1 Laboratory of Virology, Wageningen University, The Netherlands
2 Laboratory of Molecular Recognition and Antibody Technology, Wageningen University, The Netherlands
Correspondence
Marcel Prins
marcel.prins{at}wur.nl
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ABSTRACT |
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Fig. 3 is available in colour as supplementary material in JGV Online.
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INTRODUCTION |
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In the archetypical plantibody resistance paper (Tavladoraki et al., 1993), the authors demonstrated reduced virus accumulation and delay of symptom expression in plants expressing a hybridoma-derived single-chain variable antibody fragment (scFv) targeted against the coat protein of the tombusvirus Artichoke mottled crinkle virus. The mechanism by which the antibody conferred this partial resistance remained unsolved. Similarly, delay and suppression of virus symptoms were later observed against Beet necrotic yellow vein virus (Fecker et al., 1996
) and Tobacco mosaic virus (Schillberg et al., 2000
). These studies used either full-sized or recombined fragments of antibodies from hybridoma cell lines. As an alternative, the expression of antibody fragments in a phage display format (e.g. Vaughan et al., 1996
) permits rapid antibody cloning and gives great flexibility in selecting and modifying specific antibodies and, in the case of synthetic libraries, avoids the use of animals. Several successful examples of the use of phage display technology for the generation of virus-specific antibodies have been described (Ziegler et al., 1995
; Fecker et al., 1996
; Harper et al., 1997
; Boonham & Barker, 1998
; Griep et al., 2000
). Expression levels of single-chain antibodies are generally high when proteins are excreted into the apoplast by including signal sequences to the transformation construct (Owen et al., 1992
), and even higher when retained in the endoplasmic reticulum (ER) by the C-terminal addition of the KDEL retention tetrapeptide (Conrad & Fiedler, 1998
). However, expression of scFvs in the cytoplasm of plants, where the vast majority of plant viruses replicate, has long been troublesome. In an excellent recent paper, Boonrod et al. (2004)
selectively developed single-chain antibody fragments targeting the conserved replicase of TBSV.
The negative-strand Tospoviruses were the subject of this study. These viruses form a distinct genus of phytopathogenic viruses within the arthropod-borne family Bunyaviridae (Elliott et al., 2000), which is further restricted to animals. The genome of Tomato spotted wilt virus (TSWV), economically the most important tospovirus, consists of three RNA species (de Haan et al., 1990
, 1991
; Kormelink et al., 1992
). The five viral open reading frames specify a total of six mature viral proteins. These are the nucleoprotein (N); two envelope glycoproteins (G1 and G2); the viral polymerase (L); and two non-structural proteins, NSM, the viral movement protein (Storms et al., 1995
), and NSS, which is involved in suppression of RNA silencing (Bucher et al., 2003
). In natural infections, tospoviruses enter the plant cell during probing or feeding by viruliferous thrips. Upon entering the cell, the virus is relieved of its membrane and infectious nucleocapsids are released into the cytoplasm. At this stage, the viral RNA will be either transcribed or replicated. The transcription-to-replication switch is thought to be controlled by the concentration of free N protein in the cytoplasm. At low N protein concentrations, i.e. at the onset of infection, the replicase will produce viral mRNAs, by means of cap snatching (Duijsings et al., 2001
). Following translation of the N protein and its increased concentration, the polymerase switches to replicase mode at which time viral genomic RNAs are multiplied and associate with the N protein into nucleocapsids (Prins & Goldbach, 1998
). The viral replicase is important in these processes, but the N protein also plays a key role. In addition, this protein is involved in several later processes such as viral RNA packaging into nucleocapsids, cell-to-cell movement to neighbouring cells through virus-induced tubules (Storms et al., 1995
) and the formation of new virus particles by associating with viral glycoproteins at the Golgi membranes (Kikkert et al., 1999
). Early steps in the tospovirus replication cycle in plants are prime candidates for targeting by protective plant-expressed antibodies, as in these conditions the stoichiometry of the antibody versus its target antigen is most favourable. Hence the switch between replication and transcription, as well as both processes per se, are the Achilles heel of a successful virus infection and the target of the approach used here.
In our previous work (Griep et al., 2000), we isolated and characterized 12 scFv clones targeting the TSWV N gene product. These scFvs were derived from a phage display library (Vaughan et al., 1996
). Four of these scFv clones (N3, N19, N56 and N97) not only cross-reacted with TSWV, but also reacted with the related tospoviruses Tomato chlorotic spot virus (TCSV) and Groundnut ringspot virus (GRSV). As these monoclonal single-chain antibodies also recognize other tospoviruses, we reasoned that they might target conserved, essential epitopes within the N protein, making them excellent candidates for conferring resistance to transgenic plants.
In this study, we aspired to combine virological knowledge on the infection cycle of tospoviruses in plants with optimized cytosolic stability of scFvs. This was achieved using scFvs derived from a phage display library and by adding the KDEL tetrapeptide. Transgenic plants expressing these scFvs were produced and tested for virus resistance.
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METHODS |
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Transformation of tobacco.
All TSWV sequences containing pBIN19-derived vectors were introduced into Agrobacterium tumefaciens strain LB4404 (Ditta et al., 1980) by triparental mating, using pRK2013 (Horsch et al., 1985
) as a helper plasmid. N. benthamiana plants were transformed and regenerated as described by Horsch et al. (1985)
.
Western blot analysis of scFv expression in transgenic plants.
Transgenic plant lines were checked for expression levels of transgenic scFvs by Western blot analysis of leaf material. For this purpose, leaf disks of transgenic plants collected prior to virus inoculation were ground in an equal volume of PBS and boiled in dithiothreitol (DTT)-containing protein-loading buffer (50 mM Tris/HCl pH 6·8, 100 mM DTT, 2 % SDS, 10 % glycerol, 0·01 % bromophenol blue). Proteins were separated on a 12 % polyacrylamide gel and transferred to Immobilon membranes. Membranes were blocked overnight in 3 % skimmed milk powder in PBS. Transgenic (scFv) protein production could be monitored due to the C-terminal addition of the cMyc epitope. This epitope was recognized using the 9E10 monoclonal antibody (Munro & Pelham, 1986). Goat anti-mouse antibodies conjugated to alkaline phosphatase were applied in the second-round incubation; visualization was done by the addition of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
Tospovirus resistance assays in plants and protoplasts.
Inoculation of tospoviruses and ELISA tests were performed using standard conditions as first described by Gielen et al. (1991). Isolation of protoplasts and inoculation with TSWV were according to Prins et al. (1997)
.
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RESULTS |
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DISCUSSION |
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Resistance against TSWV was obtained in seven of the 23 lines, expressing either scFv N3 or N56 but not N97. Resistance was only observed in those transgenic plants that expressed high levels of scFv, but in addition depended on the significance of the interaction between the antibody and antigen. Even though lines expressing different scFvs had similar expression levels (e.g. N3-1, N56-7 and N97-3; Fig. 2), only the first two exhibited resistance. Apparently, binding of scFv N3 and N56 interfered with important functions of the N protein, such as replication of the virus, while binding of N97 did not. This showed that binding of an antibody to a viral protein, even when expressed at high levels, does not automatically imply effectiveness against the pathogen. It cannot be ruled out that the N97-targeted site in the N protein plays an important role in later phases of the infection cycle such as cell-to-cell transport or virus transmission, in which the N protein also plays a key role. During these phases, however, the high level of N protein produced by the replicating virus may saturate the expressed antibodies, thereby enabling the virus to escape their action. Protoplasts isolated from resistant plants of lines N56-1 and N56-7 completely failed to support TSWV replication and the production of N protein was completely blocked. As no N protein was produced at all in these plant cells (Fig. 4
), this suggested that the very first step in the virus life cycle, viral transcription, may be blocked by the binding of the N56 scFv, for example by interfering with the cap-snatching process (Duijsings et al., 2001
). Plants expressing the N3 scFv were also highly resistant to the virus; however, at the single-cell level limited production of N protein was observed, albeit at levels much lower than in infected protoplasts of wild-type plants. The mode of operation of the N3 scFv may therefore be different from that of N56. Since a limited amount of N protein was produced in N3 plants, this could suggest that viral mRNAs are produced from inoculated viral RNAs, but that replication of the viral RNAs may be blocked. As no extra viral RNAs become available for transcription, the amount of mRNA remains low and only limited quantities of viral proteins are produced. These amounts are insufficient to complete the infection process and thus also lead to a virus-resistant phenotype on a whole-plant level. To increase the durability of resistance in practical applications, the two resistance-conferring antibodies N3 and N56 could be combined. As both antibodies are likely to target different epitopes, it will be more difficult for the virus to mutate in such a way that recognition by the two antibodies can be prevented.
In addition to the experiments using anti-N scFvs described here, we also expressed several phage display-isolated single-chain antibodies targeting viral glycoproteins in an attempt to block virus transmission by thrips. Despite high expression of anti-G antibodies in the ER, where virus particles accumulate prior to uptake by thrips, at levels comparable to those of the N3 and N56 antibodies in resistant lines, no inhibition of virus transmission was observed (P. Maris and M. Prins, unpublished results). These findings indicate that the stoichiometry of the antigenantibody interaction may play an important role in the successful application of plantibody-mediated resistance and it is therefore paramount that an early phase of the virus life cycle is targeted when amounts of viral proteins in the cell are still low.
As was shown here, antibodies can be targeted to the cytoplasm and produce effective virus resistance. Molecular tools are available to target these proteins to other parts of the plant cell, such as the ER, Golgi apparatus or apoplast. This broadens the scope of use of scFv proteins to other types of plant pathogens, such as fungi, bacteria and nematodes. Nematodes, for example, can be targeted by neutralizing essential excretion products (Popeijus et al., 2000; Smant et al., 1998
) in either the cytoplasm or the extracellular space. As for viruses, using phage display-derived plantibodies to target proteins that are essential for early phases of the pathogenicity process could be crucial for successful applications in future resistance strategies.
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ACKNOWLEDGEMENTS |
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Received 9 February 2005;
accepted 16 March 2005.
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