated spot (based on GFP fluorescence) were included in the experiment. Plants with spots that were fluorescing beyond the cut edge of the flap, or beyond the length of isolated midvein, were discarded. The surgically isolated flaps were detached from the leaf 4 days post-inoculation (p.i.). Experiments were performed twice each for lamina flaps and midveins.
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Results |
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Kinetics of CPMV systemic spread in cowpea plants
The effect of the developmental stage of cowpea plants on vascular loading and unloading of CPMV was examined. For this, the primary leaves of plants of different developmental stages were inoculated with M19GFP2A or M19GFP7 recombinant viruses and screened for systemic infection by means of GFP fluorescence at 14 days p.i. (data not shown). Both viruses accumulated in the inoculated leaves regardless of the developmental stage of the plant at the time of inoculation. All tissues of plants were systemically invaded only when inoculated at an early developmental stage, i.e. when the first trifoliate leaf was still folded. On the contrary, when plants of later developmental stages (i.e. second trifoliate leaf present, no third trifoliate leaf) were inoculated, CPMV failed to accumulate in the first trifoliate leaf, but was unloaded and accumulated in the younger developing upper parts of the plant. Plants already having the third trifoliate leaf at the time of inoculation supported the replication of CPMV, but no systemic accumulation of the virus was observed. These results demonstrate that the developmental stage of the plant affects CPMV vascular-mediated accumulation in cowpea.
To determine the kinetics of CPMV unloading and systemic accumulation, primary leaves of cowpea plants were inoculated with M19GFP7 recombinant virus. The plants used were in a developmental stage permissive to complete systemic infection of cowpea, i.e. the first trifoliate leaf was still folded (9 days post-sowing). Plant parts were screened for virus infection at daily intervals for 14 days (three plants observed per day) (Table 1; Fig. 2
). Infection was first observed in the inoculated leaf at 2 days p.i. by the appearance of fluorescent spots which increased in number and size in the following days (Table 1
; Fig. 2d
, 2d'
and 2d'
). Systemic spread was first recorded in the stem below the inoculated leaf (Table 1
) and in the root (Table 1
; Fig. 2a
and 2a'
) at 4 days p.i., and infection developed extensively in those tissues over the following days (Table 1
; Fig. 2b
, 2b'
, 2b'
, 2a'
and 2a'''
). Remarkably, CPMV was initially transported through the petiole of the primary leaf straight to the stem and roots below the primary leaf without being unloaded in the petiole itself (Table 1
; 4 days p.i.). Only after 5 days p.i. was infection of this petiole observed (Table 1
; Fig. 2c
, 2c'
, 2c'
and 2c'''
). Initially, unloading/establishment of infection did not occur in the stem above the primary leaf or in the petiole and petiolule of first trifoliate leaf, but virus was unloaded in the first trifoliate leaf at 5 days p.i. (Table 1
; Fig. 2h
). The petiole of the first trifoliate leaf showed fluorescence only after 6 days p.i. (Table 1
; Fig. 2f
and 2f'
), whereas infection of tissues in the stem above the primary leaf and in the petiolule of the first trifoliate leaf were first observed, respectively, 10 days p.i. (Fig. 2e
) and 11 days p.i. (Fig. 2g
) and onwards (Table 1
; Fig. 2e'
and 2g'
). Similar to the first trifoliate leaf, the second trifoliate leaf was infected prior (10 days p.i. onwards; Table 1
; Fig. 2l
and 2l'
) to its petiole (13 days p.i. onwards, Table 1
; Fig. 2j
and 2j'
). Unloading and infection within the stem between the first and second trifoliate leaves and the petiolule of the second trifoliate leaf did not occur in the time-span of the experiment (Table 1
: i and k). These results demonstrate that CPMV was unloaded and accumulated first in the developing parts of the plant, which are the strongest sink tissues.
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CPMV is loaded into minor and major veins
For detailed cytological studies of CPMV vascular loading and unloading, it was essential to first establish which classes of veins were involved in these processes. The organization of the veinal structure of cowpea leaves was visualized by labelling the xylem with Texas Red dextran (Fig. 3a). The veinal network of cowpea plants is organized into a successive branching of veins (Hickey, 1979
). From the class I midvein the class II and III veins branch successively (all major veins). Class III veins occur in areoles, inside which minor veins (class IV and V) are present.
Conventional inoculation of GFP-expressing recombinants and subsequent observation of local spread by fluorescence microscopy did not reveal any clue as to the class of veins involved in CPMV loading (Fig. 3b). To establish whether there were preferred sites of virus loading, minor veins and major veins were selectively inoculated by means of the surgical isolation procedure described by Cheng et al. (2000
). Leaf lamina flaps containing only minor veins were surgically isolated from the surrounding major veins with the exception of one side that was left attached to a single class II or class III (major) vein (Fig. 3e
). The loading capability of the minor veins was determined by pinpoint-inoculation of the isolated leaf lamina flaps with GFP-recombinant virus. When local spread of CPMV was established (recorded as a fluorescent spot) but the infected area had not yet reached the connected major vein (Fig. 3f
), the flaps were detached completely. The plants were then monitored for systemic infection, indicative of successful virus loading into minor veins, during the following 14 days. Similarly, the loading capability of major veins was studied by pinpoint inoculation of a surgically isolated midvein (Fig. 3c
and 3d
). Based on the kinetics of CPMV systemic spread reported above, cowpea plants used for the surgical isolation procedure were inoculated at a developmental stage permissive for virus unloading in the first trifoliate leaf and leaf lamina flaps/midveins were removed 4 days p.i. when the virus had entered the stem. The plants were screened for systemic infection of first trifoliate leaves at 14 days p.i. For each plant, 5 to 10 leaf lamina flaps were inoculated with M19GFP7. After removing the inoculated flaps (4 days p.i.), each flap was examined for local infection in a confocal microscope (Fig. 3g
and 3h
). If virus infection on any flap had reached the fresh cut boundary (the site where the flap was attached to the class II vein) the plant was excluded from the experiment. Similar criteria were maintained for pinpoint-inoculation of surgically isolated midveins. The results of these experiments are summarized in Table 2
and demonstrate that CPMV can be loaded into minor veins of cowpea leaves, since approximately one-third of the locally infected plants (3 out of 10) became systemically infected. Comparable results were obtained when isolated midveins were pinpoint inoculated as 5 out of 7 locally infected plants became systemically infected after inoculating CPMV onto the isolated midvein (Table 2
). These results show that CPMV can be loaded into both minor veins and major veins of cowpea primary leaves to establish systemic infection.
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From each of the three samples of primary leaves and three samples of secondary leaves, five series of five sections were cut, each series at a distance of at least 20 µm from the previous. So in total approximately 75 sections from loading veins (class III and IV) and 75 sections from unloading veins (class III) were analysed, including an estimated 15 different sieve elementcompanion cell complexes for each. As an example, a primary leaf class IV vein screened for CPMV infection is shown in Fig. 5(a) and 5(b)
. In veins of both primary (source) and trifoliate (sink) leaves, plasmodesmata between bundle sheath cells (BSC) and phloem parenchyma cells (PPC), PPCPPC (data not shown) and companion cell (CC)PPC were either linear (Fig. 5e
) or branched (Fig. 5f
). Connections between sieve element (SE) and CC, in both source and sink leaves, showed the typical structure of a so-called pore-plasmodesma unit (PPU; Van Bel & Kempers, 1997
), with a single pore on the SE cell wall and branching towards the adjacent CC (Fig. 5c
and 5d
). Sometimes, SEs showed plasmodesmal connections with more than one CC (Fig. 5c
). Apparently, in cowpea plants the SEs are not symplasmically connected to cell types other than the CC (Fig. 5b
and data not shown).
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Discussion |
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By means of a surgical isolation procedure for leaf parts and pinpoint-inoculation of virus it was demonstrated that CPMV can be loaded into the phloem of both major veins and minor veins to establish systemic infection of the upper leaves. Three possible routes for entry of virus into leaf veins have been suggested (Ding et al., 1998 ; Nelson & Van Bel, 1998
). Viruses could enter the veins at the vein terminus, a gap at a vein branch or the side of a vein. The successful systemic invasion of cowpea after pinpoint-inoculation of isolated midveins suggests that CPMV is able to approach and enter the phloem stream directly from the surrounding parenchyma tissues. Studies on virus loading into plant vascular tissue are very limited. Recently, Cheng et al. (2000
) showed that TMV is loaded into minor and major veins and is able to approach and enter the midvein phloem stream directly from the surrounding parenchyma tissues. Although plant viruses apparently can be loaded into both major and minor veins, several studies suggest that minor veins are the preferred sites for photosynthate and possibly also for virus loading (reviewed in Nelson & Van Bel, 1998
).
After phloem transport the virus exits exclusively from major veins and preferentially from the class III veins in the first trifoliate leaves, as over 90% of the fluorescent foci (indicative of CPMV infection) were located adjacent to this vein type. With respect to the preferred sites of phloem unloading and accumulation, CPMV in cowpea shows a similar pattern to that of TMV (Cheng et al., 2000 ), the potyvirus Tobacco etch virus (TEV; Oparka & Santa Cruz, 2000
) and the potexvirus Potato X virus (PVX; Roberts et al., 1997
) in Nicotiana benthamiana. Remarkably, a diverse range of phloem-transported compounds such as radioactive solutes, GFP and systemic RNA signals all exit the phloem exclusively from major veins (reviewed in Oparka & Santa Cruz, 2000
), suggesting that the vein classes used for solute and macromolecule unloading are equally involved in unloading of many plant viruses. Although plant viruses (CPMV, TMV, TEV and PVX) with different mechanisms of cell-to-cell movement show the same vein preference for unloading and accumulation, this does not imply a similarity in the mechanism of unloading at the cellular level.
Careful inspection of serial sections from loading sites in class III/IV veins and unloading sites in class III veins showed a remarkable absence of CPMV replication (absence of cytopathic structures and viral antigens) in the CC of these vein types. Also, no virions or viral antigens were detected in SEs. However, CPMV replication clearly occurred in the PPC and BSC, besides the epidermal and mesophyll cells. The absence of CPMV replication in CCs in source and sink leaves cannot be explained by symplasmic isolation of the CCSE complex, as plasmodesmata, though never observed between PPCSE, were found at PPCCC, CCSE as well as at MCBSC, BSCPPC and PPCPPC interfaces. The symplasmic connection between SE via CC with surrounding vascular cells suggests a role of the CC in loading and unloading of photosynthate and also CPMV in cowpea. Absence of virus infection in CCs in inoculated source leaves was observed for the tobamovirus Sunn-hemp mosaic virus (SHMV) in Phaseolus vulgaris and Pisum sativum (Ding et al., 1998 ). For the potyviruses Potato Y virus (PVY) and Peanut stripe virus (PStV) in N. benthamiana, as well as for TMV in N. benthamiana, Capsicum annuum and Lycopersicon esculentum, a preferred infection of vascular parenchyma cells (relative to CC) was found in mature source leaves (Ding et al., 1998
). It was suggested that some viruses exploit the plasmodesmata between SE and PPC to gain access to the phloem, rather than entering the CCs directly. Considering that no plasmodesmata were ever found between PPC and SE in source leaf cowpea veins, this loading route is less likely for CPMV in this particular host.
For the cucomovirus Cucumber mosaic virus (CMV), another spherical virus that is able to form tubules (Canto & Palukaitis, 1999 ), Blackman et al. (1998
) reported the presence of virus particles in mature sieve elements in source leaves of N. clevelandii. The particles appeared in a membrane-bound viral assembly complex (VAC). Moreover, it was postulated that before CMV is loaded into the SE, virus particles disassemble in the cytoplasm of CC, move through the PPU as a ribonucleoprotein complex and reassemble in the SE (Blackman et al., 1998
). The fact that no virus was detected in CC could indicate that CPMV might be loaded from CC into SE in a non-virion form.
CPMV cellular localization in unloading vascular tissue differs from that of PVX (Roberts et al., 1997 ), BDMV (Wang et al., 1996
) and SHMV (Ding et al., 1998
), which were detected in the CC of sink leaves of systemically infected plants. PVX was detected in CC and occasionally in immature SE of N. benthamiana sink leaf veins, but vascular parenchyma cells were more heavily infected than CCs. BDMV was detected in CCs of systemically infected P. vulgaris leaves, but not in SEs. In systemically infected leaves of P. sativum, SHMV viral aggregates were detected in both vascular parenchyma cells and CCs of minor veins. In contrast to what is known for PVX in tobacco, BDMV in bean and SHMV in pea plants, CPMV was apparently unloaded from cowpea leaf veins without replicating in the CC.
The observation of virion-containing tubules in plasmodesmata between the MCBSC, BSCPPC and PPCPPC interfaces in unloading veins shows that CPMV is capable of moving through some phloem cells by means of the well-described mechanism of tubule-guided cell-to-cell movement. Interestingly, tubular structures or virus particles were never observed in the PPU connecting SECC of cowpea-infected leaves, in source or in sink tissues. The presence of virus particles in the cavity of PPUs was reported for the luteoviruses Carrot red leaf virus in Anthriscus cerefolium (Murant & Roberts, 1979 ) and Potato leafroll virus in potato (Shepardson et al., 1980
), and for the polerovirus Beet western yellows virus in sugarbeet (Esau & Hoefert, 1972
) in Thlaspi arvense (DArcy & Zoeten, 1979
) and in N. clevelandii (Mutterer et al., 1999
). Several studies have indicated that PPUs may allow the passage of large molecules (Kempers et al., 1993
; Kempers & Van Bel, 1997
; Van Bel, 1996
; Turgeon, 2000
). For the monocot Triticum aestivum, plasmodesmal channels involved in SE/CC unloading can be exceptionally large with a physical diameter of as much as 42 nm (Fisher & Cash-Clark, 2000
). Since the PPUs in several plants have a large size exclusion limit, it might be possible that CPMV is loaded into and/or unloaded from cowpea veins without gating or modifying the PPU. Whether the phloem loading and unloading of CPMV involves transportation of a virion or ribonucleoprotein complex, and whether the MP or other viral proteins play a role in this process, remain to be determined.
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Acknowledgments |
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References |
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Received 14 November 2001;
accepted 11 February 2002.