Ultrastructural studies of plasmodesmatal and vascular translocation of tobacco rattle virus (TRV) in tobacco and potato
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The studies focus on an ultrastructural analysis of the phenomenon of intercellular and systemic (vascular) transport of tobacco rattle virus (TRV) in tissues of the infected plants. TRV is a dangerous pathogen of cultivated and ornamental plants due to its wide range of plant hosts and continuous transmission by vectors—ectoparasitic nematodes. Two weeks after infection with the PSG strain of TRV, tobacco plants of the Samsun variety and potato plants of the Glada variety responded with spot surface necroses on inoculated leaf blades. Four weeks after the infection a typical systemic response was observed on tobacco and potato leaves, necroses on stems and lesions referred to as corky ringspot. Ultrastructural analysis revealed the presence of two types of TRV virions: capsidated and non-capsidated forms in tobacco and potato tissues. In the protoplast area, viral particles either occurred in a dispersed form or they formed organised inclusions of virions. We demonstrated for the first time the presence of non-capsidated-type TRV in the vicinity of and inside plasmodesmata. Capsidated particles of TRV were observed in intercellular spaces of the tissues of aboveground and underground organs. Expanded apoplast area was noted at the cell wall, with numerous dispersed non-capsidated-type TRV particles. These phenomena suggest active intercellular transport. Our ultrastructure studies showed for the first time that xylem can be a possible route of TRV systemic transport. We demonstrated that both capsidated and non-capsidated virions, of varied length, participate in long-distance transport. TRV virions were more often documented in xylem (tracheary elements and parenchyma) than in phloem. Non-capsidated TRV particles were observed inside tracheary elements in a dispersed form and in regular arrangements in potato and tobacco xylem. The presence of TRV virions inside the bordered pits was demonstrated in aboveground organs and in the root of the tested plants. We documented that both forms of TRV virions can be transported systemically via tracheary elements of xylem.
KeywordsIntercellular movement Plasmodesmata Tobravirus Vascular transport
Tobacco rattle virus (TRV) is a wide-spread pathogen in the plant world. Over 400 plant species from monocotyledon and dicotyledon classes can serve as hosts for the virus. The most common hosts are cultivated plants such as potato, sugar beet, tobacco and tomato (Allen 1963; Huth and Lesemann 1984). TRV belongs to the Tobravirus genus. It has a bipartite genome and it is made of two single strands of positive polarity (+)ssRNA. The genetic material undergoes separate encapsidation into simple, rod-shaped, helical capsids, with identical diameters of 22.5 nm. The virions of TRV vary in terms of length: longer particles (L) range from 180 to 197 nm and shorter particles (S) from 55 to 114 nm. Both types of virions have 5% RNA and 95% of protein. The virions of these two kinds fulfil different functions in the processes of viral infection and multiplication in a plant. The longer particle, containing RNA1, induces infection, whereas the shorter one, containing RNA2, is responsible for creating a coat protein (CP) for both strands (Bergh et al. 1985). If the plant is infected only by L particles, only RNA of these particles multiplies (Frost et al. 1967). Such TRV form was referred to as non-multiplying (NM). It is sensitive to ribonucleases and it is easily degradable in plant sap. Complete virions known as capsidated forms appear in the plant, when both types of TRV virions and/or their RNA are present (Harrison and Robinson 1978; MacFarlane 1999, 2010).
RNA1 has four open reading frames (ORF) which encode the proteins that participate in virus replication and transmission by vectors-nematodes, and it is believed that they also take part in cell-to-cell movement of the virus (Hamilton and Baulcombe 1989). RNA2 encodes coat protein and plays a part in the transmission by nematodes (protein 16 K) (Ploeg et al. 1993; MacFarlane and Brown 1995). The TRV strains have been classified and those isolated from potatoes that are relatively well described include: PRN (Scotland), strains from Oregon (Lister and Bracker 1969), and PSG—a strain isolated in the Netherlands and described for the first time by Cornelissen et al. (1986).
Current studies on TRV focus on strains and their recombinants that are transmitted mostly by nematodes of the Trichodorus and Paratrichodorus species. In our studies we used the method of mechanical inoculation—one of the basic possibilities of infection in field, greenhouse or laboratory conditions. The objective of these studies was to present the phenomenon of intercellular and vascular translocation of the PSG strain of TRV in potato and tobacco tissues at the ultrastructural level.
Materials and methods
Plants of Solanum tuberosum cv. Glada and Nicotiana tabacum cv. Samsun were grown in a growth chamber, at a temperature of 18°C and a 16-h light cycle with the intensity of 400 μmol m−2 s−1 PAR. Plants at four leaf stage were infected with the PSG strain of TRV. Plants were mechanically inoculated with TRV suspension using carborundum, in a 0.1 M phosphate buffer (pH 7.4). The virus isolate was received from Institute of Phytopathological Research, Wageningen (The Netherlands). Control plant material was inoculated only with phosphate buffer. The plants of tobacco cv. Samsun used as a material for infection, and the infected plants of potato were tested using the DAS-ELISA procedure at IHAR Młochów (according to Clark and Adams 1977, immunoglobulins received from D. Z. Maat, Wageningen, The Netherlands). The plant organs were collected (depending on symptoms) 2, 3 and 4 weeks after TRV infection. The investigations were repeated three times.
Analysis in transmission electron microscope (TEM)
The material was fixed in 2% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde in a 0.05 M cacodylate buffer (pH 7.2–7.4) (Karnovsky 1965) 2 h at room temperature. Next, the samples were contrasted and fixed in 2% (w/v) OsO4 in cacodylate buffer for 2 h at 4ºC. The material was rinsed with sodium cacodylate and then dehydrated in a series of increasingly strong water solutions of ethanol. The material was gradually saturated with resin Epon 812 (Fluka) and polymerized for 24 h at 60ºC. The ultrathin sections on copper grids were stained with uranyl acetate and lead citrate.
Observations were made using a Morgagni 268D (FEI) transmission electron microscope. Photographic documentation was prepared with a Morada (SIS) digital camera and the iTEM (SIS) computer programme.
Symptoms and anatomical changes
Intercellular TRV translocation
Mechanical infection with the PSG strain of TRV led to a systemic response of susceptible potato and tobacco plants. Symptoms developed in inoculated and non-inoculated leaves, in stems and potato tubers. Anatomic lesions occurred in all plant organs of the hosts. Apoplast/intercellular transport was observed in tissues of the aboveground and underground parts of plants. The Tobravirus 29 kDa MP is most closely related to the Tobamoviruses and share a common mechanism for cell-to-cell movement (Ziegler-Graff et al. 1991; Carrington et al. 1996). There has been very little research exploring the mechanism for Tobraviruses movement. However, because of the close relationships between Tobraviruses and Tobamoviruses, a model describing cell-to-cell movement has also been used to describe the mechanism for Tobravirus local transport. In this model, viruses move from cell to cell in the absence of CP. Viral movement proteins cooperatively bind viral nucleic acid forming a ribonucleoprotein complex that is transported through plasmodesma into adjacent cells.
Holeva and MacFarlane (2006) showed that RNA2-encoded protein 2b is essential for TRV transmission by nematodes, but it can be also responsible for physical interaction with coat protein. The PSG strain of TRV has RNA2, so it encodes protein 2b and CP and leads to the formation of completed and non-capsidated particles in tissues (L and S forms). While analysing transport of the GFP–2b–TRV construct, Valentine et al. (2004) showed that movement of the virus to non-inoculated leaves and roots of Arabidopsis thaliana and Nicotiana benthamiana is more efficient in the presence of protein 2b.
Our observations indicate that in the majority of cases the non-capsidated virions of TRV took part in the intercellular transport. This confirms the theory that Tobravirus TRV belongs to a small group of viruses that do not need capsid protein for transport (Swanson et al. 2002; MacFarlane 2010).
Our ultrastructural analysis of susceptible tobacco and potato plants shows that both capsidated and non-capsidated virions, of varied length, participate in long-distance transport. TRV particles were more often documented in xylem (tracheary elements and parenchyma) than in phloem. Observations inside the sieve element showed only the presence of non-capsidated particles. Our data suggest that non-capsidated type virions participate more often in transport from cell to cell than within the plant vascular system. Our observations confirm the conclusions of Swanson et al. (2002) regarding the transport of infection GFP–TRV and GFP–PEBV clones. Mutated viruses without coat protein were able to move systemically in N. benthamiana and N. clevelandii plants with the same speed as a wild-type virus. Moreover, virions without coat protein could also leave the conductive tissue in systemically infected leaves. Xylem transport by plant viruses was originally proposed in the studies of Sobemoviruses such as Rice yellow mottle virus (RYMV) and Blueberry shoestring virus (Opalka et al. 1998; Urban et al. 1989). In immunogold labelling studies using light and electron microscopy, Beet necrotic yellow vein virus (BNYVV) and Soilborne wheat mosaic virus (SBWMV) were each detected in xylem vessels or xylem parenchyma in infected plant roots (Dubois et al. 1994; Verchot et al. 2001). SBWMV inclusion bodies were also identified in xylem parenchyma and xylem vessels in infected wheat roots. Otulak and Garbaczewska (2010) detected Potato virus Y particles and capsid proteins in xylem tracheary elements and in xylem parenchyma in infected potato leaflets. RYMV and SBWMV have been detected in immature xylem elements prior to cell death. The virus is likely to move from cell to cell into immature xylem and then undergo replication. After programmed cell death, virus particles are released into the xylem and can move upward in the plant (Verchot et al. 2001; Verchot-Lubicz 2003). There is evidence that viruses may enter immature xylem elements, which have plasmodesmata connections with parenchyma cells. Thus, virus xylem loading would be developmentally regulated. We do not yet know if xylem transport is an essential component of viral long distance movement.
Grażyna Garbaczewska and Marcin Chouda designed and performed experiments, Katarzyna Otulak analysed data and prepared the manuscript, Mirosława Chrzanowska conceptual advice and instructed the experiments.
We thank inż. Ewa Znojek for expert technical assistance.
Conflict of interest
The authors declare that they have no conflict of interests.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
- Allen TC (1963) A strain of tobacco rattle virus from Oregon grown potatoes. Plant Dis Rep 47:920–923Google Scholar
- Karnovsky MJ (1965) A formaldehyde–glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27:137AGoogle Scholar
- Opalka N, Brugidou C, Bonneau C, Nicole M, Beachy RN, Yeager M, Fauquet C (1998) Movement of Rice yellow mottle virus between xylem cells through pit membrane. Proceedings of the National Academy of Science USA 95:3323–3328Google Scholar