Journal of Experimental Botany, Vol. 52, No. 354, pp. 173-177,
January 2001
© 2001 Oxford University Press
Short Communications |
Macromolecular trafficking between Nicotiana tabacum and the holoparasite Cuscuta reflexa
1 Unit of Cell Biology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
2 Lehrstuhl Botanik II, Molekulare Pflanzenphysiologie, Universität Erlangen-Nürnberg, D-91058 Erlangen, Germany
3 Martin-Luther-Universität, Halle-Wittenberg, Institut für Pflanzen- und Zellphysiologie, Weinberweg 10, D-06120 Halle/Saale, Germany
Received 2 May 2000; Accepted 5 September 2000
Abstract
Transgenic tobacco plants expressing green fluorescent protein (GFP) under the control of the companion cell-specific promoter, AtSUC2, were parasitized by the holoparasite Cuscuta reflexa (dodder). GFP, moving in the translocation stream of the host, was transferred to the Cuscuta phloem via the absorbing hyphae of the parasite. An identical pattern of transfer was observed for the phloem-mobile probe, carboxyfluorescein. Following uptake by the parasite, GFP was translocated and unloaded from the Cuscuta phloem in meristematic sink tissues. Contrary to published data, these observations suggest the presence of a functional symplastic pathway between Cuscuta and its hosts, and demonstrate a considerable capacity for macromolecular exchange between plant species.
Key words: Cuscuta, green fluorescent protein, macromolecular transport, Nicotiana, plasmodesmata, symplastic transport.
Introduction
The parasitic relationship of Cuscuta (dodder) with its hosts has been the subject of numerous investigations (De Bock and Fer, 1992
; Dörr, 1968; Haupt and Neumann, 1996; Israel et al., 1980; Jacob and Neumann, 1968; Jeschke and Hilpert, 1997; Rothe et al., 1999; Schumacher, 1934). Cuscuta has little photosynthetic activity and draws most of its nutrients from the host. For example, in parasitized faba bean plants Cuscuta is an extremely powerful competing sink for assimilates, and is capable of completely preventing fruit set and pod development (Wolswinkel, 1974).
During the infection process Cuscuta produces an haustorium, a highly elaborate adventitious root (Dawson et al., 1994) that penetrates the host tissue. From the tip of the haustorial cone, ‘searching hyphae’ grow extracellularly through host tissues in a manner similar to pollen tubes. On reaching individual sieve elements of the phloem the terminal hyphae cells differentiate into ‘absorbing hyphae’ (Dörr, 1969) which produce finger-like protrusions that surround the sieve element (Dörr, 1972). The conversion of a searching hypha into an absorbing hypha starts at the point of host sieve-tube attachment and advances towards the haustorial organ, eventually connecting with the haustorial sieve elements differentiating in the opposite direction. On the inside of the absorbing hypha cell the wall is thrown into numerous infoldings reminiscent of transfer cells while the cytoplasm develops a conspicuous smooth endoplasmic reticulum network (Dörr, 1990) resembling that found in the SE.
Although plasmodesmata occur between searching hyphae and parenchyma elements of the host cortex (Dörr, 1969), both plasmodesmata and sieve pores are thought to be absent between absorbing hyphae and sieve elements (Dörr, 1990), necessitating apoplastic transfer of solutes between the phloem systems of the host and the parasite at this interface (Tsivion, 1978; Wolswinkel, 1978). This transfer has been suggested to involve an active mechanism of solute release (Jeschke et al., 1994; Wolswinkel, 1974).
Given the apparent symplastic isolation of host sieve elements and absorbing hyphae, it appears paradoxical that Cuscuta is an effective vector for the transfer of several viruses (Bennett, 1944; Hosford, 1967; Roos and Aldrich, 1988; Schmelzer, 1958) and phytoplasmas (Heintz, 1989; Macrone et al., 1999). These studies suggest that a symplastic link occurs at some point in the host–Cuscuta interface to allow the passage of pathogenic RNAs, or RNA–protein complexes, from the phloem of one species to another.
Recently it was shown that GFP, synthesized in source tobacco companion cells under the control of the Arabidopsis SUC2 promoter, entered sieve elements and was translocated to, and unloaded within, sink tissues (Imlau et al., 1999; Oparka et al., 1999). In the transport phloem of the stem, in which the SE-CC complexes are virtually symplastically isolated, the GFP was restricted to the phloem (Oparka et al., 1999). As Nicotiana tabacum can be parasitized effectively with Cuscuta (Bennett, 1944), the authors investigated whether phloem-mobile GFP could be transferred to the phloem of Cuscuta at the sites of haustorial attachment. Here, an extensive transfer of GFP from host to parasite was demonstrated and it was shown that the protein is unloaded in sink tissues of Cuscuta. The pattern of GFP transfer was identical to the movement of the low-molecular-weight probe carboxyfluorescein, indicating a common symplastic pathway of transfer for both solutes and macromolecules. The data demonstrate a considerable capacity for macromolecular exchange between holoparasitic angiosperms and their hosts.
Materials and methods
Plant material
Plants of Nicotiana benthamiana and transgenic plants of Nicotiana tabacum expressing GFP under the control of the Arabidopsis thaliana sucrose transporter promoter (AtSUC2-GFP, see Imlau et al., 1999) were grown from seeds in a heated glasshouse and used for experiments when the plants were between 28 and 56-d-old. Cuscuta reflexa was cultivated on Vicia faba as a host under the same greenhouse conditions with 15 h light and 9 h darkness.
For experiments, Cuscuta shoots of 30–35 cm length were cut from the stock culture and carefully twisted around the stems or petioles of older source leaves. Parasitized plants were examined 16 d after infection with Cuscuta.
Phloem transport
Phloem transport between host and parasite was imaged on intact plants using the fluorescent probe carboxyfluorescein diacetate (CFDA), exactly as described earlier (Roberts et al., 1997). The plants were imaged after translocation in the light for between 1 h and 1.5 h.
Sectioning
Prior to confocal imaging parasitized petioles were cut free-hand into longitudinal or transverse sections through the middle of the haustorium. The sections were then mounted immediately in silicon oil and covered with a cover slip.
Confocal laser scanning microscopy (CLSM)
To image GFP, and to follow the movement of the fluorescent probe CFDA, a Bio-Rad MRC 1000 (Bio-Rad, Hemel Hempstead, UK) confocal laser scanning microscope (CLSM) was used. Both probes were excited by the 488 nm line produced by a 25 mW argon laser.
The individual petiole sections were ‘mapped’ using a Nikon X2 long working distance lens, and the images subsequently reconstructed using Photoshop® software (Adobe, Mountain View, CA).
Results and discussion
In the parasitic interaction between Cuscuta and its hosts, elongated ‘searching hyphae’ of the parasite penetrate the cortex and make contact with the host phloem. These ‘absorbing hyphae’ are intimately associated with sieve tubes, forming finger-like wall extensions that wrap around individual sieve elements (Dörr, 1972). When transgenic tobacco plants expressing AtSUC2-GFP were parasitized with Cuscuta, extensive movement of GFP was observed between the phloem of tobacco and that of Cuscuta at 14–16 d after attachment of the parasite (Fig. 1a, b). In addition, GFP was detected in the absorbing hyphae cells of the haustorial complex (Fig. 1b). Longitudinal sections of Cuscuta stem, taken above the point of attachment of the haustorium, revealed GFP to be restricted mainly to the phloem and to a limited extent to the neighbouring parenchyma tissues of the Cuscuta stem (Fig. 1d). Close to the apical meristem of Cuscuta considerable unloading of GFP was apparent, the protein moving from cell to cell throughout developing leaf primordia (Fig. 1f).
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When GFP is expressed in source companion cells of tobacco or Arabidopsis, it enters sieve elements and is translocated to sink regions of the plant where it is unloaded symplastically (Imlau et al., 1999
; Oparka et al., 1999). As the plasmodesmata in sink regions appear to be modified to allow the passage of small macromolecules (Fisher and Cash Clark, 2000; Oparka and Santa Cruz, 2000), the symplastic unloading of GFP in sink tissues of Cuscuta was not unexpected.
When the fluorescent probe CF was applied to source host leaves in ester form, the impermeant moiety was translocated to sink tissues, as shown previously (Knoblauch and Van Bel, 1998; Roberts et al., 1997; Wright and Oparka, 1996). In host plants parasitized by Cuscuta, the dye was unloaded extensively at the site of haustorial attachment and subsequently entered the stem phloem of Cuscuta (Fig. 1c
, e). When the haustorial complex was examined in longitudinal section, dye could be seen in the phloem of the host and also within the connecting absorbing hyphae of the parasite (Fig. 1c). A consistent feature was a lack of phloem labelling below the point of attachment of Cuscuta, suggesting that the translocating phloem of the host had been effectively ‘drained’ of solutes by the parasite (data not shown). In transverse sections, individual cell–cell contacts between the host SE-CC complexes and absorbing hyphae were apparent (Fig. 1e). In such cases dye continuity was apparent across the interface.
Because it is possible for some membrane-impermeant dyes to cross membranes (Oparka, 1991), the observations of dye transfer were considered to be equivocal evidence that a symplastic pathway was operating in the exchange of solutes between host and parasite. However, the unrestricted movement of GFP (27 kDa) from tobacco to Cuscuta provides strong evidence for a symplastic pathway between the absorbing hyphae of Cuscuta and the SE-CC complexes of the host. Ultrastructural studies of Cuscuta suggest that plasmodesmata are rare or absent at this interface (Dörr, 1972, 1990), making the extensive transfer of GFP observed here appear unusual. In other holoparasites, such as Orobanche, symplastic continuity is clearly established between the phloem of the host and parasite by direct linkage of sieve elements (Dörr and Kollmann, 1995). The observation that phloem-mobile GFP can be transferred from tobacco to Cuscuta suggests that a symplastic pathway is most probably utilized in the transport of macromolecules in this system. It seems likely that viral RNA is also transferred to other plants via this pathway (Bennett, 1944). While it remains possible that sucrose may be retrieved from the apoplast by absorbing hyphae (Jeschke et al., 1994; Wolswinkel, 1974), it seems unlikely that an apoplastic step is involved in the transport of GFP from the host SEs to the absorbing hyphae of Cuscuta for a number of reasons. First, the mature sieve elements of the host have a highly restricted endomembrane system and lack the vesicular machinery necessary to package and secrete GFP to the apoplast (Oparka and Turgeon, 1999). Second, GFP was not detected in the apoplast by confocal microscopy at any of the stages of the host–parasite interaction. At present, however, the possibility cannot be ruled out that the adjoining membranes of host SEs and absorbing hyphae are exceptionally permeable to a wide range of solutes and proteins at this contact point.
Although solute exchange between plant hosts and parasitic angiosperms has been studied extensively, little attention has been paid to the capacity for macromolecular exchange between plants via parasitic plant vectors. The capacity for GFP to exchange between Nicotiana and Cuscuta suggests that a wide range of proteins might exchange between plants of different species. The upper size exclusion limit for transport of macromolecules between Nicotiana and Cuscuta is currently being investigated.
Acknowledgments
This work was financially supported by the Scottish Executive Rural Affairs Department (SERAD) and by the county Sachsen-Anhalt (FKZ: 2213A/0085G).
Notes
4 To whom correspondence should be addressed. Fax: +49 345 5527095. E-mail: neumann{at}pflanzenphys.uni\|[hyphen]\|halle.de 
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